Antibacterial Lectins: Action Mechanisms, Defensive Roles, and Biotechnological Potential

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Antibacterial Lectins: Action Mechanisms, Defensive Roles, and Biotechnological Potential.

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CHEMICAL ENGINEERING METHODS AND TECHNOLOGY

ANTIBACTERIALS

SYNTHESIS, PROPERTIES AND BIOLOGICAL ACTIVITIES

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CHEMICAL ENGINEERING METHODS AND TECHNOLOGY

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CHEMICAL ENGINEERING METHODS AND TECHNOLOGY

ANTIBACTERIALS

SYNTHESIS, PROPERTIES AND BIOLOGICAL ACTIVITIES

ERIKA COLLINS EDITOR

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Names: Collins, Erika, editor. Title: Antibacterials: synthesis, properties and biological activities / editor, Erika Collins. Description: Hauppauge, New York: Nova Science Publisher's, Inc., [2016] | Series: Chemical engineering methods and technology | Includes index. Identifiers: LCCN 2016029817 (print) | LCCN 2016031928 (ebook) | ISBN 9781634857932 (hardcover) | ISBN 9781634858014 (ebook) | ISBN 9781634858014 (eBook) Subjects: LCSH: Antibacterial agents. Classification: LCC RM409 .A577 2016 (print) | LCC RM409 (ebook) | DDC 615.7/922--dc23 LC record available at https://lccn.loc.gov/2016029817

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CONTENTS

Preface vii Chapter 1 Synthesis and Properties of Biomass-Based Nanomaterials for Antibacterial Applications 1 Yan-Yan Dong, Ming-Guo Ma and Xue-Ming Zhang Chapter 2 Smart Metal Nanostructures for Effective Bacterial Inhibition 27 Jakub Siegel, Oleksiy Lyutakov, Vladimíra Vosmanská, Markéta Pišlová, Markéta Polívková, Yevgeniya Kalachyova and Václav Švorčík Chapter 3 Antibacterial Lectins: Action Mechanisms, Defensive Roles and Biotechnological Potential 69 Thamara F. Procópio, Maiara C. Moura, Lidiane P. Albuquerque, Francis S. Gomes, Nataly D. L. Santos, Luana C. B. B. Coelho, Emmanuel V. Pontual, Patrícia M. G. Paiva and Thiago H. Napoleão Chapter 4 Natural Antibacterials for Technical Applications 91 Thomas Grethe, Charlotte Vorneweg, Hajo Haase and Boris Mahltig Chapter 5 Antimicrobial Activities of Natural Products from Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz var. ferrea 115 Fernando Gomes Figueredo, Tania Maria Sarmento da Silva, Celso de Amorim Camara, Girliane Regina da Silva, Maria de Fátima Agra, Maynara Rodrigues Cavalcante, Jakson Gomes Figueiredo, Natalia Bitu Pinto, Rafael de Carvalho Mendes, Edinardo Fagner Ferreira Matias, Francisco Antônio Vieira dos Santos, Henrique Douglas Melo Coutinho and Marta Maria de França Fonteles

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Chapter 6 Redox-Active Metal Complexes with Cycloaminomethyl Derivatives of Diphenols: Antibacterial and SOD-Like Activity, Reduction of Cytochrome c 143 N. V. Loginova, H. I. Harbatsevich, T. V. Koval’chuk, N. P. Osipovich, Y. S. Halauko, Y. V. Faletrov and A. T. Gres Chapter 7 A 21st Century Contribution to the Antibacterial Armamentarium: A Medicinal Chemist’s Perspective 181 Stefano Biondi and Mauro Panunzio Index 269

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PREFACE

This book examines the synthesis, properties and biological activities of antibacterials. Chapter One focuses on the recent development of biomass antibacterials. Chapter Two provides comprehensive overview on smart materials based on structured noble metals covering its various forms from ultrathin layers over discrete metal islands up to isolated nanoparticles having exceptional properties applicable in prevention of bacterial infections. Chapter Three discusses general aspects regarding lectins and their purification and then reviews the state-of-art on antibacterial lectins, their action mechanisms, defensive roles and current efforts regarding their effective biotechnological application. Chapter Four reviews natural antibacterials for technical applications. Chapter Five studies antimicrobial activities of natural products from Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz var. ferrea. Chapter Six discusses redox-active metal complexes with cycloaminomethyl derivatives of diphenols. Chapter Seven analyzes recent progress in the discovery and development of novel antibiotics during the first fifteen years of the 21st Century. Chapter 1 - Biomass antibacterials could come from various types of biomass including polysaccharides, peptides and glycopeptides polymer materials, which is the main direction of future development. Biomass antibacterials act on the extracellular structural layer of microorganism, and affect the movement, transmembrane transport, and biochemical reactions of microorganism. In this chapter, the authors focus on the recent development of biomass antibacterials. The authors’ recent endeavor on the research of biomass antibacterials was summarized. Special attention was paid to the synthesis, properties, and biomedical applications of biomass antibacterials via some typical examples. The synthetic methods on the preparation of biomass antibacterials will be reviewed. Finally, the authors proposed the problems and future perspectives of biomass antibacterials in the biomedical fields. The authors wish contribute interesting content to this book “Antibacterials: Synthesis, Properties and Biological Activities.” Chapter 2 - The increasing resistance of pathogenic bacteria to conventional antibiotics is a major problem of public health in the second half of this century. Therefore, it is exceptionally desired to search non-conventional antimicrobial agents able to inhibit microbial growth. This chapter provides comprehensive overview on smart materials based on structured noble metals covering its various forms from ultrathin layers over discrete metal islands up to isolated nanoparticles having exceptional properties applicable in prevention of bacterial infections. The type of underlaying substrate, forming the support for active metal Complimentary Contributor Copy viii Erika Collins nanostructure, was carefully chosen to match the specific application of individual composites (organic polymer fibers and layers, artificial polymer foils). Engineered antimicrobial materials are tested on vast range of microorganisms of Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. First subsection of the chapter is devoted to overview on antibacterial activity of Ag and Pd nanostructures prepared by DC sputtering on polymeric foils (PI, PEN) of great medical-care importance. The possibility of managing nanostructure size via controlling the thickness of metal nanolayers prior to their annealing is considered. Antibacterial properties of such metal/polymer composites are evaluated by a drip test using Gram-positive and Gram- negative bacteria. Significant differences between antibacterial response of Ag/PI and Pd/PEN is revealed. Secondly, the system of polymer/metal composites with triggerable activity is discussed and some examples of such systems are introduced. As external stimuli the pH, temperature or light is used to activate antimicrobial activity. Thermo- or pH- sensitivities are realized using PNIPAm, its copolymer or blends, containing antibacterial agents, as potential responsive antibacterial material. The light sensitivity is achieved using PNIPAm/PMMA blends and porphyrin/silver nanoparticles dispersed in polymer matrix. Antibacterial tests on selected bacteria strains demonstrates enhanced antibacterial effects upon external stimuli. Another possibility of introducing antibacterial properties to materials is immobilization of proper metal nanoparticles onto the surface of natural or artificial polymers. Indisputable advantage of immobilization is that antimicrobials are not released into the media or environment, but still perform well against bacteria. Silver is used in the form of ions (Ag+), Ag nanoparticles or as a compound. In this particular subsection the authors introduce three types of silver immobilization onto cellulose fibres; direct reduction of silver (from silver nitrate via cellulose or chitosan), silver sputtering, and in situ precipitation of silver chloride from silver nitrate and natrium chloride. In situ precipitation of silver chloride onto cellulose surface produces evenly distributed micro-sized particles on cellulose fibres. Silver chloride is firmly bound onto the fibres and exhibits excellent antibacterial properties in liquid media, without releasing of silver. Chapter 3 - Lectins are proteins able to bind specifically and reversibly to carbohydrates, without promote any alteration in the covalent structure of the ligand. These proteins are found in microorganisms, animals, and plants. The biological functions of lectins are not fully elucidated but it is known that they are involved in defense mechanisms, for example. Lectins may recognize pathogens and consequently kill the invading cells, trigger defensive signaling pathways (in plants) as well as stimulate the release of antimicrobial peptides, phagocytosis, complement activation and melanization (in animals). Direct antibacterial activity of lectins has been demonstrated through the ability of these proteins in promoting agglutination, inhibition of planktonic growth, biofilm inhibition or eradication, and/or death of the bacteria. The growth inhibition and death induction have been credited to the interaction of lectins with bacterial cell wall components [such as N-acetylglucosamine, N-acetylmuramic acid (MurNAc), tetrapeptides linked to MurNAc, and lipopolysaccharides] as well as with membrane receptors. This may lead to permeabilization and formation of pores in the bacterial cell wall and membrane, with consequent leakage of intracellular content. These antibacterial properties have stimulated several studies on the biotechnological potential of lectins as antibiotics. In this chapter, the authors will comment on general aspects regarding lectins and their purification and then review the state-of-art on antibacterial lectins, their

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Preface ix action mechanisms, defensive roles and current efforts regarding their effective biotechnological application. Chapter 4 - Natural biocides receive increasing attention for different applications. Biocides are essential substances for antibacterial coatings on different substrates. Due to the wide variations in the fields of application, these substances reoccur in the water cycle, possibly accumulating and threatening public health. Therefore, natural based materials are beneficial, since they are usually degradable in the aquatic environment during an appropriate time scale. Technical applications for antibacterial active surfaces are for example filtration devices, medical textiles like bandages and drapes, or textiles for fiber reinforced composites made from cellulosic fibers. All these materials may suffer from colonization with bacteria or fungi leading to performance loss or even worse to a distribution of harmful germs. Since sustainable rawmaterials are of increasing importance for the growing global economy, also new approaches to bactericidal effects need to be considered. Lots of plant based substances are already known for their bactericidal properties, but suffer from market entry barriers like high prices due to the inevitable purification processes. However, some materials can be synthesized, so they can possibly become more affordable if advantages of the economics of scale can be taken. The natural bactericides can be divided into different classes by their chemical structure. Terpenoids are molecules formally derived from isoprene and contain multiple C5H8 units; known substances within this group with antibacterial properties are for example menthol, pinene, camphor, and linalool. These materials can be found in plants but can alsobe synthesized. Eugenol and cinnamaldehyde are phenylpropanoids, since they are formally derived from 1-phenylpropane. These substances show bactericidal effects and can be incorporated in coatings, which is shown for textile applications. A rarely investigated topic is the development of resistances against natural substances. Depending on the mode of action of the natural based biocides the occurrence of resistances is possible as it is for synthetic bactericides. To give a conclusive assessment of potential fields of application and long term efficiencies, this issue will also be reviewed. Chapter 5 - Studies with plants and their use in combinatorial therapies have been stimulated. In this context, Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz var. ferrea, is a leguminous tree widely distributed in the northern and northeastern regions of Brazil, where it is commonly known as “Juca” or “Pau-ferro”. The possible interactions between methanolic and aqueous extracts of Libidibia ferrea (Mart.) L.P. Queiroz fruits have been verified, combined to antimicrobial drugs against strains of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. The fruits (33.53 g) of Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz were powdered and extracted with MeOH and then with water. The extracts were filtered and concentrated using a rotary evaporator to provide methanolic (8.85 g) and aqueous (8.33 g) extracts. The chromatographic profiles of the extracts were obtained using UPLC/DAD and LC-ESI-MS. Electrospray ionization (ESI) in negative ion mode was used to analyze tannins in both extracts. The analysis of extracts by MALDI-TOF confirmed the presence of hydrolysable tannins. Antibacterial and modulating activity (on bacterial resistance) were determined by micro dilution method to identify the MIC (Minimum Inhibitory Concentration). In the antibacterial activity tests the fractions showed a MIC of ≥ 1024 µg/mL and 512 µg/mL. In regards to modulation of bacterial resistance, the products showed synergism when combined with antibiotic against bacterial strains. It was observed Complimentary Contributor Copy x Erika Collins that the products potentiated the antibiotic action from aminoglycosides class against bacterial strains of Staphylococcus aureus and Escherichia coli, with significance of p < 0.001. The results indicate that the extracts obtained from Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz fruits could represent an alternative source of natural products capable of modifying and interfering with bacterial resistance to aminoglycosides. Chapter 6 - A promising way to fight multi-drug resistant bacteria strains is to enhance chemotherapy efficiency through the use of antibacterial agents affecting simultaneously several biotargets. The authors demonstrated that among these compounds are metal complexes with redox-active ligands – cycloaminomethyl derivatives of ortho- and meta- diphenols. They are able to reduce cytochrome c (Cyt c), the key component of bacterial electron transport chain, and to act as low-molecular SOD mimics. The authors synthesized redox-active complexes of these organic ligands with Cu(II) and Zn(II) ions and estimated the level of their antibacterial activity against Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Salmonella typhimurium), and Gram-positive bacteria (Bacillus subtilis, Sarcina lutea, Staphylococcus saprophyticus, Staphylococcus aureus, Mycobacterium smegmatis) as compared to some standard antibiotics (tetracycline, streptomycin, chloramphenicol). The compounds were characterized by means of chemical, physico-chemical and pharmacological screening methods. The investigation of the molecular and electronic structure of the complexes was performed within the density functional theory framework. The reducing properties of the ligands and their metal complexes were examined by cyclic voltammetry. The kinetics of reduction of bovine heart Cyt c with these compounds in vitro was investigated spectrophotometrically as well as their SOD-like activity (using the alkaline dimethyl sulfoxide model). The biocidal effect of the hit-compounds (MIC 0.003– 0.012 µmol∙ml-1) comparable to those of commonly used antibiotics was achieved by structural modification of the ligands and complexation which purposefully change the hydrophilic-lipophilic balance, acid-base and redox properties of phenolic derivatives. Cu(II) and Zn(II) complexes of cycloaminomethyl derivatives of ortho- and meta-diphenols may be considered as potential chemotherapeutic agents with broad-spectrum antibacterial activity. Chapter 7 - Bacterial infections are a serious and growing problem. Some antibacterial agents (AAs), successfully used for decades, are no longer effective due to the emergence and spread of resistance. Multidrug-resistant strains (MDR), and extremely drug-resistant strains (XDR) are non-susceptible to all classes of antimicrobials, have compromised the clinical utility of currently available antibiotics and underscore the need for new compounds. To ensure that the supply of new antibiotics keeps pace with evolving pathogens, it is necessary to build a robust, sustainable pipeline of new drugs and innovative therapeutic approaches. Since 2000, 27 new antibiotics, i.e., less than 2 per year have been launched, covering five new drug classes for combating bacterial diseases. In this review the authors analyze recent progress in the discovery and development of novel antibiotics during the first 15 years of the 21st century.

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Chapter 1

SYNTHESIS AND PROPERTIES OF BIOMASS-BASED NANOMATERIALS FOR ANTIBACTERIAL APPLICATIONS

Yan-Yan Dong, Ming-Guo Ma, Prof, and Xue-Ming Zhang, Prof Engineering Research Center of Forestry Biomass Materials and Bioenergy, Beijing Key Laboratory of Lignocellulosic Chemistry, College of Materials Science and Technology, Beijing Forestry University, Beijing, PR China

ABSTRACT

Biomass antibacterials could come from various types of biomass including polysaccharides, peptides and glycopeptides polymer materials, which is the main direction of future development. Biomass antibacterials act on the extracellular structural layer of microorganism, and affect the movement, transmembrane transport, and biochemical reactions of microorganism. In this chapter, we focus on the recent development of biomass antibacterials. Our recent endeavor on the research of biomass antibacterials was summarized. Special attention was paid to the synthesis, properties, and biomedical applications of biomass antibacterials via some typical examples. The synthetic methods on the preparation of biomass antibacterials will be reviewed. Finally, we proposed the problems and future perspectives of biomass antibacterials in the biomedical fields. We wish contribute interesting content to this book “Antibacterials: Synthesis, Properties and Biological Activities.”

Keywords: biomass-based nanomaterials, antibacterials, synthesis, properties, applications

 Correspondence: Tel: +86-10-62337250; Fax: +86-10-62336903. Email: [email protected]. Complimentary Contributor Copy

2 Yan-Yan Dong, Ming-Guo Ma and Xue-Ming Zhang

1. INTRODUCTION

Recently, antibacterials materials have received more and more attention, and have been a research hot topic. It is reported that there are about twenty-five thousand papers by using “antibacterials” as “title” in the Web of Science over the last ten years. Biomass is the most abundant renewable natural polysaccharide found on the earth, and has unique properties such as chemical stability, mechanical strength, biocompatibility, and biodegradation. Biomass antibacterials could come from various types of biomass including polysaccharides, peptides and glycopeptides polymer materials, which is the main direction of future development. Biomass antibacterials used biomass as matrix via combining the advantages of biomass and reinforcement, and produced new properties by synergistic effect. In this chapter, we focus on the recent development of biomass antibacterials including organic biomass antibacterials, metal nanoparticles biomass antibacterials, metal oxides nanoparticles biomass antibacterials, and so on. Special attention was paid to the research of TiO2 and metal silver biomass antibacterials. Our recent endeavor on the research of biomass antibacterials was summarized via some typical examples. The synthetic methods and antimicrobial activity on the biomass antibacterials have been reviewed. Finally, we proposed the problems and future perspectives of biomass antibacterials in the biomedical fields.

2. SYNTHESIS, PROPERTIES, AND APPLICATIONS OF BIOMASS ANTIBACTERIALS

2.1. Organic Biomass Antibacterials

As early as 2008, Roy et al. reported the synthesis of antibacterial cellulose fiber via reversible addition-fragmentation chain transfer surface graft polymerization [1]. 2- (Dimethylamino)ethyl methacrylate (DMAEMA) was polymerized from cellulosic filter paper via reversible addition-fragmentation chain transfer polymerization. It was found to the PDMAEMA-grafted cellulose fiber with the highest degree of quaternization (the amount of quaternary amino groups present in the cellulose graft copolymers) and quaternized with the shortest alkyl chains with particularly high activity against E. coli. Bacterial cellulose (BC), a natural hydrogel, has unique three-dimensional network nanostructure. Cellulose-based hydrogels are biocompatible, low production costs, and nontoxic, which have wide applications in tissue engineering and controllable delivery systems. It is reported that the antimicrobial BC dry film has a high water absorption ability, which is crucial for wound dressing to absorb blood and tissue fluid on acute traumas. Dialdehyde cellulose (DABC) hydrogel membranes were obtained by acetobacter xylinum via the sodium metaperiodate method using chloramphenicol (CAP) as a model biologically active agent [2]. It observed that both the CAP-containing BC and CAP-containing DABC membranes exhibited antimicrobial activities against the three model bacteria. Antimicrobial activity was displayed on S. pneumonia, S. Aureus, and E. coli with an inhibition zone of 13, 11, and 9 mm, respectively. No inhibition zones were observed with the pure membranes against all the three model bacteria. Authors suggested that the antimicrobial effect was attributed to CAP adsorbed into BC membrane. Complimentary Contributor Copy

Synthesis and Properties of Biomass-Based Nanomaterials … 3

Joubert et al. reported the preparation of hydroxyethyl cellulose-g-poly(ionic liquid)s with different degrees of polymerization (10, 50, and 100) [3]. The graft density of poly(ionic liquid)s on the hydroxyethyl cellulose backbone had an important effect on their antibacterial activities, indicating the efficient activities of the graft copolymers against bacteria. Cationic microfibrillated cellulose (CMFC) has high surface energy, nano-scale dimensions, barrier properties, nanoporous network, and the positive charge property. More recently, it is reported the preparation of triclosan/CMFC composites with good antibacterial activity by kinetic and isotherm models [4]. It obtained the inhibition zone diameters of CMFC against E. coli of 22.1 mm and S. aureus of 29.2 mm, respectively. As for triclosan/CMFC composites, it observed the inhibition zone diameters of 15.3 mm against E. coli and 25.7 mm against S. Aureus. These results indicated the good sorption sorption capacity of CMFC to triclosan and the good antibacterial activity of triclosan after the sorption process. Moreover, it reported the as-prepared triclosan/CMFC composites with a good antibacterial effectiveness, even better than triclosan added samples. Authors suggested that this antibacterial cellulose-based material had a promising applications in antibacterial paper grades (such as paper towel and tissue paper) and other natural biomass based products. It reported the fabrication of tetracycline hydrochloride loaded regenerated cellulose composite membranes [5]. It was worth to note that the formed regenerated cellulose (RC) membrane was capable of controlled release of tetracycline hydrochloride. The as-fabricated composites displayed excellent antibacterial activities and good biocompatibility, thus confirming its utility as potential in wound dressings and other medical applications.

2.2. Metal Nanoparticles Biomass Antibacterials

Silver nanoparticles biomass antibacterials. In 2013, it reported the synthesis of quaternized cellulose/silver nanoparticles composites with high Ag content (93%) [6], which were catalytically active in the reduction of p-nitrophenol to p-aminophenol and nontoxic at concentrations sufficient enough to show good antimicrobial activity. It reported the synthesis of silver nanoparticles from native isolate of C. glutamicum with 2 + aqueous diamine silver ([Ag (NH3) ] ) containing 1 mM AgNO3 [7]. C. glutamicum strain was greatly reduced diamine silver into silver nanoparticles at the concentration of 280 mg g-1 dried biomass. The as-prepared silver nanoparticles showed maximum absorbance at 450 nm. It showed spherical silver nanoparticles with the average particle size of ~15 nm. The as- prepared silver nanoparticles showed strong antimicrobial potential against the tested bacteria. Bacterial synthesis of extra- and intracellular Ag nanoparticles had been reported using biomass, supernatant, cell-free extract, and derived components by bacteria act as natural capping and stabilizing agents [8], preventing their aggregation and providing stability for a longer time. The as-prepared bacterial Ag nanoparticles had anticancer and antioxidant properties, making them very promising as novel nanoantibiotics in therapeutic applications and water treatment. It reported the fabrication of carbon aerogels by carbonizing Ag nanoparticle BC, which was denoted as p-BC/Ag nanoparticles [9]. The unique 3D nanostructure made Ag nanoparticles dispersed uniformly onto the fibers and combining more firmly with fibers after carbonizing process. The as-prepared p-BC/Ag nanoparticles showed good mechanical properties, water reabsorption capacity, and excellent antibacterial effect on S. aureus and E. Complimentary Contributor Copy

4 Yan-Yan Dong, Ming-Guo Ma and Xue-Ming Zhang coli, for which the inhibition rate reached up to 99%. The MTT assay in Bel7402 cells showed that p-BC/Ag nanoparticles also had good cytocompatibility. Chook et al. reported the preparation of cellulose/graphene oxide/Ag composites membranes with strong antibacterial activities [10]. It reported that the presence of graphene oxide on the cellulose membranes significantly enhanced the deposition of Ag nanoparticles due to the electrostatic interaction between the positively charged Ag ammonia complex and negatively charged oxygenated functional groups of graphene oxide before the reduction to Ag nanoparticles. The Ag nanoparticles content of the membrane with 1wt% of graphene oxide was approximately 26 times greater than that of the neat cellulose membrane. The presence of graphene oxide significantly lowered the release of Ag ions and leaching of Ag nanoparticles into the aqueous solution. All of these are important in enhancing their antibacterial activities against S. aureus and E. coli. It reported the preparation of silver nanoparticles/cellulose nanocrystal (CNC) hybrids with enhance antibacterial activity [11]. CNC was first modified with dopamine, followed by in situ generation and anchoring of Ag nanoparticles on the surface of CNC through the reduction of silver ions by polydopamine coated CNC. It obtained the significantly enhance dispersion stability of Ag nanoparticles by the CNC, which in turn resulted in more than fourfold increase in antibacterial activity based on antibacterial studies using E. coli and B. subtilis. Vosmanska et al. reported the preparation of antibacterial cellulose dressing with impregnation of chitosan and precipitation of silver chloride on its surface via plasma treatment [12]. The as-prepared cellulose dressings exhibited significant growth prevention of the two representative strains of Gram-positive and Gram-negative bacteria. Electrospinning, a relatively simple and cost-effective technique, allows to fabricate polymeric micro- and nanoscale fibers, which has been widely used in synthesizing nanomaterials. Song et al. reported the fabrication of carbon nanofibers using biomass tar, polyacrylonitrile, and silver nanaoparticles through electrospinning and subsequent stabilization and carbonization process [13]. The resultant carbon nanofibers showed average diameters of ~226-507 nm, a high specific surface area (>400 m2/g), and microporosity. The pore characteristics increased the exposures and contacts of silver nanoparticles to the bacteria, leading to excellent antimicrobial performances of carbon nanofibers towards both Gram-positive S. aureus and Gram-negative E. coli. Zheng et al. reported the preparation of antibacterial cellulose fibers through the covalent bonding of silver nanoparticles to electrospun fibers [14]. Natural cotton was firstly dissolved in a room-temperature ionic liquid 1-ethyl-3-methyl acetate and wet-jet electrospun to obtain nanoscale cotton fibers, and then the cotton fibers were modified with trityl-3- mercaptopropionic acid to form sulfhydryl groups on their surfaces. With the help of sulfhydryl groups, silver nanoparticles can be covalently attached to the fibers to form stable cellulose/Ag composites, which showed significant antibacterial activities towards tested bacteria. RC can be constructed simply via physical dissolution by using “green” solvents and regeneration, which is an environmentally friendly process. In 2016, RC/nanosilver sponges were synthesized by freeze-drying of cellulose composite hydrogels, which were prepared in NaOH/urea aqueous system [15]. Cellulose solution (3 wt %) were firstly prepared in pre- cooled 7 wt% NaOH/12 wt% urea solvent. It obtained cellulose/nanosilver sponge by immersed in AgNO3 aqueous solutions with different concentrations. All of the sponges Complimentary Contributor Copy

Synthesis and Properties of Biomass-Based Nanomaterials … 5 exhibited nano and micro-porous architectures. It obtained Ag particles grew along the wall of micor-pores (Figure 1). It found that Ag+ concentrations had an important effect on the samples’ antibacterial activities (Figure 2). RC exhibited no bacterial activity against both S. aureus and E. coli. The sponges displayed the excellent antimicrobial efficacy. The average inhibition zone diameters of RC-Ag against S. aureus and E. coli enlarged from 15.5 to 26.8 mm and from 17.4 to 23.6 mm, respectively. The composite sponges had an ability to accelerate infected wound healing, especially in serious wound infection, as a result of the existence of Ag particles and absorbing capacity for wound exudate.

Figure 1. The SEM images of RCS (a,b), RCS-Ag1 (c,d), RCS-Ag2 (e,f) RCS-Ag3 (g,h) and RCS-Ag4 (i,j) and the corresponding EDX spectra (right side). From Ref. [15]. Reprinted with permission from Springer.

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6 Yan-Yan Dong, Ming-Guo Ma and Xue-Ming Zhang

Figure 2. Inhibition zones of the RCS, RCS-Ag1, RCS-Ag2, RCS-Ag3, and RCS-Ag4 against S. aureus (a-d) and E. coli (e-h) after incubation for 24 h. The diameter of sponge samples is 7 mm. From Ref. [15]. Reprinted with permission from Springer.

Gold and silver nanoparticles biomass antibacterials. Gold and silver nanoparticles were also synthesized using dried biomass of Parkia roxburghii leaf as both reductant as well as stabilizer, and HAuCl4 and AgNO3 as Au sourse and Ag source, respectively [16]. It obtained polydispersed spherical Au nanoparticles with sizes in the range of 5-25 nm and polydispersed quasi-spherical Ag nanoparticles with sizes in the range of 5-25 nm. Both as- synthesized Au and Ag nanoparticles were found to be showed pronounced antibacterial activities on S. aureus and E. coli (Figure 3), and strong and efficient photocatalytic activity towards degradation of methylene blue and rhodamine B. Cu nanoparticles biomass antibacterials. It reported the preparation of regenerated cellulose/copper nanoparticles composite films using cuprammonium solution as Cu resource and NaBH4 as reducing agent [17]. Cu nanoparticles were firmly embedded on the surface of the regenerated cellulose films. The as-obtained films showed efficient antibacterial activity against S. aureus and E. coli. It observed the dramatic reduction of viable bacteria within 0.5 h of exposure and killed all of the bacteria within 1 h.

Figure 3. Antibacterial activity of gold and silver nanoparticles against (a) S. aureus and (b) E. coli. From Ref. [16]. Reprinted with permission from Elsevier.

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Synthesis and Properties of Biomass-Based Nanomaterials … 7

2.3. Metal Oxides Nanoparticles Biomass Antibacterials

ZnO nanoparticles biomass antibacterials. It reported that ZnO nanoparticles are effective in killing Gram positive and Gram negative bacteria and also in inhibiting the growth of fungi. Antibacterial NFC/ZnO nanocomposites were obtained by the electrostatic assembly of ZnO nanoparticles onto NFC using polyelectrolytes as macromolecular linkers (Figure 4) [18]. It obtained antibacterial papers with low content of ZnO (<0.03%) using nanocomposites as fillers in starch based coating formulations for Eucalyptus globulus-based paper sheets. It found both NFC/ZnO and NFC/ZnO added papers with strong bacteriostatic and bactericidal activities towards Gram positive (S. aureus and B. cereus) and Gram negative (K. pneumoniae) bacteria even in dark conditions, associated to the generation of reactive oxgen species.

Figure 4. SEM images of NFC (a, b) and NFC/ZnO nanocomposite (c, d). From Ref. [18]. Reprinted with permission from Elsevier.

It reported the preparation of antibacterial cellulose paper coated with carboxymethyl starch-stabilized ZnO nanoparticles [19], which used carboxymethyl starch as a combined crystallizing, stabilizing and solubilizing agent and triethanolamine as the reducing agent. The paper was prepared by depositing carboxymethyl starch -ZnO nanoparticles on the cellulose paper by a wet-chemistry approach using water as the only solvent. The as-obtained papers showed higher brightness, higher whiteness, and higher stability after UV-radiation, as well as good antibacterial activity against MRSA and A. baumannii. The microwave-assisted hydrothermal method has greatly contributed to significant progress in the rapid synthesis of materials due to its remarkably reduced processing time, compared with the traditional hydrothermal processes. It was reported the preparation of hybrid nanostructured Ag/ZnO decorated powder cellulose fillers for medical plastics by stepwise microwave-assisted hydrothermal method [20]. Hexamethylenetetramine was used as precipitating agent for ZnO and reducing agent for Ag. The as-prepared Ag/ZnO showed a Complimentary Contributor Copy

8 Yan-Yan Dong, Ming-Guo Ma and Xue-Ming Zhang morphology resembling a coral reef and covered all available surfaces of cellulose particles (Figure 5). Cellulose/Ag/ZnO fillers were compounded to medical grade poly(vinyl chloride) matrix to make composites. This filler system at lower hierarchical level had relatively homogenous distribution and good dispersion, which could support the antibacterial performance of the composites. The as-prepared materials can be assessed as excellent against E. coli and very high against S. aureus, compared with other materials available nowadays.

Figure 5. SEM micrographs of Ag/ZnO on surface of cellulose substrates (a, c) ɑ-cellulose, (b, d) P- cellulose, (a, b) microwave-assisted hydrothermal method 20 min, (c, d) microwave-assisted hydrothermal method 30 min. From Ref. [20]. Reprinted with permission from Springer.

Figure 6. FE-SEM images of CNC and CNC/ZnO nanohybrids. From Ref. [21]. Reprinted with permission from Springer. Complimentary Contributor Copy

Synthesis and Properties of Biomass-Based Nanomaterials … 9

Cellulose nanocrystal/zinc oxide (CNC/ZnO) nanohybrids were reported by heat treatment using microcrystalline cellulose (MCC), NaOH, and Zn(NO3)2·6H2O [21]. The as- obtained CNC with carboxyl groups could act as both stabilizing and supporting agents to anchor ZnO nanoparticles. It was observed the CNC as rod-like monocrystals of 20 nm diameter and 240-300 nm length (Figure 6a). After the addition of Zn2+, well-dispersed ZnO nanoparticles with average diameter of 42.6 nm and narrow size distribution (15-85 nm) were observed to be anchored on the surface of CNC for CNC/ZnO-0.5 (Figure 6b). With increasing concentration of Zn2+ ions, the size of ZnO nanoparticles were increased to 126.6 nm (CNC/ZnO-1.0, 50-210 nm) (Figure 6c), and 143.1 nm (CNC/ZnO-1.5, 55-265 nm) (Figure 6d), respectively. Higher Zn2+ concentration can provide a greater opportunity for CNC to anchor more Zn2+, resulting in the growth of larger ZnO nanoparticles on the surface of CNC. A suitable concentration of Zn2+ ions was beneficial for growth of smaller ZnO nanoparticles with narrow size distribution. It was observed that the inhibition zones of samples were 4.5 mm (S. aureus) and 4.3 mm (E. coli, CNC/ZnO-0.5), 3.2 mm (S. aureus) and 3.4 mm (E. coli, CNC/ZnO-1.0), 2.3 mm (S. aureus) and 2.2 mm (E. coli, CNC/ZnO-1.5). After 15 h of incubation, highest antibacterial rations of 100.0 and 100.0% for viable E. coli and S. aureus were found for CNC/ZnO-0.5, respectively (Figure 7). Such nanohybrids with improved antibacterial properties showed great potential for use as biomedical materials. Fu et al. reported the preparation of RC-based ZnO nanocomposite films from cellulose carbamate-NaOH/ZnO solutions through one-step coagulation in Na2SO4 aqueous solutions [22]. It obtained ZnO nanoparticles with a hexagonal wurtzite structure and contents ranging from 2.7 to15.1 wt% embedded in the cellulose matrix. The antibacterial tests towards S. aureus and E. coli showed that a dramatic reduction in viable bacteria was observed within 3 h of exposure, while all of the bacteria were killed within 6 h. The results demonstrated potential application as functional biomaterials of regenerated cellulose nanocomposites.

Figure 7. Antibacterial ability of CNC/ZnO nanohybrids against S. aureus and E. coli. From Ref. [21]. Reprinted with permission from Springer.

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10 Yan-Yan Dong, Ming-Guo Ma and Xue-Ming Zhang

Ag ion release (ppm) release ion Ag

Figure 8. Silver ion release from paper as a function of immersion time. The mass fraction of Ag/TiO2 nanobelt or Ag/acid-corroded nanobelt in paper is 40%. From Ref. [24]. Reprinted with permission from Elsevier.

TiO2 nanoparticles biomass antibacterials. It reported the preparation of hybrid cellulose/titania/chitosan/Ag composites [23]. TiO2/chitosan composite films were firstly coated on the cellulose microfibril bundles by a self-assembly process. Silver ions were adsorbed on the cellulose/TiO2/chitosan composite films by the strong chelating ability, and were in situ reduced to Ag under UV irradiation. It obtained Ag nanoparticles with 4-20 nm in diameter and well distributed in the hierarchically fibrous structure of the composites. The as- prepared composites showed excellent antibacterial activities against both Gram-positive and Gram-negative bacteria, assigned to the intrinsic biocidal effect of titania composition, positively charged chitosan component and high loading content of Ag nanoparticles with small sizes. Papers with cellulose fiber-TiO2 nanobelt-silver nanoparticle hierachically structure were prepared by combining the photocatalytic property of TiO2 with the antibacterial property of Ag [24]. It fabricated papers by adsorb TiO2 nanobelts on the surface of cellulose fibers and assemble silver nanoparticles on the surface of TiO2 nanobelt nanobelts. It found that the papers with 40 wt% of TiO2 nanobelt displayed high photocatalytic activity and kept a high value after three photocatalytic cycles. Antimicrobial activity of papers increased with high silver ion concentration (Figure 8). It observed the great antibacterial effect (inhibition zone) of paper filled with Ag/acid-corroded/TiO2 nanobelt, compared with that of paper filled with Ag/TiO2 nanobelt (Figure 9), which was closely related to the release of Ag ion from the paper. Chauhan et al. reported the preparation of titania immobilized cellulose fibers by a hydrothermal method [25], containing the contents of TiO2 nanoparticles (10-20 nm) in the range from 2.5 to 21.0 wt%. These paper matrices can degraded similar to 82% of formaldehyde and completely degraded methyl orange in 180 min in sunlight and showed promising antibacterial activity against E. coli in visible light. Complimentary Contributor Copy

Synthesis and Properties of Biomass-Based Nanomaterials … 11

Figure 9. Antibacterial test photograph of paper added with 40 wt% of (a) TiO2 nanobelts, (b) acid- corroded TiO2 nanobelt, (c) Ag/TiO2 nanobelt and (d) Ag/acid-corroded TiO2 nanobelts. From Ref. [24]. Reprinted with permission from Elsevier.

It is also reported the preparation of paper matrices with the decoration of TiO2 nanoparticles (diameter ca. 40-250 nm) on the surface of the cellulose fibers using commercial TiO2, NaOH, and ethanol [26]. It was observed that paper matrices with 0.0 wt% of the TiO2 nanaoparticles showed no antibacterial activity. At 3 h of visible light exposure, paper matrices decorated with 1.0 and 3.5 wt% TiO2 nanaoparticles showed around 20 and 25% antibacterial activity, respectively. It observed around 62 and 97% reduction in bacterial growth for paper matrices decorated with 6.0 and 10.0 wt% of TiO2 nanoparticles. At 6 h of visible light exposure, it observed a substantial decrease in the bacterial concentration of 62, 65, 70, and 97% for the paper matrices decorated with 1.0, 3.5, 6.0, and 10.0 wt% TiO2 nanaoparticles, respectively. At 9 h of visible light exposure, it observed the decrease in the bacterial concentration of 85, 85, 95, and 100% (no growth) for the paper matrices decorated with 1.0, 3.5, 6.0, and 10.0 wt% TiO2 nanaoparticles. The as-prepared TiO2/cellulose fiber had efficient antimicrobial activities towards E. coli for multiple activities ideal for household applications. Galkina et al. reported the preparation of cellulose nanofiber-titania nanocomposites loaded with two different types of antibiotic medicines, tetracycline and phosphomycin, in aqueous media [27]. The samples showed excellent antibacterial effects towards S. aureus and E. coli, while tetracycline-loaded materials showed enhanced stability after UV irradiation. Luo et al. reported the preparation of anatase-titania/cellulose composite and Ag- nanoparticles/anatase-titania/cellulose composites with enhanced photocatalytic performance towards degradation of a methylene blue dye and antibacterial activities against both Gram- positive and Gram-negative bacteria by a solvo-co-hydrothermal treatment using titanium butoxide as the precursor to grow anatase-titania nanocrystallites (2-5 nm) uniformly on the cellulose nanofiber surfaces [28]. Complimentary Contributor Copy

12 Yan-Yan Dong, Ming-Guo Ma and Xue-Ming Zhang

Figure 10. FE-SEM images of (A) pristine paper and (B-D) CuO paper. From Ref. [29]. Reprinted with permission from Elsevier.

CuO nanoparticles biomass antibacterials. An in situ technique was developed to deposite copper oxide nanoparticles on the cellulose paper [29]. It obtained CuO particles with a form of nanoleaves stably anchored on the substrates (Figure 10). The as-prepared CuO papers showed good and stable antibacterial activities against both gram positive (S. aureus) and gram negative bacteria (E. coli). It found the stable antibacterial activity of CuO paper against E. coli over the four cycles of disinfection, and only around 12% reductions in copper content after the fourth run. Fe2O3/Ag nanoparticles biomass antibacterials. Maleki et al. reported the design and development of cellulose/gamma-Fe2O3/Ag nanocomposite combing the advantages of magnetic ability and antibacterial activity [30]. The as-prepared cellulose/gamma-Fe2O3/Ag nanocomposite could be used as a catalyst for the synthesis of trisubstituted imidazoles and a- aminonitriles and could be easily separated from the reaction mixture without considerable loss of catalytic activity. The nanocomposites had good antibacterial properties towards S. aureus as the representative of Gram-positive bacteria and E. coli as the representative of Gram-negative bacteria.

2.4. Other Biomass Antibacterials

ZnS-cellulose nanocomposite was fabricated using zinc nitrate and sodium sulphide [31]. The as-obtained nanocomposite displayed the ctystalline nature of ZnS nanoparticle with the average size of 4.3 nm. The ZnS-cellulose particles were formed in spherical shape with sizes ranging from 8 to 10 nm. The as-prepared nanocomposites showed inhibition zone of 12 mm with concentration at 2 mg mL-1, indicating the excellent antibacterial activities. The antibiotic tetracyline showed 22 mm diameter zones of inhibition as positive control. Complimentary Contributor Copy

Synthesis and Properties of Biomass-Based Nanomaterials … 13

3. OUR RECENT ENDEAVOR ON THE RESEARCH OF BIOMASS ANTIBACTERIALS

Among of various types of inorganic antibacterials, Ag crystals display excellent antimicrobial activities. But Ag nanoparticles are easy congregated and washed away, which are expensive antibacterials. Using biomass as matrix, biomass antibacterials can improve the congregated and washed away properties, and brings the price down. Our group does system work on the biomass antibacterials. We synthesized various biomass antibacterials including cellulose/Ag, cellulose/AgCl, cellulose/Ag/AgCl, cellulose/AgBr, and lignocelluloses/Ag, investigated the influences of reducing reagent such as ascorbic acid, NaBH4, ethylene glycol, fructose, glucose, and (holo)cellulose on the shape, size, and microstructure of biomass antibacterials, and explored the advantages of various synthetic methods of microwave- assisted method, hydrothermal method, microwave-assisted ionic liquids method, ultrasound agitation method, and precipitation methods.

Figure 11. XRD patterns of the cellulose-based nanocomposites prepared by microwave heating the o DMAc solution of AgNO3, cellulose solution, and different ascorbic acid concentrations at 150 C for 40 min. The amounts of ascorbic acid used were: (a) 0.352 g; (b) 1.0 g; (c) 2.0 g; (d) 3.0 g. From Ref. [32]. Reprinted with permission from Elsevier.

Our group investigated the influence of ascorbic acid on the synthesis of cellulose-silver nanocomposites using cellulose solution, AgNO3 and ascorbic acid in N,N-dimethylacetamide (DMAc) via the microwave-assisted method, in which silver nanoparticles were homogeneously dispersed in the cellulose matrix [32]. In this work, experimental results indicated that the ascorbic acid concentration played an important role in the phase of the nanocomposites, as shown in Scheme 1. We dissolved microcrystalline cellulose in a solvent system of lithium chloride (LiCl)/DMAc to obtain the cellulose solution. When the AgNO3 was added into the solution, Ag+ was reacted with Cl- to fabricate AgCl crystals. Therefore, with the ascorbic acid amount of 0.352 g, only litter Ag+ was reduced to Ag crystals and Complimentary Contributor Copy

14 Yan-Yan Dong, Ming-Guo Ma and Xue-Ming Zhang cellulose-AgCl-silver nanocomposites were obtained (Figure 11a). With increasing ascorbic acid amount, the reducing ability of ascorbic acid increased, the Ag amount increased. Using 1.0 g ascorbic acid as reducing agent, the product was still cellulose-AgCl-silver nanocomposites (Figure 11b). However, the peaks intensities of AgCl decreased and the peaks intensities of silver increased. When the ascorbic acid amount was increased to 2.0 g, the major phase was silver and the AgCl existed as a minor phase (Figure 11c). Further increased ascorbic acid amount to 3.0 g, the cellulose-silver nanocomposites were obtained (Figure 11d). Obviously, the ascorbic acid concentration decided the fabrication of silver from AgCl.

Scheme 1. Illustration for the formation of cellulose-based nanocomposites. From Ref. [32]. Reprinted with permission from Elsevier.

Figure 12. Antimicrobial activities of the cellulose-Ag nanocomposites: (a,c) E. coli and (b,d) S. aureus. From Ref. [33]. Reprinted with permission from Elsevier.

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Synthesis and Properties of Biomass-Based Nanomaterials … 15

Microwave-assisted method exhibited unique effects including rapid volumetric heating, high reaction rate, short reaction time, enhanced reaction selectivity, and energy saving. Our group developed the microwave-assisted method to fabricate cellulose-silver nanocomposites by reducing silver nitrate in ethylene glycol (EG) [33]. In this paper, EG was acted as a solvent, a reducing reagent, and a microwave absorber. We investigated the antibacterial activity of the cellulose-silver nanocomposites with low concentration (0.075 g) and high concentration (0.150 g). Obviously, pure microcrystalline cellulose does not display antibacterial activity as control (Figure 12), and the antibacterial activity is only due to the silver nanoparticles. It observed that the inhibition zones of the cellulose-silver nanocomposites with low concentration for E. coli and S. aureus were 10 mm and 2.5 mm, respectively (Figure 12a,b), while inhibition zones of the cellulose-silver nanocomposites with high concentration for E. coli and S. aureus were 12.5 mm and 6.5 mm, respectively (Figure 12c,d). The inhibition zone experiment indicated that the cellulose-silver nanocomposites possess a high antimicrobial activity against the model microbes E. coli (Gram-negative) and S. aureus (Gram-positive), implying that the cellulose-silver nanocomposites are a promising material for the application in the biomedical field. Our group also evaluated the effect of reducing reagents on the hybrids from cellulose and Ag using fructose and glucose as reducing reagents via a hydrothermal method [34]. Types of reducing reagents were found to play an important role in the shape and dispersion of silver structures in hybrids. It obtained dispersed silver particles using fructose as reducing agent in water, and congregated silver particles using glucose as reducing agent in water. It carried out silver with particles and sheets using fructose as reducing agent in NaOH/urea solution, and silver with particles and spheres using glucose as reducing agent in NaOH/urea solution. It obtained the high percent of cellulose and low percent of silver in the hybrids using glucose as reducing reagent. The different shapes of silver structures in hybrids were due to the different reducing ability of glucose and fructose. In this paper, the (holo)cellulose was pretreated in NaOH/urea solution by the hydrothermal method. During the synthetic procedure, hemicellulose was released easily, displaying a reducing ability and inducing the synthesis of silver with complex shapes, compared with the microcrystalline cellulose. The more interesting results are the cellulose solution as reducing agent, which played a vital role in the reduction process of Ag+ to Ag particles. In the present article, Ag particles- filled cellulose hybrids with high antimicrobial activity were carried out using AgNO3, AlCl3·6H2O, cellulose solution, and ethylene glycol (EG) by the microwave-assisted method [35]. Cellulose solution was obtained by dissolution the microcrystalline cellulose (MCC) in NaOH/urea solution. It obtained well-crystallized Ag with a cubic structure with the volume ratios of EG to MCC solution of 5 mL/15 mL, 10 mL/10 mL, and 15 mL/5 mL, confirming the completely reducing of AgCl to Ag crystals (Figure 13b-d). However, it observed AgCl with a cubic structure using only EG as solvent without MCC solution, indicating that EG has relatively weak reducing ability for the reduction of AgCl to Ag crystals (Figure 13e). It carried out the phase of Ag with only MCC solution without EG, demonstrating that the MCC solution displayed a strong reducing ability for the reduction of AgCl to Ag crystals (Figure 13a). All these results displayed that MCC solution played a vital role in the formation process of Ag crystals.

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16 Yan-Yan Dong, Ming-Guo Ma and Xue-Ming Zhang

Figure 13. XRD patterns of the samples synthesized by microwave heating using different ratio of EG to MCC solution at 90 oC for 20 min: (a) EG (0 mL)/MCC solution (20 mL); (b) EG (5 mL)/MCC solution (15 mL); (c) EG (10 mL)/MCC solution (10 mL); (d) EG (15 mL)/MCC solution (5 mL); (e) EG (20 mL)/MCC solution (0 mL). From Ref. [34]. Reprinted with permission from Elsevier.

200

111 220

222

311 (c)

(b) Intensity (a.u.)

(a)

10 203040506070 2(degree)

Figure 14. XRD patterns of the samples synthesized by microwave heating at 90 oC for 20 min: (a) 20 mL EG/0.324 g MCC; (b) 20 mL H2O/0.500 g MCC; (c) 20 mL NaOH/urea solution (without MCC). From Ref. [35]. Reprinted with permission from Elsevier.

We tried our best to explore the possible reducing mechanism of MCC solution during the reduction of AgCl to Ag crystals in detail via a list of compared experiments. It observed well-crystallized AgCl in EG using MCC without dissolution the MCC in NaOH/urea solution, indicating that MCC does not has a strong reducing ability to for the synthesis of Ag crystals (Figure 14a). It also obtained well-crystallized AgCl in water using MCC without dissolution the MCC in NaOH/urea solution, demonstrating that the MCC does not display a

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Synthesis and Properties of Biomass-Based Nanomaterials … 17 strong reducing ability in water (Figure 14b). The product was still AgCl in NaOH/urea aqueous solution without dissolution of MCC, implying that NaOH/urea aqueous solution also does not has a strong reducing ability (Figure 14c). It accorded to the mixed phases of AgCl and Ag with MCC dosages of 0.10, 0.20, 0.40, and 0.81 g, in which the peaks intensities of the AgCl decreased gradually and the peaks intensities of the Ag increased gradually with the increasing dosages of MCC in NaOH/urea solution, indicating that with the incresed dosages of MCC dissoluted in NaOH /urea aqeous solution, the stronger reducing ability of MCC solution, and more Ag crystals were obtained. Undoubtedly, all these mentioned above results confirmed the MCC solution with strong reducing ability for the synthesis of Ag crystals in the cellulose hybrids. Based on the above results on the MCC solution with strong reducing ability, we further synthesized silver particles filled cellulose hybrids with excellent antimicrobial activities against E. coli (Gram-negative) and S. aureus (Gram-positive) using microcrystalline cellulose solution, AgNO3, and AlCl3·6H2O by a hydrothermal method [36]. Experimental results further indicated that the microcrystalline cellulose solution played an important role in the synthesis of silver crystals.

Figure 15. XRD patterns of the hybrids synthesized by hydrothermal method for 12 h: (a) NaOH/urea solution without dissolution of MCC; (b) 0.324 g MCC; (c) 2 mL cellulose solution (dissolution the MCC in NaOH/urea solution); (d) 1 mL cellulose solution (dissolution the MCC in NaOH/urea solution). From Ref. [36]. Reprinted with permission from Elsevier.

It observed mixed phases of well-crystallized AgCl and silver using AgNO3, AlCl3·6H2O, and NaOH/urea solution without dissolution the MCC (Figure 15a). It carried out AgCl crystals as a major phase and silver crystals as a minor phase using AgNO3, AlCl3·6H2O, and MCC without dissolution in NaOH/urea solvent (Figure 15b). It observed AgCl crystals as the major phase and silver as the minor phase with 1 mL of the cellulose solution (Figure 15d), and silver crystals as the major phase and AgCl as the minor phase with 2 mL of the cellulose solution (Figure 15c). However, it exhibited crystallized silver with a cubic structure using AgNO3, AlCl3·6H2O, and MCC solution via dissolution the MCC in NaOH/urea solution by the hydrothermal method. These results further demonstrated that microcrystalline cellulose solution is considered to play a vital role in the synthetic process of silver crystals in Complimentary Contributor Copy

18 Yan-Yan Dong, Ming-Guo Ma and Xue-Ming Zhang the hybrids. The silver peaks intensities were reported to increase obviously with increasing cellulose solution volumes. Obviously, there existed the interactions between MCC and NaOH/urea solution. Taking MCC for instance, firstly, MCC was dissoluted in NaOH/urea solution and formed cellulose solution. When cellulose solution was mixed with AgNO3 + - + solution and AlCl3·6H2O solution, Ag reacted with Cl to form AgCl crystals and some Ag diffused into the matrix and anchored on the surface of cellulose probably via electrostatic interactions by the electron-rich oxygen atoms of hydroxyl and ether groups of cellulose. After that, the absorbed silver ions inside the cellulose matrix were reduced to the metallic silver crystals under the hydrothermal reaction condition. Of course, the intrinsic mechanism at the molecular level is also needed to be further studied in detail in the near future.

Figure 16. Antimicrobial activities of cellulose/Ag/AgCl hybrids synthesized at 160 oC for 12 h: (a,b) cellulose solution; (c, d) NaOH/urea solution; (e, f) 0.324 g MCC. From Ref. [36]. Reprinted with permission from Elsevier.

Similar with previous results, pure cellulose alone as control had no antibacterial properties (Figure 16). It observed the inhibition zones of 1.0 mm for the cellulose-silver hybrids synthesized using cellulose solution by the dissolution of MCC in NaOH/urea solution) for E. coli and S. aureus (Figure 16a,b). It obtained the inhibition zones of 1.8 mm and 2.0 mm for the cellulose/Ag/AgCl hybrids synthesized using NaOH/urea solution (without MCC) for E. coli and S. aureus (Figure 16c,d). As for the cellulose/Ag/AgCl hybrids Complimentary Contributor Copy

Synthesis and Properties of Biomass-Based Nanomaterials … 19 synthesized using MCC without dissolution in NaOH/urea solution, it carried out the inhibition zones of 1.5 mm/3.0 mm (inner/outer) and 2.0 mm for E. coli and S. aureus, respectively (Figure 16e,f). It is worth noting that the two inhibition zones of cellulose/Ag/AgCl hybrids were observed for E. coli, which could be due to the releasing behavior of silver and AgCl crystals in the hybrids. It was thought that the silver and AgCl crystals located in the surface region of the cellulose matrix were ready to release when incubated with bacteria, while the silver and AgCl crystals impregnated in the inner part of the composite were hard to diffuse out due to the special structure of cellulose, thus the silver and AgCl crystals went into a sustained release way. The size and size distribution of silver particles have significant influence on their antibacterial properties. The mean size of as- prepared corresponding hybrids particles were 85.2 nm, 5.3 µm, and 1.7 µm, respectively. It reported that the cellulose/Ag/AgCl hybrids with relatively small size displayed high antibacterial property. What’s more, the Ag+ releasing concentrations were 0.091 ppm, 0.073 ppm, and 0.080 ppm in solutions (using H2O as solvent) for 4, 12, and 24 h, respectively, clearly demonstrating that the antibacterial activity of hybrids was due to the existence of silver crystals. Of course, the intrinsic mechanism of effects on the antibacterial properties of the as-prepared hybrids needs to be further investigated in the near future. It reported that silver chloride (AgCl) is a photosensitive material, which was extensively used in photometry, plating, electrochemical and biomedical fields. We dissolved microcrystalline cellulose and lithium chloride (LiCl) in DMAc. The cellulose/AgCl nanocomposites were synthesized using cellulose solution and AgNO3 in N,N- dimethylacetamide (DMAc) solvent using DMAc simultaneously as a solvent and a microwave absorber, and LiCl as the reactant to fabricate AgCl [37]. The cellulose/AgCl nanocomposites were reported to display high-purity, good thermal stability and antimicrobial activity. One can observe that AgCl nanoparticles were dispersed in the cellulose matrix (Figure 17). It found the cellulose with fiber-like morphology. In this article, it should be noticed that the simultaneous formation of AgCl nanoparticles and precipitation of the cellulose lead to a homogeneous distribution of AgCl nanoparticles in the cellulose matrix. With the help of microwave, the cellulose rapidly precipitated from the DMAc solvent and Ag+ ions rapidly react with Cl- to fabricate AgCl, leading to a rapid nucleation and well- dispersed AgCl nanoparticles in the cellulose matrix. It also observed the inhibition zone of the cellulose/AgCl nanocomposites for E. coli and S. aureus of 4.5 mm and 4.5 mm, respectively. These results implied that the cellulose/AgCl nanocomposites had good antibacterial activities against both E. coli (Gram-negative) and S. aureus (Gram-positive) due to the AgCl nanoparticles. Our group also applied microwave-assisted ionic liquids method to fabricate the hybrids from cellulose and AgX (X = Cl, Br) using cellulose and AgNO3 [38]. In this paper, the ionic liquids were reported to be acted simultaneously as a solvent, a microwave absorber, and a reactant. For example, ionic liquids provided Cl- or Br- to the synthesis of AgCl or AgBr crystals. Using cellulose-AgCl and cellulose-AgBr hybrids as precursors, it obtained the cellulose-Ag/AgCl hybrid and cellulose-Ag/AgBr hybrid. More importantly, we discovered the different chemical stability on the hybrids.

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20 Yan-Yan Dong, Ming-Guo Ma and Xue-Ming Zhang

Figure 17. SEM micrographs of the cellulose/AgCl nanocomposites prepared by microwave heating at 150oC for 20 min. From Ref. [37]. Reprinted with permission from Elsevier.

Figure 18. XRD patterns of the cellulose-AgX/Ag hybrids prepared by microwave heating using different types of ionic liquids: (a), (b) [Amim]Cl; (c), (d) [Bmim]Br. From Ref. [38]. Reprinted with permission from Elsevier.

Using cellulose-AgCl hybrid as precursor and ascorbic acid as the reducing reagent in [Amim]Cl via microwave heating, the product was still cellulose and AgCl (Figure 18a), demonstrating that the as-obtained cellulose-AgCl hybrid had highly chemical stability. However, it reported the synthesis of cellulose-AgCl/Ag hybrids using [Amim]Cl, AgNO3 and ascorbic acid via microwave heating (Figure 18b). These results indicated that Ag+ reacted with ascorbic acid to form Ag crystals, and AgCl crystals did not react with ascorbic acid to form Ag crystals after adding ascorbic acid. Complimentary Contributor Copy

Synthesis and Properties of Biomass-Based Nanomaterials … 21

Using cellulose-AgBr hybrid as precursor and ascorbic acid as the reducing reagent in [Bmim]Br via microwave heating, it observed AgBr and Ag (Figure 18c), indicating that the AgBr crystals were reduced to form Ag crystals by adding ascorbic acid. Furthermore, it obtained cellulose-AgBr hybrid using [Bmim]Br, AgNO3 and ascorbic acid via microwave heating (Figure 18d). These results indicated that Ag was not obtained from AgBr via ascorbic acid. This is an interesting result. Obviously, the detail mechanism still need to be further explored in the near future. Ultrasound agitation has important applications in organic synthesis, materials and organometallic chemistry, and industrial manufacturing process. It reported that the acoustic cavitation induced the most notable effects during the formation, growth, and implosive collapse of bubbles. The ultrasound effects could be categorized as primary sonochemistry, in which gas-phase chemistry occurring outside the bubbles, and physical modifications, which was caused by high-speed jets or shock waves derived from bubble collapse. We reported the preparation of cellulose/Ag/AgCl hybrids using the cellulose solution, AgNO3, AlCl3·6H2O with ultrasound agitation method [39]. The cellulose solution was synthesized by the dissolution of the microcrystalline cellulose in NaOH/urea aqueous solution. It observed the inhibition zones of the cellulose/AgCl/Ag hybrids synthesized by ultrasound agitation method for 10 min for E. coli and S. aureus of 2.0 and 3.0 mm, respectively (Figure 19a,b). As for 30 min, the inhibition zones of the cellulose/AgCl/Ag hybrids for E. coli and S. aureus were 2.0 and 2.5 mm, respectively (Figure 19c,d). These results confirmed that the AgCl/Ag nanoparticles had good antimicrobial activities. Antimicrobial activity indicated that the cellulose/AgCl/Ag hybrids exhibited good antibacterial activity, which may be a promising antibacterial candidate for the applications in biomedical field and public health area.

Figure 19. Antimicrobial activities of the cellulose/AgCl/Ag hybrids synthesized for (a, b) for 10 min and (c and d) 30 min: (a, c) E. coli and (b, d) S. aureus. From Ref. [39]. Reprinted with permission from Elsevier.

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22 Yan-Yan Dong, Ming-Guo Ma and Xue-Ming Zhang

NaOH/Urea solution AgNO3

ultrasonic irradiation pretreatment dispersed lignocelluloses solution lignocelluloses

Ag nanoparticles 10 min

20 min with NaBH4 Ag nanosheets 40 min

60 min

Ag nanoparticles 60 min without NaBH4

using different reducing agents

fructose C6H12O6 ascorbic acid

Ag needles

lignocelluloses support

Ag nanowires Ag nanorods Ag nanosheets

Figure 20. Schematic illustration of the formation process of hybrids from lignocelluloses and Ag with different ultrasonic time and with different reducing agents. From Ref. [40]. Reprinted with permission from Elsevier.

Our group investigated the types of reducing reagents on the lignocelluloses/silver hybrids in detail via an environmentally-friendly sonochemistry method [40]. Dewaxed cotton straw powder was pretreated with NaOH/urea aqueous solution. As shown in Figure 20, using NaBH4 as reducing reagent, it achieved Ag nanoparticles and nanosheets. Without NaBH4, it observed Ag nanopartciles with small size. Moreover, it carried out Ag needles with the addition of fructose or C6H12O6, and Ag sheets with the addition of ascorbic acid. The silver with various shapes including nanosheets, nanowires, and nanorods grew on the lignocelluloses matrix with C6H12O6 as reducing agent for 60 min. These experimental results demonstrated that the types of reducing reagents had dramatically effects on the shape, size, and dispersion of silver crystals in the lignocelluloses/silver hybrids. Obviously, during the synthesis of silver crystals in the hybrids, we should paid more attention to the types of reducing reagents. It should be pointed out that, however, we are still far away from a full understanding mechanism on the synthesis of Ag via various reducing reagents. The mechanism at the molecular level is also needed to be further studied in detail, which is important for the applications of antibacterial hybrids. Complimentary Contributor Copy

Synthesis and Properties of Biomass-Based Nanomaterials … 23

CONCLUSION

In summary, this chapter described the recent development of biomass antibacterials via some typical examples. More attentions have paid to the research of biomass antibacterials. Based on our knowledge, these issues should be solved in the near future. First of all, the fabrication of biomass antibacterials should be carried out from experimental scale to large scale or industry scale. Then, the interaction mechanism between the biomass and inorganic antibacterials should be further explored. Washability is key index for representing the property of biomass antibacterials used for fabrics. Therefore, development of biomass antibacterials with high quality, washable, broad spectrum, low-cost, and safe stability is of great importance for broadening and improving their industrial applications. We should combine the advantages of biomass and inorganic antibacterials, inducing some new properties and opening up new applications in various fields.

ACKNOWLEDGMENTS

Financial support from the Fundamental Research Funds for the Central Universities (No. 2015ZCQ-CL-03, JC2013-3) is gratefully acknowledged.

REFERENCES

[1] Roy, D., Knapp, J.S., Guthrie, J.T., Perrier, S., 2008. Antibacterial cellulose fiber via RAFT surface graft polymerization. Biomacromoleculars, 9, 91-99. [2] Laçin, N.T., 2014. Development of biodegradable antibacterial cellulose based hydrogel membranes for wound healing. Int. J. Biol. Macromol., 67, 22-27. [3] Joubert, F., Yeo, R.P., Sharples, G.J., Musa, O.M., Hodgson, D.R.W., Camer on, N.R., 2015. Preparation of an antibacterial poly(ionic liquid) graft copolymer of hydroxyethyl cellulose. Biomacromoleculars, 16, 3970-3979. [4] Zhang, H.J., Zeng, X., Xie, J.L., Li, Z.Q., Li, H.L., 2016. Study on the sorption process of triclosan on cationic microfibrillated cellulose and its antibacterial activity. Carbohyd. Polym., 136, 493-498. [5] Shao, W., Wang, S.X., Liu, X.F., Liu, H., Wu, JM., Zhang, R., Min, H.H., Huang, M., 2016. Tetracycline hydrochloride loaded regenerated cellulose composite membranes with controlled release and efficient antibacterial performance. RSC Adv., 6, 3068-3073. [6] You, J., Xiang, M.X., Hu, H.Z., Cai, J., Zhou, J.P., Zhang, Y.P., 2013. Aque ous synthesis of silver nanoparticles stabilized by cationic cellulose and their catalytic and antibacterial activities. RSC Adv., 3, 19319-19329. [7] Gowramma, B., Keerthi, U., Rafi, M., Rao, D.M., 2015. Biogenic silver nano particles production and characterization from native stain of Corynebacterium species and its antimicrobial activity. 3 Biotech, 5, 195-201. [8] Singh, R., Shedbalkar, U.U., Wadhwani, S.A., Chopade, B.A., 2015. Bacte riagenic silver nanoparticles: synthesis, mechanism, and applications. Appl. Microbiol. Biotechnol., 99, 4579-4593. Complimentary Contributor Copy

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[9] Yan, N., Zhou, Y.B., Zheng, Y.D., Qiao, S., Yu, Q., Lia, Z.Z., Lua, H.Y., 2015. Antibacterial properties and cytocompatibility of bio-based nanostructured carbon aerogels derived from silver nanoparticles deposited onto bacterial cellulose. RSC Adv., 5, 97467-97476. [10] Chook, S.W., Chia, C.H., Zakaria, S., Ayob, M.K., Huang, N.M., Neoh, H.M., Jamal, R., 2015. Antibacterial hybrid cellulose-graphene oxide nanocomposite immobilized with silver nanoparticles. RSC Adv, 5, 26263-26268. [11] Shi, Z.Q., Tang, J.T., Chen, L., Yan, C.R., Tanvir, S., Anderson, W.A., Berry, R.M., Tam, K.C., 2015. Enhanced colloidal stability and antibacterial performance of silver nanoparticles/cellulose nanocrystal hybrids. J. Mater. Chem. B, 3, 603-611. [12] Vosmanská, V., Kolárová, K.; Rimpelova, S.; Kolska, Z.; Svorcik, V. 2015. Antibacterial wound dressing: plasma treatment effect on chitosan impregna-tion and in situ synthesis of silver chloride on cellulose surface. RSC Adv., 5, 17690-17699. [13] Song, K.L., Wu, Q.L., Zhang, Z., Ren, S.X., Lei, T.Z., Negulescu, I.I., Zhang, Q.G., 2015. Porous carbon nanofibers from electrospun biomass tar/polyacrylonitrile/silver hybrids as antimicrobial materials. ACS Appl. Mater. Interfaces, 7, 15108-15116. [14] Zheng, Y.Y., Cai, C., Zhang, F.M., Monty, J., Linhardt, R.J., Simmons, T.J., 2016. Can natural fibers be a silver bullet? Antibacterial cellulose fibers through the covalent bonding of silver nanoparticles to electrospun fibers. Nanotechnology, 27, DOI: 10.1088/0957 4484/27/5/055102. [15] Ye, D.D., Zhong, Z.B., Xu, H., Chang, C.Y., Yang, Z.X., Wang, Y.F., Ye, Q.F., Zhang, L.N., 2016. Construction of cellulose/nanosilver sponge materials and their antibacterial activities for infected wounds healing. Cellulose, 23, 749-763. [16] Paul, B., Bhuyan, B., Purkayastha, D.D., Dhar, S.S., 2016. Photocatalytic and antibacterial activities of gold and silver nanoparticles synthesized using biomass of Parkia roxburghii leaf. J. Photoch. Photobio. B, 154, 1-7. [17] Jia, B.Q., Mei, Y., Cheng, L., Zhou, J.P., Zhang, L.N., 2012. Preparation of copper nanoparticles coated cellulose films with antibacterial properties through one-step reduction. ACS Appl. Mater. Interfaces, 4, 2897-2902. [18] Martins, N.C.T., Freire, C.S.R., Neto, C.P., Silvestre, A.J.D., Causio, J., Baldi, G., Sadocco, P., Trindade. T., 2013. Antibacterial paper based on composite coatings of nanofibrillated cellulose and ZnO. Colloid Surface A, 417, 111- 119. [19] Cheng, F., Betts, J.W., Kelly, S.M., Wareham, D.W., Kornherr, A., Dumestre, F., Schaller, J., Heinze, T., 2014. Whiter, brighter, and more stable cellulose paper coated with antibacterial carboxymethyl starch stabilized ZnO nanoparticles. J. Mater. Chem. B, 2, 3057-3064. [20] Bazant, P., Kuritka, I., Munster, L., Machovsky, M., Kozakova, Z., Saha, P., 2014. Hybrid nanostructured Ag/ZnO decorated powder cellulose fillers for medical plastics with enhanced surface antibacterial activity. J. Mater. Sci. Mater. Med., 25, 2501-2512. [21] Yu, H.Y., Chen, G.Y., Wang, Y.B., Yao, J.M., 2015. A facile one-pot route for preparing cellulose nanocrystal/zinc oxide nanohybrids with high antibacterial and photocatalytic activity. Cellulose, 2015, 22, 261-273. [22] Fu, F.Y., Li, L.Y., Liu, L.J., Cai, J., Zhang, Y.P., Zhou, J.P., Zhang, L.N., 2015. Construction of cellulose based ZnO nanocomposite films with antibacterial properties through one-step coagulation. ACS Appl. Mater. Interfaces, 7, 2597-2606.

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[23] Xiao, W., Xu, J.B., Liu, X.Y., Hu, Q.L., Huang, J.G., 2013. Antibacterial hybrid materials fabricated by nanocoating of microfibril bundles of cellulose substance with titania/chitosan/silver-nanoparticle composite films. J. Mater. Chem. B, 1, 3477-3485. [24] Wang, J.X., Liu, W.X., Li, H.D., Wang, H.L., Wang, Z., Zhou, W.J., Liu, H., 2013. Preparation of cellulose fiber-TiO2 nanobelt-silver nanoparticle hierarchocally structured hybrid paper and its photocatalytic and antibacterial properties. Chem. Eng. J., 228; 272-280. [25] Chauhan, I., Mohanty, P., 2014. Immobilization of titania nanoparticles on the surface of cellulose fibres by a facile single step hydrothermal method and study of their photocatalytic and antibacterial activities. RSC Adv., 4, 57885-57890. [26] Chauhan, I., Mohanty, P., 2015. In situ decoration of TiO2 nanoparticles on the surface of the cellulose fibers and study of their photocatalytic and antibacterial activities. Cellulose, 2015; 22; 507-519. [27] Galkina, O. L., Onneby, K., Huang, P., Ivanov, V. K., Agafonov, A. V., Seisenbaeva, G. A., Kessler, V. G., 2015. Antibacterial and photochemical properties of cellulose nanofiber-titania nanocomposites loaded with two different types of antibiotic medicines. J. Mater. Chem. B, 3, 7125-7134. [28] Luo, Y., Huang, J. G., 2015. Hierarchical-structured anatase titania/cellulose composite sheet with high photocatalytic performance and antibacterial activity. Chem.-A Europ. J., 21, 2568-2575. [29] Booshehri, A.Y., Wang, R., Xu, R., 2015. Simple method of deposition of CuO nanoparticles on a cellulose paper and its antibacterial activity. Chem. Eng. J., 262, 999-1008. [30] Maleki, A., Movahed, H., Paydar, R., 2016. Design and development of a novel cellulose/gamma-Fe2O3/Ag nanocomposite: A potential green catalyst and antibacterial agent. RSC Adv., 6, 13657-13665. [31] Pathania, D., Kumari, M., Gupta, V.K., 2015. Fabrication of ZnS-cellulose nanocomposite for drug delivery, antibacterial and photocatalytic activity. Mater. Des., 87, 1056-1064. [32] Li, S.M., Jia, N., Zhu, J.F., Ma, M.G., Xu, F., Wang, B., Sun R.C., 2011. Rapid microwave-assisted preparation and characterization of cellulose-silver nanocomposites. Carbohyd. Polym., 83, 422-429. [33] Li, S.M., Jia, N., Ma, M.G., Zhang, Z., Liu, Q.H., Sun, R.C., 2011. Cellulose-silver nanocomposites: Microwave-assisted synthesis, characterization, their thermal stability, and antimicrobial property. Carbohyd. Polym., 86, 441-447. [34] Dong, Y.Y., Li, S.M., Ma, M.G., Yao, K., Sun, R.C., 2014. Compare study cellulose/Ag hybrids using fructose and glucose as reducing reagents by hydrothermal method. Carbohyd. Polym., 106, 14-21. [35] Dong, Y.Y., Yao, K., Bian, J., Ma, M.G., Yi, L.J., 2015. Ag particles-filled cellulose hybrids: Microwave-assisted synthesis, characterization and antibacterial activity. Sci. Adv. Mater., 7, 1028-1038. [36] Dong, Y.Y., Fu, L.H., Liu, S., Ma, M.G., Wang, B., 2015. Silver-reinforced cellulose hybrids with enhanced antibacterial activity: Synthesis, characterization, and mechanism. RSC Adv., 5, 97359-97366.

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[37] Li, S.M., Fu, L.H., Ma, M.G., Zhu, J.F., Sun, R.C., Xu, F., 2012. Simultane ous microwave-assisted synthesis, characterization, thermal stability, and antimicrobial activity of cellulose/AgCl nanocomposites. Biomass Bioenerg., 47, 516-521. [38] Dong, Y.Y., He, J., Sun, S.L., Ma, M.G., Fu, L.H., Sun, R.C., 2013. Envi ronmentally- friendly microwave ionic liquids synthesis of hybrids from cellulose and AgX (X=Cl, Br). Carbohyd. Polym., 98, 168-173. [39] Dong, Y.Y., Deng, F., Zhao, J.J., He, J., Ma, M.G., Xu, F., Sun, R.C., 2014. Environmentally friendly ultrosound synthesis and antibacterial activity of cellulose/Ag/AgCl hybrids. Carbohyd. Polym., 99, 166-172. [40] Dong, Y.Y., Li, S.M., Ma, M.G., Zhao, J.J., Sun, R.C., Wang. S.P., 2014. Environmentally-friendly sonochemistry synthesis of hybrids from lignocelluloses and silver. Carbohyd. Polym., 102, 445-452.

BIOGRAPHICAL SKETCH

Prof. Dr. Ming-Guo Ma

Prof. Dr. Ming-Guo Ma received his B.S. (2001) and M.S. (2005) in Chemistry from Shandong University. He completed his PhD (2008) in Materials Physical Chemistry at the Institute of Shanghai Ceramic, Academic of Chinese Science. He was a postdoctoral fellow at Beijing Forestry University from 2009 to 2012. Then, he worked as a visiting professor at University of Konstanz from 2013 to 2014. Now, he is a full professor and a group leader at Beijing Forestry University. His current research focuses on the synthesis, properties, and applications of the polymer nanocomposites, biomaterials, and biochar via green strategies. He is lecturer and co-author around 99 international scientific publications.

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Chapter 2

SMART METAL NANOSTRUCTURES FOR EFFECTIVE BACTERIAL INHIBITION

Jakub Siegel*, Oleksiy Lyutakov, Vladimíra Vosmanská, Markéta Pišlová, Markéta Polívková, Yevgeniya Kalachyova and Václav Švorčík Department of Solid State Engineering, University of Chemistry and Technology Prague, Prague, Czech Republic

ABSTRACT

The increasing resistance of pathogenic bacteria to conventional antibiotics is a major problem of public health in the second half of this century. Therefore, it is exceptionally desired to search non-conventional antimicrobial agents able to inhibit microbial growth. This chapter provides comprehensive overview on smart materials based on structured noble metals covering its various forms from ultrathin layers over discrete metal islands up to isolated nanoparticles having exceptional properties applicable in prevention of bacterial infections. The type of underlaying substrate, forming the support for active metal nanostructure, was carefully chosen to match the specific application of individual composites (organic polymer fibers and layers, artificial polymer foils). Engineered antimicrobial materials are tested on vast range of microorganisms of Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. First subsection of the chapter is devoted to overview on antibacterial activity of Ag and Pd nanostructures prepared by DC sputtering on polymeric foils (PI, PEN) of great medical-care importance. The possibility of managing nanostructure size via controlling the thickness of metal nanolayers prior to their annealing is considered. Antibacterial properties of such metal/polymer composites are evaluated by a drip test using Gram-positive and Gram-negative bacteria. Significant differences between antibacterial response of Ag/PI and Pd/PEN is revealed. Secondly, the system of polymer/metal composites with triggerable activity is discussed and some examples of such systems are introduced. As external stimuli the pH, temperature or light

* Corresponding author: Email: [email protected]. Complimentary Contributor Copy

28 Jakub Siegel, Oleksiy Lyutakov, Vladimíra Vosmanská et al.

is used to activate antimicrobial activity. Thermo- or pH-sensitivities are realized using PNIPAm, its copolymer or blends, containing antibacterial agents, as potential responsive antibacterial material. The light sensitivity is achieved using PNIPAm/PMMA blends and porphyrin/silver nanoparticles dispersed in polymer matrix. Antibacterial tests on selected bacteria strains demonstrates enhanced antibacterial effects upon external stimuli. Another possibility of introducing antibacterial properties to materials is immobilization of proper metal nanoparticles onto the surface of natural or artificial polymers. Indisputable advantage of immobilization is that antimicrobials are not released into the media or environment, but still perform well against bacteria. Silver is used in the form of ions (Ag+), Ag nanoparticles or as a compound. In this particular subsection we introduce three types of silver immobilization onto cellulose fibres; direct reduction of silver (from silver nitrate via cellulose or chitosan), silver sputtering, and in situ precipitation of silver chloride from silver nitrate and natrium chloride. In situ precipitation of silver chloride onto cellulose surface produces evenly distributed micro-sized particles on cellulose fibres. Silver chloride is firmly bound onto the fibres and exhibits excellent antibacterial properties in liquid media, without releasing of silver.

Keywords: sputtering, metals, nanostructure, reduction, nanoparticle, polymers, bactericidal agent, triggerable antimicrobials, bacteria

INTRODUCTION

In recent years the risk of bacterial infection during or in close relation to conventional surgery considerably increases namely due to enhanced resistance of broad spectrum of pathogens to common antibiotics. This disturbing fact drives researchers all over the world to find new, perspective materials effective in microbial inhibition with minimal side effects against living tissues [1, 2]. Successful development of such materials safely applicable in health industry, however, comprises multistage process with precisely defined steps. Nowadays, especially thanks to rapid development of nanotechnology, the major part of newly developed antimicrobial materials have the nature of composites. They usually consist of skeleton (carrier material or matrix with excellent biocompatibility) and functional particles or structures which imparts them exceptional antimicrobial properties. The skeleton, however, does not necessarily stand for functional element with its own structure and shape such as catheters, blood-vessels, artificial joint, or compensation of urinary tract, but it may also represents basic tools and supports for medical-care (bandages, disinfecting solutions, or material for storage of blood and blood-derivatives) [3, 4]. While extremely loaded components suitable for joint replacement are exclusively made from metals; soft, flexible, and light components are commonly based on polymers both synthetic and natural. Especially the latter mentioned group of carrier materials (polymers) provide great application potential in development of second generation antimicrobials suitable for health-care industry. Besides tissue polystyrene, there are many other synthetic or natural biocompatible polymers which can be used in development of antibacterial composites e.g., polyethylenenaphthalate, polytetraflouroethylene, polymethylemethacrylate, polylactideacid, polyglicoleacid, chitosan, polyethyleneglycole, glycerol, hydrogels, etc. [5, 6] As it has been mentioned above, the engineering process leading to successful construction of functional antibacterial material comprises from carefully considered steps. Firstly, suitable type of skeleton material must be chosen to match its final application. If, for Complimentary Contributor Copy

Smart Metal Nanostructures for Effective Bacterial Inhibition 29 example, one is developing antibacterial material with triggerable activity [7, 8], the carrier must be able to tolerate external stimuli without unwanted changes of its biocompatibility. Therefore such polymers must be resistant to elevated temperature in case of temperature responsive material or to different kind of electromagnetic stimuli in case of light or electrical responsive materials. These requirements restrict numerous groups of suitable polymeric materials to only units which, in addition, must be resistant to all next processing steps. Secondly, carrier material must be properly modified to achieve required antibacterial properties. This may in principle be done by several ways: (i) inclusion of particles capable of microbial inhibition, (ii) preparation of antimicrobial-active coatings or (iii) combination of (i) and (ii) in complex structure enabling controlled switching between two states – antimicrobial active or inactive [9, 10]. Generally, the polymer/metal nanocomposites can be prepared by (i) mechanical mixing of a polymer matrix with metal nanoparticles above the melting point of the polymer [11], (ii) in situ polymerization of a monomer in the presence of metal nanoparticles [5], (iii) in situ reduction of metal ions (in the form of salts or complexes) in a polymer matrix [12] or by (iv) immobilisation/anchoring of metal particles with antibacterial effect on/to polymer surface [13]. Resulting antimicrobial properties of polymer/metal nanocomposites are attributed to polymer matrix itself and type, size, content and dispersion level of metal nanoparticles or clusters incorporated in polymer as well as quality of nanocomposite interface (i.e., surface morphology and roughness) [14-17]. Necessary prerequisite for successful nanoparticle embedding into the polymer matrix and/or their anchoring to single polymer chain is proper functionalization of either the polymer or nanoparticle itself. Thus, the adhesion of nanoparticles with polymer matrix as well as the dispersion of nanoparticles can be significantly increased [18]. Another form of polymer/metal composites, in which noble metal nanoparticles have found considerable application are nanocomposite polymeric membranes used in separation processes, especially in desalination. Nanocomposite metal-embedded membranes, showing pronounced biofouling resistance, have been frequently fabricated on polymers such as polysulfone (PS) [19], polyethersulfone (PES) [20, 21], and cellulose acetate (CA) [22]. Most common process of membrane preparation is inversion technique which yields asymmetric membranes. Multicomponent solution containing polymer, solvent inorganic and organic additives is flooded by coagulant bath resulting in membrane formation with a dense thin top layer and porous support layer [23]. Yuksel et al. [22] studied the effect of polymer types on AgNP location in membrane matrix. PS, PES and CA polymers were used for fabrication of flat-sheet bare and nanocomposite membranes. It was found that AgNPs should mostly accumulate onto the top and skin layers of PES and PS nanocomposite membranes while it settled down the sublayer of CA nanocomposite membrane. The location of AgNP in membrane matrix changed to the bacteriostatic effects of nanocomposite membranes since the interaction between AgNP and bacteria depends on the release of ionic silver from AgNP embedded in membrane. The nanocomposite membranes which stored AgNP at surface exhibited the best antibacterial properties. The bacteriostatic effect of the silver nanocomposite membranes depended on their ability to leach out biotoxic silver ions (Ag+) to the medium and also bacterial cells accessibility to embedded nanoparticles. The antibacterial tests showed that the diffusivity of ionic silver (Ag+) from membrane matrix determines the antibiofouling properties and the location of AgNP in membrane matrix can change the diffusivity of Ag+. The antibacterial properties were related to the diffusivity or release of Ag+ Complimentary Contributor Copy

30 Jakub Siegel, Oleksiy Lyutakov, Vladimíra Vosmanská et al. from membrane matrix and depended on the polymer type. PS polymer membrane provided the best antibacterial nanocomposite membrane surface. Comprehensive review on antimicrobial effect of the most frequently used metals was published by Hobman et al. [24]. Antibacterial action of silver, mercury, copper, arsenic, and antimony is considered upon specific metal ion release and production of reactive oxygen species (ROS) to which the mechanism of bacterial inhibition is often attributed. Mechanisms of metal toxicity are generally agreed to be a consequence of metal ion affinity for cellular components and biomolecules, or the stability of metal–biomolecule complexes formed, although the consequences are varied. Especially in case of silver, dissolved silver ions are responsible for the inhibition of respiration, membrane damage and destruction of the proton motive force. The interaction of Ag+ with SH- groups in membrane proteins/enzymes is thought to be a major mechanism of toxicity, with data suggesting that the key toxicity event is interactions between Ag+ and respiratory chain enzymes [25]. Proteomic studies have shown that ionic silver and AgNPs cause destabilization of the outer membrane, collapse of the cytoplasmic membrane potential and depletion of intracellular ATP levels in E. coli, consistent with interference with the respiratory chain [26, 27]. Today modern medicine put the emphasis on prevention instead of antibiotic therapy. In this course, physico–chemical modifications of the surfaces preventing the bacterial adhesion and/or materials with antimicrobial coatings are developed to an increasing extent. Currently, the most effective antimicrobial coatings are nanostructured noble metals in the form of thin coatings supported to biocompatible carrier or isolated NPs itself, such as silver [28], palladium [29] and gold [30]. Before intensive research of nanostructured coatings, medical devices have been coated by conventional antibiotics and antiseptics, such as sulfadiazine and chlorhexidine [31]. Nevertheless, conventional antibiotics have many disadvantages, including cytotoxicity towards human cells and/or development of bacterial resistance. There are two types of bacterial resistance (i) primary (determined by the type of bacteria) and (ii) secondary (acquired as a result of the bacterial genome evolution). In view of these disadvantages, non- conventional antimicrobial coatings were investigated. For example silver sulfadiazine is well known to produce silver ions which can inhibit DNA replication of bacteria and deactivate their metabolic enzymes [32]. This fact led to examination of silver and then other noble metals as antimicrobial coatings [33]. Currently, metal nanoparticles (NPs) were repeatedly used as the antimicrobial coatings in health-care industry (antimicrobial treatment of medical devices). Their advantages against bulk metals are (i) larger surface area-to-volume ratios, (ii) higher antimicrobial efficiency, and (iii) lower human toxicity [33]. Billions of nanoparticles can be distributed over the polymeric matrix. Thus prepared materials demonstrate very strong antimicrobial effects against a variety of organisms irrespective of their resistance to antibiotics [3]. By this method, Samuel et al. [3] prepared silver NPs coatings of catheters and determined the silver release, and antibacterial response with artificial urine contaminated with E. coli. They found that this catheter type is suitable for clinical trials. Adams et al. [34] studied antibacterial effects of PdNPs coatings against Gram-negative bacteria E. coli and Gram-positive bacteria S. aureus. Strong antibacterial response was observed against S. aureus; E. coli was inhibited only by higher concentrations of PdNPs.

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Smart Metal Nanostructures for Effective Bacterial Inhibition 31

1. Ag AND Pd COATINGS ON BIOCOMPATIBLE POLYMERS

This subsection gives insight into the problematic of the preparation process and antibacterial activity tuning of metal coatings in the form of permanent thin layers of various thicknesses. Possibility of low-temperature transformation of metal nanolayers into discrete nanoislands with high specific surface area is also discussed. One can assume that as the metal surface area increases the antibacterial activity increases accordingly. Especially silver- and palladium-based coatings of biocompatible polymers (polyimide, polyethylene naphthalate) used in medical instrumentation are discussed. Antibacterial response of these nanostructures, both before and after annealing, using Escherichia coli (E. coli) and Staphylococcus epidermidis (S. epidermidis) as model organisms is shown. Surface characterization of these composites was performed by AFM, XPS, and the measurement of sheet resistance, and correlated with their resulting antibacterial effects. Metal/polymer composites were prepared by DC sputtering of Ag and Pd (99.999% pure of targets) on two types of biocompatible polymers, polyimide (Kapton®, PI) and polyethylene naphthalate (PEN). Thermal annealing (in air, at 250°C and for 1 h) was applied to post-deposition transformation of prepared metal layers into discrete nanoislands. As- sputtered and annealed samples were characterized by AFM, XPS, and the measurement of sheet resistance (Rs). Antibacterial properties of these nanostructures were studied by the drip test using two bacterial strains: gram-negative E. coli (naturally occurring on mucous membranes) and gram-positive S. epidermidis (human skin).

1.1. Surface Morphology and Chemical Structure

The surface morphology of pristine and metal-coated samples, both as-sputtered and annealed, was characterized by 3D AFM scans. The effective metal thicknesses were measured by AFM scratch method [35] and surface roughness (Ra) was carried out (see Figure 3). Figure 1 represents AFM scans of Ag-coated PI samples with effective thicknesses of 0, 2 and 19 nm. Figure 2 shows the surface morphology of Pd-coated PEN samples of 0, 1 and 22 nm thick layers. In general, the surface morphology of pristine and as-sputtered samples demonstrated a soft corrugation. The value of surface roughness remained practically unchanged regardless of specific thickness of metal coating, except of as-sputtered PI/Ag sample for 200 s. The annealing caused a mild increase of the surface roughness of the pristine PI due to a soft corrugation of sample surface in the course of annealing (Figure 1). Compared to that, the surface morphology of PEN after the annealing underwent no significant changes (Figure 2). Noticeable changes in the surface morphology were observed for both types of composites (PI/Ag and PEN/Pd) in the case of increasing metal thickness of annealed samples (deposition times 20 and 200 s). One can see that the annealing resulted in a complete rearrangement of the surface; the formation of “spherolytic and hummock-like structures” – discrete nanoislands, homogeneously distributed over the polymeric surface. This phenomenon might be caused by thermally induced changes in the amorphous phase of polymers (transition between the glassy and elastic state of the polymer), while a thicker metal coatings exhibit a thermal accumulation. Enhanced diffusion of metal at elevated temperature leads to its aggregation into larger structures. The formation of island-like Complimentary Contributor Copy

32 Jakub Siegel, Oleksiy Lyutakov, Vladimíra Vosmanská et al. structures after annealing have been repeatedly observed in case of the thin silver [36] and gold [37] structures in several different studies. The morphology change was supported by a remarkable increase of Ra. The Ra value of increased by two orders of magnitude for PEN/Pd samples (Figure 2), but this increase was considerably less significant for PI/Ag samples (Figure 1). Formed Ag nanoislands were smaller, but more regular than Pd structures. Nevertheless, as the thickness of the layer increases, the size of metal islands increases and thus the size of islands might be effectively controlled by the thickness of metal coating preceding the annealing process. The transformation of layers into island-like structures leads to the increase of the specific surface area of metal; for this reason, the antibacterial effects of both metals might be intensified.

Reproduced from Ref. 16 with permission from Elsevier.

Figure 1. AFM images of pristine and silver-coated PI (sputtering time 0, 20 and 200 s) before (As- sputtered) and after annealing (Annealed) at 250°C. Ra represents average surface roughness in nm. Complimentary Contributor Copy

Smart Metal Nanostructures for Effective Bacterial Inhibition 33

Reproduced from Ref. 17 with permission from the Royal Society of Chemistry.

Figure 2. AFM images of pristine and palladium-coated PEN (sputtering time 0, 20 and 200 s) before (As-sputtered) and after annealing (Annealed) at 250°C. Ra represents average surface roughness in nm.

Table 1. Atomic concentrations (at. %) of Ag (3d), O (1s), C (1s), N (1s) and Si (1s) measured by XPS (pristine PI, as-sputtered and annealed silver-coated samples) for various sputtering times

Sputtering Atomic concentrations of elements (at. %) Sample time (s) Ag O C N Si As-sputtered 0 - 18.3 73.2 7.1 1.4 20 10.5 19.0 65.3 6.1 1.0 200 24.4 5.7 69.8 - - Annealed 0 - 13.9 78.8 5.3 2.3 20 2.4 14.2 76.3 5.1 2.0 200 7.4 15.9 71.6 4.2 1.0

Atomic concentrations (at. %) of chemical elements were determined from XPS spectra (pristine, as-sputtered, and annealed samples). Typical accessible depth of XPS is 6-8 atomic Complimentary Contributor Copy

34 Jakub Siegel, Oleksiy Lyutakov, Vladimíra Vosmanská et al. layers. One can see the values for PI/Ag and PEN/Pd samples in Table 1 and Table 2, respectively. The concentrations of C, N and O originate from PI stoichiometry (Table 1) and concentrations of C and O correspond to PEN stoichiometry (Table 2). In both cases, the concentration of C might be increased by the presence hydrocarbon impurities, which are frequently adsorbed on the surfaces of polar polymeric materials and are generally adsorbed from air [38]. Silicon, a common impurity in PI, which can be introduced into the polymeric foils during the manufacturing process, was found in pristine PI, PI/Ag samples of 20 s (both as- sputtered ad annealed), and 200 s (annealed samples) (see Table 1) [39]. Due to the effect of lateral growth [40], the increase of the thickness of Ag layer causes the increase of its density, and the layer becomes more homogeneous. The cracks presented in layers are shrinked, and the number of inhomogeneities is reduced by shielding electrons from the underlying polymer. Due to this phenomenon, neither Si nor N was detected in the thicker silver layer (sputtering time of 200 s). One can see (Table 1 and 2) that the detected concentrations of metal increased as the thicknesses of the metal layer increased (for both as-sputtered and annealed samples). However, the detected metal concentrations decreased after annealing at the expense of the concentrations of elements originating from underlying polymer, most likely due to the diffusion and aggregation of metal (see Figures. 1 and 2); the formation of island-like structure determined by AFM was affirmed by XPS measurements. The electrical continuity of thin metal coatings was carried out by the measurement of the sheet resistance (Rs). The dependence of the Rs values of metal layers on the deposition time before and after annealing is shown in Figure 3: (a) PI/Ag samples and (b) PEN/Pd samples. For the as-sputtered samples, the Rs decreases rapidly in the narrow range of the deposition times from 80 to 140 s and 60 to 140 s for PI/Ag and PEN/Pd samples, respectively. An electrically continuous metal layer on underlying polymer is formed. Finally, in case of longer deposition times (thicker layers) the Rs value is saturated at the level of ca 140 Ω, which is the typical value for nanometer-scale metal coatings (Matthiessen’s rule) [41]. For the annealed samples, one can see a significant shift of the resistance curve towards longer sputtering times (thicker coatings), which corresponds with the structural changes observed by AFM (transformation of nanolayers into isolated Pd nanoislands) (see Figures 1 and 2). The annealed samples were electrically discontinuous up to the sputtering time of 400 s (Ag effective thickness of 23 nm) in the case of PI/Ag samples (Figure 3a), and 160 s (Pd effective thickness of 18 nm) in the case of PEN/Pd samples (Figure 3b). From these deposition times metal layers become continuous and a percolation limit is overcome. For the longer sputtering times, the Rs decreased gradually and it achieved the saturation at same level, which was observed for the as-sputtered layers.

1.2. Antibacterial Tests

Since the quality of metal/polymer interface essentially influences resulting bactericidal properties, the assessment of bactericidal properties of PI/Ag and PEN/Pd composites was performed. Antibacterial response was evaluated by the drip method using two bacterial strains; gram-positive S. epidermidis (Figure 4a), and gram-negative E. coli (Figure 4b), frequently involved in hospital-acquired infections associated with a biofilm formation. The bacteria were in a close contact with tested samples in physiological solution either under Complimentary Contributor Copy

Smart Metal Nanostructures for Effective Bacterial Inhibition 35 sterile conditions for 3 h and then inoculated on solid agar plates. The inhibition effect of Ag- coated and annealed PI sample after 3h contact with S. epidermidis is shown in Figure 5 (positive control on the left, PI/Ag/200 s on the right).

Table 2. Atomic concentrations (at. %) of Pd(3d), O(1s) and C(1s) measured by XPS (pristine PEN, as-sputtered and annealed palladium-coated samples) for various sputtering times

Sputtering Atomic concentrations of elements (at. %) Sample time (s) Pd O C As-sputtered 0 - 15.9 84.1 20 2.6 15.1 82.3 200 25.3 1.2 73.5 Annealed 0 - 26.9 73.1 20 1.3 25.1 73.6 200 16.1 18.3 65.6

Reproduced from Ref. 16 with permission from Elsevier. Reproduced from Ref. 17 with permission from the Royal Society of Chemistry.

Figure 3. Dependence of the electrical sheet resistance (Rs) on the sputtering time for the as-sputtered and annealed samples: (a) PI/Ag, (b) PEN/Pd.

In Figure 4 (up), one can see that all Ag-coated PI samples (as-sputtered and annealed) inhibited bacterial growth of both strains (S. epidermidis (a), E. coli (b)), contrary to pristine PI, which exhibited no antibacterial activity. It is evident that as the thickness of Ag layer increased (increasing sputtering time), the antibacterial effect increased too. Surprisingly, Ag- coated samples demonstrated a similar antibacterial response of gram-negative and gram- positive strains, which is in contrast to a generally accepted phenomenon that gram-positive bacteria are more susceptible to the inhibition effect of antimicrobial agents [42], presumably caused by more facile penetration of agent through their thinner cell walls. For example, it Complimentary Contributor Copy

36 Jakub Siegel, Oleksiy Lyutakov, Vladimíra Vosmanská et al.

was shown that Ag co-functionalized with TiO2 forming nano-Ag-TiO2 composite, resulted in the significantly increased antibacterial effects towards gram-positive bacteria compared to gram-negative ones [43]. The increased antibacterial effects of annealed samples (both sputtering times) were observed, when we compared these results with the as-sputtered samples, which were notably more pronounced in the case of S. epidermidis (for details see Figure 4 (up) a, b). This increase of antibacterial response might be caused by the formation of island-like structures on the surface of PI, and subsequently, the increase of the specific surface area of Ag (see Figure 1). In this connection, it should be noted that nature-inspired (on the base of lotus leaf effect) coatings with rough surfaces have been repeatedly successfully designed for medical applications [44].

Reproduced from Ref. 17 with permission from the Royal Society of Chemistry.

Figure 4. Relative viability (number of CFU for examined microorganisms divided by number of CFU for control sample) of the colonies of (a) S. epidemidis and (b) E. coli on deposition time for pristine and metal-coated polymer. Green line represents reference level (number of CFU in physiological solution) for corresponding bacterial strains together with its uncertainty (dash line). (up) Ag/PI samples, (down) Pd/PEN samples.

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Smart Metal Nanostructures for Effective Bacterial Inhibition 37

Figure 5. The inhibition effect of Ag-coated PI on S. epidermidis - PCA agar plate: (a) control sample, (b) annealed sample of 200 s.

The antibacterial properties of PEN/Pd composites are shown in Figure 4 (down). It is apparent that pristine PEN exhibited no antibacterial activity in the case of both bacterial strains (S. epidermidis and E. coli, see Figure 4a, b, respectively); the values of CFU were comparable with the control sample. The same trend was observed for the annealed samples of 20 s. The most noticeable inhibition effect of Pd, in the case of S. epidermidis, was determined for the samples of 200 s, both as-sputtered and annealed, in which all CFU were inhibited. As-sputtered sample of 20 s was also highly effective. In the case of E. coli, the most effective inhibition effect was found for the as-sputtered sample of 200 s (total inhibition of CFU). The annealed sample of 200 s exhibited very low antibacterial effect. Generally, in case of PI/Ag samples it may be concluded that hand in hand with increasing metal thickness (increasing sputtering time), the antibacterial effect increases too. However, it is obvious that annealed samples were less effective than as-sputtered ones. These findings are in conflict with the increase of the specific surface of Pd after annealing, which was observed in the case of Ag. In the light of the results of surface ablation (XPS) and Pd release (ICP-MS) published elsewhere [24], we attribute the morphology changes not only to the coalescence of Pd into discrete nanoislands, but also to embedding of these clusters into the polymer interior. This incorporation is, however, very superficial most likely reminding ultrathin (ones of nm) polymer overlay reaching almost the top of individual Pd-islands (for more details see ref. [17] “curtain” effect). Overall, we may attribute the nature of antibacterial action to the contact of bacteria with metal itself and the release of metal ions into a physiological solution. To provide further insight into the process of bacterial inhibition and determine which phenomenon predominates, it will be necessary to focus further investigations on a deeper examination of the mechanism of antibacterial action. Prepared Ag and Pd composites based on biocompatible polymers are potentially applicable in the tissue engineering for antibacterial treatment in the artificial replacement of the outer skin layer. Preliminary tests showed that these antibacterial coatings are neutral from the cytocompatibility point of view; it prevents

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38 Jakub Siegel, Oleksiy Lyutakov, Vladimíra Vosmanská et al. microbial infections while the smooth vascular cells or keratinocytes may proliferate normally on such carriers.

2. IN SITU GENERATION OF SILVER CHLORIDE PARTICLES ON CELLULOSE

Wound healing is a complex process, where bacterial infection is detrimental to a proper wound healing, especially in a case of chronic wounds [45]. Therefore a wound dressing with antibacterial properties is an advantageous option as a wound cover. Our aim was to prepare cellulose with antibacterial properties that could potentially serve as a wound dressing for chronic wounds. More specifically, the research target was to immobilize antibacterial agens – silver chloride on cellulose fibres to introduce antibacterial properties. Immobilization of silver onto cellulose surface was investigated in our previous research together with effect of plasma treatment and chitosan coating on the immobilization process [46], here we present a relevant part of our previous research – chitosan coating effect on silver chloride (AgCl) immobilization and antibacterial properties. AgCl was immobilized on cellulose fibres via in situ precipitation with or without prior coating by chitosan. Silver in the form of AgCl↓ precipitate exerts sufficient antibacterial activity, but low toxicity in comparison to Ag+ ions [47], which are suspected to be toxic for mammalian cells [48-51]. Moreover, AgCl is poorly soluble in water, so we expected that immobilized AgCl precipitate would be firmly bonded onto the wound dressing and not released into the aqueous environment. AgCl can be precipitated directly on cellulose fibres from silver nitrate and natrium chloride, such in situ precipitation of AgCl onto cellulose surface produced evenly distributed micro-sized particles on cellulose fibres. This experiment was based on the premise that aldehyde and carboxyl groups of cellulose, would serve as anchoring sites for silver ions due to oxygen attraction forces [52, 53]. Since oxygen does not show as high attraction of silver as nitrogen [52, 53], chitosan was used for this experiment as a cellulose coating to increase the amount of immobilized silver, because it is well known to chelate metals [54]. Silver cations can be bound by chelation on amine groups of chitosan in near neutral solutions. However, the binding mechanism of Ag+ by chitosan is pH-dependent; the amino groups get easily protonated in acidic environment, thus turning the chelation into the electrostatic attraction of anions [55]. Chitosan is not only a chelating agent, it is also a biopolymer with wide spectrum of biological activities, including antifungal and antibacterial activities owing to its polycationic character [56, 57]. Cellulose (β-1,4-glucopyranose) and chitosan (β-1,4-linked 2-acetamido-2-deoxy-D- glucopyranose and β-1,4-linked 2-amino-2-deoxy-D-glucopyranose) are both polymers of natural origin and their structural similarity induces high affinity between both polymers. There is a difference in ionic character of these polymers; cellulose has anionic character meanwhile chitosan has cationic character [58, 59], leading to adsorption of chitosan on cellulose via electrostatic interaction [53]. Irreversible adsorption of chitosan on cellulose is preferred at low pH conditions, when amino groups are charged. For irreversible binding, cellulose carboxyl and aldehyde groups are the reaction sites for amino groups of chitosan [60]. Complimentary Contributor Copy

Smart Metal Nanostructures for Effective Bacterial Inhibition 39

Non-woven cellulose dressing was subjected to a two-step modification: (i) coating with chitosan, (ii) in situ precipitation of AgCl from silver nitrate (AgNO3) and natrium chloride (NaCl) solutions. Schematic illustration of the modifications performed on cellulose is shown on Figure 6. Each layer in the sample scheme represents one modification step, chronologically from the bottom to the top. Two sets of the samples were obtained, cellulose with immobilized AgCl with or without prior coating with chitosan. Pristine cellulose served as a control. Prepared samples were then characterized by optical microscopy, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), goniometry, absorption atomic spectroscopy (AAS) and zeta potential. The antibacterial properties were evaluated using environmental bacterial strains of Gram-negative Escherichia coli and Gram- positive Staphylococcus epidermidis. The prepared samples with immobilized AgCl were tested by a drip [61] and disc test [62] for antibacterial activity. Both tests were carried out using E. coli and S. epidermidis bacterial strains. All experiments were accomplished under sterile conditions and each sample was prepared in a triplicate by inoculating three separate samples. In the drip test, samples were incubated with bacteria suspension, dripped on agar plates, incubated and number of grown bacteria was then counted. The disc test, also called disc diffusion method, consisted in an assessment of the inhibition zone formed around those samples possessing antibacterial activity, similarly to Bauer et al. [62].

2.1. Chitosan Coatings

The presence of chitosan on the sample surface was first investigated by ninhydrin reaction which gives purple colour if the chitosan is present [63], ninhydrin reacts with primary amino groups forming purple colour, so called Ruhemann´s purple. Reaction takes 2 min at 100°C. The primary amino groups of chitosan readily reacted with ninhydrin and the samples turned purple, thus reliably pointing to the presence of chitosan. The precise amount of chitosan on the sample surface was determined by the XPS, characteristic nitrogen peaks (N 1s) were searched, see Table 3. Nitrogen was found in all samples that were impregnated with chitosan, nitrogen peaks were recorded for cellulose/chitosan and cellulose/ chitosan/AgCl samples. Otherwise, no nitrogen was found in other samples, which led to the conclusion that nitrogen detected by the XPS in the superficial layer of the samples was originating from chitosan. Apart from nitrogen, we focused on the concentration of carbon (C 1s), oxygen (O 1s), and silver (Ag 3d3/2, 3d5/2), see Table 3, hydrogen cannot be detected due to the technique limitation. The Table 1 also contains the elemental composition and the O/C ratio of pure cellulose [64], showing a difference of carbon and oxygen content in comparison to the pristine cellulose sample. The O/C ratio of the pristine cellulose sample was 0.33, considerably different from the theoretical value of 0.83, owing to non-cellulosic components (proteins, lignins, pectins, waxes, etc.), which naturally occur in the superficial layer of cotton [65, 66].

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40 Jakub Siegel, Oleksiy Lyutakov, Vladimíra Vosmanská et al.

Reproduced from Ref. 46 with permission from the Royal Society of Chemistry.

Figure 6. Modifications of the cellulose surface: (A) cellulose and precipitated AgCl, (B) cellulose coated with chitosan and precipitated AgCl.

Concerning the manner of chitosan binding to cellulose, similarities between cellulose and chitosan enable their high affinity and the intermolecular interactions are very likely based on H-bonds and Van der Waals forces [67], for irreversible attachment of chitosan to cellulose it is important to introduce carboxyl or aldehyde groups onto the fibres´ surface which are then anchoring sites for chitosan [60, 68]. The polar functional groups acted as nucleophilic binding centres for chitosan [69], that was attracted to cellulose via positively charged amino groups (the impregnation done under the acidic conditions). Taking into account the aforementioned, it should be noted that cellulose fibres (including wound dressings) are usually weakly acidic due to the pre-treatments such as scouring and bleaching allowing effective chitosan adsorption as well [70].

Table 3. Element concentration of C, O, N, Ag and Cl determined by the XPS in the surface layer of samples: cellulose (theoretical), pristine cellulose, cellulose/chitosan, cellulose/AgCl, cellulose/chitosan/AgCl

Element composition (at. %) O/C Sample C(1s) O(1s) N(1s) Ag(3d5/2) Cl(2s) ratio Cellulose (theoretical) 54.5 45.5 - - - 0.83 Pristine cellulose 75.2 24.8 - - - 0.33 Cellulose/chitosan 69.5 29.6 0.9 - - 0.46 Cellulose/AgCl 69.2 30.7 - 0.05 * 0.44 Cellulose/chitosan/AgC 70.5 28.5 0.7 0.11 0.11 0.40

Tabel 4. Zeta potential results

Sample Zeta potential (mV) Pristine cellulose -18.0 Cellulose/chitosan -8.5

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Smart Metal Nanostructures for Effective Bacterial Inhibition 41

Reproduced from Ref. 46 with permission from the Royal Society of Chemistry.

Figure 7. SEM images of the samples with in situ prepared AgCl particles: (A) cellulose and precipitated AgCl, (B) cellulose coated with chitosan and precipitated AgCl.

To investigate a change of electrokinetic potential caused by the chitosan coating, the zeta potential measurement was done. The electrokinetic potential (zeta potential) at the interface of sample surface-liquid is caused, among others, by ionization or dissociation of surface functional groups [71]. The negative charge of the cellulose samples resulted from the adsorption of chloride and hydroxide ions from the KCl water solution during the measurement [72]. The positive charge (meaning the positive increment to the overall zeta + potential) was caused by the presence of chitosan amino groups that were ionized (–NH3 ) and therefore the surface was less negatively (more positively) charged than cellulose without impregnation. Furthermore, the dissociation of acidic or basic groups during the zeta potential measurement is considered to be equivalent to the adsorption of hydroxide or hydroxonium ions [73]. In the case of the chitosan coated samples, they showed less negatively (more Complimentary Contributor Copy

42 Jakub Siegel, Oleksiy Lyutakov, Vladimíra Vosmanská et al. positively) charged surface than pristine cellulose samples (Table 4) with a difference of almost 10 mV. The difference between the chitosan coated samples and non-coated samples depended on the amount of positively charged groups on the surface, the amino groups. The XPS results show nitrogen presence (Table 3), which is consistent with the zeta potential measurement.

2.2. AgCl Precipitation

AgCl particles were formed on the sample´s surface via in situ reaction in AgNO3 and NaCl solutions. The particles were visible in the SEM images (Figure 7) and were spread homogenously on the surface, the amount and size varied. The cellulose/AgCl samples (Figure 7A) had low amount of AgCl particles, on the other hand the sample that were coated with chitosan prior to the precipitation (Figure 7B) had a significant amount of AgCl particles. Size of the particles was 256 nm for cellulose without prior coating by chitosan and 417 nm with prior coating (standard deviation was counted to ± 4 nm). Compared to the work of Hu et al. [74], the time factor of the precipitation process played a crucial role in the size of AgCl particles. Hu et al. [74] immersed samples of bacterial cellulose for 1 min into AgNO3 and for 1 min into NaCl solution, resulting in AgCl formation with the mean size of 25.8 nm. The immersion time in our work was 10× longer and the particles were approximately 10× bigger. The difference in the particle size and precipitation homogeneity can be discussed as follows. Silver has high affinity for nitrogen, lower for oxygen and much lower for carbon [52]. Nitrogen atoms have free electron doublets that are able to react with silver cations, because they have lone electron pairs in orbitals. Oxygen is able to attract the silver via electrostatic interactions, but, the attraction is weaker than in the case of nitrogen [55]. In the case of cellulose, silver ions are attracted to hydroxyl and ether groups [75]. In the case of chitosan, amine groups in chitosan are strongly attractive for silver cations. In another words, the affinity of silver to nitrogen prevailed over the affinity to oxygen However, it must be pointed out that the uptake mechanism of silver cations by amine groups is pH-dependent; the + amine groups get easily protonated (-NH3 ) in acidic environment, thus turning the chelation of cations into the electrostatic attraction of anions [55]. Therefore it was important to rinse the chitosan impregnated samples in distilled water because the impregnation was done under acidic conditions. In the case of cellulose/AgCl and cellulose/chitosan/AgCl, up to twice more silver was found on the samples that were coated with chitosan prior to the silver precipitation. The difference was caused by the presence of nitrogen that attracted and chelated silver. One of the major tasks was to examine whether the silver was irreversibly bounded on cellulose or not, therefore we performed a leaking test. An aqueous extracts of the samples with AgCl were analysed, and the AAS results showed that leaking of AgCl from the samples was considerably low, owing to the fact that AgCl is poorly soluble in water. AgCl had to be securely bounded on the samples. Results from the AAS were 0.054 mg/l for cellulose/AgCl and 0.056 mg/l for cellulose/chitosan/AgCl. Standard error of the AAS measurement is 0- 10% and the detection limit for silver is 0.03 mg/l, thus concluding that the release of silver from the samples to the aqueous environment was negligible.

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Smart Metal Nanostructures for Effective Bacterial Inhibition 43

Reproduced from Ref. 46 with permission from the Royal Society of Chemistry.

Figure 8. Growth of bacteria in the presence of the samples with and without chitosan coating and AgCl particles, (A) E. coli and (B) S. epidermidis.

2.3. Antibacterial Tests

The drip test showed 100% reduction of E. coli within 24 h for all samples containing silver (Figure 8A). Clearly, the AgCl particles were responsible for the antibacterial effect on E. coli. The cellulose/chitosan samples showed confluent growth due to the lower bactericidal activity of chitosan on gram-negative bacteria strains [76]. For results of the drip test performed on S. epidermidis see Figure 8B. Generally, the growth of S. epidermidis was diminished from 50 up to 75% for all samples within 24 h, in comparison to the control. Concerning the antibacterial effect of chitosan on S. epidermidis, data were consistent with the literature [56, 57]. As regards the efficiency of antibacterial properties, it is evident that AgCl reduced the bacterial growth more than chitosan; see the respective bars in Figure 8B. However, the situation gets interesting when it comes to the combination of both chitosan and AgCl. When the cellulose/chitosan, cellulose/AgCl samples and cellulose/chitosan/AgCl samples were compared, it was evident that there was a trend of rising efficiency against bacterial growth. Based on these results, the combination of chitosan and AgCl treatment increased the antibacterial efficiency against S. epidermidis. When comparing the drip test for E. coli and S. epidermidis, our results were consistent with the work of Xia et al., that chitosan generally showed stronger bactericidal effects on gram- positive bacteria than gram-negative bacteria [57]. In the disc test, inhibition zones were observed around the samples with AgCl and no inhibition zones around other samples. The inhibition zones around the samples were 2 mm wide, caused by firm immobilization of AgCl to the sample and its poor solubility in water. In the disc test the antibacterial effect of the samples impregnated with chitosan was not observed, although it should have resulted in the elimination of the bacterial growth [76]. For both bacterial strains, E. coli and S. epidermidis, the same result was experienced. The two-step modification of the standard cellulose wound dressing presented in this study is cost-effective and does not require large and complicated laboratory set up. Chitosan was used here due to silver-binding, antibacterial and healing promoting activity. The Complimentary Contributor Copy

44 Jakub Siegel, Oleksiy Lyutakov, Vladimíra Vosmanská et al. chitosan coating also positively affected the amount of AgCl in the superficial layer of the sample and finally, AgCl along with chitosan were responsible for the most effective antibacterial properties. Interesting result was that the combination of chitosan impregnation and AgCl precipitation showed better results than each of them used separately. AgCl was firmly bound onto the fibres and exhibited excellent antibacterial properties in liquid media in the case of S. epidermidis, without release of silver.

3. DIRECT REDUCTION OF SILVER FROM SILVER NITRATE VIA CELLULOSE OR CHITOSAN

Silver nanoparticles may exist in multiple forms, e.g., as cubes, triangles, rods, spheres or polyhedrons in sizes up to 100 nm. The nanoparticles have a large surface area, which allows better contact with microorganisms, so the silver nanoparticles are effective in an antibacterial action [77]. Nowadays, there are a lot of methods how to make the silver nanoparticles. They can be prepared for example via chemical reduction, electrochemical or thermic decomposition or decomposition using radiation [78]. Lately, the trend is to prepare nanoparticles by a simple, low-cost and environmentally friendly method. Huang T. et al. [79] prepared various metal-chitosan nanocomposites in an aqueous + solution by reduction of corresponding salts with NaBH4. Firstly, ions of Ag were converted to Ag0 then they began to agglomerate into clusters, and eventually formed colloidal silver nanoparticles. Such reducing agents like NaBH4 may be dangerous to the environment and therefore, finding less harmful techniques were a challenge. Huang H. et al. [80] were the first who have used chitosan in the synthesis of gold and silver nanoparticles. They found out that chitosan is more than a protecting agent and it can reduce gold nanoparticles without any additional reducing agent. So they have developed a simple, green method using polysaccharides as reducing/stabilizing agents for synthesis of gold and silver nanoparticles. Most importantly, the green synthesis is a method of preparation nanoparticles which is environmentally friendly and using non-toxic reducing agents that are harmless to the environment. Wei et al. [81] tested chitosan as a non-toxic reducing agent for preparation silver nanoparticles by reduction of silver nitrate salts. Polymers are often studied as stabilizing agents for nanoparticles. The number of scientists, who are involved in the synthesis of the silver nanoparticles stabilized by polymers, is still increasing. Chitosan stabilized silver nanoparticles show very promising antibacterial properties [7]. Apart from the above mentioned chitosan, polyethylene glycol is a stabilizer for silver nanoparticles, as demonstrated by numerous works [78, 82-84]. Ahmad et al. [78] used chitosan for reduction of silver nanoparticles and polyethylene glycol as stabilizer. Based on the above mentioned works [78, 82-84] it can be concluded, that polymers with higher molecular weight might be more appropriate for forming stable silver nanoparticles. Using additives for chitosan films usually comes with changes in material properties [85, 86] and therefore a water absorption was studied. The water absorption is an important attribute when aiming on tissue engineering and a contact with living tissue. Ability of exudate absorption and maintenance of moist environment in the tissue is a desired combination of the material properties when used in wound healing. Apart from water

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Smart Metal Nanostructures for Effective Bacterial Inhibition 45 absorption, surface wettability is another important feature, that influences adherence to the wound [87].

3.1. Silver Precipitation

In our research [12, 88], three different stabilizers of silver nanoparticles were used – chitosan, microcrystalline cellulose and PEG (Mr 400 and 20,0000). In all experiments, silver nitrate was used as a source of silver. Aliquots of film forming solutions (varying in composition) were dried to yield a polymeric film with silver nanoparticles, see Figure 9. These films can be used as a solid stock of silver nanoparticles for later reconstitution – once they are dissolved, they form a homogeneous solution of silver nanoparticles. PEGs were used as a stabilizing agents of silver nanoparticles in chitosan matrix, and we found out that polyethylene glycol significantly altered the material properties of the prepared films [88]. Samples with addition of both PEG 400 and PEG 20,000 exhibited lower water absorption and lower contact angle than the control samples without PEG. This means that after the addition of PEG, the water permeability was reduced and at the same time surface wettability was increased. Samples without use of PEG (chitosan-cellulose films) exhibited higher water absorption and contact angle compared to samples which were additionally doped with silver. It was demonstrated that films with the addition of silver significantly reduced the wettability and water absorption. The results of absorptivity measurement and the contact angle measurement are shown in Table 5. The films were then dissolved in acetate buffer – chitosan is a polysaccharide, which is soluble in acidic media, and the presence of silver nanoparticles was studied by the UV-Vis spectroscopy. After the dissolution, the respective solutions were pale yellow to orange which is typically connected to the shape and concertation of silver nanoparticles with characteristic absorption at 400 nm. Stability of the solution containing nanoparticles can be affected a number of factors, e.g., light-fastness, pH, temperature, just to name a few. In our case, the prepared solutions were stored both in the presence of light and in the dark, at laboratory temperature.

A B C

Figure 9. Images of prepared films: A- chitosan–cellulose–Ag, B- chitosan–cellulose–PEG 400–Ag, C- Chitosan–cellulose–PEG 20,000–Ag.

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46 Jakub Siegel, Oleksiy Lyutakov, Vladimíra Vosmanská et al.

Table 5. Water absorption and contact angle measurement

Sample Water absorption Contact angel (°) Chitosan-cellulose-Ag 8.6 ± 0.9 82 ± 2 Chitosan-cellulose-PEG 400-Ag 5.0 ± 1.5 48 ± 9 Chitosan-cellulose-PEG 20,000-Ag 4.7 ± 1.4 35 ± 8

Figure 10. UV-Vis spectra of dissolved chitosan-cellulose films in acetate buffer stored in day light and in dark: A- chitosan–cellulose–Ag, B- chitosan–cellulose–PEG 400–Ag, C- Chitosan–cellulose–PEG 20,000–Ag.

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Smart Metal Nanostructures for Effective Bacterial Inhibition 47

To examine the effect of light on the stability of reconstituted solutions of silver nanoparticles, three solutions stored in light and three solutions stored in the dark were measured on the UV-Vis spectrophotometer. The compositions of the film forming solutions were cellulose-chitosan-Ag, cellulose-chitosan-PEG 400-Ag, and cellulose-chitosan-PEG 20,000-Ag. Figure 10 shows that during the measurement a slight decrease of absorbance occurred and together with a shift of peak wavelength towards the higher wavelength. The most striking peaks were observed for samples chitosan-cellulose-PEG 20,000-Ag regardless the storage conditions [88]. After 21 days after dissolution of films, the colour of the tested solutions remained the same no effect of the storage conditions was observed on the UV-Vis spectra. From literature it is known that solutions of silver nanoparticles absorb about wavelength of 400 nm, and according to the maximum wavelength the particles size can be determined [12]. Huang T. et al. [79] have also dealt with the determination of the nanoparticle size in according to the maximum wavelength, and they have proved that different colour of solution means a different shape and nanoparticle size. Therefore, according to the work of Huang T. at al., it is very likely that the chitosan solutions will contain nanoparticles sized tens of nanometres. Solutions were stored (three of them on day light and another three in dark) and measured again after one year. The maximum wavelength shifted to higher values in the case of solutions stored in the presence of day light. In the case of solutions stored in the dark, only minor changes in the maximum wavelength were observed. High increase in the peak area of silver was at sample A (chitosan-cellulose-Ag, Figure 10) was observed and no change for samples containing PEGs. That is in consistent with Luo et al. [83], where it was found out that the reducing activity of PEG was increased with the increase of its chain length. In order to get specific data on the size and shape of nanoparticles, transmission electron microscopy (TEM) was used, see Figure 11. In the solution, silver nanoparticles of various shapes were observed; spherical shape was the prevailing one. Minor frequency was observed for triangular and rod-shaped particles of silver. Chitosan-cellulose films obtained silver nanoparticles in the range of 10 to 80 nm, chitosan-cellulose-PEG 400-Ag 9 to 50 nm, and chitosan-cellulose-PEG-20,000 Ag 7-40 nm. By the TEM imaging, it was confirmed that the PEGs affect particle size. Films with addition of PEGs yielded smaller silver nanoparticles than films without them [88]. For analysis of concentration of elements on the surface of sample was used X-ray photoelectron spectroscopy (XPS) was used for elemental analysis of the prepared films. The XPS is showing the elemental composition of the superficial layer where we expected the silver, results are summarised in the Table 6. As expected, surface of samples contained elements typical for chitosan, cellulose and polyethylene glycol (carbon, oxygen and nitrogen). For all samples with the addition of silver nitrate was silver detected on the surface. Table 6 shows that the highest concentration of silver was detected in the film of chitosan- cellulose-Ag. The highest amount of oxygen was present in the samples containing PEGs, which was expected result for the use of PEGs [88]. The silver content on surface layers of these chitosan films was ranging 2.5 to 3.8 at.% of silver. The highest percentage of silver on the surface was found for chitosan-cellulose-Ag film and the lowest has chitosan-cellulose- PEG 20 000-Ag film [88].

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48 Jakub Siegel, Oleksiy Lyutakov, Vladimíra Vosmanská et al.

Figure 11. TEM images of dissolved chitosan films in acetate buffer: A- chitosan–cellulose–Ag, B- chitosan–cellulose–PEG 400–Ag, C- Chitosan–cellulose–PEG 20,000–Ag.

Table 6. XPS data on the elemental composition of chitosan films

Concentration of elements (at. %) Sample C(1s) O(1s) N(1s) Ag(3d) Chitosan-cellulose-Ag 57.3 32,6 6.2 3.9 Chitosan-cellulose-PEG 400-Ag 58.8 34.2 4.0 3.0 Chitosan-cellulose-PEG 20,000-Ag 56.3 34.9 6.3 2.5

4. STIMULI-RESPONSIVE ANTIBACTERIAL MATERIALS

The creation of an antibacterial material with triggerable properties enables us to avoid the overuse or misuse of antibacterial substances and, thus, prevent the emergence of resistant bacterial strains. Among the great amount of physico-chemical stimuli, which can be used for the initiation of antimicrobial activity, the main attention was paid on the temperature, pH, and light responsive materials. Light-responsive antimicrobial materials with antimicrobial response are very promising candidates for overcoming the bacterial resistance and phototreatment of different diseases (see Figure 12). Depending on the wavelength, which is used to initiate material response, these materials are constructed to be used in-vitro (nearinfrared wavelength) or ex -vivo (usually visible light spectrum). Especially interesting is the application of differently modified metal nanoparticles, which usually show the plasmon resonance and thus can very efficiently absorb the wide range of wavelengths.

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Smart Metal Nanostructures for Effective Bacterial Inhibition 49

Figure 12. Schematic representation of the AuNUs light triggerable antimicrobial activity.

4.1. Near Infrared Sensitive Functionalysed Metal Nanoparticles

Synthesis of surface-modified gold urchin-like nanoparticles (AuNUs) with plasmon resonance in the near IR range was performed by simple chemical reduction of gold chloride aqueous solution followed by modification using water soluble arenediazonium tosylates with different functional organic groups (OFGs). Schematic representation of AuNUs synthesis and surface modification is depicted in the Figure 13. AuNUs were prepared in aqueous solution according to well known procedure [89]. Presence of silver ions induced the anisotropic growth of gold explain formation of branches on the gold surface. Then, AuNUs surface was modified by freshly prepared suspension of arenediazonium tosylates (ADTs): 4- nitrobenzenediazonium tosylate, 4-aminobenzenediazonium tosylate, and 4- carboxybenzenediazonium tosylate [90]. The AuNUs shape and size distribution are presented in the Figure 14. Here, the TEM (14a) and SEM (14b) microscopies were applied to study AuNUs after preparation and purification procedure. It is evident, that the AuNUs size distribution lies in the 30-60 nm range. Grafting of OFGs was confirmed using the surface enhanced Raman spectroscopy (SERS). AuNUs contain a large number of plasmonic hot spots and thus the significant enhancement of Raman response can be detected. SERS spectra in Figure 14c identify the presence of all grafted OFGs: 4-nitrophenyl (-C6H4-NO2), 4- aminophenyl (-C6H4-NH2) and 4-carboxyphenyl (-C6H4-COOH). Wavenumber position peak at ≈ 400 cm-1 appears on all functionalized samples and affirms the formation of covalently attached organic functional groups [91, 92].

Figure 13. Scheme of synthesis of modified gold multibranched nanoparticles (mod-AuMs-R; R = - COOH, -NH2, NO2).

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Figure 14. A, B – TEM and SEM images of modified gold multibranched nanoparticles; C - Raman spectra of multibranched nanoparticles (AuMs) and surface-modified gold multibranched nanoparticles (mod-AuMs) by ADTs with -NO2, -NH2, -COOH functional groups (aromatic ring Ar).

Figure 15 gives the results of the antibacterial test - a number of survival bacteria after their incubation with pristine and surface functionalized AuNUs with and without IR illumination. It is evident, that the light treatment itself results in only slight decrease of survival bacteria. Latent period of bacterial strains with nonfunctionalized AuNUs results in the bacteria death (Figure 15). This effect is visible in the case of gramm-positive S. epidermis (Figure 15b) but it is very slight in the case of gramm-negative E.coli (Figure 15a). However, grafting of OFGs can significantly increase the antimicrobial effectivity. Moreover, the antibacterial activities of functionalized AuNUs were found to be dependent on the grafted organic functional groups. Grafting with amino groups leads to the best antibacterial activity, grafting with carboxyl groups also results to the increase of antibacterial activity, but in case of nitro OFGs does not change the antibacterial activity of AuNUs. Gramm negative E. coli in all cases exhibited better resistance than gramm-positive S. epidermis to antibacterial triggering. Functional AuNUs under light triggering increase their efficiency in the killing of both, gramm-negative and gramm-positive bacteria. Applying of effective plasmon excitation together with AuNUs concentrates the light energy into so-called hot- spots on the NPs cusp and conducts to degradation of neighbored organic compounds and destroying of bacterial membrane. AuNUs cups have strong plasmon resonance and this leads Complimentary Contributor Copy

Smart Metal Nanostructures for Effective Bacterial Inhibition 51 to significant reduction of the light power needed and to apparent bacteria killing under continuous illumination, comparing with previously published work, where a powerful laser beam was used to initiate the antimicrobial activity.

4.2. Visible Light Responsive Polymer Thin Films

Visible light activated antimicrobial materials in combination with a simple light system can be utilized in the development of a range of photobactericidal polymeric medical devices and coatings. One of the possible examples of such materials is the antimicrobial light- activated polymethylmethactylate thin films doped with silver nanoparticles and tetraphenylporphyrin (TPP) [93]. Porphyrine molecules can serve as light absorber with dual antimicrobial response - under illumination they produce reactive oxygen (RO) and affect kinetics of silver release from polymer. Reactive oxygen kills Gram-positive bacteria and silver is responsible for antimicrobial effect against Gram-negative bacteria. Additionally, porphyrine stability increases with interaction of AgNPs. In general, antimicrobial response of the material can be controlled by blue light illumination and activated through local heating of polymer matrix, transfer of excited state from porphyrin to silver and synergetic effect of reactive oxygen and silver. The possibility of TPP to produce of RO to was checked using iod (I-) reduction. Production of I2 manifests itself by yellow coloring of initially opaque potassium iodide KI solution (appearance of pronounced absorption band at 330 nm). Figure 16A presents absorption spectra of KI solution illuminated in the presence of pristine PMMA (iodine reduction by light), TPP/PMMA (iodine reduction by RO and light), and TPP-AgNPs/PMMA films. Fresh samples and samples previously treated by light illumination were examined in water during 72 h. Absorption peak below 400 nm is attributed to I2. Light irradiation causes iodine reduction it is evident from Figure 16A. But iodine reduction is more apparent when TPP/PMMA and TPP-AgNPs/PMMA samples are irradiated. However at the other hand, the creation of RO by TPP-AgNPs/PMMA does not change significantly compared to untreated samples. AgNPs affect the RO production and this phenomenon is completely compensated in the presence of AgNPs.

Figure 15. Antimicrobial activity: CFU per mililiter after bacteria incubation (A – E. coli; B – S. epidermis) with AuMs and differently modified AuMs (with -NO2, -NH2, -COOH) with and without light triggering.

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Figure 16. A - Absortion spectra of I2 solution created under illumination of PMMA, TPP/PMMA, and TPP-AgNPs/PMMA films in KI solution; B - time dependence of silver concentrations in extracted solution released under light illumination and in dark; C - depth profile of silver concentration in pristine, illuminated and non-illuminated TPP-AgNPs/PMMA films (72 h).

Figure 16B shows the cumulative release of silver from the TPP-AgNPs/PMMA films with and without illumination. Surface morphologies of AgNPs doped PMMA films before and after silver releasing were studied and reported in our previous work [94]. From Figure 16B it is apparent, that at early stages, AgNPs are located close to the surface and are released predominantly. This effect completely disappears in the later illumination stages. Light illumination speeds AgNPs release from near surface region and slightly slows down release from deeper regions. Figure 16C depicts silver concentration for pristine, illuminated and non-illuminated samples. Silver distribution after the sample preparation is non-uniform; the concentration being sufficiently higher near the surface. Soaking of samples lead to decrease of silver concentration especially in near surface region. When the sample is simultaneously irradiated this effect is more pronounced. The excitation of TPP in TPP-AgNPs/PMMA films enhances AgNPs release form the sample surface that reaffirms results of XPS and AAS studies.

Figure 17. Photos illustrating inhibitory effect on P. aeruginosa in the presence of illuminated/non- illuminated TPP/PMMA, AgNPs/PMMA, and TPP-AgNPs/PMMA films.

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Smart Metal Nanostructures for Effective Bacterial Inhibition 53

The antimicrobial properties of AgNPs/PMMA, TPP/PMMA, and TPP-AgNPs/PMMA were examined against gram-positive and gram-negative bacteria. Studies were performed in solution. Results are presented in the Figure 17, where the positive sign specifies the case of fully destroyed bacteria, negative sign indicates absence of antimicrobial effect, plus/minus sign corresponds to an interim case (see Figure 17). In light control experiments only weak reduction of bacteria growth was observed, apparently due to RO production by intracellular chromophores. In dark control experiments no bacteria growth inhibition was observed. AgNPs/PMMA samples were efficient in the case of gram-negative bacteria. In the case of S. aureus AgNPs caused only weak antimicrobial effect. TPP/PMMA films were more effective against S. aureus. In the case of TPP, gram-positive bacteria were inactivated faster than gram-negative ones [95]. At the other hand AgNPs are highly active against gram-negative bacteria [94]. As to TPP-AgNPs/PMMA films, they were effective against both, gramm- negative and gramm-positive bacteria, but mainly in “active”, illuminated state. So, it can be concluded, that activation by light is needed for sufficient TPP-AgNPs/PMMA antimicrobial activity. Light-switchable properties of TPP-AgNPs/PMMA films were also examined by in- contact method. The irradiation was applied with light-emitting diode for 0.5, 1, and 3 hours. Non-illuminated sample (3 hours) was chosen as a control. Results are presented in the Figure 18, where the lighter areas correspond to the development of many bacterial microcolonies and to the absence of antimicrobial properties. Lighter areas in both controls and samples irradiated during 0.5 h were observed. The antimicrobial properties are completely manifested, but some bacteria still survived. In the case of samples irradiated during 2 h, the same situation was observed. Complete destruction of S. aureus was found after irradiation for 3 h, but in the case of P. aeruginosa only partial destruction was observed. So, it can be concluded that the antimicrobial properties were entirely controlled by light switch-on/off. Additionally, Figure 18E shows antimicrobial properties of samples stored at ambient conditions (sunlight, normal air) for three months. Both samples were light-activated for 3 h. Antimicrobial activity of the aged samples slightly decreased in the case of P. aeruginosa and remained fully unchanged in the case of S. aureus.

4.3. Visible Light Responsive Polymer Nanofibers

Antimicrobial activity of the previously describe thin films can be significantly enhanced by increasing of the surface to volume ration through the deposition of protective coating in the nanofiber form [96]. The PMMA nanofibers doped with AgNPs and TPP were prepared by electrospinning procedure and tested for their activity. The electrospinning was performed using a vertical set up with a 0.01 mL h-1 feeding rate, 40 mm tip-to-collector distance and potential 14 kV. Figure 19A shows SEM images of as spun Ag/TPP/PMMA nanofibers. It is evident, that relatively broad distribution of nanofiber diameters (from 100 nm to 2.7 µm) was obtained (determined on ≥10 SEM images from randomly selected 10 places from each image using image analysis software). Additionally, light emitted diode (LED, λ=405 nm) treated nanofibers are presented in the Figure 19B. It is evident, that after LED treatment for 20 h the nanofibers structure did not change significantly.

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Figure 18. Antimicrobial activity against (i) P.aeruginosa and (ii) S.aureus. Ag/TPP-PMMA films were in contact with bacteria on agar for 1 (A), 2 (B), and 3 (C) hours under illumination. Control sample was placed in contact with bacteria but not activated by external light (D). Parts E exhibit light- activated antimicrobial activity of the samples aged for 3 months under environmental conditions.

Creation of AgNPs as well as their release under the light illumination was checked using the TEM technique. Figure 20 shows the TEM images of AgNPs embedded in polymer nanofibers before (Figure 20A) and after (Figures. 20B) light treatment. As is evident from the Figure 20A, the AgNPs were successfully synthesized and well-distributed within polymer nanofibers. It is also seen that the AgNPs have average diameter of about 2 nm with narrow size distribution. The effect of the irradiation with light from LED leads to pronounced release of AgNPs from polymer nanofibers. Released AgNPs are visible on the carbon layer of the TEM grid (dark spots in the proximity of pristine nanofiber). As could be expected a simultaneous decrease of AgNPs concentration inside the nanofibers occurred. Probably, upon LED irradiation a heating of polymer chains occurred, which led to an increase of the mobility of segments and, as a result, to movement of AgNPs within the polymer matrix.

Figure 19. Scanning electron microscope images of nanofibers prepared by electrospinning from solutions in chloroform: A – as prepared Ag/TPP/PMMA nanofibers; B – Ag/TPP/PMMA nanofibers after LED treatment.

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Figure 20. Transmission electron microscopy images of AgNPs embedded TPP/PMMA polymer nanofibers and distribution of AgNPs in TPP/PMMA matrix before treatment: A – fibers as prepared by electrospinning; B – effect of LED treatment after 20 h.

Releasing of AgNPs under the light illumination must significantly affect the nanofibers antimicrobial activity. Results of corresponded test, performed by in-contact method with and without samples illumination are present in the Figure 21. Here, the antibacterial activity are presented as the “protected” agar area, where the bacterial growth do not occurs (the area, which was covered by sample, is depicted by dotted line in the Figure 21). Generally, two typical examples were observed: killing of bacteria under the sample only (when the column height matches the dotted line) or additional appearance of an inhibition zone (when the column height exceeds dotted line). It is evident, that the antibacterial activity of AgNPs/TPP/PMMA nanofibers under light irradiation is the highest due to the release of nanoparticles from the polymer nanofibers promoted by LED irradiation. In this case the light absorbed by TPP was transferred to heat, which induced polymer chains motion and AgNPs elimination. The antibacterial activity of nanofibers was also compared with AgNPs/TPP/PMMA films containing the same amount of AgNPs/TPP (Figure 21). It is evident, that the AgNPs/TPP/PMMA films can effectively kill bacteria under the sample, but the formation of the inhibition zone was almost not observed. We should also note the lack of a similar effect in the thin films that exhibit antibacterial activity only upon lighting and the zone of inhibition is much smaller than a similar one in the case of AgNPs/TPP/PMMA nanofibers.

4.4. Temperature and pH Responsive Materials

One of the most studied stimuli-responsive polymers is PNIPAm, which undergoes volume phase transition near 31°C (LCST – lower critical solution temperature). PNIPAm is fully water-soluble below the LCST and loses its solubility above this temperature. From the practical point of view it is sometimes necessary to tune the LCST into a different temperature range. A basic way to shift LCST value is copolymerization with other monomers [97]. The most common comonomer is acrylic acid (AAc), which adds responsivity to acidity. At pH > 4.25 the AAc groups are deprotonated and the polymer swells. When the pH decreases below 4.25 again, the polymer returns back to its initial state [98-100].

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Figure 21. Protected areas of the AgNPs/TPP/PMMA and TPP/PMMA nanofibers against S. epidermidis, E. faecalis mats determined by disc diffusion method in the dark and under LED irradiation (24 h).

Confinement effect was also reported to influence the polymer phase transition and the release of small molecules from thin films [101-103]. Relative reflection of light from 1 µm thick PNIPAm-co-AAc films deposited on the Si substrate was used to detect the phase transition of PNIPAm-co-AAc thin films, deposited on the silicon substrate in dependence on the films thicknesses. Results are presented in the Figure 22 for previously heated polymer films immersed into distilled water and gradually cooled. Values of the central temperature and the temperature range of PNIPAm-co-AAc phase transition are given here as the central point and two small symbols (beginning and end of the phase transition). It is apparent that with decreasing thickness of polymer, phase transitions are shifted to higher values, especially for polymer film with thicknesses below 200 nm. Simultaneously, the temperature range broadens significantly. Environmental pH also affects the thickness dependences – under acidic pH the observed phenomena are more significant.

Table 7. Dependences of the size of normed inhibition zone (NIZ size) on the thickness of PNIPAm-co-AAc films doped with Crystal Violet spin-coated on Si substrate

Time of bacteria growth (h) 20 22 24 Thickness (m) 1.0 0.5 0.05 1.0 0.5 0.05 1.0 0.5 0.05 NIZ size (cm) 7.6 9.6 78 5.3 6.4 46 5.8 7.0 44 Complimentary Contributor Copy

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55 55

Si substrate Si substrate

C) o 50 o 50 45 45

pH=7,0

40 40 pH=4,0

35 35 Temperature ( 30 Temperature ( 30 25 25 0,0 0,40,81,2 0,00,40,81,2 Thickness (m) Thickness (m)

Figure 22. Temperature dependence of phase transition at pH = 4 and pH = 7 of PNIPAm-co-AAc film deposited on Si substate.

Observed thickness dependences were applied to control the release of crystal violet (CV) from thin and ultrathin PNIPAm-co-AAc films deposited on Si substrates. It must be noted that CV was chosen not only because of its strong absorption peak at 590 nm for release measurement, but also due to its activity against gram-positive bacteria. Results of antibacterial tests performed in contact arrangement are presented in Table 7. The inhibition zone sizes were normalized for determination of antibacterial effect per thickness unit. Samples were in contact with bacteria at the temperature of 37°C. This temperature is above the phase transition temperature of the thick polymer, but below the transition temperature of the thin one. Thus it was initially expected that thick polymer film will not create a zone of inhibition. However, in this case inhibition zones were well visible. The Table 7 illustrates that with the decrease of polymer thickness the normalized size of inhibition zone increases. On the base of the in-contact antibacterial tests it can be concluded that the antibacterial activity of CV doped PNIPAm-co-AAc films increases with decreasing thickness. This effect can be attributed to the shift of PNIPAm-co-AAc phase transition from “critical” thickness.

ACKNOWLEDGMENT

Financial support of this work from the GACR projects Nos. 14-18131S, 15-19209S and 15-19485S and Ministery of Health of CR under the project 15-33459A is gratefully acknowledged.

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[51] Siegel, J., Kolářová, K., Vosmanská, V., Rimpelová, S., Leitner, J. and Švorčík, V. (2013). Antibacterial properties of green-synthesized noble metal nanoparticles. Mater. Lett., 113, 59-62. [52] Grazhulene, S. S., Red’kin, A. N., Telegin, G. F., Bazhenov, A. V. and Fursova, T. N. (2010). Adsorption properties of carbon nanotubes depending on the temperature of their synthesis and subsequent treatment. J. Anal. Chem., 65, 682-689. [53] Myllytie, P., Salmi, J. and Laine, J. (2009). The influence of ph on the adsorption and interaction of chitosan with cellulose. BioResources., 4, 1647-1662. [54] Qin, Y. (1993). The chelating properties of chitosan fibers. J. Appl. Polym. Sci., 49, 727-731. [55] Guibal, E. (2004). Interactions of metal ions with chitosan-based sorbents: A review. Sep. Purif. Technol., 38, 43-74. [56] Jayakumar, R., Menon, D., Manzoor, K., Nair, S. V. and Tamura, H. (2010). Biomedical applications of chitin and chitosan based nanomaterials—a short review. Carbohydr. Polym., 82, 227-232. [57] Xia, W., Liu, P., Zhang, J. and Chen, J. (2011). Biological activities of chitosan and chitooligosaccharides. Food Hydrocolloids., 25, 170-179. [58] Fei Liu, X., Lin Guan, Y., Zhi Yang, D., Li, Z. and De Yao, K. (2001). Antibacterial action of chitosan and carboxymethylated chitosan. J. Appl. Polym. Sci., 79, 1324-1335. [59] Lim, S. H. and Hudson, S. M. (2004). Synthesis and antimicrobial activity of a water- soluble chitosan derivative with a fiber-reactive group. Carbohydr. Res., 339, 313-319. [60] Strnad, S., Sauperl, O. and Fras Zemljič, L. (2010). Cellulose fibres funcionalised by chitosan: Characterization and application. In: Elnashar M, editor. Biopolymers: InTech. [61] Kolarova, K., Vosmanska, V., Rimpelova, S. and Svorcik, V. (2013). Effect of plasma treatment on cellulose fiber. Cellulose., 20, 953-961. [62] Bauer, A. W., Kirby, W. M., Sherris, J. C. and Turck, M. (1966). Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol., 45, 496-496. [63] Jelly, R., Lewis, S. W., Lennard, C., Lim, K. F. and Almog, J. (2010). Substituted naphthoquinones as novel amino acid sensitive reagents for the detection of latent fingermarks on paper surfaces. Talanta., 82, 1717-1724. [64] Topalovic, T., Nierstrasz, V. A., Bautista, L., Jocic, D., Navarro, A. and Warmoeskerken, M. M. C. G. (2007). Xps and contact angle study of cotton surface oxidation by catalytic bleaching. Colloid. Surf. A., 296, 76-85. [65] Calvimontes, A., Mauersberger, P., Nitschke, M., Dutschk, V. and Simon, F. (2011). Effects of oxygen plasma on cellulose surface. Cellulose., 18, 803-809. [66] Karahan, H. A. and Özdoğan, E. (2008). Improvements of surface functionality of cotton fibers by atmospheric plasma treatment. Fibers Polym., 9, 21-26. [67] Thakur, V. K. and Thakur, M. K. (2015). Handbook of sustainable polymers: Processing and applications. Pan Stanford. [68] Da Róz, A. L., Leite, F. L., Pereiro, L. V., Nascente, P. A. P., Zucolotto, V., Oliveira, O. N. and Carvalho, A. J. F. (2010). Adsorption of chitosan on spin-coated cellulose films. Carbohydr. Polym., 80, 65-70. [69] Zemljič, L. F., Peršin, Z., Stenius, P. and Kleinschek, K. S. (2008). Carboxyl groups in pre-treated regenerated cellulose fibres. Cellulose., 15, 681-690. Complimentary Contributor Copy

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[70] Zemljič, L., Fras, Peršin, Z. and Stenius, P. (2009). Improvement of chitosan adsorption onto cellulosic fabrics by plasma treatment. Biomacromolecules., 10, 1181-1187. [71] Kolska, Z., Makajova, Z., Kolarova, K., Kasalkova, N., Trostova, S., Reznickova, A., Siegel, J. and Svorcik, V. (2013). Electrokinetic potential and other surface properties of polymer foils and their modifications. In: Yılmaz F., editor. Polymer Science: InTech. [72] Elimelech, M., Chen, W. H. and Waypa, J. J. (1994). Measuring the zeta (electrokinetic) potential of reverse osmosis membranes by a streaming potential analyzer. Desalination., 95, 269-286. [73] Stana-Kleinschek, K., Strnad, S. and Ribitsch, V. (1999). Surface characterization and adsorption abilities of cellulose fibers. Polym. Eng. Sci., 39, 1412-1424. [74] Hu, W., Chen, S., Li, X., Shi, S., Shen, W., Zhang, X. and Wang, H. (2009). In situ synthesis of silver chloride nanoparticles into bacterial cellulose membranes. Mater. Sci. Eng: C., 29, 1216-1219. [75] Son, W. K., Youk, J. H., Lee, T. S. and Park, W. H. (2004). Preparation of antimicrobial ultrafine cellulose acetate fibers with silver nanoparticles. Macromol. Rapid Commun., 25, 1632-1637. [76] Kim, J. S. and Kim, Y. (2007). The inhibitory effect of natural bioactives on the growth of pathogenic bacteria. Nutr. Res. Pract., 1, 273-278. [77] Sharma, V. K., Yngard, R. A. and Lin, Y. (2009). Silver nanoparticles: Green synthesis and their antimicrobial activities. Adv. Colloid Interface Sci., 145, 83-96. [78] Ahmad, M. B., Tay, M. Y., Shameli, K., Hussein, M. Z. and Lim, J. J. (2011). Green synthesis and characterization of silver/chitosan/ polyethylene glycol nanocomposites without any reducing agent. Int. J. Mol. Sci., 12, 4872-4884. [79] Huang, T. and Xu, X. H. N. (2010). Synthesis and characterization of tunable rainbow colored colloidal silver nanoparticles using single-nanoparticle plasmonic microscopy and spectroscopy. J. Mater. Chem., 20, 9867-9876. [80] Huang, H., Yuan, Q. and Yang, X. (2004). Preparation and characterization of metal– chitosan nanocomposites. Colloids Surf. B-Biointerfaces., 39, 31-37. [81] Wei, D., Sun, W., Qian, W., Ye, Y. and Ma, X. (2009). The synthesis of chitosan-based silver nanoparticles and their antibacterial activity. Carbohydr. Res., 344, 2375-2382. [82] Shkilnyy, A., Soucé, M., Dubois, P., Warmont, F., Saboungi, M. L. and Chourpa, I. (2009). Poly (ethylene glycol)-stabilized silver nanoparticles for bioanalytical applications of sers spectroscopy. Analyst., 134, 1868-1872. [83] Luo, C., Zhang, Y., Zeng, X., Zeng, Y. and Wang, Y. (2005). The role of poly(ethylene glycol) in the formation of silver nanoparticles. J. Colloid Interface Sci., 288, 444-448. [84] Panáček, A., Kolář, M., Večeřová, R., Prucek, R., Soukupová, J., Kryštof, V., Hamal, P., Zbořil, R. and Kvítek, L. (2009). Antifungal activity of silver nanoparticles against candida spp. Biomaterials., 30, 6333-6340. [85] Zhang, M., Li, X. H., Gong, Y. D., Zhao, N. M. and Zhang, X. F. (2002). Properties and biocompatibility of chitosan films modified by blending with peg. Biomaterials., 23, 2641-2648. [86] Yoshida, C. M. P., Oliveira Junior, E. N. and Franco, T. T. (2009). Chitosan tailor- made films: The effects of additives on barrier and mechanical properties. Packag. Technol. Sci., 22, 161-170.

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[87] Sopuch, T., Drahovzalová, R., Rýdl, J., Bureš, I., Milichovský, M., Vytřasová, J., Moťková, P., Švorčík, V., Podlaha, J. and Horáková, M. (2013). Celulózové materiály v ošetřování ran. Abstrakt z: Mezinárodní spolupráce při léčbě ran a kožních defektů. 24.–25.1. 2013. Hojení ran 7, 1. 24. [88] Pišlová, M., Kateřina, K., Vladimíra, V. and Ondřej, K. (2015). Příprava polysacharidových filmů na bázi chitosanu a celulosy. Chem. Listy., 109, 942-945. [89] Cheng, L. C., Huang, J. H., Chen, H. M., Lai, T. C., Yang, K. Y., Liu, R. S., Hsiao, M., Chen, C. H., Her, L. J. and Tsai, D. P. (2012). Seedless, silver-induced synthesis of star- shaped gold/silver bimetallic nanoparticles as high efficiency photothermal therapy reagent. J. Mater. Chem., 22, 2244-2253. [90] Kalachyova, Y., Olshtrem, A., Guselnikova, O. A., Postnikov, P. S., Elashnikov, R., Ulbrich, P., Rimpelova, S., Svorcik, V. and Lyutakov, O. (2016). Synthesis, characterization and antimicrobial evaluation of a near-IR photoactive surface functionalysed gold multibranched tags, Nanoscale., submitted. [91] Ahmad, R., Boubekeur-Lecaque, L., Nguyen, M., Lau-Truong, S. p., Lamouri, A., Decorse, P., Galtayries, A., Pinson, J., Felidj, N. and Mangeney, C. (2014). Tailoring the surface chemistry of gold nanorods through au–c/ag–c covalent bonds using aryl diazonium salts. J. Phys. Chem. C., 118, 19098-19105. [92] Mesnage, A., Lefèvre, X., Jégou, P., Deniau, G. and Palacin, S. (2012). Spontaneous grafting of diazonium salts: Chemical mechanism on metallic surfaces. Langmuir., 28, 11767-11778. [93] Lyutakov, O., Hejna, O., Solovyev, A., Kalachyova, Y. and Svorcik, V. (2014). Polymethylmethacrylate doped with porphyrin and silver nanoparticles as light- activated antimicrobial material. RSC Adv., 4, 50624-50630. [94] Lyutakov, O., Kalachyova, Y., Solovyev, A., Vytykacova, S., Svanda, J., Siegel, J., P. Ulbrich, P. and Svorcik, V. (2015) One-step preparation of antimicrobial silver nanoparticles in polymer matrix. J. Nanopart. Res., 17, 120 (11). [95] Tom, R. T., Samal, A., Sreeprasad, T. and Pradeep, T. (2007). Hemoprotein bioconjugates of gold and silver nanoparticles and gold nanorods: Structure-function correlations. Langmuir., 23, 1320-1325. [96] Elashnikov, R., Lyutakov, O., Ulbrich, P. and Svorcik, V. (2016). Light-activated polymethylmethacrylate nanofibers with antibacterial activity. Mater. Sci. Eng. C., 64, 229-235. [97] Zhang, J., Chu, L. Y., Li, Y. K. and Lee, Y. M. (2007). Dual thermo-and ph-sensitive poly (n-isopropylacrylamide-co-acrylic acid) hydrogels with rapid response behaviors. Polymer., 48, 1718-1728. [98] Huang, H. and Serpe, M. J. (2015). Poly (n‐isopropylacrylamide) microgel‐based etalons for determining the concentration of ethanol in gasoline. J. Appl. Polym. Sci., 132. [99] Johnson, K. C., Mendez, F. and Serpe, M. J. (2012). Detecting solution ph changes using poly (n-isopropylacrylamide)-co-acrylic acid microgel-based etalon modified quartz crystal microbalances. Anal. Chim. Acta., 739, 83-88. [100] Parasuraman, D. and Serpe, M. J. (2011). Poly (n-isopropylacrylamide) microgel-based assemblies for organic dye removal from water. ACS Appl. Mater. Interfaces., 3, 4714- 4721.

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[101] Elashnikov, R., Radocha, M., Rimpelova, S., Švorčík, V. and Lyutakov, O. (2015). Thickness and substrate dependences of phase transition, drug release and antibacterial properties of pnipam-co-aac films. RSC Adv., 5, 86825-86831. [102] Keddie, J. L., Jones, R. A. and Cory, R. A. (1994). Size-dependent depression of the glass transition temperature in polymer films. EPL (Europhysics Letters)., 27, 59. [103] Keddie, J. L., Jones, R. A. L. and Cory, R. A. (1994). Interface and surface effects on the glass-transition temperature in thin polymer films. Faraday Discuss., 98, 219-230.

BIOGRAPHICAL SKETCHES

Jakub Siegel, PhD

Affiliation: Department of Solid State Engineering, University of Chemistry and Technology Prague, Technicka 5, 166 28 Prague 6, Czech Republic

Education: 2003-2007 MSc., University of Chemistry and Technology Prague, Material engineering 2007-2010 Ph.D., University of Chemistry and Technology Prague, Material engineering

Research and Professional Experience: 2007 Institut für Angewandte Physik, Johannes Kepler Universität Linz, Ostereich 2008 – 2014 Department of Solid State Engineering, University of Chemistry and Technology Prague, Czech Republic

Professional Appointments: 2008 – 2010 Research Fellow, Department of Solid State Engineering, University of Chemistry and Technology Prague 2010 – 2014 Assistant Professor, Department of Solid State Engineering, University of Chemistry and Technology Prague 2014 Associate Professor of Materials Science, University of Chemistry and Technology Prague

Honors: Werner von Siemens Excellence Award 2010. Award of the Rector of UCT Prague for outstanding young academic staff¨, 2015.

Publications Last 3 Years: Publications According to Web of Science – publications 58, citations 542, H index 14 [1] M. Staszek, J. Siegel, S. Rimpelova, O. Lyutakov, V. Svorcik, Mater Lett. 158 (2015) 351-354. [2] P. Slepicka, N.S. Kasalkova, J. Siegel, Z. Kolska, L. Bacakova, V. Svorcik, Biotechnology Advances. 33 (2015) 1120-1129. [3] J. Siegel, M. Polivkova, M. Staszek, K. Kolarova, S. Rimpelova, V. Svorcik, Mater Lett. 145 (2015) 87-90. Complimentary Contributor Copy

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[4] M. Polivkova, M. Valova, J. Siegel, S. Rimpelova, T. Hubacek, O. Lyutakov, V. Svorcik, Rsc Advances. 5 (2015) 73767-73774. [5] O. Lyutakov, Y. Kalachyova, A. Solovyev, S. Vytykacova, J. Svanda, J. Siegel, P. Ulbrich, V. Svorcik, Journal of Nanoparticle Research. 17 (2015). [6] M. Staszek, J. Siegel, K. Kolarova, S. Rimpelova, V. Svorcik, Micro and Nano Letters. 9 (2014) 778-781. [7] P. Slepicka, O. Nedela, J. Siegel, R. Krajcar, Z. Kolska, V. Svorcik, Express Polym Lett. 8 (2014) 459-466. [8] J. Siegel, M. Staszek, V. Svorcik, Chem Listy. 108 (2014) 1102-1112. [9] R. Krajcar, J. Siegel, P. Slepicka, P. Fitl, V. Svorcik, Mater Lett. 117 (2014) 184-187. [10] R. Krajcar, J. Siegel, O. Lyutakov, P. Slepicka, V. Svorcik, Mater Lett. 137 (2014) 72- 74. [11] Y. Kalachyova, O. Lyutakov, V. Prajzler, J. Tuma, J. Siegel, V. Svorcik, Polym Compos. 35 (2014) 665-670. [12] R.A. Barb, C. Hrelescu, L. Dong, J. Heitz, J. Siegel, P. Slepicka, V. Vosmanska, V. Svorcik, B. Magnus, R. Marksteiner, M. Schernthaner, K. Groschner, Appl Phys A- Mater Sci Process. 117 (2014) 295-300. [13] J. Tuma, O. Lyutakov, I. Huttel, J. Siegel, J. Heitz, Y. Kalachyova, V. Svorcik, J Mater Sci. 48 (2013) 900-905. [14] V. Svorcik, Z. Kolska, J. Siegel, P. Slepicka, J Nano Res. 25 (2013) 40-48. [15] J. Siegel, M. Polivkova, N.S. Kasalkova, Z. Kolska, V. Svorcik, Nanoscale Res Lett. 8 (2013) 1-10. [16] J. Siegel, O. Kvitek, O. Lyutakov, A. Reznickova, V. Svorcik, Vacuum. 98 (2013) 100- 105. [17] J. Siegel, K. Kolarova, V. Vosmanska, S. Rimpelova, J. Leitner, V. Svorcik, Mater Lett. 113 (2013) 59-62. [18] J. Siegel, P. Jurik, Z. Kolska, V. Svorcik, Surf Interface Anal. 45 (2013) 1063-1066. [19] J. Siegel, J. Heitz, A. Reznickova, V. Svorcik, Appl Surf Sci. 264 (2013) 443-447. [20] O. Lyutakov, J. Tuma, I. Huttel, V. Prajzler, J. Siegel, V. Svorcik, Applied Physics B- Lasers and Optics. 110 (2013) 539-549. [21] O. Kvitek, J. Siegel, V. Hnatowicz, V. Svorcik, J Nanomater. (2013). [22] Z. Kolska, N.S. Kasalkova, J. Siegel, V. Svorcik, J Nano Res. 25 (2013) 31-39. [23] T. Hubacek, J. Siegel, R. Khalili, N. Slepickova-Kasalkova, V. Svorcik, Appl Surf Sci. 275 (2013) 43-48. [24] T. Hubacek, Z. Kolska, J. Siegel, V. Svorcik, J Mater Sci. 48 (2013) 819-824.

Oleksiy Lyutakov, PhD

Affiliation: Department of Solid State Engineering, University of Chemistry and Technology Prague, Technicka 5, 166 28 Prague 6, Czech Republic

Education: 1999-2004 MSc., Donetsk National University, Inorganic Chemistry 2005-2009 Ph.D., University of Chemistry and Technology Prague, Material engineering

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66 Jakub Siegel, Oleksiy Lyutakov, Vladimíra Vosmanská et al.

Professional Appointments: 2009 – Research worker, Department of Solid State Engineering, University of Chemistry and Technology Prague

Publications Last 3 Years: Publications According to Web of Science – publications 50, citations 251, H index 10 [1] Kalachyova Y,; Mares D,; Jerabek V,; Zaruba K.; Ulbrich P.; Lapcak, L.; Svorcik, V.; Lyutakov O.; The Effect of Silver Grating and Nanoparticles Grafting for LSP–SPP Coupling and SERS Response Intensification J. Phys. Chem. C, 2016, DOI: 10.1021/acs.jpcc.6b01587. [2] Svanda, J.; Kalachyova, Y.; Slepicka, P.; Svorcik, V.; Lyutakov, O.; Smart Component for Switching of Plasmon Resonance by External Electric Field, ACS Appl. Mater. Interf., 8, 225-231, 2016. [3] Kalachyova, Y.; Mares, D.; Lyutakov, O.; Kostejn, M.; Lapcak, L.; Svorcik, V.; Surface Plasmon Polaritons on Silver Gratings for Optimal SERS Response, J. Phys. Chem. C, 119, 9506-9512, 2015. Citations: 0. [4] Kalachyova, Y.; Alkhimova, D.; Kostejn, M.; Machac, P.; Svorcik, V.; Lyutakov, O. Plasmooptoelectronic tuning of optical properties and SERS response of ordered silver grating by free carrier generation RSC Adv. 5, 92869-92877, 2015. [5] Kalachyova, Y.; Lyutakov, O.; Goncharova, I.; Svorcik, V. "Artificial" chirality induced in doped polymer by irradiation with circularly polarized excimer laser light, Opt. Mater. Express, 5, 2761-2767, 2015.

Václav Švorčík, PhD

Affiliation: Department of Solid State Engineering, University of Chemistry and Technology Prague, Technicka 5, 166 28 Prague 6, Czech Republic

Education: 1969-1973 MSc., University of Chemistry and Technology Prague, Material engineering 1979-1981 Ph.D., University of Chemistry and Technology Prague, Material engineering

Research and Professional Experience: 1980 – now Department of Solid State Engineering, University of Chemistry and Technology Prague, Czech Republic

Professional Appointments: 2001 – present: Head of the Department of Solid State Engineering, University of Chemistry and Technology Prague 2000 Professor of Materials Science and Engineering, University of Chemistry and Technology Prague

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Smart Metal Nanostructures for Effective Bacterial Inhibition 67

Honors: Werner von Siemens Excellence Award 2010, best dissertation thesis in technical field. 2015 Award of the Rector of UCT Prague for outstanding young academic staff.

Publications Last 3 Years: Publications According to Web of Science – publications 336, citations 3610, H index 29

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Chapter 3

ANTIBACTERIAL LECTINS: ACTION MECHANISMS, DEFENSIVE ROLES AND BIOTECHNOLOGICAL POTENTIAL

Thamara F. Procópio1, Maiara C. Moura1, Lidiane P. Albuquerque2, Francis S. Gomes3, Nataly D. L. Santos1, Luana C. B. B. Coelho1, Emmanuel V. Pontual4, Patrícia M. G. Paiva1 and Thiago H. Napoleão1, 1Departamento de Bioquímica, Centro de Biociências, Universidade Federal de Pernambuco, Recife, Brazil 2Departamento de Bioquímica e Farmacologia, Universidade Federal do Piauí, Teresina, Brazil 3Instituto de Química e Biotecnologia, Universidade Federal de Alagoas, Maceió, Brazil 4Departamento de Morfologia e Fisiologia Animal, Universidade Federal Rural de Pernambuco, Recife, Brazil

ABSTRACT

Lectins are proteins able to bind specifically and reversibly to carbohydrates, without promote any alteration in the covalent structure of the ligand. These proteins are found in microorganisms, animals, and plants. The biological functions of lectins are not fully elucidated but it is known that they are involved in defense mechanisms, for example. Lectins may recognize pathogens and consequently kill the invading cells, trigger defensive signaling pathways (in plants) as well as stimulate the release of antimicrobial peptides, phagocytosis, complement activation and melanization (in animals). Direct antibacterial activity of lectins has been demonstrated through the ability of these proteins in promoting agglutination, inhibition of planktonic growth, biofilm inhibition or eradication, and/or death of the bacteria. The growth inhibition and death induction have been credited to the interaction of lectins with bacterial cell wall components [such as N-

 Corresponding author: Thiago H. Napoleão. E-mail: [email protected]. Tel: +55 81 21268540, fax: +55 81 21268576. Complimentary Contributor Copy

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acetylglucosamine, N-acetylmuramic acid (MurNAc), tetrapeptides linked to MurNAc, and lipopolysaccharides] as well as with membrane receptors. This may lead to permeabilization and formation of pores in the bacterial cell wall and membrane, with consequent leakage of intracellular content. These antibacterial properties have stimulated several studies on the biotechnological potential of lectins as antibiotics. In this chapter, we will comment on general aspects regarding lectins and their purification and then review the state-of-art on antibacterial lectins, their action mechanisms, defensive roles and current efforts regarding their effective biotechnological application.

1. LECTINS: GENERALITIES AND PURIFICATION STRATEGIES

Lectins consist of a highly diverse class of carbohydrate-binding proteins with non- immune origin. These proteins possess at least one non-catalytic carbohydrate-recognizing domain (CRD), which is able to bind sugars reversibly and with high specificity, without modifying the covalent structure of them (Sharon and Lis, 2004; Fu et al., 2011; Karnchanatat, 2012; Macedo et al., 2015). The specificity of the carbohydrate-binding site is determined by the spatial arrangement of both the amino acids that constitute the CRD and the neighboring amino acids; in addition, metal ions may contribute for correct positioning of the carbohydrate for binding (Paiva et al., 2010). The CRDs of lectins may bind to simple (monosaccharides and dissacharides) or complex (polysaccharides, glycoproteins and glycolipids) sugar structures through hydrogen bonds as well as van der Waals and hydrophobic interactions (Figure 1A) (Paiva et al., 2012; Singh et al., 2015). The ability of lectins to detect even slight variations in carbohydrate structures present at the surface of cells and tissues has established the concept of protein-carbohydrate recognition. According to the carbohydrate specificity, the lectins can be classified as: glucose/ mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine, fucose or sialic acid binding lectins (Wu et al., 2001; Sharon and Lis, 2007). However, this classification cannot be applied to all lectins since some of them are only able to bind complex sugars or glycoconjugates, for example (Ahmed et al., 2015; Shanmugavel et al., 2016). These proteins were first discovered in plants but they are widely distributed in virus, bacteria, fungi and animals (Albuquerque et al., 2014; Sousa et al., 2016; Sun et al., 2016). The plants are the most exploited source of lectins due to the ease of extraction and the relatively high yields that can be obtained. Moreover, several families of plants as well as different tissues from the same plant can contain distinct lectins, with different carbohydrate- binding specificities and bioactivities. Lectins are certainly the most versatile group of proteins used in biological and biomedical researches. The recognition of cell surface carbohydrates by lectins has broad implications in biorecognition technologies aiming to investigate the structure and function of complex carbohydrates and to map changes in cell surface during physiological and pathological processes (Lam and Ng, 2010; Madariaga et al., 2015; Sousa et al., 2015). The carbohydrate-lectin interaction is also behind a number of biological properties of lectins, including immunomodulatory, anti-inflammatory, antitumor, hypotensive, insecticidal, antiviral, antifungal, and antibacterial activities (Paiva et al., 2012; Carvalho et al., 2015; Macedo et al., 2015; Campos et al., 2016).

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Antibacterial Lectins 71

In order to investigate the biotechnological potential of lectins, including antimicrobial properties, it is necessary to perform the isolation of these biomolecules. The first step in lectin purification is the protein extraction, which is usually achieved using water, saline solution or buffers (Coelho et al., 2009; Vaz et al., 2010; Rafiq et al., 2014; Ramos et al., 2014). The extraction must take place under established conditions of time and temperature in order to optimize the yield and preserve the lectin activity. From the crude extracts, the proteins may be pre-purified by different methods, such as the fractionation using salts. The preciptation using ammonium sulfate at saturation conditions is among the most employed methods for pre-purification because this technique preserves the native structure of the protein. The ammonium sulfate is a highly hydrophilic salt that removes the solvation layer around the protein, leading to increase of protein-protein interactions and then precipitation (Green and Hughes, 1955). Pre-purified lectin samples obtained by salt fractionation must undergo a dialysis process using semipermeable membranes in order to remove the salt. The proteins are retained within the membrane while smaller molecules (such as carbohydrates or salts) present in the sample pass to the solvent solution. The pre-purification can also involve the removal of non-protein contaminants, such as phenolic compounds. Gomes et al. (2013) used activated charcoal to remove these compounds from a saline extract from leaves of Schinus terebinthifolius, and this was the single pre-purification step in the lectin isolation procedure described by them. Occasionally, no pre-purification step is necessary and the crude extract can be directly submitted to chromatographic steps, such as in the procedure for isolation of Apuleia leiocarpa seed lectin (Carvalho et al., 2015).

A B

Figure 1. Schematic representations of the interaction between lectins and carbohydrates. (A) A lectin interacting with two monosaccharides through hydrogen bonds, van der Waals and/or hydrophobic interactions. CRD: carbohydrate-recognizing domain. (B) Agglutination of bacterial cells resulting from the crosslinking between the lectin molecules and carbohydrates found in cell surface.

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Lectins can be purified by chromatographic methods using matrices whose choice will depend on the molecular size, net charge, and/or carbohydrate specificity of the protein (Pohleven et al., 2012; Van Damme, 2014). The ion exchange chromatography is based on the protein net charge at a particular pH value. When the protein is negatively charged (anionic), it is used a positively charged matrix (cationic) or vice versa. In this sense, if the lectin possess a liquid net charge opposite to that of the matrix, it will bind while the other molecules will be eliminated in washing step. Examples of cationic matrices (anion exchangers) are those containing the diethylaminoethyl (DEAE) or quaternary ammonium (Q) groups. Among the anionic matrices (cation exchangers), the carboxymethyl (CM) is one of the groups most frequently present. The gel filtration (size exclusion) chromatography is based on the separation of the proteins according to their molecular mass. This method consists of a stationary phase composed by a crosslinked porous matrix. The proteins with high molecular mass are collected more quickly than those with low size because of the smaller molecules come inside the pores and pass throughout a greater number of channels. Some examples of gel filtration matrices are Sephadex, Superdex, Sepharose (glucose polymers), Sephacryl (a dextran- polyacrylamide polymer) and Bio-Gel (polyacrilamide). Affinity chromatography is broadly used for lectin purification due to the specificity of these proteins for carbohydrates. Several affinity matrices composed by polysaccharides may be used for lectin purification. The lectins adsorb to these matrices through non-covalent bonds and are usually eluted with high purity by changing the pH value, ionic strength or using a competitor (free carbohydrates that compete with the matrix for the lectin CRD) (Pohleven et al., 2012). For example, galactose-binding lectins can be isolated using guar gel column (Campos et al., 2016) and N-acetylglucosamine-binding lectins can be isolated by chitin column (Coelho et al., 2009; Napoleão et al., 2011). Matrices containing immobilized monosaccharides, disaccharides or glyconconjugates (such as Mannose-Sepharose 4B, Lactose-Sepharose, Mucin-Sepharose) may also be used to purify lectins (Singh et al., 2014; Zhou et al., 2014). The traditional gel filtration matrix Sephadex may also be used as an affinity support for purification of glucose-binding lectins (Correia and Coelho, 1995). Different chromatographic procedures have been used for purification of antimicrobial lectins (Table 1). For a purification process reach satisfactory results, four variables should be taken into account. The first is the capacity of the chromatographic system, i.e., the amount of sample that the stationary phase can hold. If this amount exceeds the capacity of the column, the resolution (the second variable) will be impaired. The resolution corresponds to the separation degree achieved using a chromatography: the lectin of interest should be concentrated in a smaller amount of fractions and separated from other proteins. The third variable is the speed because methods requiring much time and/or many chromatographic steps may be not cost-effective. Finally, the fourth criterion is the recovery/yield in terms of amount or biological activity; thus the techniques used during purification should keep the lectin at its native state.

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Table 1. Examples of chromatography matrices used for isolation of antimicrobial lectins

Lectin source Carbohydrate specificity Matrices Animals Bothrops leucurus (venom) Galactose Guar gel and Superdex 75 Colossoma macropomum (serum) Fucose, galactose and Con A–Sepharose 4B methyl-α-galactopyranoside Microorganisms Aspergillus gorakhpurensis Chondroitin-6-sulphate Mucin-Sepharose Gymnopilus spectabilis Sialic acid Mannose-Sepharose and DEAE-cellulose Lichen Cladonia verticillaris N-acetylglucosamine Sephadex G-100 (GlcNAc) Plants Apuleia leiocarpa (seeds) GlcNAc Chitin Bauhinia monandra (roots) Galactose Guar gel Euphorbia helioscopia (leaves) Fructose DEAE-cellulose and Sephadex G-100 Eugenia uniflora (seeds) Glycoproteins DEAE-Sephadex Microgramma vacciniifolia GlcNAc, glucose, mannose Chitin (rhizome) Moringa oleifera (seeds) GlcNAc, fructose, glucose Chitin Myracrodruon urundeuva GlcNAc Chitin (heartwood) Phaseolus vulgaris (seeds) Glucosamine Blue Gel, Q Sepharose and Superdex 75 10/300 Phthirusa pyrifolia (leaves) Fructose-1-6-biphosphate Sephadex G-100 and CM- cellulose Schinus terebinthifolius (leaves) N-acetylglucosamine Chitin Sebastiania jacobinensis (bark) Glycoproteins CM-cellulose and Sephadex G-100 References: Oliveira et al. (2008), Sá et al. (2009), Costa et al. (2010), Vaz et al. (2010), Nunes et al. (2011), Souza et al. (2011), Carvalho et al. (2012), Gomes et al. (2013), Alborés et al. (2014), Albuquerque et al. (2014), Ang et al. (2014), Singh et al. (2014), Rafiq et al. (2014), Ramos et al. (2014), Carvalho et al. (2015), Moura et al. (2015).

2. MECHANISMS OF ACTION OF LECTINS ON BACTERIAL CELLS

Bacterial cell surfaces are covered with abundant and diverse carbohydrates, such as lipopolysaccharides (glycoconjugates found in Gram-negative bacteria, composed by a lipid, an oligosaccharide core, and an O-antigen polysaccharide chain) and peptidoglycans [present on cell wall of Gram-positive and Gram-negative bacteria, formed by β(1→4) linked N-

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74 Thamara F. Procópio, Maiara C. Moura, Lidiane P. Albuquerque et al. acetylmuramic (MurNAc) and N-acetylglcosamine alternated residues, with tetrapeptides associated with the MurNAc] (Vollmer et al., 2008; Lannoo and Van Damme, 2014). The interaction between lectins and bacterial cell surfaces may result in the agglutination and inhibition of cell growth. Lectins are usually able to agglutinate bacterial cells (Figure 1B) at concentrations lower than that needed for growth inhibition; the agglutination results in bacteria immobilization, which may facilitate the action of the own lectin molecules or other antibiotic agents on a high number of cells (Costa et al., 2010; Moura et al., 2015). The binding of lectins to glycoconjugates at bacterial surface may lead to growth inhibition through several mechanisms, such as alteration in cell permeability leading to reduction in nutrient uptake and/or interaction with membrane receptors that trigger intracellular responses (Karnchanatat, 2012). A secreted lectin present in Andrias davidianus skin was able to inhibit the respiration of Escherichia coli, Enterobacter aerogenes, Staphylococcus aureus, Bacillus subtilis and Shewanella sp. (Qu et al., 2015). Among other mechanisms, the death of bacterial cells promoted by lectins may be consequence of damage to cell wall and membrane. According to Talas-Ogras et al. (2005), lectins may induce or promote the formation of pores in bacterial cell, resulting in leakage of cellular contents (Figure 2). The lectin REG3α, localized at the human intestinal epithelium, recognizes the bacterial peptidoglycan and then causes the cell permeabilization, which happens due to electrostatic interaction between cationic residues of the lectin and the negatively charged bacterial membranes (Cash et al., 2006; Lehotzky et al., 2010, Mukherjee et al., 2014, Mukherjee and Hooper, 2015). Another examples are the lectin from Moringa oleifera seeds, which was able to induce the leakage of proteins from bacterial cells as well as to damage the integrity of the cell wall (Moura et al., 2015), and the Araucaria angustifolia seed lectin, which promoted bubbling and formation of pores in bacterial membrane, which could be visualized by electron microscopy (Santi-Gadelha et al. 2006).

A B

Figure 2. Schematic representation of an entire bacterial cell membrane (A) and of the leakage of intracellular content due to the action of a pore-forming lectin (B).

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Antibacterial Lectins 75

Figure 3. The adhesion of bacteria to membrane of host cell (1) may be impaired due to blocking of interaction by an antibacterial lectin that recognizes and binds to bacterial surface carbohydrates (2).

The recognition of carbohydrates present in the bacterial surface may also block the sites for the interaction with host cells and thus preventing infections (Figure 3) (Iordache et al., 2015; Fang et al., 2016). As mentioned before, the lectins constitute a heterogenous group and even those that showing sequence homologies may differ in both carbohydrate-binding affinity and biological properties due to minor variations in their structures. Thus, there is no standard on the degree and spectrum of the antimicrobial action of lectins; in addition, the effects of a same lectin on different microrganisms may also be distinct because of the variety of carbohydrate organization in the surface of microbial cells (Cavada et al., 2001; Islam et al., 2009). However, the absence of an individual type of carbohydrate-lectin interaction responsible for antimicrobial property is interesting, because there is a large number of simultaneous and specific contacts, resulting in high efficacy and specificity in pathogen recognition (Bouvier, 2016).

3. THE DEFENSIVE ROLE OF ANTIBACTERIAL LECTINS

The previously mentioned ability of lectins to detect small differences in the configuration of carbohydrate molecules makes these proteins important in several biological processes that involve recognition of cell patterns, including the defense against pathogens (Hirabayashi, 2008; Cavalcante et al., 2013). Plant and animal lectins are commonly associated with defense responses and immune system, acting through the recognition of carbohydrates present at the cell surface of invading pathogen (Dias et al., 2015). The recognition of pathogen carbohydrates by lectins is among the most important defense strategies in plants and in animals that did not possess an adaptive immune response fully developed, such as some invertebrates and fishes (Zelensky and Gready, 2005; Sun et al., 2016).

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Plants produce a variety of lectins that are constitutively expressed or others that are inducible as responses toward environmental stress and pathogen attack (Singh et al., 2012; Lannoo and Van Damme, 2014). Plant lectins are found in several tissues such as bark, bulb, fruit, latex, leaf, rhizome, root, seed, stem, tuber, flowers and ovaries (Dias et al., 2015). Lannoo and Van Damme (2014) defined the following classes of antibacterial lectins from plants: membrane-bound lectins (lectin receptor kinases – LecRK) and soluble lectins (amaranthins, calreticulin/calnexin, EUL-related lectins, jacalin-related lectins, nictaba- related lectins and ricin-B lectins). A wide variety of lectin domains have been described in proteins attached to cell membranes, playing an important role in plant defense against phytopathogens. In general, the constitutively expressed plant lectins are synthesized with a signal peptide and remain sequestered in vacuoles before being secreted to the extracellular space. Soluble antibacterial lectins are usually present at the middle areas of the cytosol and are synthesized in response to multiple abiotic and biotic stress factors (Lannoo and Van Damme, 2010, 2014). These lectins may act directly as antibacterial agents or may act as part of intracellular signaling pathways linked to plant defense. For example, the calreticulin – a glucose-binding lectin present on the endoplasmic reticulum of Arabidopsis cells – is divided in the proteins CRTs 1/2 and 3 after pathogen invasion, with the CRTs 1 and 2 acting as general chaperones and the CRT 3 acting in the folding of receptors for bacterial peptides (Jin et al., 2009; Thelin et al., 2011). In addition, in some specific cases, the lectin is involved in the defense mechanisms but not preventing the infection: the Arabidopsis thaliana LecRK-V.5 regulates negatively the ROS biosynthesis in stomates and it was reported that the loss of LecRK-V.5 function increases the resistance to surface inoculation with the bacteria Pseudomonas syringae pv. tomato (Desclos-Theveniau et al., 2012). Animal lectins with antibacterial role are more often found in tissues that are in direct contact with the external environment, such as skin, respiratory tract and intestinal tract, acting directly or indirectly against the pathogen via different mechanisms (Mukherjee and Hooper, 2015). The recognition of a pathogen by animal lectins may lead to the rapid clearance of the microorganism by opsonization, phagocytosis and oxidative burst by immune system cells (Tateno et al., 2002; Dias et al., 2015). Lakhtin et al. (2011) classified the animal lectins in C-type and S-Lac-type lectins, whose carbohydrate binding abilities are calcium- dependent and independent, respectively. The C-type lectins possess more than one carbohydrate recognition domain and include collectins and selectins (Dam and Brewer, 2009; Sun et al., 2016). The selectins are membrane-associated receptors while the collectins are soluble (Dam and Brewer, 2009). The soluble C-type lectins act directly on the pathogen by different mechanisms, such as through a cross-linking between the lectin and bacterial surface which causes the immobilization and facilitates the phagocytosis (Zelensky and Gready, 2005; Wang et al., 2014b). The action of these lectins could also activate the complement in vertebrates (Epstein et al., 1996) or the melanization cascade in invertebrates (Yu et al., 1999; Wu et al., 2013). Dendritic cell- associated lectin-2 (Dectin-2) is one of the most well characterized members of the C-type lectin family and is able to recognize a wide variety of pathogens, including bacteria; when bound to a pathogen, Dectin-2 induces the secretion of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and TNF-α (Yabe and Saijo, 2016). The C-type lectin from the shrimp Marsupenaeus japonicus (MjHeCL) modulates the expression of antimicrobial peptides, which inhibit the proliferation of microbes in the Complimentary Contributor Copy

Antibacterial Lectins 77 hemolymph (Wang et al., 2014a). These authors proved the importance of MjHeCL for the immunity of this shrimp since they observed a desequilibration in hemolymph microbiota homeostasis, resulting in death of the animal, when the expression of this lectin was silenced by using interference RNA. Sun et al. (2016) described Bulb-type mannose-specific lectins from the fish Cynoglos sussemilaevis that possess antibacterial activity against Gram-negative and Gram-positive bacteria. These lectins are also Ca+2-dependent and possess a bactericidal capacity that is not highly selective and that enable them to reduce bacterial infection under in vivo conditions. The S-Lac-type lectins, also called galectins, are specific for β-galactoside (β-Gal) and N- acetyl-D-lactosamine (LacNAC) residues and are expressed in both vertebrate and invertebrate animals (Yamaura et al., 2008; Cummings and Liu, 2009). Most of them are bivalent proteins and their antibacterial activity is due to binding to glycan epitopes, like LacNAc moieties present in lipopolysaccharides, lipooligosaccharides and capsular oligosaccharides of bacteria (Dam and Brewer, 2009). According to Farnworth et al. (2008) galectins-3 are produced by resident alveolar macrophages in response to Streptococcus pneumoniae infection leading to activation of neutrophils that phagocyte bacteria and secrete cytotoxic mediators. In vitro assay performed with HeLa cells infected with Salmonella enterica serovar Typhimurium showed that the galectin-8, which is present in the cytosol of human cells, is able to sense bacterial invasion by binding glycans exposed on the membrane of bacteria-containing vacuoles, which induces a cascade of events that culminates in autophagy activation (Thurston et al., 2012). The bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strains. They share a common ancestor with plant lectins as suggested by extensive structural homology. An example is the pyocin L1 from Pseudomonas aeruginosa that inhibits susceptible strains through recognition of a homopolymer of D- rhamnose present in P. aeruginosa lipopolysaccharides (McCaughey et al., 2014).

4. BIOTECHNOLOGICAL POTENTIAL OF ANTIBACTERIAL LECTINS

The in vitro effects of lectins isolated from plants and animals against planktonic and biofilm lifestyles of several bacteria species has been largely determined, and some examples are listed in Table 2. The chitin-binding lectin from Schinus terebinthifolius leaves (SteLL) showed inhibitory action against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus aureus and Salmonella enteritidis, with minimal inhibitory concentrations (MIC50) ranging from 0.45 to 28.75 µg/mL (Gomes et al., 2013). The authors also observed bactericidal effect against all these species, with the best result obtained for S. aureus (minimal bactericidal concentration, MBC, of 7.18 µg/mL); in addition, SteLL could be considered a potential bactericidal drug against E. coli, S. aureus and P. mirabilis since the MBC values were not fourfold higher than the MIC50 value. The lectin isolated from the heartwood of Myracrodruon urundeuva was also active against human pathogenic bacteria, such as Bacillus subtilis, Corynebacterium callunae, Streptococcus faecalis, S. aureus, E. coli, K. pneumoniae and P. aeruginosa, with MIC90 ranging from 0.58 to 9.37 µg/mL; this lectin was also able to promote the agglutination of bacterial cells (Sá et al., 2009). Complimentary Contributor Copy

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Table 2. Examples of lectins isolated from plants and animals with antibacterial and/or antibiofilm activities

Lectin source Bacteria affected Activity and Reference mechanisms Animals Andrias davidianus Bacillus subtilis Bacteriostatic and Qu et al. (2015) (skin, secreted) Enterobacter aerogenes respiratory block Escherichia coli effects Shewanella sp. Staphylococcus aureus Bothrops jararacussu Staphylococcus aureus Antibiofilm activity Klein et al., (venom) Staphylococcus epidermidis (2015) Bothrops leucurus Enterococcus faecalis Bacteriostatic and Nunes et al. (venom) S. aureus bactericide effects (2011) Clarias gariepinus Aeromonas hydrophila Bacteriostatic and Olayemi et al. Alcaligenes faecalis bactericide effects (2015). Bacillus cereus Bacillus polymxa E. faecalis Klebsiella edwardsii Pseudomonas aeruginosa Vibrio metschnikovi Cliona varians B. subtillis Bacteriostatic and Moura et al. S. aureus bactericide effects (2006) Eriocheir sinensis S. aureus Bacteriostatic and Fang et al. (hemolymph) E. coli agglutinating effects, (2016) Pichia pastoris activation of prophenoloxidase cascade Oncothynchus mykiss B. subtilis Bacteriostatic and Tateno et al. (eggs) E. coli bactericide effects (2002) Algae Bryothamnion Streptococcus mitis Anti-adhesion activity Teixeira et al. seaforthii and Streptococcus mutans (2007) Bryothamnion Streptococcus oralis triquetrum Streptococcus sanguis Streptococcus sobrinus Plants Apuleia leiocarpa Bacillus cereus Bacteriostatic and Carvalho et al. (seeds) B. subtilis bactericide effects (2015) E. faecalis E. coli Klebsiella pneumoniae Micrococcus luteus Pseudomonas aeruginosa Salmonella enteritidis S. aureus Streptococcus pyogenes Xanthomonas campestris pv. campestris Xanthomonas campestris pv. malvacearum Xanthomonas campestris pv. viticola Complimentary Contributor Copy

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Lectin source Bacteria affected Activity and mechanisms Reference Bauhinia variegata (seeds) S. mutans Inhibition of the cell adhesion Klafke et al. S. sanguis and antibiofilm activity (2013) Streptococcus sobrinus Canavalia ensiformis (seeds) S. mutans Bacteriostatic, inhibition of Islam et al. cell adhesion and antibiofilm (2009) activity Canavalia maritima (seeds) S. mutans Bacteriostatic, inhibition of Cavalcante et cell adhesion and antibiofilm al. (2013) activity by disruption of quorum sensing signaling Eugenia uniflora (seeds) B. subtilis Bacteriostatic, bactericide Oliveira et al. Corynebacterium bovis and agglutinating effects (2008) E. coli Klebsiella sp. P. aeruginosa S. aureus Streptococcus sp. Moringa oleifera (seeds) Bacillus sp. Bacteriostatic and bactericide Ferreira et al. Bacillus cereus effects (2011); Moura Bacillus pumillus et al. (2015) Bacillus megaterium E. coli Micrococcus sp. Pseudomonas sp. Pseudomonas fluorescens Pseudomonas stutzeri Serratia marcescens S. aureus Myracrodruon urundeuva B. subtilis Bacteriostatic and Sá et al. (2009) Corynebacterium callunae agglutinating affects E. coli K. pneumoniae S. aureus Streptococcus faecalis P. aeruginosa Schinus terebinthifolius (leaf) E. coli Bacteriostatic and bactericide Gomes et al. K. pneumoniae effects (2013) Proteus mirabilis P. aeruginosa Salmonella enteritidis S. aureus Trigonella foenumgraecum S. mutans Bacteriostatic, inhibition of Islam et al. (seeds) cell adhesion and eradication (2009) of preformed biofilms

Ferreira et al. (2011) showed that the water-soluble lectin from Moringa oleifera seeds (WSMoL) was able to reduce the number of colony-forming units in eutrophic water both by killing and promoting sedimentation of bacterial cells. This result suggests a potential application of this lectin for water disinfection. Moura et al. (2015) also evaluated the antibacterial properties of WSMoL but focusing on corrosive bacteria (Bacillus sp., Bacillus cereus, Bacillus pumillus, Bacillus megaterium, Complimentary Contributor Copy

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Micrococcus sp., Pseudomonas sp., Pseudomonas fluorescens, Pseudomonas stutzeri and Serratia marcescens). This lectin was able to inihibit the growth (MIC50 from 5.2 to 167 µg/mL), agglutinate the bacterial cells at very low concentrations (0.0005 to 17 µg/mL), and cause the leakage of intracellular proteins; also it was able to damage the membrane integrity of S. marcescens, which was the most sensitive bacteria. The authors stated that these results open windows for the investigation of WSMoL as a biocorrosion controlling agent. Carvalho et al. (2015) reported the bacteriostatic action of a lectin from Apuleia leiocarpa seeds (ApulSL) against a variety of human pathogenic bacteria, including B. cereus and B. subtilis (MIC50 of 45.12 µg/mL). However, this lectin was more effective against phytopathogenic bacteria, showing bacteriostatic and bactericide activities against three varieties of Xanthomonas campestris: X. campestris pv. campestris (MIC50: 11.2 µg/mL; MBC: 22.5 µg/mL), X. campestris pv. malvacearum and X. campestris pv. viticola (MIC50 and MBC of 22.5 µg/mL for both strains). The authors highlighted the potential of ApulSL for further studies on control of phytopathogens that cause plant diseases of great economical impact. Bacteriostatic and bactericide effects were also determined for lectins isolated from animal source. Three different L-rhamnose-binding lectins (STL1, STL2 and STL3) isolated from Oncothynchus mykiss eggs were able to inhibit the growth and agglutinate cells of E. coli and B. subtilis. STL3 showed the best effect on E. coli, inhibiting 50% of growth when used at 60 µg/mL, compared with STL1 (20%) and STL2 (15%) at 250 µg/mL. For B. subtilis, STL1, STL2 and STL3 at 250 µg/mL promoted 40, 30 and 30% of growth inhibition, respectively (Tateno et al., 2002). The authors suggested that lipopolysaccharides and lipoteichoic acids appear to be involved in these activities due to the ability of these three lectins to bound O-antigens moieties in bacterial cells. A galactose-specific lectin from skin mucus of Clarias gariepinus was able to agglutinate only Gram-negative bacteria (Aeromonas hydrophila, Alcaligenes faecalis, Klebsiella edwardsii and Vibrio metschnikovii), while inhibition of growth could be observed against Gram-negative and Gram-positive strains (Bacillus polymxa, B. cereus, A. hydrophila, V. metschnikovii, P. aeruginosa, Klebsiella sp. and Enterococcus faecalis) (Olayemi et al., 2015). A marine sponge (Cliona varians) is also source of lectin (CvL) with antibacterial activity. CvL was active against Gram-positive bacteria but was demonstrated to be inactive against Gram-negative bacteria; the percentage of inhibition of B. subtilis growth ranged from 75% to 90% after 4-h incubation with the lectin at 25-100 µg/mL; for S. aureus, CvL reduced the cell growth in 90% when used at 50 µg/mL (Moura et al., 2006). Nunes et al. (2011) purified a lectin from the Bothrops leucurus snake venom (BlL) with antibacterial activity against Gram-positive bacteria: S. aureus (MIC of 31.5 and MBC of 500 µg/mL), E. faecalis (MIC of 62.5 and MBC of 330 µg/mL) and B. subtilis (MIC of 125 and MBC of 250 µg/mL). The authors suggested that this lectin probably interacts with the bacterial peptidoglycan and described that the antibacterial activity was abolished when the assay was performed in the presence of galactose. The antibacterial potential of lectins has been evaluated through the years but their effective application as new therapeutic strategies has been underexplored. However, an example is the human mannose-binding lectin (MBL), which is a collectin of the immune system that binds mannose, N-acetylglucosamine, fucose and glucose residues present in bacterial cells (Shi et al., 2004; Dzwonek et al., 2008). The deficiency of MBL in plasma is Complimentary Contributor Copy

Antibacterial Lectins 81 associated with a range of infectious and autoimmune diseases (Summerfield, 2003; Dzwonek et al., 2008) and thus therapy using plasma or recombinant MBL has been used in lupus erythematosus (Jacobsen et al., 2013), rheumatoid arthritis (Carroll et al., 2013), and cystic fibrosis (Garred et al., 2002). According to Garret et al. (2002) the introduction of MBL (twice a week for 3 months) lead to the improvement of clinical conditions of patients with cystic fibrosis, MBL deficiency and severe bronchpulmonary P. aeruginosa infection. Antiadhesion therapy using lectins appears to be a promising strategy against biofilm development in biotic and abiotic surfaces, preventing the early adhesion of bacterial cells and thus the growth of biofilms (Teixeira et al., 2007; Cavalcante et al., 2013). The researches on the biotechnological potential of lectins for combating biofilms are stimulated by the need of nontoxic, biocompatible and effective compounds for prevent device-associated infections, mainly in hospital environments (Teixeira et al., 2007; Cavalcante et al., 2011; Hasan et al., 2014). The antibiofilm activity of lectins could not be attributed only to the mechanisms discussed in the section 2 of this chapter but also to the ability of these proteins to interact with quorum sensing signals (Figure 4), surfactants, enzymes and polysaccharides involved in biofilm formation (Cavalcante et al., 2011; 2013; Hasan et al., 2014). Additionally, it has been reported the ability of lectins in eradicating preexisting biofilms of a pathogenic bacteria, in synergism or not with antibiotics (Islam et al., 2009; Klein et al., 2015). A chitin-binding lectin from Solanum tuberosum inhibited biofilm formation of P. aeruginosa and this activity was attributed to binding to N-acetylglucosamine residues (Hasan et al., 2014). A glucose/mannose specific lectin from Trigonella foenumgraecum seeds prevented the adhesion and subsequent biofilm formation of S. mutans at subinibitory concentrations; in addition, the treatment of preformed biofilms with this lectin altered the morphology of the cells, causing invaginations, and destroyed the biofilm matrix (Islam et al., 2009). According to the authors, the eradication property of this lectin may be due to interference with the arrangement and structural integrity of the biofilm. The antibiofilm effect of a lectin from Bothrops jararacussu venom was demonstrated in a dose-dependent manner against S. aureus and Staphylococcus epidermidis cells, without interference on bacterial growth; this lectin also presented activity in preformed biofilms when used at 100 µg/mL during 2 h of incubation, disrupting more than 50% of the biofilms (Klein et al., 2015). Teixeira et al. (2007) studied two lectins from the red algae Bryothamnion seaforthii and Bryothamnion triquetrum and reported that they were able to form a strong and uniform coating onto saliva-coated hydroxyapatite beads, preventing the initial attachment of Streptococcus cariogenic species (S. oralis, S. sanguis, S. mitis, S. mutans and S. sobrinus).

CONCLUSION

Antibacterial lectins play essential roles in the defense of plants and animals against pathogens. The action mechanisms of these lectins are variable, being the interaction with carbohydrates present at bacterial cell surfaces an event that may lead to growth inhibition, cell wall damage and cellular responses, among other effects. The investigation of the biotechnological potential of antibacterial lectins is supported by several studies describing their bacteriostatic, bactericidal and antibiofilm activities in vitro. Complimentary Contributor Copy

82 Thamara F. Procópio, Maiara C. Moura, Lidiane P. Albuquerque et al.

Figure 4. Antibiofilm activity of lectins. (A) The biofilm formation begins with the adsorption of free- floating bacteria to a surface through weak and reversible forces including van der Waals interactions. Next, the bacteria anchore more firmly to the surface and produce quorum sensing molecules that allow the biofilm formation. (B) The antibiofilm activity of a lectin attached to a surface may be due to interaction with glycosylated molecules at bacterial cell surface, preventing the adhesion, or by interaction with the quorum sensing signals impairing the communication between bacterial cells. (*) Death of a bacterium by pore formation induced by the lectin.

ACKNOWLEDGMENTS

The authors express their gratitude to the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) for financial support. T.F. Procópio and M.C. Moura would like to thank FACEPE for graduate scholarships. N.D.L. Santos would like to thank CAPES for postdoctoral scholarship. L.C.B.B. Coelho and P.M.G. Paiva would like to thank CNPq for research grants.

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Chapter 4

NATURAL ANTIBACTERIALS FOR TECHNICAL APPLICATIONS

Thomas Grethe1,∗, Charlotte Vorneweg1, Hajo Haase2 and Boris Mahltig1 1Research Institute of Textile and Clothing, Niederrhein University of Applied Sciences, Moenchengladbach, Germany 2Department of Food Chemistry and Toxicology, Berlin Institute of Technology, Berlin, Germany

Abstract Natural biocides receive increasing attention for different applications. Biocides are essential substances for antibacterial coatings on different substrates. Due to the wide variations in the fields of application, these substances reoccur in the water cycle, possibly accumulating and threatening public health. Therefore, natural based materials are beneficial, since they are usually degradable in the aquatic environment during an appropriate time scale. Technical applications for antibacterial active surfaces are for example filtration devices, medical textiles like bandages and drapes, or textiles for fiber reinforced composites made from cellulosic fibers. All these materials may suffer from colonization with bacteria or fungi leading to performance loss or even worse to a distribution of harmful germs. Since sustainableraw materials are of increasing importancefor thegrowing global economy, also new approaches to bactericidal effects need to be considered. Lots of plant based substances are already known for their bactericidal properties, but suffer from market entry barriers like high prices due to the inevitable purification processes. However, some materials can be synthesized, so they can possibly become more affordable if advantages of the economics of scale can be taken. The natural bactericides can be divided into different classes by their chemical structure. Terpenoids are molecules formally derived from isoprene and contain multiple C5H8 units; known substances within this group with antibacterial properties are for example menthol, pinene, camphor, and linalool. These materials can be found in plants but can also

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be synthesized. Eugenol and cinnamaldehyde are phenylpropanoids, since they are formally derived from 1-phenylpropane. These substances show bactericidal effects and can be incorporated in coatings, which is shown for textile applications. A rarely investigated topic is the development of resistances against natural substances. Depending on the mode of action of the natural based biocides the occurrence of resistances is possible as it is for synthetic bactericides. To give a conclusive assessment of potential ﬁelds of application and long term efﬁciencies, this issue will also be reviewed.

1. Introduction

Biocides are a wide range of products to control and mitigate different pests and para- sites. The non-exhaustive list includes mice, rats, mosquitoes, flies, algae, shells, fungi and bacteria. The group with probably the biggest number of individual organisms are microorganisms like bacteria and fungi. Besides the former are prokaryotes and the latter are eukaryotes, both differ significantly in the composition of the cellular membrane. While bacterial cell membranes contain peptidoglycans, fungal membranes are made of glucans or chitin. Most fungi, but not all, also utilize ergosterol in their cell walls [82]. Therefore different antimicrobials may be effective against one or the other. Against fungi most commonly agents inhibiting the cell wall synthesis are applied [27], but also DNA replication[20] and mitosis [58] are targeted. ATP synthesis in fungi can be inhibited by quinol oxidation inhibitors which is a common agricultural measure to protect crop plants [23]. Since mammalian cells are also eukaryotic, most fungicides are also harmful to animals and humans, which manifests in different side effects. Thus, bactericides can act more specific and the treatment of bacterial infections is often accompanied with less secondary effects compared to mitigation of fungal infections. Antibacterials often target cell wall synthesis, protein biosynthesis, DNA replication or RNA synthesis. Since bacteria exhibit higher cell division rates, they are more sensitive to antimicrobial substances than fungi. Another approach to achieve bactericidal and fungicidal effects is the use of heavy metal ions. This effect was first described by Naegeli in 1893 [78]. However, even up to now the bactericidal mechanism of heavy metal ions could not be fully clarified. Some modes of actions are proposed discussing the impact of silver ions on cellular respiration of bacteria [68, 70] and copper ions acting in a Fenton-type reaction leading to the release of hydroxyl radicals[30]. For example, such metal ions can be used in coatings to create antimicrobial textiles [31]. However these biocides are usually not of a natural origin and are only partial or even not biodegradable. This is an issue of increasing public concern, because of the accumulation of such substances in the water cycle and of the observation of resistances of common antibiotics. For clinical applications, alternatives are rare due to the necessity of little side effects, high therapeutic index and efficiency. However, technical applications allow more degrees of freedom to develop and apply different biocides. This allows the use of natural substances to achieve bactericidal properties, if they can be applied in common production processes.

Complimentary Contributor Copy Natural Antibacterials for Technical Applications 93 2. Natural Biocides: Synthesis and Effectiveness

Natural bactericidal and fungicidal substances can be obtained by different methods. The most common one is usually the extraction from plant materials. Some materials are easy commercially available and the concentration of the target substance is high, so this way is favorable. On the other hand, extraction needs concentration and puriﬁcation steps which increase the effort to obtain plant materials in this manner. Furthermore, the concentration of the desired ingredients may be dependent on the growing conditions of the plant. Thus, chemical synthesis of the natural substance can be beneﬁcial, depending on the individual substanceand requirements of application. Thisreport willbe focused on natural substances like terpenoids, phenylpropanoids, tannins, and natural dyes, their production in technical scale, as well as their application and effectiveness against microorganisms.

2.1. Terpenoids, Phenylpropanoids and Tannins Terpenoids are formally derived from isoprene and are therefore often called isoprenoids. While the latter are commonly understood to be pure hydrocarbons, the definition of terpenoids is wider and includes also organic substituted isoprenoids. In numerous plants a number of different terpenoids occur which can be used for chemotaxonomic identification of different strains of a species [14, 6]. This can be an important toolfor plant materials used e.g. in the pharmaceutical industry [11]. In plant organisms two pathways of biosynthesis are discussed in the literature: The longest known variant is the mevalonate pathway which starts with acetyl coenzyme A and results in the production of dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) which both can be understood as phospho- rylated isopropenoids. These molecules are quite reactive due to the phosphorous groups and most mammals and a few bacteria synthesize terpenoids and isopropenoids from these building blocks [38]. However, it was found that most bacteria and plants employ an alternative pathway to synthesize terpenoids, the mevalonate-independent or deoxyxylulose pathway [64]. This variant starts with pyruvate and glyceraldehyde 3-phosphat to form deoxy d-xylulose. It results in the same terpenoid precursors as the former mentioned pathway [46, 38]. The precursors can than react to different terpenes, as depicted in Figure 1. By repeating the process and intramolecular additions, more complex and cyclic structures can be obtained. While in nature these pathways are fundamental and yield in even complex molecules like hormones, this chapter focuses on more simple terpenoids with bactericidal properties useful for technical and industrial applications. For example, substances with a high turnover in the industrial market are menthol, pinene, and camphor (Figure 2). Some of the terpenoids can also be synthesized in a technical manner, however, these processes are complex and involve different reaction steps, so in most cases the substances are purified from natural plant materials. The total synthesis of Pinene was reported in 1976 starting from Hagemann’s ester (ethyl-2-methyl-4-oxo-2-cyclohexenecarboxylate) [75]. The authors report a sophisticatedmulti step synthesiswith different special chemicals involved (like LAH, ethanedithiol, pyridine, etc.) leading to different enantiopure products. Although this is a remarkable work, industrial applications need more cost efficient approaches probably without the need for enantioselective materials. Pinene can be obtained Complimentary Contributor Copy 94 Thomas Grethe, Charlotte Vorneweg, Hajo Haase et al.

Figure 1. Synthesis of terpenes from IPP and DMAPP.

Figure 2. Structure of menthol (left), pinene (center), and camphor (right). from the byproduct turpentine of the paper production from wooden materials. This liquid is distilled to remove high volatile compounds and obtain α-pinene and β-pinene. The former is converted into pine oil, while the latter is used to produce camphor and other substances [81]. Menthol can be obtained by extraction of different species of Mentha, most commonly Mentha arvensis. Also different synthetic pathways are known. The Takasago process uses myrcene as starting material, which is aminated to diethylgeranylamine. The latter is isomerized by the use of a rhodium catalyst to citronellaldiethylamine. This is hydrolyzed to citronellal which is converted catalytically by zinc bromide to isopulegol which can be reduced to menthol (Figure 3) [74, 25]. A similar approach was patented by BASF, where geraniol and nerol are used as starting materials [5]. The German Haarman & Reimer company published a method to obtain menthol from m-cresole: M-cresole is alkylated with propylene to yield thymole; the latter is than reduced to menthol [67]. The phenylpropanoids eugenol and cinnamaldehyde are very common substances exhibiting bactericidal properties. For industrial purposes they are isolated from plant materials. Cinnamaldehyde is obtained by distillation of cinnamon bark. Syntheticoptions to produce cinnamaldehydeare also known for a long time, but play no signiﬁcant role for industrial applications. Eugenol, a constituent of the ﬂower buds of Complimentary Contributor Copy Natural Antibacterials for Technical Applications 95

Figure 3. Takasago process for menthol synthesis.

Figure 4. Chemical structure of cinnamaldehyde (left) and eugenol (right). the clove tree (Syzygium aromaticum), is isolated from clove oil by alkaline extraction. Lots of other terpenoids and phenylpropanoids exist in essential oils from plant materials, but most of them are not favorable for large scale applications, since the extraction and purification process is quite inefficient. Griffin et al. investigated a large amount of terpenoids on their antibacterial activity against different bacteria. Their goal was to detect relationships between chemical structure and antibacterial activity. They concluded, a sufficient H-bonding capacity and water solubilityof the molecules is needed for good bactericidal effects. Pure hydrocarbons or acetates were less active. For activity against gram negative bacteria, molecular size was also found to be an important factor [69]. Tannins are polyhydroxyphenols and are secondary plant compounds mostly providing protection against herbivores. Tannins can be divided into two classes, hydrolyzable and condensed tannins. The former are derived formally from gallic acid. For example, gallic acid can be bound via ester linkages to glucose forming pentagalloyl-glucose (Figure 5). The molecules exhibit the ability to bind to proteins which depends on the specific structure of the tannin. Condensed tannins are more complex molecules and incorporate flavonoid structures. They also condensate and form polymers from usually flavonoid monomers (Figure 6). These polymeric tannins occur also in some crop plants and need for further processing to increase or modify these tannins since their nutritional role is still under investigation [2]. For use in technical applications, gallic acid (Figure 5) is an inter- Complimentary Contributor Copy 96 Thomas Grethe, Charlotte Vorneweg, Hajo Haase et al.

Figure 5. Chemical structure of a pentagalloyl-glucose (left) and gallic acid (right).

Figure 6. Chemical structure of ﬂavan (top right), catechin (bottom right), a ﬂavonoid, and sorghum procyanidin (left).

esting compound, since it can be acquired easily in a commercial scale and is known for antimicrobial effects [8].

2.2. Mode of Action of Natural Biocides In general, bactericidal substances either infringe the reproductive capability of germs by blocking proteins involved in DNA replication, the protein biosynthesis or, more often found in topical antibiotics, the cell membrane integrity. Blocking specific proteins is also a possible mode of action, especially with compounds capable of forming complexes like metal ions or phenolics. As mentioned in the preceding section, molecular size and polarity are factors influencing the antibacterial activity. These properties need to be quite specific, to allow bactericidal molecules to pass through the partially lipophilic cell membrane but also to accommodate the aqueous cytoplasm. In technical applications most substances are applied as coatings and theirmode of action relies mostly on alteration of thecell membrane structure. Widely known materials are quaternary ammonium compounds, substituted with Complimentary Contributor Copy Natural Antibacterials for Technical Applications 97 an alkyl chain. These chains can penetrate the cell membrane which leads to depolarization of the cell. However, these substances are not of natural origin and are therefore not in focus here. Modes of action for natural biocides can be diverse and are summarized for the most economic interesting ones as follows.

2.2.1. GallicAcid Gallic acid like other phenolic compounds alters the surface properties of bacteria. Borges et al. observed a change in bacterial hydrophobicity. Depending on the gram type of the bacteria, exposition to gallic acid led to increased electron acceptor capabilities of the cell membrane for gram positive bacteria and vice versa for gram negative cells [8]. Further- more, the presence of gallic acid increases the K+ leakage of bacteria. This effect was found to be more signiﬁcant in gram negative bacteria, whereas gram positive bacteria did not show such increase in leakage. Phenols can also infringe the synthesis of nucleic acids in gram positive and negative bacteria.

2.2.2. Eugenol The phenylpropanoid eugenol seems to increase cell membrane permeability [36] and therefore leads to increased K+ leakage out of bacterial cells. Walsh et al. also found that EDTA1 can increase the bactericidal efficiency of the compound. The authors conclude a better accessibility of eugenol to intracellular sites due to the presence of EDTA at least for gram negative bacteria. The increased action of EDTA against the gram positive S. aureus was explained by the binding of cations to EDTA [80]. However, the most prominent effect is the disruption of the cell membrane, which can be observed by measuring the efflux of cytoplasm components into the cell exterior. Eugenol was also be found to decrease the proton gradient in the cell membrane, which drives the motion of the flagellum of E. coli. [29].

2.2.3. Cinnamaldehyde Cinnamaldehyde as a phenolic propenoid comprises a non polar propenoid side chain which enables such molecules to penetrate the cell wall and diffuse into the cytoplasm [10]. It is also reported that a good solubility of phenolic acids in lipid and aqueous phase is beneﬁ- cial for antibacterial effectiveness [37]. This seems to be plausible since most detergents also show bactericidal properties due to the ability to dissolve the cell membrane, but am- phiphilic properties may also help to diffuse through the lipid based cell membrane into the cytoplasm without the need for micelle formation properties. It was also found that cinnamaldehyde can inhibit the ATP production in E. coli and L. monocytogenes [29].

2.2.4. Terpenoids Information about terpenoids are less specific, however, their mode of action primarily relies on the permeabilization of the cell membrane, which was demonstrated for linalyl acetate, menthol, and thymol [15]. This could also be verified by determining the K+ efflux

1ethylenediaminetetraacetate Complimentary Contributor Copy 98 Thomas Grethe, Charlotte Vorneweg, Hajo Haase et al. of S. aureus during treatment with the terpene alcohols farnesol, nerolidol, and plaunotol [39]. Terpenoids and also phenylpropanoids exhibit the ability to inﬂuence the composition of the cell membrane particular the fatty acid components of the lipids [59]. This gives rise to the idea, that such components not only interfere with the existing cell membrane, but also impair the synthesis of new membrane parts. Therefore these substances might affect the metabolism of bacterial cells.

2.3. Natural Dyes and Their Antimicrobial Action on Textiles The coloration of textiles by using natural dye stuffs is probable one of the oldest treatment of textile materials performed since thousands of years [66, 44, 50]. For this, dyeing processes could be understood as probable one of the first textile functionalization processes. Naturally occurring colorants can be natural pigments and natural dyes. Natural pigments are mostly of inorganic nature and originated from mineral sources. Prominent examples of such pigments are iron oxideyellow Fe2O3 (CI Pigment Yellow 42) or iron oxide red Fe2O3 (CI Pigment Red 101) [66]. However, this chapter is related to the antimicrobial properties of natural compounds and not of inorganic minerals, so the properties of these natural pigments are here not further discussed. In contrast to the inorganic pigments, natural dyes are originally produced from living organism, as plants or animals. These natural dyes from biological resources are the main focus of the current overview. Leather, wood and papers but also textile materials, as silk, wool, cotton or synthetic fibers can be colored by application of natural dyes. Especially important is the use to apply certain coloration to food and drinks [7]. Nowadays, in industrial textile production and treatment the use of natural dyes are uncommon. In most processes natural dyes are replaced by synthetic dyes. Mainly two reasons could be mentioned for that replacement. First, compared to natural dyes, the synthetic dye can be produced in nearly unlimited amounts independent from natural resources, as e.g. land which is needed to grow crops for dye production. The production of synthetic dyes is also mostly more cost effective [60]. Second, the fastness properties and especially the light fastness of many synthetic dyes are advantageous compared to the fastness gained with natural dyes [57]. Nevertheless, natural dyes can be part of the actual discussion and the trend of using materials from renewable resources while avoiding materials based of fossil resources [76]. There are many well reported recipes and application processes for natural dyes onto textile available. These descriptions of dyeing processes are often very detailed and related to simple devices. So they can be reproduced even by persons, which are not educated experts in textile finishing processes. It can be understood as part of a ”do it yourself” community, may be better described with the term ”dye it yourself” [24, 45, 13, 61]. However, it must be remarked that many dye plants used for production of natural dyes contain toxicity or even high toxicity [61]. From a chemical point of view, natural dyes are not pure substances. They are mixtures often containing different colored and uncolored substances [66]. The amounts of different components vary in a certain range depending, e.g., on the type of plant, the country of origin or climate conditions. Often one main component or only few compounds can be identified to be responsible for the color properties [66]. To give in this current chapter a Complimentary Contributor Copy Natural Antibacterials for Technical Applications 99 certain systematic view, following the natural dyes are mentioned together with their main component and its chemical structure. Also the natural source, different used names and terminations as the CI index are given. Of course, this is only possible and performed for view often reported natural dyes. The reader show keep in mind that there are hundreds of plant that can be used for dye production,e.g. for India the number of more than 450 plants are mentioned [72]. Due to the fact that natural dyes are mixtures containing different substances from natural resources, it should be clear that possible antimicrobial activities reported in literature for natural dyes are may be of quantitative low reproducibility. In some cases it might be even not clear, if the colored main component would be the substance responsible for the antimicrobial action of the natural dye. From that point it can be expected that the antimicrobial effects gained by natural dyes are also transferred after the dyeing process to the dyed textile. Pathogenic bacteria can be defended by antimicrobial effect of applied natural dyes [9, 18, 62, 51]. Positive dermato- logic properties may be established by this. Natural fiber materials like wool or cotton can contain a humid content of around 10 wt-% and are for this an ideal place for bacteria to growth. Antibacterial properties gained by dyeing with natural dyes could be here an advantageous property to prolong the life-time of these textile materials. In recent years, many investigations are published related to natural dyes and their interaction with many types of different bacteria, health issues and their medicinal importance [12]. Also the application for realization of protective clothing is mentioned [71]. Beside deodorizing properties also UV-protective functions can be established by natural dye applications [61]. One important point for application of natural dyes is the use of a mordant as pretreatment with a metal salt before the dye application. This mordant increases color properties and wash fastness, however some of the used metal salts can exhibit an antimicrobial property by themselves. For this, it should be distinguished between the antimicrobial property of the natural dye and the metal salt of the mordant. An interesting study in this area is given by Mirjalili and Abbasipour comparing the antimicrobial properties of three different natural dyes applied with and without mordant in comparison to a very antimicrobial active nanosilver application [52]. The application of the pure natural dyes leads to a certain antimicrobial action. However in combination with dichromate containing mordant excellent antimicrobial properties comparable to the one gained with the nanosilver are reached [52]. Probably in this application the toxic dichromate component is responsible for the strong antimicrobial action. To response here to the natural dye is obviously a misinterpretation of data. In literature very often it is referred to the antimicrobial properties of five very prominent natural dyes, which are summarized below in table 1. To give an adequate structure for the current overview following each of these dyes is presented one after the other. Also the natural dyes are referred to their plant of origin and the main components for the color properties are given. After this overview on the five prominent natural dyes, also other antimicrobial natural dyes and pigments are discussed and special applications are mentioned.

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Table 1. Overview on some natural dyes with reported antimicrobial properties

Dye Plantoforigin Color Maincomponent C.I. Natural Yellow 3 Turmeric yellow Curcumin CI75300 C.I.NaturalOrange6 Henna orange LawsoneCI75480 C.I. Natural Yellow 18 Barberry yellow Berberine CI751600 C.I.NaturalBrown7 Walnut brown JuglonCI75500 C.I.NaturalRed8 Madderroot red Differentanthraquinonecompounds: Alizarin CI75330; Pseudopurpurin CI75420; Rubiadin CI75350; Munjistin CI75370

Figure 7. Chemical structure of curcumin (enol type) - Mw=368.38 g/mol.

2.3.1. Natural Yellow 3 Natural Yellow 3 is originated from the turmeric plant. The dyeing is usually performed with the root of this plant. The main chemical component responsible for the coloration is curcumin (Figure 7). The application of natural yellow 3 as antibacterial dyeing on wool and polyamide fabrics as textile from synthetic ﬁber is reported in combination with different mordant agents [35, 53]. The antimicrobial properties of curcumin and its wash fastness on the polyamide is signiﬁcantly improved by the mordant. Especially effective is a mordant containing iron sulfate [53]. For the dye curcumin a phototoxic effect is reported. In this investigation the gram- positive bacteria are reported to be more sensitive against the phototoxicity compared to gram-negative bacteria [17]. To enable the phototoxic effect of curcumine the presence of oxygen is necessary, so it can be also named as photooxidative process [17]. These processes could be of same origin as similar photoactivityreported for the dye Bengal rose applied on viscose fabrics [49].

2.3.2. Natural Orange 6 Natural Orange 6 is originated from the henna plant. The dyeing is usually performed with the leaves of this plant. The main chemical component responsible for the coloration is the naphthoquinone lawsone (Figure 8). The dyeing of polyamide fabrics and wool by henna extracts are well investigated and antimicrobial properties are determined against bacteria and fungi [3, 85]. The dye up-take of the textile and the antimicrobial activityfor henna can be improved by a combined application with chitosan [19].

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Figure 8. Chemical structure of lawsone which is main dye component of Henna leaves (C.I. Natural Orange 6) 2-Hydroxy-1,4-naphthoquinone - Mw=174.15 g/mol.

Figure 9. Chemical structure of the cationic dye berberine.

2.3.3. Natural Yellow 18 Natural Yellow 18 is originated from barberry plant. The dyeing is usually performed with leaves and roots of this plant. One of the main chemical components responsible for the coloration is the cationic dye berberine, shown in figure 9 as cation without mentioning the accompanying anion. It is expected that the antimicrobial property of berberine are related to its cationic nature [34] comparable to the antimicrobial effect of the cationic natural polymer chitosan or other antimicrobial active cationic dyes based on modified anthraquinone structure [40, 47]. Remarkable is as well, that derivatives of berberine are supposed to act as antimalarial compounds [77]. An intensive investigation of the antibacterial properties of berberine on wool was done by Haji, especially in respect to combination with mordant containing copper ions, aluminum ions or dichromate [34]. Here a synergetic behavior of active natural dye and active metal ions are observed leading in combination to high effective antibacterial effect. An interesting alternative approach could be the application of a biomordant in combination with berberine. Even if the described biomordant does not exhibit any antimicrobial effect, in combination with berberine the antibacterial effect is significant [33]. Analogously intensive investigation of berberine on polyamide textiles are performed by Son et al. [73].

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Figure 10. Chemical structure of Juglone (5-Hydroxy-1,4-naphthoquinone) - Mw=174.15 g/mol.

2.3.4. Natural Brown 7 Natural Brown 7 is originated from the walnut-tree. The dyeing is usually performed with leaves and the shells of the fruit. The main chemical component responsible for the coloration is the naphthoquinone compound juglon (Figure 10) [66]. However, also many other compounds responsible for the color properties are extracted and identiﬁed in walnut green husks [54, 22]. For these components antioxidative and antimicrobial properties are described [22].

2.3.5. Natural Red 8 Natural Red 8 is originated from the root of madder. For the color properties different anthraquinone components are responsible [66]. An overview on the chemical structures of these anthraquinone components (alizarin, pseudopurpurin, rubiadin and munjistin) is given in figure 11. The dyeing properties on protein fibers of madder can be significantly influenced by an alkaline pH during application or the presence of liposomes [55, 56]. The antimicrobial properties of extracts from madder roots and other parts of this plant is intensively reported for different bacteria and fungi. However, not every type of microbe is defeated effectively [41]. Structural related to the anthraquinonecompounds in dye natural red 8 is the yellownat- ural dye from Rheum emodi L [42]. The active components of this dye are anthraquinone derivatives with different functional groups and side chains (Figure 6). The antimicrobial activity was investigated and observed for bacteria and fungi. This investigation was done as function of dye concentration and in combination with and without an aluminum mordant. Interesting is especially the result, that by the mordant the fastness properties of the dye on wool can be improved but the antimicrobial effect is significantly decreased in presence of the mordant [42] A suitable explanation of this result could be the strong fixation of the active dye onto the fiber surface by the mordant, so the availability of the dye for surrounding bacteria is probable reduced.

2.3.6. Fiber-Additive Interaction The property of an antimicrobial additive in a textile application is not only determined by the type of additive alone. Significant is also the interaction of the additive with the textile substrate. In the field of textile dyeing, this kind of interaction is known as fiber / dye relationship. The main conclusion from this relationship is that for certain types of fibers Complimentary Contributor Copy Natural Antibacterials for Technical Applications 103

Figure 11. Chemical structures of some main dye components in the madder root.

Figure 12. Chemical structure of active components in Rheum emodi L / R1 is −H or −CH3; R2 is −CH3,-CH2OH, −COOH, −OH or −OCH3 [42].

only a certain type of dyestuff is useful. Only a certain group of dyes can guarantee a good wash fastness on a certain type of fiber. The reason for the different wash fastness reached is the different attractive interaction between dye and fiber leading in best case to a permanent fixation of the dye onto the fiber surface. Permanent fixation is the main precondition to reach a good wash fastness. The easiest example can be given with polyamide fibers. These polyamide fibers are supposed to contain a positive surface charge, which is caused by protonation of the terminated amino groups of the polyamide polymer (see the schematic description in figure 13) [21]. Because of this positive charge an additive or dye stuff applied on polyamide fibers should exhibit a negative net charge. In that case an electrostatic attraction of positively charged fiber and negatively charged additive is responsible for the fixation of the additiveonto the fiber. In case of dyeing processes, this is related to so-called acid dyes which are as well named as anionic dyes. More general, it can be spoken from an anionic additive. The opposite case are acrylic fibers commonly containing a negative surface charge [84]. Usually acrylic fibers used for clothing application are built up by copolymers of acrylonitrile monomer units and another negatively charged monomer unit, as it is for example depicted in figure 14. For this type of anionic fiber, obviously a positively charged cationic additive should be used for application to guarantee best fixation of the additive Complimentary Contributor Copy 104 Thomas Grethe, Charlotte Vorneweg, Hajo Haase et al.

Figure 13. Schematic view on the polyamide ﬁber and its interaction with negatively charged additives.

Figure 14. Chemical structure of a copolymer with acrylic monomer unit and negatively charged unit, as it is typical for acrylic ﬁbers.

onto the fiber surface. This can be a cationic dye stuff but also every other kind of cationic additive, as e.g. cationic antimicrobial compounds [43]. The most prominent synthetic fibers are polyester fibers with a ratio of 74 % of total world production of synthetic fibers [1]. Common polyester fibers do not contain a surface charge or polar functional groups (as e.g. hydroxyl- or amino-groups) onto their fiber surface. Polyesteris also mentioned to be a hydrophobicfiber. For this,the fixation of additives on polyester fibers cannot be caused by attractive electrostatic interaction [63]. Usually the dyeing and finishing of polyester fibers are performed using so-called high temperature processes (HT-processes). The process temperature in such HT-processes is in the range of 120 ◦C to 130 ◦C, right above the glass temperature of polyester. At that temperature the polymer chains of polyester gain a certain movability, so hydrophobic molecules can easily diffuse from a finishing bath into the matrix of amorphous areas of the polyester fibers. After this finishing process, the fiber is cooled down to temperatures significantlybelow the glass temperature, so the embedded additives are not able to remove from the fiber matrix by simple diffusion processes. For this, a high wash fastness at common temperatures of usage is reached. Complimentary Contributor Copy Natural Antibacterials for Technical Applications 105

Figure 15. Schematic drawing of functional additive bonded by metal complex onto a cellulosic ﬁber.

A completely different behavior compared to synthetic fibers is shown by natural fibers. Natural fibers are of high polarity and very hydrophilic with good water up-taking properties. Roughly natural fibers can be distinguished into two types of fibers related to their main chemical structure. These are cellulosic fibers, e.g. as cotton, and protein fibers, as e.g. wool or silk. The textile chemistry of cellulosic fibers is characterized by the huge amount of hydroxyl (alcohol) groups responsible for the high polarity and high hydrophilic- ity of the cellulosic fiber. Agents with as well high polarity contain a high attraction to the cellulosic fiber. The interaction of polar fiber to the as well polar additive yields in a good fixation of the additive onto the fiber surface. This fixation can be strengthened by using a mordant [65]. The mordant is a textile pretreatment usually containing metal salts. By this, metal ions can form complex bonds to the fiber surface and the afterwards applied finishing agent. Roughly spoken, the metal ions resulting from the mordant can be understood as kind of anchor for this functional agent on the fiber. A schematic example is give in figure 15. Usually metal mordant are aluminum, copper, iron or chromium [83]. Beside the improvement of wash fastness, the mordant leads also to other advantages as improved color depth, the modification of color shade and also an improved light fastness is possible [65, 28]. Protein fibers are structurally characterized by amide groups connecting the aminoacid units to the whole protein chain. Further, the textile chemistry of protein fibers is influenced by side groups of the protein chain which can contain different types of functional groups of hydrophobic or hydrophilic nature. Therefore, the surface of protein fibers offers many different bonding sides or polar finishing agents. Also on protein fibers mordant agents are used to improve the fixation and wash fastness. The use of an aluminum salt containing mordant is reported to be an effective method to improve the wash fastness of antimicrobial active natural dyes on wool [26].

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The development of resistant germs is of increasing concern especially for the treatment of bacterial infections. Beside the continuous development of novel antibacterial drugs another strategy to mitigate the occurrence of resistances is the reduced application of antimicrobials. Especially in the case of non-medical applications more degrees of freedom in choosing a bactericidal substance are available since no interaction with the human metabolism needs to be considered. Therefore, substances with a broader effect spectrum can be utilized. For example, peroxides, ethylene oxide, or halides destroy bacterial cells quite effective by oxidation,so the development of resistances is rather unlikely. It has to be noted, that the development of resistances cannot categorically be excluded in any case, but the probability of such a development is reduced the more fundamental the mode of action of a bactericide is. As described in section 2.2 natural biocides affect mostly the permeability of the cell membranes. However, the cause for this maybe just electrostatic or Van-der-Waals interactions, but there are also signs, that the bacterial metabolism is affected. This was proved by Pasqua et al., who investigated the lipid composition of bacteria treated with essential oils [59]. Also ATP-Synthesis is affected, and the blocking of enzymes is considered. This renders natural antimicrobials obviously more specific than the aforementioned oxidizing agents but less specific than antibiotics for medical applications. Therefore, the development of resistances seems to be a reasonable possibility. Although, research on that topic is quite rare, some information points in the direction. Becerril et al. investigated the influence of repeated sublethal stress by cinnamon and origanum oil on different bacteria [4]. They found that cinnamon oil had no effect, but origanum oil increased the MIC 2 of M. morganii and P. mirabilis by a factor of four, and in the case of P. aeruginosa by a factor of two. With S. marcescens no effect was found. Walsh et al. investigated the development of bacterial resistances against eugenol, thymol, and other synthetic antibacterials and were able to isolate E. coli bacteria with elevated MICs against eugenol and thymol [79]. How- ever, they stated that these stems were nevertheless susceptible to higher concentrations of the bactericidal substance. On the other hand, da Silva Luz et al. confirmed no significant development of resistances for Listeria monocytogenes against origanum essential oil or carvacrol in their study [16]. It can be concluded, that the question of development of bacterial resistances against natural antibacterials needs further research. Signs for possible accommodation of bacteria to these substances are evident and plausible, but currently it is difficult to access the risk of the occurrence of resistances. It is important to consider, that technical applications of natural bactericides may bring these substances into contact with bacterial stems which did not encounter these substances in the natural environment e.g. human pathogens. Therefore, the development of resistances might be more likely than in the natural environment where a long co-evolution could already take place.

2Minimum inhibitory concentration

Complimentary Contributor Copy Natural Antibacterials for Technical Applications 107 4. Natural Substances for Antimicrobial Textile Finishing

4.1. Sample Preparation and Analysis The discussed substances can be utilized to obtain antimicrobial effects on textile surfaces. The active material was diluted in a solvent (water or ethanol) which was applied on cotton fabrics by padding. After the ﬁnishing process, the textile specimens were dried at 100 ◦C. As active agents camphor, linalool, pinene, and cinnamaldehyde were used. The latter three substances were diluted in a concentration of 200 g/l in water with the additional use of sodium oleate. Camphor was investigated in three different concentrations, 70 g/l, 200 g/l, and 1400 g/l (oversaturated) in mixtures of ethanol and water 2%, 40%, and 80% (vol. ethanol) respectively without the use of an additional detergent. The antimicrobial properties of the prepared textile samples against E. coli and S. warneri are investigated as previously described [48]. Brieﬂy, textile samples (circles of 5 mm diameter) are placed in sterile 96-multiwell cell culture plates together with 200 µl bacterial suspension (diluted 1:250 in LB medium) per well. After incubation for 3 h at 37 ◦C in an orbital incubator rotating at 120 rpm, cellular viabilityis tested by incubation with 0.01 % (w/v) MTT in culture medium, followed by lysis in isopropanol and determination of the absorption at 570 nm and a reference wavelength of 700 nm. Data are shown as % viability relative to bacteria in the absence of fabric samples. For each textile sample, three independent measurements are conducted with different cutouts from the same sample.

4.2. Results and Discussion Finishing with camphor leads to an antimicrobial activity at least against the gram negative E. coli bacterium. For the gram positive S. warneri no significant effect was achieved. Fur- thermore, only the highest liquor concentration of camphor led to an antimicrobial effect, while the lower ones seemed to be ineffective (Figure 16). For linalool, pinene, and cinnamaldehyde a significant decrease in bacterial viability of E. coli was found. The viability decreased to approx. 20 % in every of the three cases. For S. warneri a reduction of the cell viabilitywas also recorded, but in a less extent. Measured rates were between 40 % and 57 % (Figure 17). The viability on textile specimens without a biocide was measured at 134 % for E. coli and 157 % for S. warneri It can be concluded, that the gram negative species was more susceptible to the antimicrobial agents, although both germs were affected by pinene, linalool, and cinnamaldehyde. Camphor was only active against E. coli and only for the highest applied liquor concentration, which might be not ideal for industrial applications. S. warneri showed no susceptibility on camphor. While cinnamaldehyde exhibited the best bactericidal results, it is also advantageous due to its lower vapor pressure compared with the other three tested substances [32]. Since the textile material needs to be dried after finishing, high volatile compounds may evaporate during the process. This can also affect storage conditions of the finished material.

Complimentary Contributor Copy 108 Thomas Grethe, Charlotte Vorneweg, Hajo Haase et al.

Figure 16. Viability results of camphor on cotton woven fabrics.

Figure 17. Viability results of linalool, pinene, and cinnamaldehyde treated cotton fabrics, concentrations were 200g/l in the ﬁnishing agent.

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Chapter 5

ANTIMICROBIAL ACTIVITIES OF NATURAL PRODUCTS FROM LIBIDIBIA FERREA (MART. EX TUL.) L.P. QUEIROZ VAR. FERREA

Fernando Gomes Figueredo1,4, Tania Maria Sarmento da Silva2, Celso de Amorim Camara2, Girliane Regina da Silva2, Maria de Fátima Agra3, Maynara Rodrigues Cavalcante4, Jakson Gomes Figueiredo4, Natalia Bitu Pinto4, Rafael de Carvalho Mendes4, Edinardo Fagner Ferreira Matias5, Francisco Antônio Vieira dos Santos4,5, Henrique Douglas Melo Coutinho5,* and Marta Maria de França Fonteles1 1Departamento de Farmácia/FFOE, Universidade Federal da Ceará, Fortaleza-CE, Brazil 2Laboratório de Bioprospecção fitoquímica, Universidade Federal Rural de Pernambuco, Recife, PE, Brazil 3Departamento de Biotecnologia, Universidade Federal da Paraíba, João Pessoa, PB, Brazil 4Departamento de microbiologia/FMJ, Faculdade de Medicina Estácio de Juazeiro do Norte, Brazil 5Departamento de Química Biológica, Universidade Regional do Cariri - URCA, Crato-CE, Brazil

ABSTRACT

Studies with plants and their use in combinatorial therapies have been stimulated. In this context, Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz var. ferrea, is a leguminous tree

* Corresponding author at: Laboratório de Microbiologia e Biologia Molecular, Universidade Regional do Cariri, 63105-000 Crato, CE, Brazil. Tel.: +55 (88) 31021212. E-mail address: [email protected] (H.D.M. Coutinho). Complimentary Contributor Copy

116 F. Gomes Figueredo, T. Maria Sarmento da Silva, C. de Amorim Camara et al.

widely distributed in the northern and northeastern regions of Brazil, where it is commonly known as “Juca” or “Pau-ferro”. The possible interactions between methanolic and aqueous extracts of Libidibia ferrea (Mart.) L.P. Queiroz fruits have been verified, combined to antimicrobial drugs against strains of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. The fruits (33.53 g) of Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz were powdered and extracted with MeOH and then with water. The extracts were filtered and concentrated using a rotary evaporator to provide methanolic (8.85 g) and aqueous (8.33 g) extracts. The chromatographic profiles of the extracts were obtained using UPLC/DAD and LC-ESI-MS. Electrospray ionization (ESI) in negative ion mode was used to analyze tannins in both extracts. The analysis of extracts by MALDI-TOF confirmed the presence of hydrolysable tannins. Antibacterial and modulating activity (on bacterial resistance) were determined by micro dilution method to identify the MIC (Minimum Inhibitory Concentration). In the antibacterial activity tests the fractions showed a MIC of ≥ 1024 µg/mL and 512 µg/mL. In regards to modulation of bacterial resistance, the products showed synergism when combined with antibiotic against bacterial strains. It was observed that the products potentiated the antibiotic action from aminoglycosides class against bacterial strains of Staphylococcus aureus and Escherichia coli, with significance of p < 0.001. The results indicate that the extracts obtained from Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz fruits could represent an alternative source of natural products capable of modifying and interfering with bacterial resistance to aminoglycosides.

1. INTRODUCTION

The use of plants as a therapeutic alternative is a millennial practice worldwide, and the potential that these possess, stimulate researchers to an intense and insistent health promotion. South America has received incentives from the world health organization, due to its valuable ethnobotanical knowledge and ample natural biodiversity, for the practice of scientific research regarding medicinal plants with therapeutic goals (Haraguchi; Carvalho, 2010). Bearing in mind the incidence of human infectious diseases, the significant increase of infections caused by bacteria and the successive bacterial resistance (Aguiar et al., 2012; Ahmed et al., 2012; Diab et al., 2012; Spellberg et al., 2013), it has become eminently necessary in the last years, to search for new substance with antimicrobial activity, even with the high diversity of antimicrobials already formulated, that act over diverse pathogenic organisms (Pazhani et al., 2004). On this account, this significant need is also related to the inadequate use of antibiotics, which reveals a significant increase to resistance and alerts society worldwide to the care of health (Goodman et al., 2010). In this way, with the objective to develop substances that act through inhibiting microbial resistance or bacterial growth, the vegetables became a therapeutic alternative for possessing chemical compounds with antimicrobial properties which have contributed in significant and efficient results in therapeutic treatments (Albuquerque; Hanazaki; 2006; Oliveira et al., 2007). The subfamily Caesalpinioideae (Fabaceae) consists of around 150 genera. The genus Caesalpinia possesses around 2200 species (Lewis et al., 2005; Queiroz et al., 2005; Simpson, 2006; Cavaheiro, et al., 2009). The species previously denominated Caesalpinia ferrea Mart. today called the Libidibia férrea (Mart. Ex Tul.) L.P. Queiroz. Popularly known as “porjucá” Complimentary Contributor Copy

Antimicrobial Activities of Natural Products from Libidibia ferrea … 117 or “pau-ferro”, an endemic species of Brazil, found in the Caatinga, Cerrado and Atlantic Forest in the Northeast (Alagoas, Bahia, Ceará, Maranhão, Paraíba, Pernambuco, Piauí, Rio Grande do Norte and Sergipe) and Southeast (Espírito Santo, Minas Gerais, Rio de Janeiro). Found in almost all of Ceará, being, however, more frequent in the Serra do Araripe, Serra do Apodi, eastern, western and southern part of the state (Maia, 2004). Pharmacological studies register the use of L. Férrea as an antifungal, antimicrobial, modulator of antibiotic activity, anti-inflammatory and as an analgesic, indicated for combating mouth ulcers known as canker sores, as well as in the control of gastrointestinal problems (Vieria, 1992; Di Stasi; Hiruma-Lima, 2002; Borrás, 2003; Cavalcante, 2008, Ferreira et al., 2016). They also possess numerous therapeutic properties which include the use of the bark to treat wounds, bruises, combat asthma and chronic cough (Maia, 2004). The fruits are anti-diarrheal, anticatarral and healing, while the roots are anti-thermals (Braga, 1976). Green beans and roots of the L. férrea species have been used for many years, such as infusions, for the treatment of diverse problems like injuries, rheumatism, hemoptysis, fever, enterocolitis and diabetes (Nakamura, 2002; Frasson, 2003; Gomes, 2003). In this way, such research can contribute to the development or the discovery of new herbal drugs for the significant promotion of the health field, at a global level, detecting more effective substances with less toxic side effects against multiresistant and pathogenic microorganisms (Barbosa-Filho et al., 2008).

2. ANTIBIOTICS AND BACTERIAL MECHANISMS OF RESISTANCE

The discovery of antibiotics provided a great advance in medicinal therapeutics, thus reducing the morbidity and mortality of people affected by infectious diseases. These are natural or synthetic compounds capable of inhibiting the growth or causing death to microorganisms (Guimarães, Momesso and Pupo, 2010). The discovery of sulfonamides and penicillin marked the history of antibiotics, however these discoveries did not occur at the same velocity as the events in bacterial resistance. New antibiotic classes have emerged since 1935; up until the year 2003, 27 classes of antimicrobials exist (Butler; Buss, 2006). The most utilized antibiotics in the clinic have a natural or semi-synthetic origin, they can be classified into β-lactams, tetracyclines, aminoglycosides, macrolides, cyclic peptides, streptogramins, among others (lincosamides, chloramphenicol, cephalosporins, rifamycins etc.) (Guimarães; Momesso, Pupo, 2010). Aminoglycosides are a class of antibiotics originating from streptomycin, isolated in 1944 from Streptomyces griseus. They possess a bactericidal effect, acting at the 30S subunit of bacterial ribosomes, interrupting protein synthesis. Aminoglycosides possess a synergistic effect with β-lactams and act effectively against aerobic Gram-negative bacteria (Durante- Mangoni, Grammatikos, Utili and Falagas, 2009). The β-lactam antibiotics, however, are effective in therapy and have a low toxicity, in this group lies the penicillins, carbapenems, monobactams, β-lactamase inhibitors and cephalosporins (Baptista, 2013). Cephalosporins are classified by generations in accordance with antimicrobial activity. The first generation cephalosporins are characterized by a narrower antibacterial action spectrum, acting over Gram-positive bacteria and staphylococci producing penicillinase. The second generation cephalosporins have greater activity against Gram-negative enteric Complimentary Contributor Copy

118 F. Gomes Figueredo, T. Maria Sarmento da Silva, C. de Amorim Camara et al. bacteria. The third generation cephalosporins possess the greatest action spectrum against gram-negative bacteria, including a certain stability in the presence of β-lactamases, however, are less active in Gram-positive bacteria. Fourth generation cephalosporins reunite the advantages of the first and third generations and present good activity both over Gram- positive as well as Gram-negative microorganisms (Spinosa; Górniak, Bernardi, 2006; Climeni et al., 2009). The carbapenem antibiotics (imipenem [IPM] and meropenem [MEM]) are broad spectrum β-lactams, derived from thienamycin, with bactericidal activity in the treatment of infections provoked by multiresistant P. aeruginosa isolates. They possess considerable stability against the majority of beta-lactamases, including those of broad spectrum (ESBL); for this reason, carbapenems are considered reserve drugs, frequently used as a last resort in the treatment of hospital acquired infections caused by Gram-negative bacteria resistant to other beta-lactams or to other antibacterials (Rupp; FEY, 2003; Neves et al., 2011). Despite the existence of all these antibiotic classes, bacterial resistance has become a challenge, as for decades the microorganisms have adapted to the antibiotics (Tavares, 2000). The increasing advance in resistance has been intensely related and its constant change must be taken into consideration during the choice of antimicrobial treatment (Dias; COELHO; Dorigon, 2015). Resistance is considered an ecological phenomenon which occurs as a response from bacteria to the broad use of antibiotics and its presence in the environment (Guimarães et al., 2010). The antibiotic age during the XX century substantially reduced the risk of infectious diseases. However, over the years, a reduction in microbial sensitivity to existing antimicrobial agents, responsible for the critical resistance point to medicines in hospitals and communities has occurred (SAVOIA, 2012). The development of resistance to antibiotics can be natural (intrinsic) or acquired and can be transmitted to the same or to different bacterial species (Hemaiswarya, 2008). The bacterial genome is dynamic, small, economic and singular, characteristics which aid the microorganism in its survival and consequently, resistance to current drugs. The singular chromosome codes for the essential activities, while the non-essential activities, such as the defense against drugs and genetic transfer, are conducted through mobile elements (plasmids, transposons and integrin’s) (Souza, 1998). These allow the bacterial cell to express resistance mechanisms such as: alterations of the antibiotic receptors, elimination of the antibiotic through efflux pumps, drug inactivation, reduction in cellular membrane permeability (Craig, 2005; Lim, 2005). There are also bacterial enzymes which inactivate the antibiotic (Wright, 2005; Happi et al., 2005) through a cleavage process in which bacterial resistance to β-lactams stands out through steric impedance, which promote the opening of the β-lactam ring and the addition of carbon groups impeding the access of antibiotics to the active site of β-lactamase enzymes (Suaréz et al., 2009). Another mechanism of resistance that functions as drug throwers, are proteins whose function is to expel antimicrobials present in the plasma membrane to the outside of the cell, being denominated as an efflux pump and responsible for multiple resistance to drugs (Piddock, 2006). In this context, the abusive and indiscriminate use of antibiotics promote a selective effect in the appearance and maintenance of bacterial resistance. Regarding the Gram-positive bacteria Staphylococcus aureus, for example, methicillin, penicillin, lincomycins, Complimentary Contributor Copy

Antimicrobial Activities of Natural Products from Libidibia ferrea … 119 tetracyclines, rifampicin and vancomycin resistant strains exist (LIU, 1999). Pseudomonas aeruginosa is also a resistance reference, especially in in nosocomial infections (Kobayashi; Sadoyama; Vieira, 2009). The main mechanisms related to its multiresistant phenotypes are the production of metalobetalactamase (MBL) of the SPM-1 type, the presence of 16S rRNA methylase, RmtD, loss of OprD pores and superexpression of efflux pumps, which can explain the high incidence of resistance to carbapenems and aminoglycosides (Neves et al., 2011). In Brazil, infectious outbreaks caused by P. aeruginosa have been related with a clonal dissemination of the specie. Currently, the therapeutic options for the treatment of infections caused by this organism are limited, often restricted to the use of carbapenems (for example, imipenem). This way, the resistance to imipenem is a public health matter, once this antibiotic is used as a last resort in the treatment of infections of hospital origin, caused by multiresistant Gram-negative bacteria (Neves et al., 2011). To combat this resistance, the use of antimicrobials in combination is widely used, with the probability that at least one of the selected agents is active against the pathogenic infector, this because of an additive or even a synergistic effect in combination, however, this is not common practice to reduce resistance (Mizuta et al., 2006). The advantage of this therapy is that the chance of having a mutant strain resistant to both antibiotics is very small, as long as the mechanisms of resistance are independent (Mouton, 1996).

3. NATURAL PRODUCTS AND ANTIBACTERIAL ACTIVITY

Since ancient times, traditional medicine was used in the treatment of diverse pathologies, however, a great part of this knowledge does not contain the endorsement of scientific studies which prove its therapeutic properties. Besides the popular used, the plants have helped, throughout the years, in the obtainment of diverse drugs used in the clinic. Examples of these are the cases of rutin, colchicine, morphine, ementina, etc (Alonso, 2004). The use of plants with a therapeutic goal, known as phytotherapy, is a rich medicinal tradition conserved by people from different countries, where its use in the treatment of venereal diseases, inflammation, infections, wounds and gastroenteritis stand out. Approximately 25% of drugs launched on the market are derived from phytotherapy. Phytotherapy is considered an alternative to the use of synthetic medicines, normally considered more expensive and aggressive to organisms (Ramirez; Diaz, 2007). The use of empirical knowledge on the so called medicinal plants, especially in Latin America, contributed greatly to health care. For the treatment of mild infections, many species are used in the form of infusions, poultices or crude extracts without any scientific evidence surrounding its efficacy. The therapeutic property of a medicinal plant is associated with the active elements that it produces. These active elements are called secondary metabolites, substances produced which are not directly connected to the life maintenance of the producting organism, but which confer advantage to the perpetuation and survival of the specie (Wink, 2008). Depending on the metabolite produced, the plant can present, for example, anti- inflammatory, healing, hepatoprotective, antimicrobial activity, etc (Wink, 2008). Phytochemical and biological studies can be directed through the previous analyses of Complimentary Contributor Copy

120 F. Gomes Figueredo, T. Maria Sarmento da Silva, C. de Amorim Camara et al. chemical compounds present in the plants, which in addition to identifying the presence of determined chemical groups can also be related to the active elements (Lôbo et al., 2010). Secondary metabolites can be influenced by various factors which can coordinate or alter the rate of production of secondary metabolites. Some of these factors are: seasonality, temperature, water availability, ultraviolet radiation, nutrients (macronutrients and micronutrients), air pollution, altitude, and induction by mechanical stimuli or pathogen attack. These factors may lead to variations in the secondary metabolite content, requiring strict quality control by modern analytical techniques, to ensure constancy in the composition of secondary metabolites in the prepared herbal medicine on an industrial scale (Gobbo-Neto, 2007). Microorganisms, vegetables and, in smaller proportions, animals present a metabolic arsenal with potential for production, transformation and accumulation of diverse substances not directly related to life maintenance of the producer. In this arsenal, substances where the production is restricted to the number of organisms with unique and specific metabolism and biochemistry can be found (Semionatto et al., 2007). Some plants are known as producers of bioactives with a toxic nature against bacteria and fungi. Diverse studies show a wide diversity of plants possess antimicrobial activity in vitro. Many classes of plant secondary metabolites are related to an anti-microbial activity: terpenoids, alkaloids, phenolic compounds (quinones, flavonoids and tannins), saponins and carotenoids. Secondary metabolites are present, frequently, in complex combinations, presenting diverse functionalities. This strategy guarantees its action in multiple molecular targets in herbivores and microorganisms, protecting the plant of a range of enemies (Tapiero, 2013). In this context, the selection of microbial strains resistant to current antibiotics is considered a global concern. This panorama requires the investigation of novel antimicrobial substances. A possible source to minimize this problem are the denominated medicinal plants. The higher antimicrobial activity of vegetable extracts and natural products is related, mainly, with the production of secondary metabolites with biocidal activity (bactericidal, fungicidal and insecticidal), highlighting the importance of scientific investigations of popularly used species (Carneiro et al., 2015). Therefore, the search for therapeutic substances became the purpose of great scientific interest in research of chemical, biological and pharmacological constituents, possibly present in products of natural origin (Barbosa-Filho et al., 2008; Biavatti et al., 2007; Coutinho et al., 2008). The antimicrobial properties of substances are desirable tools in the control of pathogenic microorganisms, especially in the different treatments of infectious diseases and in the deterioration of foods. The active components generally interfere with the growth and metabolism of the microorganisms and prevent contamination (Dash et al., 2008). The combination of two or more compounds are generally superior to the use of a single compound, especially for the treatment of serious infectious diseases, caused by bacterial resistance to antibiotics (El-Kalek; Mohamed, 2012). Synergism can be obtained through the combination of antibiotics with extracts at sub inhibitory concentrations applied directly to the culture medium (Coutinho et al., 2008a, 2010a). This strategy is called “shotgun herbs” or “synergistic effect of various segmentation” and refers to the use of plants and drugs in an approach using mono or multi-extract of combinations, which can affect not only a single target, but various targets, where the Complimentary Contributor Copy

Antimicrobial Activities of Natural Products from Libidibia ferrea … 121 different therapeutic components collaborate in a synergistic-antagonistic form. This approach is not only for the combination of extracts; combinations between natural products or extracts and synthetic products or antibiotics are also possible (Wagner, Ulrich-Merzenich, 2009; Coutinho et al., 2010b). In this way, substances of vegetable origin and its derivatives become a viable and efficient alternative (Oliveira et al. 2007a, Silva et al. 2007), since the antimicrobial activity of the drug can be amplified or reduced by the action of natural products (Coutinho et al. 2008a) which hamper antimicrobial mechanisms of resistance due to the complexity of its structures, consequently, avoiding microbial adaptations (Daferera et al., 2003). According to Coutinho et al., (2010a), many studies have been performed with different natural products and demonstrated an antibacterial and synergistic effect with antibiotics against multiresistant bacteria. Having analyzed the antifungal and synergic effect, this presents less susceptibility to natural products, as based in the results of studies by Morais- Braga, et al., 2013.

4. LIBIDIBIA FERREA (MART.) L.P. QUEIROZ

The Caesalpinia ferrea (Libidibia ferrea) Mart. is a tree species belonging to the family Leguminosae, within the dicotyledons it is one of the biggest families with around 650 genera, reuniting more than 18 thousand species, its subfamily is the Caesalpinoidae which consists of approximately 150 genera and 2200 species (Cronquist, 1981). It is a big native tree in Brazil, found mainly in the North and Northeast of the country, popularly known as “pau-ferro” or “Jucá” (Figure 1). It is easily recognized because of the clear spots present in its trunk (Figure 2), and the presence of yellow flowers (Figure 3), small follicles and its hard vegetables (Rizzini, 1995). It is an evergreen or semi-deciduous plant, of wide dispersion and low population density, widely used in civil and joinery construction, due to its hard core, in the form of stakes, beams, logs and supports, on top of its use in ornamentation and in popular medicine. Due to its uses, it is at a threat from extinction, for being devastated (Scalon et al., 2011). The presence of secondary metabolites in the pau ferro (Caesalpinia ferrea M.) provide it with therapeutic activity, these compounds are saponins, coumarins, tannins, phenols, flavonoids, anthra derivatives, alkaloids, lactones, sesquiterpenes, triterpenes and quinones (Cavalheiro et al., 2009, Souza et al., 2006). In phytochemical studies of the hydroalcoholic extract from leaves (Figure 4) and of the bark of C. ferrea, flavonoids, tannins, phenolic compounds, steroids, saponins and coumarins were observed (LORENZI; MATOS, 2008). The tannins are weighted as the main compounds of the fruit extract, but anthra derivatives, sugars, alkaloids, depsidonas, Depsides, saponins, triterpenes, flavonoids, and sesquiterpene lactones are also present(SAMPAIO et al., 2009).

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122 F. Gomes Figueredo, T. Maria Sarmento da Silva, C. de Amorim Camara et al.

Figure 1. Tree of Caesalpinia ferrea. http://legumminosae.blogspot.com.br/2013_01_01_archive.html.

Figure 2. Stem of Caesalpinia ferrea. http://www.clickmudas.com.br/media/catalog/product/cache/1/image/800x800/9df78eab33525d08d6e5 fb8d27136e95/p/a/pau-ferro-tronco_1_1_1.jpg.

They possess a variety of therapeutic properties, already described in the literature, due to the components present in its structure, such as antiulcerogenic (Bacchie et al., 1995), anti- inflammatory, analgesic and anticoagulant activity (Thomas et al., 1998), which can be used to eliminate Aedes aegypti larvae (Cavalheiro, et al., 2009). C. ferrea roots are used as antipyretics and anti-diarrheal (Lewis, 1987), the fruits are used for the treatment of diabetes and for cancer prevention and the decoction of its wood possesses ant secretory activity. The bark of the stem is used in the treatment of enterocolitis, anti-diarrheal (Balbach, 1972)., Complimentary Contributor Copy

Antimicrobial Activities of Natural Products from Libidibia ferrea … 123 decongestant, in addition to the treatment of rheumatism and beneficial cardiovascular system (Menezes et al., 2007). Studies have been performed with the intuition of analyzing the therapeutic function of isolated and identified C. ferrea active compounds, the ellagic acid and ellagic acid 2- (2,3,6-trihydroxy-4-carboxyphenyl) present the capacity to decrease complications caused by diabetes (Ueda et al., 2001), while the anticarcinogenic activity is connected to the presence of chalcone (Nozaki et al., 2007), gallic acid and ellagic acid possessing prophylactic activity against neoplasms (Nakamura et al., 2002). The fruit extracts have been demonstrated activity against the oral pathogens Streptococcus sp. and Candida albicans (Sampaio et al., 2009.). According to Sampaio et al. (2009) the partial inhibition of biofilm formation of multiple Candida albicans, S. mutants, Streptococcus salivarius, S. oralis and Lactobacillus casei species have been reported after the application of C. ferrea extracts.

Figura 3. Flowers of Caesalpinia ferrea. http://professoralucianekawa.blogspot.com.br/2014_05_01_archive.html.

Figure 4. Leaves of Caesalpinia ferrea. ttp://www.cnip.org.br/banco_img/Pau%20Ferro/caesalpiniaferreamartextul.html.

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124 F. Gomes Figueredo, T. Maria Sarmento da Silva, C. de Amorim Camara et al.

Chemical Analysis

Plant Material Mature or ripe fruits of Libidibia ferrea were collected in Brazil, in State of Paraíba, municipality of Vieirópolis at the Sítio Riacho, which is an area of the Caatinga, a Brazilian biome, in April of 2015. The species was identified by Maria de Fátima Agra, Biotechnology Department of the Federal University of Paraiba (UFPB) and deposited at the Herbarium Prof. Lauro Pires Xavier (JPB), UFPB.

Extraction The fruits (33.53 g) of Libidibia ferrea were powdered and extracted with ethanol and then with water. The extracts were filtered and concentrated using a rotary evaporator to provide ethanolic (8.85 g) and aqueous (8.33 g) extracts.

UPLC/XEVO-G2XSQTOF Analysis of Phenolics The XEVO-G2XSQTOF mass spectrometer (Waters, Manchester, UK) was connected to the ACQUITY UPLC system (Waters, Milford, MA, USA) via an electrospray ionization (ESI) interface. Chromatographic separation of compounds was performed on the ACQUITY UPLC with a conditioned autosampler at 4°C, using an Acquity BEH C18 column (50 mm × 2.1 mm i.d., 1.7 μm particle size) (Waters, Milford, MA, USA). The column temperature was maintained at 40 °C. The mobile phase consisting of water with 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) was pumped at a flow rate of 0.4 mL min−1. The gradient elution program was as follows: 0-5min, 5-10% B; 5-9min, 10-95% B. The injection volume was 10 μL. MS analysis was performed on a Xevo G2 QTOF (Waters MS Technologies, Manchester, UK), a quadrupole time-of-flight tandem mass spectrometer coupled with an electrospray ionisation source in the negative ion mode. The scan range was from 50 to 1200 m/z for data acquisition. In addition, MSE experiments were carried which allows both precursor and product ion data to be acquired in one injection. Source conditions as follows: capillary voltage, 2.0 kV; sample cone, source temperature, 100 °C; desolvation −1 −1 temperature 250 C; cone gas flow rate 20Lh ; desolvation gas (N2) flow rate 600Lh . All analyses were performed using the lockspray, which ensured accuracy and reproducibility. Leucine–enkephalin (5 ngmL−1) was used as a standard or reference compound to calibrate mass spectrometers during analysis and introduced by a lockspray at 10 μLmin−1 for accurate mass acquisition. All the acquisition and analysis of data were controlled using Waters MassLynx v 4.1 software.

5. TECHNIQUE FOR THE EVALUATION OF ANTIBACTERIAL ACTIVITY AND THE MODULATION OF ANTIBIOTIC ACTIVITY

In the initial preparation of the solution, each extract was solubilized in distilled sterile water, with the following proportions being observed: 10 mg of solubilized extracts in 1mL of distilled water, to obtain an initial concentration of 10 mg/mL. Then, this solution was diluted in distilled water reaching a concentration of the extract of 1024μg/mL and reducing the concentration of DMSO to 10% and thereafter, 1:1 serial dilutions were performed, during the Complimentary Contributor Copy

Antimicrobial Activities of Natural Products from Libidibia ferrea … 125 microdilution test, obtaining the extract concentrations varying from 512 to 8 μg/mL and DMSO to 5% of the concentration. The following culture media were utilized in the biological assays: Heart Infusion Agar – HIA (DifcoLaboratories Ltda.), Brain Heart Infusion broth – BHI at 10% (AcumediaManufacturers Inc.). All the culture media were prepared according to the manufacturers specifications. The microorganisms used in the tests were obtained through the National Institute of Control and Quality in Health (INCQS) of the Oswaldo Cruz Foundation, Ministry of Health. Three standard strains of the bacteria Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 25923 were used. Bacterial cultures were maintained at 4ºC in Heart Infusion Agar (HIA). The strains were transferred to the HIA media and incubated at 35ºC for 24 horas. The passaged strains to be tested were diluted in saline solution until reaching a concentration of 0.5 McFarland equivalent to 105 cells/mL (NCCLSI, 2012). The assays for the determination of antimicrobial activity and MIC of the extracts were performed through the broth microdilution method, with concentrations ranging from 512 to 8 μg/mL. This method uses small media and sample volumes, distributed in sterile microplate wells. The samples were prepared at a doubled concentration (1024 µg/mL) in relation to the initial defined concentration and volumes of 100 µL and are subsequently serially diluted 1: 2 in 10% BHI broth. In each well with 100 µL the culture media, a bacterial suspension sample diluted at 1:10 will be inoculated. Negative controls with the culture media, positive controls (media + inoculum) and inhibition controls using the extract in concentrations from 1024 to 0.5 µg/mL were included in the assays. The filled plates were incubated at 35ºC for 24 horas (NCCLSI, 2012). To evidence the MIC of the sample, an indicatory solution of sodium Resazurin (Sigma) was prepared in sterile distilled water in the concentration of 0.01% (w/v). After the incubation, 20 µL of the indicatory solution were added in each well and the plates went through an incubation period of 1 hour at room temperature. The change in blue color to pink due to the reduction of Resazurin indicates bacterial growth (Mann; Markham, 1998; Palominoet al., 2002), aiding the visualization of the MIC, defined as the smallest concentration capable of inhibiting microbial growth, as evidenced by the unaltered blue color. For the modulation of antibiotic action test, the method proposed by Coutinho et al. (2008b) was adopted. Eppendorf® tubes were prepared, each one supporting 1.5 mL of solution filled with 10% BHI culture medium, 150 μL of bacterial suspension and the natural product at the concentration MIC/8. For the control, eppendorf® tubes with 1.5mL of solution containing 1350 μL of 10% BHI and 150 μL of microorganismal suspension were prepared. The microdilution plate was filled by adding 100 μL of this solution in each well, proceeding to serial microdilution, up until the penultimate well. The concentration of antibiotics varied gradually from 1024 to 1 μg/mL. The test results were obtained in triplicates and expressed as the geometric mean. For statistical analysis a Two-Way ANOVE followed by Bonferroni’s post hoc test will be applied. Considering significance with p < 0.05 (Matias et al., 2013).

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126 F. Gomes Figueredo, T. Maria Sarmento da Silva, C. de Amorim Camara et al.

Table 1. Mass spectrometric data of phenolic constituents from EtOH extract of Libidibia ferrea

Peak Retention [M-H]- MS2 (m/z) Compound tentatively Reference time identified 1 0.35 191.0547 171.0380, 155.0434, 127.0387, Quinic acid SANTOS et al., 2011. 85.0325 2 0.41 951.0720 933.0997, 915.0924, 463.0706, Trisgalloyl HHDP KAHKONEN et al., 433.0622, 301.0106, 169.0125 glucose 2012. NEGRI; TABACH, 2013. 3 0.53 343.0648 281.8370, 234, 8995, 203.0090, Galloyl quinic acid WYREPKOWSKI 191.0644, 169.0180, 125.0822 derivative et al., 2014 4 0.79 325.0552 239.0746, 225.0592, 185.0090, Galloyl shikimic acid WYREPKOWSKI 183.0109 et al., 2014 5 2.39 1119.0767 1075.1300, 1057.1190, 950.0888, Mallotusinic acid BUENO et al., 2014 931.0840. 913.0738, 425.0330, 301.0126, 169.0277 6 3.66 951.0709 933.1044, 915.0978, 463.0755, Trisgalloyl HHDP KAHKONEN et al., 301.0122, 167.0125 glucose 2012. NEGRI; TABACH, 2013. 7 4.32 967.0663 951.0764, 905.0706, 721.0906, Unknown ellagitannin 635.0414, 483.0297, 300.9986, 271.1646 8 4.68 1109.0928 1049.1201, 935.1221, 463.0663, Alaeocarpusin YANG et al., 2012 301.0172 9 5.16 1133.0947 951.1129, 933.1024, 913.0762, Unknown ellagitannin 483.0375, 301.0169 10 6.52 1147.1074 1129.1484, 951.1121, 933.1015, Unknown ellagitannin 931.0888, 763.0867, 301.0111 11 5.94 1117.0625 1099.0517, 1073.0754, 951.0764, Unknown ellagitannin LIBERAL et al., 913.0419, 300.9978, 271.1663 2015. 12 5.96 1117.0605 1099.0517, 1073.0754, 951.0764, Unknown ellagitannin LIBERAL et al., 913.0419, 300.9978, 271.1663 2015. 13 5.98 1117.0615 1099.0517, 1073.0754, 951.0764, Unknown ellagitannin LIBERAL et al., 913.0419, 300.9978, 271.1663 2015. 14 6.37 951.0706 933.1044, 915.0978, 765.0922, Trisgalloyl HHDP NEGRI; TABACH, 463.0755, 301.0122 glucose isomer 2013. KAHKONEN et al., 2012 16 6.38 637.0372 423.0248, 301.0072, 169.0224 Unknown ellagitannin 17 6.74 1073.0735 1055.1097, 1029.1277, 737.0944, Unknown ellagitannin 905.1060, 317.0111, 301.0082, 273.0182, 167.0013 18 6.77 1117.0630 1099.0975, 1081.0869, 300.9978 Unknown ellagitannin LIBERAL et al., 2015. 19 6.81 953.0881 935.1125, 301.0087 Chebulagic acid YANG et al., 2012 20 6.83 469.0523 431.0672, 313.0557, 241.0358, Valoneic acid dilactone WYREPKOWSKI 169.0132 et al., 2014 21 6.59 1147.1591 1129.1484, 951.1121, 933.1015, Unknown ellagitannin 913.0888, 301.0111 22 7.15 723.5027 677.4868, 595.1730, 505.1803, Unknown ellagitannin 300.9943, 169.0135 23 7.46 513.2164 467.2125, 339.1980, 325.1825, 311.1661, 149.9927 24 7.63 375.1996 331.2155, 329.2288, 343.8339, Unknown ellagitannin 229.1447, 211.1419, 181.0844, 171.1005 25 8.30 331.2465 331.2360, 277.2148, 215.1281, Unknown Ellagitannin 185.1172, 169.1229

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Antimicrobial Activities of Natural Products from Libidibia ferrea … 127

6. RESULTS AND DISCUSSION

UPLC-PDA phenolic profile from etanolic and aqueous extracts of Libidibia ferrea were recorded at 270 nm (Figure 5) and the compounds were tentatively identified by ultra high pressure liquid chromatography along with quadrupole time of flight mass spectrometry (UHPLC–QTOF-MS/MS) as hydrolyzable tannins (gallotannins, galloylquinic and galloylshikimic acids, and ellagitannins), based on their characteristic UV and mass detection as well as accurate mass measurementof both precursor and product ions. Two groups of compounds could be distinguished on the basis of their UV-vis spectra, resembling that of ellagic acid (with two absorbance maxima around 252 and 366 nm), of galloyl and hexahydroxydiphenoyl (HHDP) derivatives (maximum around 275 nm). The substances found in ethanol and aqueous extracts from L. ferrea are listed in Tables 1 and 2 and showed in Figure 5. In both extracts the most abundant substances are the ellagitannins.

Table 1. Minimum Inhibitory Concentration (MIC) of the extracts and antimicrobials (μg/mL)

Extracts and EC AS PA Antimicrobials Methanolic Ex. ≥1024 ≥1024 512 Aqueous Ex. ≥1024 ≥1024 512 Gentamicin 16 64 32 Amikacin 32 256 32 EEAM – Aqueous Extract of Libidibia ferrea; EEAC – Methanolic extract of Libidibia ferrea. EC – Escherichia coli, SA – Staphylococcus aureus.

According to Table 1, a total of 22 polyphenolic compounds could be at least tentatively identified from ethanol extract. All of them were classified as ellagitannins and gallotannins with the exception of peaks 1, which belong to the quinic acid. From aqueous extract were analyzed 12 polyphelolic. These polyphenolics, the peaks 1, 8 and 12 also were showed in the ethanolic extract. Ellagitannins and gallotannins were also observed in the aqueous extract with exception the peak 5 corresponding to syringic acid. Particular fragmentation patterns are described as follows: Two peaks 1 (extract ethanol and aqueous extracts) and 3 (etanol extract) exhibited a [M−H]− at 191.0545 and 343.0648 showed the characteristic fragmentation of quinic acid and galloylquinic acid, respectively. The product ion in the MS/MS spectrum from m/z 343.0648 was at m/z 191.0644 corresponding to quinic acid in structure, a fragment ion at m/z, 169.0180 and 125.0822, which were fragments characteristic of gallic monomer. Mass spectrometric data of peaks 2, 5-19, 21 and 22 (ethanol extract) and 2, 4, 6, 8, 11 and 12 (aqueous extract) may be correspond to those of galloyl-HHDP glucose derivatives (Gordon et al., 2011). Molecule at m/z 951 which after fragmentation yielded a peak at m/z 933.1082, that could be assigned to the loss of 18 u (H2O), while the fragment ion at m/z 463.0756 indicates the presence of HHDP-glucose. The m/z 301.0132 corresponding to HHDP unit after lactonization to ellagic acid (Santos et al., 2011). The ion at m/z 169.0125 corresponds to gallic acid anion. The fragmentation pathway of trisgalloyl HHDP glucose isomer is illustrated in the Figure 6 and mass fragmentation pattern is in Figure 7. The peaks 2, 6, 14 extract ethanol and 4 extract aqueous were trisgalloyl HHDP glucose isomer. Compounds with the same fragmentation Complimentary Contributor Copy

128 F. Gomes Figueredo, T. Maria Sarmento da Silva, C. de Amorim Camara et al. pattern were suggested to be HHDP glucose derivatives or ellagitannin (7, 9-13, 16-18, 21 and 22 ethanol extrac and 2, 6, 8, 11, 12 aqueous extract). The m/z 1119.0767 was assigned to mallotusinic acid and the fragments MS/MS were according BUENO et al. (2014). The peaks 8 (1109.0928), 19 (953.0881) and 20 (469.0523) from ethanol extract were tentatively identified as elaeocarpusin, chebulagic acid (YANG et al., 2012) and valoneic acid dilactone (WYREPKOWSKI et al., 2014) respectively. The aqueous extract showed the peaks 3, 5, 7, 9 and 10 that were identified as methyl gallate, syringic acid, ellagic acid, ellagic acid hexose and digalloylglucose, respectively. Peak 3 was correlated with the structure of gallic acid methyl ester (methyl gallate) with m/z 183.0277 and typical fragments observed for subsequent demethylation (m/z 169.0202) followed by decarboxylation (m/z 125.0254). Syringic acid on the other hand first lost a water molecule generating a fragment ion at m/z 169.0220 followed by a loss of carbon dioxide producing the other fragment at m/z 125.0226. The peak at m/z 463.0871 (9) with a fragment at m/z 300.0433 has been assigned to ellagic acid hexoside. Peak 10 provides parental [M-H]- ions at m/z 483.0871 and the fragment typically corresponding to HHDP m/z 301.0136. Peak 11 evidenced a [M-H]- ion at m/z 497.0346, MS2 fragments at m/z 451.3675 from the cleavage of trigalloyl unit and spontaneous lactonization and 301.0139 which corresponds to ellagic acid residue.

Table 2. Mass spectrometric data of phenolic constituents from aqueous extract of Libidibia ferrea

Peak Retention [M-H]- MS2 (m/z) Compound time tentatively identified 1 0.35 191.0545 171.0427, 155.0501, 127.0509, Quinic acid SANTOS et al., 109.0298, 93.0389, 85.0335 2011. 2 0.39 1133.0923 951.1166, 933.1059, 931.0924, Unknown 763.0800, 483.0438, 461.0587, ellagitannin 301.0135 3 1.40 183.0277 169.0202, 145, 3153, 125.0254, Methyl gallate BUENO et al., 124.0220 2014 4 2.95 951.0706 933.1082, 915.0997, 463.0756, Trisgalloyl HHDP 301.0132, 167.0058 glucose isomer 5 3.92 197.0435 169.0220, 125.0293, 124.0226 Syringic acid SUN et al., 2007. 6 4.73 1133.0898 1133.1514, 951.1208, 933.1103, Unknown 913.0866, 763.0820, 483.0439, ellagitannin 301.0143 7 5.95 300.9964 245.0213, 229.0255, 185.0339, Ellagic acid WYREPKOWSK 145.0367 I et al., 2014 8 6.45 1147.1086 1129.1602, 951.1236, 933.1121, Unknown 913.0856, 483.0450, 301.0113 ellagitannin 9 6.66 463.0871 445.0755, 300.0433, 271.0398, Ellagic acid hexose 179.0093, 151.0115 10 6.92 483.0187 301.0136, 271.0022, 151.0120 Unknown ellagitannin 11 7.11 497.0346 451.3675, 301.0139, 271.0029, Unknown 169.0243, 151.0074 ellagitannin 12 7.14 723.5020 451.3598, 367.2935, 313.2270, Unknown 301.0195, 257.0213, 225.1801, ellagitannin 169.0201 13 7.45 513.2179 467.2175, 341.1040, 301.0204, Unknown 185.1168, 157.1228, 143.1069 ellagitannin 14 7.63 375.2001 331.2020, 329.2583, 229.1520, Unknown 181.0935, 171.1134, 167.0450 ellagitannin 15 3.30 331.2473 313.2665, 215.1407, 185.1280, Unknown 169.1229 ellagitannin Complimentary Contributor Copy

17-Mar-2016 20:05:18 VC EtOHc rep 4: Diode Array x16 0.34 270.532 0.0500Da 288.5320 Range: 3.301 3.0 A 2.8

2.6

2.4

2.2

2.0

1.8

AU 1.6

1.4

1.2 6.73 1.0 3.60 219.5320 219.5320 8.0e-1 6.54 2.32 219.5320 219.5320 6.0e-1 5.91 220.5320 4.0e-1 6.33 216.5320 2.0e-1

0.0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 VC EtOHc rep 1: TOF MS ES- x2 0.35 3.66 BPI 191.0547 951.0709 1.64e6 B 0.41

951.0720 % 2.39 1119.0767

5.96 6.58 1117.0605 637.0372

6.38 6.74 951.0706 1073.0735 7.10 6.23 321.1529 300.9967 6.86 497.3329 0.79 2.81 4.68 5.16 7.24 325.0552 638.0468 1109.0928 1133.0947 450.9929

0 Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 17-Mar-2016 20:05:18 VC EtOHc rep 4: Diode Array x24 0.34 270.532 0.0500Da 288.5320 Range: 3.301 3.0 C 2.8

2.6

2.4

2.2

2.0

1.8

AU 1.6 6.73 219.5320 1.4 3.60 219.5320 1.2 6.54 1.0 2.32 219.5320 219.5320 8.0e-1 5.91 220.5320 6.48 6.0e-1 217.5320 6.33 4.0e-1 216.5320

2.0e-1

0.0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 VC H2Os rep 1: TOF MS ES- x2 0.35 BPI 191.0545 1.57e6

0.39 1133.0923 D

1.40

% 183.0277

6.92 483.0187 5.95 300.9964

0.90 183.0275 4.73 1133.0898

2.95 7.14 3.92 951.0706 6.45 723.5020 197.0435 1147.1086 6.66 463.0871

0 Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20

Figure 5. UPLC-DAD (270 nm) of extract ethanol (A) and aqueous (C) extracts from L. ferrea fruits. Base peak ion chromatograms of extract ethanol (B) and aqueuos (D) extracts obtained by an MSE data collection technique method (UPLC-Q-TOF/MSE) in negative mode.

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18-Mar-2016 17:17:32 VC EtOHc MS MS 117 (4.199) 15: TOF MSMS 951.00ES- ! 1.74e5 100 933.0997

! 301.0106 !

934.1039 %

! 951.1126

! 298.9954 ! 952.1177

! ! 273.0158 302.0147 ! ! 463.0708 ! 445.0599 613.0735 ! ! 915.0924 ! ! 765.0900 ! 317.0070 ! ! ! ! ! 953.1187 275.0307 ! ! ! ! ! ! ! 443.0446 464.0740 ! ! 766.0951 ! 907.1219 ! ! ! ! ! ! 631.0838 ! ! ! 78.1375 103.6223 167.0055 203.0452 !;318.0138343.0238 417.0753 569.0856 603.0950 729.0710 843.1119 !;954.1313 985.5819 123.0112 219.0392 247.0323 505.0833 531.1046 685.0696 763.0739 861.1204 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940 960 980

Figure 6. Mass fragmentation pattern of Trisgalloyl HHDP glucose under ESI-MS in negative mode.

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OH O OH OH OH O OH -H O OH -H OH OH -H O O OH OH OH O O OH -H O OH -HOH O OH O OH O O O O O O O OH O OH OH OH O OH O O O O O OH O OH OH O OH OH O OH OH O O O OH OH O OH OH O OH OH OH OH m/z 951 OH OH OH OH m/z 933 OH OH OH OH OH OH OH OH -HOH m/z 301 m/z 633 OH OH O O -H OH OH -H OH OH O OH O OH + O O OH O OH OH O O O O O O OH O O O OH O OH O OH OH OH O O OH OH O O O OH OH m/z 915 OH OH OH OH OH OH m/z 465 m/z 451 -HH OH -H O OH OH -H O O OH O O OH O OH O + O OH O O OH OH O O O O O OH OH O O OH OH O O OH m/z 463 OH m/z 613 OH OH OH m/z 301 OH

Figure 7. Negative-ion mode fragmentation pathways of Trisgalloyl HHDP glucose m/z 951.

Complimentary Contributor Copy 132 F. Gomes Figueredo, T. Maria Sarmento da Silva, C. de Amorim Camara et al.

The substances isolated galloyl quinic acid derivative, galloyl shikimic acid, valoneic acid, ellagic acid and ellagic acid hexoside compounds had been detected in the ethanol extract from the bark stem bark of L ferrea (Wyrepkowski et al., 2014). To the best of our knowledge, this is the first characterisation of these compounds in L. ferrea fruits. The determination of the Minimum Inhibitory Concentration (MIC) of the methanolic and aqueous Libidibia ferrea extracts tested against reference E. coli, P. aeruginosas and S. aureus. Comparatively, the AELF and MELF presented the same MIC (Table 1). They did not demonstrate clinically relevant activity according to limits established by the protocol (Houghton et al., 2007). The use of extracts and essential oils from plants as antimicrobial agents presents a low possibility of microorganism acquiring resistance to its action, because they are complex mixtures, causing the microbial adaptability very difficult (Daferera et al., 2003). Various medicinal plants were used as sources of prime material for the isolation and purification of many antimicrobials which are used in the treatment of infectious diseases, including treatment against bacteria multiresistant to antibiotics (MATIAS et al., 2010). In this way, it is probable that considering the use of plants as sources of new drugs against bacteria resistant to traditional antimicrobials (Buttler, 2006). Figures 5 and 6 represent the results of the evaluation of the modulatory activity of antibiotics of the aminoglycoside class (amikacin and gentamicin) when in combination with the AELF and MELF. The results demonstrated that the sample combinations with the antibiotics presented a synergistic effect against the tested strains with significant p < 0.001, with the exception of the combinations with the AELF against P. aeruginosa and with MELF against S. aureus, in addition to the combination of amikacin with MELF against P. aeruginosa which presented significance p > 0.05. The results corroborate with the work by Ferreira et al., (2016), which evidenced modulatory activity of the ethanolic extract from L. férrea leaves against bacterial strains. The synergistic action of the natural products together with antimicrobials used in therapeutic treatment was evaluated, determining a reduction in its MIC, when compared to the control where the product to be tested is absent (Coutinho et al., 2008b; Matias et al., 2013; Figueredo et al., 2013., Sousa et al., 2011). When the substance used in combination intervenes in a positive manner, that is, increasing the activity of the antibiotic, it is said to provoke a synergistic effect (Canton; Onofre, 2010). Due to the absorption into the intracellular space, cellular toxicity is common in all aminoglycosides (except with streptomycin). Nephrotoxicity, ototoxicity and neuromuscular block are the most important toxic effect of aminoglycosides (Vallejo et al., 2001; Oliveira, 2006). The related frequency of these collateral effects are very variable due to the different criteria used for diagnostics. Neuromuscular blocks are rare, the ototoxicity varies between 0 to 62% (cochlear) and 0 to 19% (vestibular) and nephrotoxicity varies between 0 to 50% (Gilbert, 1995). The cellular toxicity is a common characteristic of aminoglycosides (except streptomycin), in function of its absorption through intracellular means. The combination of the extracts with the aminoglycosides can be an alternative to decrease the side effects of this antibiotic class, once the association leads to a synergistic effect significantly reducing the MIC of these drugs, decreasing the necessary dose for therapeutic success (Figueredo et al., 2013).

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Antimicrobial Activities of Natural Products from Libidibia ferrea … 133

250 *** EA + Antimicrobial

200 Control of the Antimicrobial

g/mL)  150 *** 100 ns ns *** **

50 Concentrations ( Concentrations 0 Amik Gent Amik Gent Amik Gent E. coli S. aureus P. aeruginosa

Figure 8. Graph demonstrating the modulatory activity of antibiotics of the aminoglycoside class against standard bacterial strains, in the presence and absence of the Aqueous Extract of Libidibia férrea (AE).

The observed synergism can be related to the constitution of secondary metabolites present in the extracts such as tannins, flavonoids and alkaloids which are synthesized by plants in response to microbial infections (Dixon et al., 1983; Ho et al., 2001), being capable of altering the cellular wall or destroying the plasma membrane facilitating the absorption of drugs (Matias et al., 2010; Sousa et al., 2011). The mechanisms through which the extracts can inhibit the growth of microorganisms are varied, and can be in part due to the hydrophobic nature of some components. These components can interact with the double lipid layer of the cellular membrane and affect the respiratory chain and energy production of the cell (Nicolson et al., 1999), or even make the cell more permeable to antibiotics, leading to an interruption of vital cellular activities (Burt, 2004). The interference with the enzymatic system of the bacteria can also be a potential mechanism of action (Wendakoon; Sakaguchi, 1995). The increase in antibiotic activity when in association with two or more drug classes can be attributed to chelation (Barreiros; David, 2006). The discovery of new agents of natural origin which act synergistically, reducing the possibility of occurrence of undesirable effects and which potentiate the antimicrobials used in the clinic thus become of great importance (Leite, 2014; Lima et al., 2015).

250 ns EM + Antimicrobial

200 Control of the Antimicrobial

g/mL)  150 ns 100 ns *** ** **

50 Concentrations ( Concentrations 0 Amik Gent Amik Gent Amik Gent E. coli S. aureus P. aeruginosa

Figure 9. Graph demonstrating the modulatory activity of antibiotics of the aminoglycoside class against standard bacterial strains, in the presence and absence of the Methanolic Extract of Libidibia férrea (EM). Complimentary Contributor Copy

134 F. Gomes Figueredo, T. Maria Sarmento da Silva, C. de Amorim Camara et al.

CONCLUSION

The use of antibiotics is a common and necessary practice in clinical routine, however, the occurrence of resistance hampers the promising results for many patients, and when there is a risk of toxicity as a result of the use of some classes, especially with the aminoglycosides. In this context, the use of aqueous and methanolic extracts of L. férrea can provide potentiation of the activity of antibiotics against multiresistant strains. Therefore, it is suggested that the methanolic and aqueous L. férrea extracts can be used as a source of natural products with the goal of aiding its possible use in antimicrobial therapy and in the control of bacterial Multiresistance.

ACKNOWLEDGMENTS

This work was financially supported by grants from FINEP, FUNCAP, CNPq, FACEPE (Grant no. PRONEM APQ-0741106/2014) and CAPES.

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Goodman, L; Gilman, A(2010). As bases farmacológicas da terapêutica. 11ª. Porto Alegre: Rio de Janeiro: Editora McGraw Hill. p. 1821. Gordon, A; Jungfer, E; Silva, BA; Maia, JG; Marx, F(2011). Phenolic Constituents and Antioxidant Capacity of Four Underutilized Fruits from the Amazon Region. Guimarães DO, Momesso LDS(2010). Pupo MT. Antibióticos: importância terapêutica e perspectivas para a descoberta e desenvolvimento de novos agentes. Quim. Nova. v. 33. n.3, p. 667-679. Happi, C T; Gbotosho, G O; Folarin, OA; Akinbove, DO; Yusuf, BO.; Ebong, OO.; Sowunmi, A: Kyle, DE; Milhous, W; Wirth, DT.; Oduola, AMJ (2005) Polymorphisms in Plasmodium falciparum dhfr and dhps genes and age related in vivo sulfadoxine– pyrimethamine resistance in malaria-infected patients from Nigeria. Acta Tropical. v.95, p. 183. Haraguchi, LMM; Carvalho, OBD (2010). Plantas Medicinais. 1ª. São Paulo. Hemaiswarya, S; Kruthiventi, AK; Doble, M (2008). Synergism between natural products and antibiotics against infectious diseases. phytomedicine. v.15, n.8, p. 639-652. Ho, KY; Tsai, CC; Huang, JS; Chen, CP; Lin, TC; Lin, CC (2001). Antimicrobial activity of tannin components from Vaccinium vitisidaea L. Journal of Pharmacy and Pharmacology, v.53, p. 91-187. Houghton, PJ; Howes, MJ; Lee, CC; Steventon, G (2007): Uses and abuses of in vitro tests in ethnopharmacology: Visualizing an elephant. Journal Ethnopharmacol, v.110, p.391- 400. Journal of Agricultural and Food Chemistry, v.59, p.7688-7699. Kahkonen, M; Kylli, P; Ollilainen, VJP; Heinonen, Ma(2012). Antioxidant Activity of Isolated Ellagitannins from Red Raspberries and Cloudberries. Journal of Agricultural and Food Chemistry, v.60, p.1167−1174. Kluytmans, JAJW; Mouton, JW; VandenBergh, MFQ; Manders, MJAAJ, Maat, APWM; Wagenvoort, JHT; Michel, MF; Verbrugh, HÁ. (1996). Reduction of Surgical-Site Infections in Cardiothoracic Surgery by Elimination of Nasal Carriage of Staphylococcus aureus. Infection Control and Hospital Epidemiology. v. 17, n.12,p.780-785. Kobayashi CCBA, Sadoyama G, Vieira JDG(2009). Determination of associated antimicrobial resistance in clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa in a public hospital in Goiânia, State of Goiás. Rev. Soc. Bras. Med. Trop. v. 42, p. 404-410. Leite, NF (2014). Atividade citoprotetora de extratos hidroalcoólicos de Psidum sobraleanum Proença Landrum e Psidium mysinites DC. Contra danos tóxicos causados pelo mercúrio. Dissertação (Mestrado em Bioprospecção Molecular). Crato-CE, Brasil: Universidade Regional do Cariri–URCA. p.32-3. Lewis GP, Schire BD, Mackinder BA, Lock JM (2005). Legumes of the World. Royal Botanic Gardens, Kew. p. 1-3. Lewis; GP (1987). Legumes of Bahia. Inglaterra: Royal Botanic Garden. Liberal, J; Costa, G; Carmo, A; Vitorino, R; Marques, C; Domingues, MR; Domingues, P; Goncalves, AC; Alves, R; Sarmento-Ribeiro, AB; Girão, H; Cruz, MT; Batista, MT(2015). Chemical characterization and cytotoxic potential of an ellagitannin-enriched fraction from Fragaria vesca leaves. Arabian Journal of Chemistry, in press.

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Lim, SM; Webb, SA(2005). Nosocomial bacterial infections in Intensive Care Units. I: Organisms and mechanisms of antibiotic resistance. Anaesthesia Sep. v.60,n.9, p.887- 902. Lim, SM; Webb, SA (2005). Nosocomial bacterial infections in Intensive Care Units. I: Organisms and mechanisms of antibiotic resistance. Anaesthesia Sep. v.60. n.9. p. 887- 902. Lima, LF; Figueredo, F. G.; Martins, G. M.; Flaviana, M.; Ferreira, J. V.; Carneiro, J.; Brito, D.; Lavor, A.; Matias, E.; Machado, A.; Menezes, I.; Coutinho, H. D. M (2015). Ferns as protective agents against the contamination with mercurium chloride: The example of Pityrogramma calomelanos (L.) Link and a short review. In: Jerald tuft. (Org.). Ferns and Shrubs: Diversity, Cultivation and Implications for the environment. 1ed.New York: New York: Nova Science Publishers Inc, v. 1, p. 1-30. Lôbo, KMS; Athayde, ACR.; Silva, AMA.; Rodrigues, FFG. Lôbo, IS; Bezerra, DAC.; Costa, JGM (2010). Avaliação da atividade antibacteriana e prospecção fitoquímica de SolanumpaniculatumLam. eOperculinahamiltonii(G. Don) D. F. Austin and Staples, do semiárido paraibano.Revista Brasileira de Plantas Medicinais, v.12, n.2. Lorenzi, H; Matos, FJA (2008). Plantas medicinais do Brasil: nativas e exóticas. 2.ed. Nova Odessa, SP: Instituto Plantarum, p. 544. Maia, GN. (2004). Caatinga: árvores e arbustos e suas utilidades. São Paulo-SP, D&Z Computação Gráfica, Leitura and Arte. Mann, C; Markham, JA(1998). new method for determining the minimum inhibitory concentration of essential oils. Journal of Applied Microbiology. v. 84, n. 4, p. 538-544. Matias, EFF, Alves, EF, Santos, BS, Souza, CES, Ferreira, JVA, Lavor, AKLS, et al. (2013). Evidence•Based Complementary and Alternative Medicine. v.2013, n.1, p.1-7. Matias, EFF; Santos, KKA; Almeida, TS; Costa, JGM; Coutinho, HDM(2010). Enhancement of antibiotic activity by Cordia verbenacea DC. Lat Am J Pharm. v.29, p.52-1049. Menezes, IAC; Moreira, IJA; Carvalho, AA; Antoniolli, AR; Santos, MRV (2007). Cardiovascular effects of the aqueous extract from Caesalpinia ferrea Mart.: Involvement of ATP- sensitive potassium channels. Vasc Pharmacol. v.47, p. 41-47. Mitsugui, CS; Tognim, CB; Carrara-Marrone, FE; Garcia LB (2008). Efeito antimicrobiano in vitro da associação de polimixina B e ceftazidima em amostras clínica de Pseudomonas aeruginosa. Ciência cuidado saúde. v.7, p. 76-81. Morais-Braga, MFB; Souza, TM; Santos, KKA; Guedes, GMM.; Andrade, JC; Tintino, S R; Costa, JGM; Menezes, IRA; Saraiva, AÁF; Coutinho, HDM (2013). Atividade antibacteriana, antifúngica e moduladora da atividade antimicrobiana de frações obtidas de Lygodium venustum SW. Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, v.12, n.1. Nakamura, ELS; Kurosaki, F; Arisawa, M; Mukainaka, T; Okuda, M; Tokuda, H; Nishino, H; Pastore, Jr (2002). Cancer chemopreventive effects of constituents of Caesalpinia ferrea Mart and related compounds. Cancer Lett. v.177, p. 119-124. NCCLSI. National Comittee for Clinical Laboratory Standards (2012). Performance standards for antimicrobial susceptibility testing; 22nd informational supplement. Document M100- S22. In: Clinical Laboratory Standards Institute, Wayne, PA. Negri, G; Tabach, R(2013). Saponins, tannins and flavonols found in hydroethanolic extract from Periandra dulcis roots. Revista Brasileira de Farmacognosia, v. 23, n.6, p.851-860.

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Neves P R(2010). Alterações da permeabilidade e expressão de bombas de efluxo em isolados clínicos de Pseudomonas aeruginosa resistente ao imipenem. São Paulo. 116 p. Tese (Doutorado), Faculdade de Ciências Farmacêuticas, Universidade de São Paulo. São Paulo. Neves, PR; Mamizuka, EM; Levy, CE; Lincopan, N (2011). Pseudomonas aeruginosa multirresistente: um problema endêmico no Brasil. Bras. Patol. Med. Lab. v.47:.p. 409- 420. Nicolson, K; Evans, GO; Toole, PW(1999). Potentiation of methicillin activity againstmethicillin-resistant Staphylococcus aureus by diterpenes. FEMS Microbiol Lett, v.179, p.9-233. Oliveira, FQ; Gobira, B; Guimarães, C; Batista, J; Barreto, M; Souza, M2007a. Espécies vegetais indicadas na odontologia. Revista Brasileira de Farmacognosia, v. 17, p. 466- 476. Oliveira, J.F.P.; Cipullo, J.P.; Burdmann, E.A (2006). Nefrotoxicidade dos aminoglicosídios. Brazilian Journal of Cardiovascular Surgery., v. 21, n. 4, p. 444-452. Palomino, JC; Martin, A; Camacho, M; Guerra, H; Swings, J; Portaels, F (2002). Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy. v. 46, n. 8, p. 2720-2722. Pazhani, GP; Sarkar, B; Ramamurthy, T; Bhattacharya, SK.; Takeda, Y; Nivogi. SK (2004). Clonal multidrug-resistant Shigella dysenteriae Type 1 strains associated with epidemic and sporadic dysenteries in Eastern India. Antimicrobial Agents Chemotherapy, v.48, p.4- 681. Piddock, LJV (2006). Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacterial. Clinical Microbiology and Infection, v.19, p. 382-402. Queiroz, LP(2009). Leguminosas da Caatinga. 1. ed. Feira de Santana: Universidade Estadual de Feira de Santana. v. 1. P. 443. Ramirez, LS; Diaz, HE (2007). Actividad antibacteriana de extractos y fracciones del ruibarbo (Rumex conglomeratus). Scientia et Technica. v. 1, n. 33. Rizzini, CT (1995). Botânica econômicabrasileira. 2.ed. Rio de Janeiro: Âmbito Cultural, p. 248. Rupp, ME; Fey PD (2003). Extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae: considerations for diagnosis, prevention and drug treatment. Drugs, v.63, n.4, p.65- 353. Sampaio, FC; Pereira, MCV; Dias, CS; Costa, VCO; Conde, NCO; Buzalaf, MAR(2009). Journal of Ethnopharmacology. v.124, p. 289–294. Sampaio, FC; Pereira, M,S; Dias,CS; Costa,VC; Conde, NC; Buzalaf, MA(2009). Invitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens. J.Ethnopharmacol. v.124, p.289–294. Santos, SAO; Freire, CSR; Domingues, MRM; Silvestre, AJD; Pascoal Neto, C (2011).Characterization of Phenolic Components in Polar Extracts of Eucalyptus globulus Labill. Bark by High-Performance Liquid Chromatography-Mass Spectrometry. Journal of Agricultural and Food Chemistry, v.59, n.17, p. 9386-9393. Savoia, D. (2012). Plant-derived antimicrobial compounds: alternatives to antibiotics. Future Microbiology, v. 7,n. 8, p. 979-990.

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Chapter 6

REDOX-ACTIVE METAL COMPLEXES WITH CYCLOAMINOMETHYL DERIVATIVES OF DIPHENOLS: ANTIBACTERIAL AND SOD-LIKE ACTIVITY, REDUCTION OF CYTOCHROME C

N. V. Loginova1,*, H. I. Harbatsevich1, T. V. Koval’chuk1,2, N. P. Osipovich2, Y. S. Halauko1, Y. V. Faletrov2 and A. T. Gres1 1Faculty of Chemistry, Belarusian State University, Minsk, Belarus 2Research Institute for Physico-Chemical Problems of the Belarusian State University, Minsk, Belarus

ABSTRACT

A promising way to fight multi-drug resistant bacteria strains is to enhance chemotherapy efficiency through the use of antibacterial agents affecting simultaneously several biotargets. We demonstrated that among these compounds are metal complexes with redox-active ligands – cycloaminomethyl derivatives of ortho- and meta-diphenols. They are able to reduce cytochrome c (Cyt c), the key component of bacterial electron transport chain, and to act as low-molecular SOD mimics. We synthesized redox-active complexes of these organic ligands with Cu(II) and Zn(II) ions and estimated the level of their antibacterial activity against Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Salmonella typhimurium), and Gram- positive bacteria (Bacillus subtilis, Sarcina lutea, Staphylococcus saprophyticus, Staphylococcus aureus, Mycobacterium smegmatis) as compared to some standard antibiotics (tetracycline, streptomycin, chloramphenicol). The compounds were characterized by means of chemical, physico-chemical and pharmacological screening methods. The investigation of the molecular and electronic structure of the complexes was performed within the density functional theory framework. The reducing properties of the ligands and their metal complexes were examined by cyclic voltammetry. The kinetics of reduction of bovine heart Cyt c with these compounds in vitro was

* Corresponding author: Phone: +375 172 09-51-99, fax: +375 17 2095464, E-mail address: [email protected]; [email protected]. Complimentary Contributor Copy

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investigated spectrophotometrically as well as their SOD-like activity (using the alkaline dimethyl sulfoxide model). The biocidal effect of the hit-compounds (MIC 0.003–0.012 µmol∙ml-1) comparable to those of commonly used antibiotics was achieved by structural modification of the ligands and complexation which purposefully change the hydrophilic- lipophilic balance, acid-base and redox properties of phenolic derivatives. Cu(II) and Zn(II) complexes of cycloaminomethyl derivatives of ortho- and meta-diphenols may be considered as potential chemotherapeutic agents with broad-spectrum antibacterial activity.

Keywords: antibacterial activity, diphenols, redox-active metal complexes, cytochrome c, SOD-like activity

1. INTRODUCTION

Demand for metallotherapeutic drugs and metal-based diagnostic agents in contemporary medicine is a matter of common knowledge [1], although the discovery and wide use of antibiotics as well as the toxicity and low bioavailability of salts of transition metals were the reasons for the substantially limited possibility and range of their functioning. At the same time systematic abuse of antibiotics, especially in agriculture, resulted in appearance and propagation of resistant strains [2]. In this connection the development of novel antibacterial agents differing from antibiotics in their action mechanism became a burning problem. Unlike many antibiotics, compounds of transition metals are able to realize several mechanisms of biocidal action (caused mainly by the metal ions) involving such targets as functional groups (specifically sulfhydryl ones) in cellular walls of microorganisms and in various enzymes, nucleoproteins, nucleic acids and others [3]. To form a strain highly tolerant to metallotherapeutic drugs, mutations should occur in genes of all their principal targets. But the probability of such multiple mutations is negligibly small in comparison to that of individual ones. Besides, metallotherapeutic drugs have also other advantages over antibiotics: a low induction of resistance in many pathogenic microorganisms against ions of transition metals, a broad spectrum of action (bacteria, fungi and viruses), and safety in therapeutic doses (due to oligodynamic effect of transition metal ions) [1, 3]. It should also be emphasized that in clinical practice there are no antibiotics active both against bacteria and fungi. One of strategic directions of searching for novel chemotherapeutic agents active against multi-drug resistant bacteria strains is synthesis of coordination compounds of metals with organic ligands, pharmaceuticals included, because complexation can result not only in overcoming the resistance of microorganisms and revealing new biological targets, but in increasing antimicrobial activity, decreasing metal ion toxicity and broadening the spectrum of action of organic substances as well [4]. The toxic action of metals on the cells of microorganisms can be due to several principle mechanisms: i) induction of formation of active oxygen forms, specifically highly reactive hydroxyl radicals causing damage to cellular organelles and vital enzymes [5]; ii) inactivation of enzymes as a result of generation of active oxygen forms oxidizing lateral chains of amino acids in enzymes and disrupting the conformation of the latter [6] as well as because of metal ions in the enzyme active sites being substituted [7]; iii) disruption of membrane bearing a big number of negatively charged

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Redox-Active Metal Complexes with Cycloaminomethyl Derivatives … 145 groups, which ends in its failure [8]; iv) disruption of assimilation of nutritive elements causing the intracellular stores of metabolites to exhaust [9]; v) genotoxicity, when replication of DNA is inhibited as a result of the latter being damaged, for instance, by products of the Fenton reaction involving ions of transition metals [10] as well as by the molecules of a metal compound intercalating between DNA nitrogen bases [11]. The concepts listed above allow one to predict antimicrobial properties of transition metal complexes synthesized, but they do not take into account the role of their redox properties in realization of bioactivity and do not consider a possibility of correlation between these properties. Meanwhile the study of complexes of some transition metals affecting the structure and functions of cellular organelles in microorganisms and mammals demonstrated that these compounds can disrupt mitochondrial functions and uncouple respiration [12, 13]. When it is considered that some antimicrobials act in vivo as electron transfer agents in the production of radical species or disruption of normal electron transport [14], it may be suggested that the redox-active metal complexes which are able to affect the electron transport system of cellular respiration can possess a potential for anti-infective activity. Thus redox-active metal complexes with sterically hindered derivatives of diphenols may become a promising direction of a search for novel hit-compounds to produce anti-infective agents with a broad spectrum of action [15, 16]. Derivatives of o-diphenols are known to suppress synthesis of DNA due to its alkylation and oxidative damage [17]. Sulfhydryl groups of protein molecules and glutathione as well as amino groups of lysine in proteins make other targets for these compounds [18]. One of the possible types of their biological macromolecular targets can be oxidoreductases  electron transport chain components, specifically cytochrome c (Cyt c): owing to subcellular localization, bacterial cytochromes c are among the first target enzymes for antimicrobials on their way into the cell [19]. It is common knowledge that active oxygen forms (superoxide, hydrogen peroxide, singlet oxygen and hydroxyl radical) which are generated in phagolisosome and are involved in bactericide mechanisms can produce an antimicrobial effect. But some microorganisms, being active producers of extracellular superoxide themselves, are able to prevent their action [20–22]. Superoxide can directly interact with biomolecules and also precede other active oxygen forms. Superoxide dismutases (SOD) are oxidoreductases catalyzing transformation of superoxide (one of the main factors of oxidative stress) into hydrogen peroxide and oxygen [23]. SOD are demanded as pharmaceuticals [24], but the use of native SOD in medicine is limited by their thermolability, low ability to penetrate cells and others [1]. Many of the limiting factors could be overcome by developing synthetic, low-molecular-weight mimics of the SOD enzyme and by using a direct method to measure the SOD-like activity of these complexes [25]. SOD isoforms are distinguished by the nature of cofactor metal in the active site of the enzyme: Cu/Zn-SOD, Mn-SOD as well as Fe-SOD and Ni-SOD of limited occurrence. The radical mechanism of interaction with superoxide of the Zn-containing enzyme where the role of zinc consists only in steric stabilization of the active site differs from the mechanism of action of SOD which comprises a redox-active metal (copper, manganese, iron) involved in the catalytic process [26]. Besides, it was found that Zn-containing low-molecular analogues of Cu/Zn-SOD can interfere in the mechanism of inactivation of active oxygen forms of fungi and some bacteria and exhibit antimicrobial activity through competition with the native SOD [27]. Thus there are facts suggesting that redox-active metal complexes with antioxidants as ligands possess antimicrobial activity together with SOD-like one. Complimentary Contributor Copy

146 N. V. Loginova, H. I. Harbatsevich, T. V. Koval’chuk et al.

In the earlier investigations we have shown that antimicrobial action of ortho-diphenols can be optimized through incorporation of substituents into benzene ring and complexation with metal ions purposefully changing hydrophilic-lipophilic balance, acid-base and redox properties of molecules as well as allowing the toxicity of phenolic derivatives to be reduced [28, 29]. It was also found that some derivatives of sterically hindered ortho-diphenols as well as their metal complexes exhibit a pronounced reducing ability correlating with their antimicrobial activity and the rate of the reduction of bovine heart Cyt с in a limited series of these compounds [16, 30]. We extended our research to novel modified derivatives of diphenols and their complexes in order to examine how structural modifications of substituents in benzene ring and transition metal complexation can influence the biological activity and reducing ability of the corresponding complexes. For this purpose we chose Mannich reaction, because it is one of the main tools for the development of bioactive compounds [31, 32]. Using this synthetic method, we were able to produce novel redox-active ligands – cycloaminomethyl derivatives of ortho- and meta-diphenols for complexation with transition metals and further biological evaluation of both sterically hindered diphenols and their metal complexes. (Figure 1) [33].

Figure 1. Cycloaminomethyl derivatives of ortho- and meta-diphenols – the ligands for synthesizing redox-active complexes of transition metals. Complimentary Contributor Copy

Redox-Active Metal Complexes with Cycloaminomethyl Derivatives … 147

In the present investigation a procedure was developed of synthesizing redox-active complexes of the above-mentioned ligands with specially selected transition metals – copper and zinc. These metals are recognized as essential elements participating in many biological redox-processes, and the role of Cu(II) and Zn(II) complexation in enhancing the pharmacological profile of the antimicrobial activity of some drugs and bioactive compounds is noted [34, 35]. In this connection it is of interest to study various aspects of coordination chemistry of these transition metal ions interacting with cycloaminomethyl derivatives of ortho- and meta-diphenols, physico-chemical and biological properties of the complexes synthesized. Redox properties of the above-mentioned ligands and their Cu(II) and Zn(II) complexes were determined electrochemically, and the reduction of bovine heart Cyt c with these compounds as well as their SOD-like activity were investigated spectrophotometrically. The results obtained are discussed based on a supposed relationship between the capability of the compounds under study for reducing Cyt c, their antibacterial and SOD-like activity, redox properties determined electrochemically, and lipophilicity.

2. COMPLEXATION OF COPPER(II) AND ZINC(II) IONS WITH CYCLOAMINOMETHYL DERIVATIVES OF DIPHENOLS. PHYSICOCHEMICAL CHARACTERIZATION OF REDOX-ACTIVE METAL COMPLEXES

To produce the metal complexes, the organic compounds HLI–HLXV were used for ligands (Figure 1). These compounds were synthesized according to the procedure described in [33, 36]. The Mannich bases HLI–HLXV are of interest as ligands, because they contain several nucleophilic centers: oxygen atoms of hydroxyl groups and the nitrogen atom of the cycloaminomethyl fragment. Because of the presence of two phenolic hydroxyls incorporated into the compounds they are able to dissociate stepwise as weak acids, all being characterized by close pKa1 values due to their similar structure and low differentiating action of water/organic mixtures on diphenols. Dissociation constants of the compounds HLI–HLXV in water-organic medium were determined by the method of potentiometric titration; their values are given in Table 1, save for HLXI–HLXV, since on titrating them the upper limit was reached of the range of pH values which can be registered by the glass electrode [37]. The compounds HLI–HLXV are weak bases in water-organic medium (Table 1): HLI–III, HLVI–VIII, XI–XIII and HL have close pKb values (5.4–6.0) for they are characterized by a similar environment of the nitrogen atom in the cycloaminomethyl fragment. However the compounds containing the morpholine (HLIV, HLIX, and HLXIV) and piperazine (HLV, HLX, XV and HL ) moieties demonstrate weaker basic properties (pKb= 6.9–7.7) which result from the (–I)-effect of the nitrogen and oxygen atoms incorporated into heterocycles. Potentiometric titration by the Bjerrum method showed that in the water-organic medium Cu(II) and Zn(II) ions form the complexes М(II):L=1:2 with the compounds HLI–HLXV. The stability constants of these complexes are given in Table 2, the Cu(II) complexes being more stable than the Zn(II) ones. Cu(II) and Zn(II) complexes with Mannich bases HLI–HLXV were synthesized as described elsewhere [16] (yield: 80–85%). The complexes are differently coloured

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148 N. V. Loginova, H. I. Harbatsevich, T. V. Koval’chuk et al. precipitates virtually insoluble in water and soluble mainly in low-polarity solvents (tetrahydrofuran, chloroform). According to the elemental analysis data, the composition of Cu(II) and Zn(II) complexes I XV with the compounds HL –HL in the solid state corresponds to ML2 (Table 3), and the results of the X-ray powder diffraction analysis are indicative of their amorphous structure. Thermal analyses in air flow with identification of the final products by X-ray powder diffraction demonstrated all the complexes to be anhydrous and unsolvated, because their DTA curves lack any endothermic peaks over a wide range from 60 to 120°C. A summary of the results is given in Table 4. An exothermic peak in the range of 120–210°C corresponds to the stage of the complex molecules condensing to form oligomers [38]. Peaks in the range of 210–700°C may be assigned to destruction of the ligands followed by thermal decomposition of the complexes above 700°C with the formation of metal oxides (CuO, ZnO) as the final products. The agreement between the experimental and theoretical weight losses for the above processes confirms the formulas of the Cu(II) and Zn(II) complexes. The low values of the molar conductivity in acetonitrile for the Cu(II) and Zn(II) complexes indicate their being essentially non-electrolytes in this solvent (Table 5) [39]. Thus, the conductivity data suggest that the ligands may be coordinated to the metal(II) ion as uninegatively charged ligands. The complexes are characterized by high values of the n-octanol/water partition coefficient (Table 6) which was determined as reported in [40]. Of the ligands, the compounds containing the bulky non-polar trytil group (HLVI–HLX) and the azepan moiety (HLIII, HLVIII, and HLXIII) are the most lipophilic. The partition coefficients of the ligands are lower than those of their complexes, which is related to the polar hydroxyl and amino groups of the ligands taking part in complexation. In the series of the ligands of the same kind the lipophilicity of the Zn(II) complexes is higher than that of the Cu(II) ones. The lipophilicity parameters of these complexes suggest their potential ability for transmembrane transfer and are also important for assessment of their bioavailability as well as their bioactivity [41].

Table 1. Dissociation constants of sterically hindered diphenols HLI–HLXV

Compound Solvent pKа1 pKа2 pKb HLI water-ethanol 11.60 ± 0.31 – 5.5 HLII water-ethanol 11.92 ± 0.39 – 5.9 HLIII water-ethanol 11.90 ± 0.34 – 6.0 HLIV water-ethanol 11.94 ± 0.35 – 7.7 HLV water-ethanol 11.59 ± 0.28 – 7.1 HLVI water-tetrahydrofuran 10.76 ± 0.27 – 5.4 HLVII water-tetrahydrofuran 12.04 ± 0.56 – 5.6 HLVIII water-tetrahydrofuran 11.98 ± 0.46 – 5.6 HLIX water-tetrahydrofuran 11.45 ± 0.31 – 7.5 HLX water-tetrahydrofuran 11.49 ± 0.18 – 6.8 HLXI water-acetone 10.87 ± 0.39 12.01 ± 0.45 5.4 HLXII water-acetone 11.10 ± 0.33 12.07 ± 0.31 5.5 HLXIII water-acetone 11.68 ± 0.28 12.12 ± 0.29 5.7 HLXIV water-acetone 11.76 ± 0.17 12.20 ± 0.16 7.5 HLXV water-acetone 11.04 ± 0.72 12.02 ± 0.28 6.9

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Redox-Active Metal Complexes with Cycloaminomethyl Derivatives … 149

Table 2. Stepwise (Ki) and overall () stability constants of the metal complexes

Stability Metal ion Ligand Solvent constant Cu(II) Zn(II) I HL water-ethanol lgKI 9.03 ± 0.45 8.3 ± 0.42 lgKII 8.57 ± 0.26 7.35 ± 0.37 lgβ 17.60 ± 0.52 15.65 ± 0.56 II HL water-ethanol lgKI 9.57 ± 0.48 8.66 ± 0.35 lgKII 8.60 ± 0.43 7.83±0.39 lgβ 18.17 ± 0.64 16.49 ± 0.52 III HL water-ethanol lgKI 9.42 ± 0.38 8.46 ± 0.34 lgKII 8.77 ± 0.44 7.74 ± 0.23 lgβ 18.19 ± 0.58 16.2 ± 0.41 IV HL water-ethanol lgKI 9.24 ± 0.37 8.3 ± 0.33 lgKII 9.34 ± 0.37 8.33 ± 0.25 lgβ 18.58 ± 0.52 16.63 ± 0.41 V HL water-ethanol lgKI 9.27 ± 0.46 8.24 ± 0.25 lgKII 8.07 ± 0.40 7.57 ± 0.23 lgβ 17.34 ± 0.61 15.81 ± 0.34 VI HL water-tetrahydrofuran lgKI 8.24 ± 0.25 7.76 ± 0.31 lgKII 7.8 ± 0.23 6.82 ± 0.34 lgβ 16.04 ± 0.34 14.58 ± 0.46 VII HL water-tetrahydrofuran lgKI 9.22 ± 0.28 8.66 ± 0.35 lgKII 8.82 ± 0.44 8.36 ± 0.25 lgβ 18.04 ± 0.52 17.02 ± 0.43 VIII HL water-tetrahydrofuran lgKI 9.57 ± 0.48 8.70 ± 0.44 lgKII 8.76 ± 0.44 8.12 ± 0.32 lgβ 18.33 ± 0.65 16.82 ± 0.54 IX HL water-tetrahydrofuran lgKI 9.1 ± 0.46 8.04 ± 0.40 lgKII 8.66 ± 0.35 7.93 ± 0.32 lgβ 17.76 ± 0.58 15.97 ± 0.51 X HL water-tetrahydrofuran lgKI 8.45 ± 0.34 8.27 ± 0.33 lgKII 7.17 ± 0.22 7.66 ± 0.38 lgβ 15.62 ± 0.40 15.93 ± 0.50 XI HL water-acetone lgKI 9.11 ± 0.27 8.21 ± 0.25 lgKII 8.68 ± 0.26 7.52 ± 0.23 lgβ 17.79 ± 0.37 15.73 ± 0.34 XII HL water-acetone lgKI 9.69 ± 0.39 8.31 ± 0.42 lgKII 8.20 ± 0.41 8.12 ± 0.24 lgβ 17.89 ± 0.57 16.43 ± 0.48 XIII HL water-acetone lgKI 9.99 ± 0.50 8.86 ± 0.27 lgKII 7.96 ± 0.24 8.45 ± 0.34 lgβ 17.95 ± 0.55 17.31 ± 0.43 XIV HL water-acetone lgKI 10.54 ± 0.42 9.22 ± 0.46 lgKII 9.15 ± 0.46 8.54 ± 0.34 lgβ 19.69 ± 0.62 17.76 ± 0.57 XV HL water-acetone lgKI 9.38 ± 0.28 8.87 ± 0.44 lgKII 8.15 ± 0.33 8.09 ± 0.40 lgβ 17.53 ± 0.43 16.96 ± 0.59 Complimentary Contributor Copy

150 N. V. Loginova, H. I. Harbatsevich, T. V. Koval’chuk et al.

Table 3. Analytical data for the metal complexes with the ligands HLI–HLXV

Found/Calculated (%) Complex Formula С Н N Metal I Zn(L )2 C30H44N2O4Zn 64.15/64.11 7.72/7.89 4.88/4.98 11.55/11.63 II Zn(L )2 C32H48N2O4Zn 65.26/65.13 8.01/8.20 4.81/4.75 11.19/11.08 III Zn(L )2 C34H52N2O4Zn 66.18/66.06 8.31/8.48 4.43/4.53 10.38/10.58 IV Zn(L )2 C30H44N2O6Zn 60.42/60.65 7.32/7.47 4.78/4.72 11.20/11.01 V Zn(L )2 C32H50N4O4Zn 61.21/61.98 8.00/8.13 8.94/9.03 10.71/10.54 VI Zn(L )2 C60H56N2O4Zn 77.01/77.12 6.08/6.04 3.07/3.00 6.91/7.00 VII Zn(L )2 C62H60N2O4Zn 77.45/77.37 6.34/6.28 2.80/2.91 6.69/6.79 VIII Zn(L )2 C64H64N2O4Zn 77.49/77.60 6.43/6.51 2.81/2.83 6.55/6.60 IX Zn(L )2 C60H56N2O6Zn 74.51/74.56 5.79/5.84 3.01/2.90 6.78/6.77 X Zn(L )2 C62H62N4O4Zn 75.11/75.03 6.28/6.30 5.66/5.64 6.54/6.59 XI Zn(L )2 С38H60O4N2Zn 67.55/67.69 8.88/8.97 4.08/4.15 9.61/9.70 XII Zn(L )2 C40H64O4N2Zn 68.31/68.40 9.07/9.18 3.90/3.99 9.22/9.31 XIII Zn(L )2 C42H68O4N2Zn 68.05/69.06 9.26/9.38 3.77/3.84 8.83/8.95 XIV Zn(L )2 C38H60O6N2Zn 64.52/64.61 8.45/8.56 3.88/3.97 9.15/9.26 XV Zn(L )2 C40H66O4N4Zn 65.50/65.59 8.97/9.08 7.53/7.65 8.80/8.93 I Cu(L )2 C30H44N2O4Cu 64.26/64.32 8.01/7.92 4.87/5.00 11.41/11.34 II Cu(L )2 C32H48N2O4Cu 65.16/65.33 8.37/8.22 4.65/4.76 10.87/10.80 III Cu(L )2 C34H52N2O4Cu 66.37/66.26 8.41/8.50 4.61/4.55 10.44/10.31 IV Cu(L )2 C30H44N2O6Cu 60.99/60.84 7.60/7.49 4.66/4.73 10.81/10.73 V Cu(L )2 C32H50N4O4Cu 62.25/62.16 8.09/8.15 8.99/9.06 10.17/10.28 VI Cu(L )2 C60H56N2O4Cu 78.04/77.27 6.11/6.05 3.03/3.00 6.74/6.81 VII Cu(L )2 C62H60N2O4Cu 78.29/77.51 6.23/6.29 2.95/2.92 6.68/6.61 VIII Cu(L )2 C64H64N2O4Cu 76.97/77.75 6.45/6.52 2.8/2.83 6.49/6.43 IX Cu(L )2 C60H56N2O6Cu 73.96/74.71 5.91/5.85 2.93/2.90 6.66/6.59 X Cu(L )2 C62H62N4O4Cu 74.42/75.17 6.37/6.31 5.6/5.66 6.35/6.41 XI Cu(L )2 С38H60O4N2Cu 67.19/67.87 8.90/8.99 4.21/4.17 9.36/9.45 XII Cu(L )2 C40H64O4N2Cu 69.28/68.59 9.30/9.21 4.04/4.00 9.16/9.07 XIII Cu(L )2 C42H68O4N2Cu 69.93/69.24 9.50/9.41 3.89/3.85 8.63/8.72 XIV Cu(L )2 C38H60O6N2Cu 65.44/64.79 8.67/8.58 4.02/3.98 9.11/9.02 XV Cu(L )2 C40H66O4N4Cu 66.43/65.77 9.02/9.11 7.75/7.67 8.61/8.70 * Elemental analyses were carried out with an instrument Vario EL (CHNS mode). The metals were determined using an atomic emission spectrometer with an inductively coupled plasma excitation source (Spectroflame Modula).

There are several reasons for carrying out an electrochemical investigation of redox properties of the compounds HLIHLXV and their Cu(II) and Zn(II) complexes. First, in connection with the fact that the phenolic ligands in transition metal complexes can be in different redox states depending on conditions, formal consideration of their electronic structure is conventionally carried out in the frame of the triad of redox ligand forms [42–44]: diamagnetic single- or double-charged anions, neutral o-benzoquinones or paramagnetic o- benzosemiquinone anion-radicals. Second, in parallel with the key problem of coordination

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Redox-Active Metal Complexes with Cycloaminomethyl Derivatives … 151 chemistry related to description of electronic structure of the central complexing ion, an additional problem arises of determining the redox state of the phenolic ligands in the complexes synthesized. Third, on the basis of our previous data it is safe to assume that redox properties of the phenolic derivatives under study and their transitional metal complexes can affect their biological activity [33].

Table 4. Thermal analysis data for decomposition of the metal complexes

Mass loss (%), Complex Temperature°C (Process) Found/Calc. I Cu(L )2 140–160, 180–200, 210–240, 260–275 (endo) 86.49/85.80 II Cu(L )2 120–160, 220–260 (exo), 270–350 (endo) 86.99/86.48 III Cu(L )2 120–150, 180–220 (exo), 280–350 (endo) 86.76/87.09 IV Cu(L )2 130–150, 220–290 (exo), 350–450 (endo) 87.18/86.57 V Cu(L )2 120–160, 160–180 (exo), 200–240, 260–360 (endo) 86.31/87.14 VI Cu(L )2 210–300, 420–450 (endo) 90.88/91.47 VII Cu(L )2 190–225, 240–390 (endo) 91.99/91.72 VIII Cu(L )2 180–220 (exo), 240–320 (endo), 410–450 (exo) 93.04/91.96 IX Cu(L )2 190–225, 230–390 (endo) 91.01/91.75 X Cu(L )2 200–310, 440–450 (endo) 93.15/91.97 XI Cu(L )2 120–170 (exo), 200–380, 400–500 (endo) 86.92/88.17 XII Cu(L )2 120–150, 160–190 (exo), 220–320, 380–480 (endo) 88.98/88.64 XIII Cu(L )2 160–180 (endo), 260–320 (exo) 87.93/89.08 XIV Cu(L )2 150–210 (endo), 220–320 (exo), 380–480 (endo) 88.01/88.71 XV Cu(L )2 120–160, 210–230 (exo), 250–300, 310–380, 460–550 (endo) 90.11/89.11 I Zn(L )2 120–160 (exo), 260–270, 320–360 (endo) 86.01/85.52 II Zn(L )2 120–190 (exo), 260–280, 310–410 (endo) 86.42/86.21 III Zn(L )2 120–140, 180–210, 270–310 (exo), 360–400 (endo) 87.12/86.83 IV Zn(L )2 130–150, 180–220 (exo), 240–280, 320–370 (endo) 86.56/86.30 V Zn(L )2 120–140, 200–240 (exo), 280–330, 370–400 (endo) 86.42/86.88 VI Zn(L )2 160–210 (exo), 220–300, 300–370 (endo) 90.65/91.29 VII Zn(L )2 160–170 (exo), 210–220, 250–290, 400–500 (endo) 90.92/91.54 VIII Zn(L )2 150–170 (exo), 210–270, 280–290, 400–500 (endo) 90.31/91.78 IX Zn(L )2 190–200 (exo), 210–300, 320–360 (endo) 90.78/91.58 X Zn(L )2 190–220 (exo), 230–280, 320–370 (endo) 91.01/91.80 XI Zn(L )2 130–150, 200–220 (exo), 260–300, 420–450 (endo) 88.32/87.93 XII Zn(L )2 170–190 (exo), 200–270, 450–460 (endo) 89.01/88.41 XIII Zn(L )2 170–180 (exo), 210–300, 400–550 (endo) 89.41/88.86 XIV Zn(L )2 170–210 (endo), 240–270 (exo), 500–560 (endo) 89.12/88.48 XV Zn(L )2 180–210 (exo), 220–270, 500–570 (endo) 87.99/88.89 * Thermal analysis was performed using a Simultaneous Thermal Analyzer STA 449 C with identification of the final products by X-ray powder diffraction. The heating rates were suitably controlled at 5°C min−1 under argon atmosphere, and the mass loss was measured from ambient temperature up to 700°C.

The derivatives of ortho-diphenols HLI–HLX are electrochemically inactive upon cathodic polarization and are oxidized upon anodic one. There are two oxidation peaks in voltammograms (about 0.42–0.73 and 0.90–1.42 V), corresponding to successive transfer of the first and second electrons with respective formation of benzosemiquinone and benzoquinones (as an example Figure 2 presents a voltammogram for HLIII, and the data for Complimentary Contributor Copy

152 N. V. Loginova, H. I. Harbatsevich, T. V. Koval’chuk et al. these compounds are summarized in Table 7). The peak potentials of all these compounds are close, some variation of their values may be related to the influence of substituents. The sole exception is provided by HLV and HLX having the second anodic peak positioned 0.3–0.4 V more cathodically as compared to other ortho-diphenols. The peaks of reduction of the respective products of anodic oxidation are observed on the reverse scan (about 0.12–0.32 and 0.43–0.84 V, Table 7). The participation of the first electron and then the second one in the anodic electrochemical process was confirmed by the results of coulometry at controlled potentials. It should be noted that the molecules of these compounds contain yet another possible center for the anodic oxidation – the nitrogen atom of the amino group with a lone electron pair. However, the anodic oxidation for the amino nitrogen atom proceeding irreversibly, with no peaks of the reverse process being observed on the reverse scan, these processes appear not to be realized for the ortho-diphenols HLI–HLX. Electrochemical oxidation of the derivatives of meta-diphenols HLXI–HLXV proceeds similarly to that of ortho-diphenols HLI–HLX: in their voltammograms two peaks at 0.78– 0.93 and 1.27–1.61 V are observed, but they are anodically shifted by 200–300 mV as compared to ortho-diphenols (Table 7). On the reverse scan a small peak for reduction of products of anodic oxidation is observed only for HLXV, being absent for HLXI–HLXIV. After prolonged electrochemical oxidation of meta-diphenols HLXI–HLXV a cathodic wave with several peaks of reduction is observed in cathodic voltammograms, suggesting that several products are formed on anodic oxidation. Evidently, meta-orientation of hydroxyls in HLXI– HLXV does not favour the resonance interaction of functional groups, resulting in the redox properties of meta-diphenols HLXI–HLXV and ortho-diphenols HLI–HLX being noticeably different.

Table 5. Molar conductivity of the metal complexes (in acetonitrile)

–1 2 –11 –1 2 –1 Complex mol,  cm mol Complex mol,  cm mol I I Cu(L )2 22.1 Zn(L )2 10.5 II II Cu(L )2 28.2 Zn(L )2 14.4 III III Cu(L )2 29.9 Zn(L )2 17.0 IV IV Cu(L )2 25.1 Zn(L )2 13.9 V V Cu(L )2 26.9 Zn(L )2 18.1 VI VI Cu(L )2 67.9 Zn(L )2 5.4 VII VII Cu(L )2 62.3 Zn(L )2 7.3 VIII VIII Cu(L )2 73.8 Zn(L )2 10.9 IX IX Cu(L )2 86.3 Zn(L )2 8.1 X X Cu(L )2 56.3 Zn(L )2 10.3 XI XI Cu(L )2 62.0 Zn(L )2 21.5 XII XII Cu(L )2 74.0 Zn(L )2 25.4 XIII XIII Cu(L )2 59.7 Zn(L )2 48.7 XIV XIV Cu(L )2 81.2 Zn(L )2 22.4 XV XV Cu(L )2 60.2 Zn(L )2 37.4 * The molar conductivity of 10–3 M solutions of the metal(II) complexes in acetonitrile was measured at 20 C using a TESLA BMS91 conductometer (cell constant 1,0).

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I XV Table 6. Octanol/water partition coefficients (logPow*) of the ligands HL –HL and their metal complexes

Compound logPow Compound logPow Compound logPow I I I HL 1.1 Cu(L )2 2.1 Zn(L )2 2.7 II II II HL 1.2 Cu(L )2 2.2 Zn(L )2 2.3 III III III HL 1.3 Cu(L )2 2.7 Zn(L )2 2.8 IV IV IV HL 0.9 Cu(L )2 2.4 Zn(L )2 2.2 V V V HL 1.0 Cu(L )2 1.9 Zn(L )2 2.5 VI VI VI HL 2.1 Cu(L )2 2.9 Zn(L )2 3.9 VII VII VII HL 2.2 Cu(L )2 2.9 Zn(L )2 3.7 VIII VIII VIII HL 2.3 Cu(L )2 3.1 Zn(L )2 3.3 IX IX IX HL 1.8 Cu(L )2 3.3 Zn(L )2 3.2 X X X HL 1.9 Cu(L )2 3.4 Zn(L )2 3.3 XI XI XI HL 1.3 Cu(L )2 2.2 Zn(L )2 2.6 XII XII XII HL 1.4 Cu(L )2 2.1 Zn(L )2 3.2 XIII XIII XIII HL 1.5 Cu(L )2 2.3 Zn(L )2 3.5 XIV XIV XIV HL 1.2 Cu(L )2 2.4 Zn(L )2 2.9 XV XV XV HL 1.2 Cu(L )2 2.7 Zn(L )2 2.5 1 *Pow=Coctanol-1/Сwater (C, mol·l ).

Table 7. Cyclic voltammetry data for the ortho-and meta-diphenols

1 1 1 2 2 2 Compound E pa , V E pc ,V E 1/2, V E pa , V E pc , V E 1/2, V HLI 0.44 0.18 0.31 1.37 0.84 1.11 HLII 0.45 0.24 0.35 1.30 0.71 1.01 HLIII 0.42 0.25 0.34 1.32 0.77 1.05 HLIV 0.57 0.32 0.45 1.27 0.71 0.99 HLV 0.44 0.12 0.28 0.90 0.43 0.07 HLVI 0.47 0.16 0.32 1.36 0.64 1.00 HLVII 0.52 0.21 0.37 1.39 0.56 0.98 HLVIII 0.41 0.17 0.29 1.31 0.61 0.96 HLIX 0.73 0.24 0.49 1.42 1.21 1.32 HLX 0.55 0.10 0.33 1.04 0.41 0.73 HLXI 0.78 – – 1.56 – – HLXII 0.79 – – 1.54 – – HLXIII 0.78 – – 1.60 – – HLXIV 0.93 – – 1.61 – – HLXV 0.81 0.48 0.65 1.27 0.93 1.10 1 * The formal potential of the redox system E 1/2 used as a criterion for reducing ability (according to [45]) 1 was calculated as the average potential of the peaks found by the cyclic voltammetry method: E 1/2 = 1 1 (Еpa + Еpc )/2).

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154 N. V. Loginova, H. I. Harbatsevich, T. V. Koval’chuk et al.

Table 8. Cyclic voltammetry data for the Zn(II) complexes

1 1 1 2 3 4 Compound E pa, V E pc, V E 1/2, V E pa , V E pa, V E pa, V I Zn(L )2 0.52 0.11 0.32 1.03 1.32 – II Zn(L )2 0.54 0.13 0.34 0.99 1.33 – III Zn(L )2 0.52 0.14 0.33 0.98 1.35 – IV Zn(L )2 0.71 0.23 0.47 1.30 – – V Zn(L )2 0.65 0.01 0.33 0.99 1.60 – VI Zn(L )2 0.49 0.13 0.31 0.80 1.38 – VII Zn(L )2 0.51 0.18 0.35 0.80 0.94 1.40 VIII Zn(L )2 0.49 0.20 0.35 0.78 1.41 – IX Zn(L )2 0.65 0.27 0.46 0.84 1.39 – X Zn(L )2 0.56 0.06 0.31 0.94 1.40 – XI Zn(L )2 0.69 – – 1.56 – – XII Zn(L )2 0.82 – – 1.14 1.66 – XIII Zn(L )2 0.81 – – 1.67 – – XIV Zn(L )2 1.13 – – 1.58 – – XV Zn(L )2 0.91 – – 1.35 1.55 –

Redox potentials of the Zn(II) complexes with these phenolic ligands are summarized in Table 8. A sweep from open circuit potential to 2.0 V shows that anodic oxidation of the Zn(II) complexes proceeds in the same potential regions as for the ligands. Since Zn(II) ion in its complexes does not undergo any electrochemical transformations, the anodic processes can be attributed to ligand-centered processes. In voltammograms of the complexes from two to four anodic peaks are observed, presumably accounting for successive processes of III electron transfer (Table 8, a voltammogram for Zn(L )2 is given in Figure 2 as an example). Unlike HLI–HLXV and the Zn(II) complexes, in voltammograms of the Cu(II) complexes on cathodic polarization one or two cathodic waves are observed (Table 9). They may be related to realization of two-step reduction of Cu(II) to Cu(I) and then to Cu(0). For the I VII IX XI complexes Cu(L )2, Cu(L )2, Cu(L )2–Cu(L )2 the second reduction step possibly lies IV beyond the accessible potential range. An anodic peak (a double anodic peak for Cu(L )2 and V Cu(L )2) about –0.15÷–0.30 V on the reverse scan corresponds to oxidation of the products of III V Cu(L )2–Cu(L )2 reduction (Figure 2, Table 9). On anodic polarization in voltammograms for the Cu(II) complexes with HLI–HLXV from one to four oxidation peaks are observed (Table 9). Generally the Cu(II) complexes show a lower reducing ability as compared with that of the respective ligands: oxidation of the Cu(II) complexes begins at more positive potential values than in the case of the respective ligands. I II IV V VIII XI XII The complexes Cu(L )2, Cu(L )2, Cu(L )2, Cu(L )2, Cu(L )2, Cu(L )2, Cu(L )2 are the III VI VII X least active as reductants, while Cu(L )2, Cu(L )2, Cu(L )2, Cu(L )2 are the most active ones. Thus, the electrochemical investigation substantiated the results of the spectroscopic one, according to which the coordination cores of the complexes are formed by Zn(II) or Cu(II) ions and the ligands in monoanionic form. Furthermore, it was found that the Cu(II) complexes rank below the ligands in their reducing ability, while the Zn(II) ones show the reducing ability comparable to that of the ligands.

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–1 III –1 III Figure 2. Voltammograms (50 mV s ) of the compound HL (1.36 mmoll ) and its Cu(L )2 and III –1 Zn(L )2 complexes in 0.1 moll (C2H5)4NClO4 acetonitrile solution under dry nitrogen on glassy- carbon working electrode (dotted line  background cyclic voltammogram of glassy-carbon electrode). Solid line – polarization in the anodic direction from the open circuit potential; dashed line – polarization in the cathodic direction from the open circuit potential.

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Table 9. Cyclic voltammetry data for the Cu(II) complexes

Processes under anodic polarization Processes under cathodic polarization Compound 1 1 1 2 2 3 3 4 1 1 2 2 Е pa, V Е pc, V E 1/2, V Е pa, V Е pc, V Е pa, V Е pc, V Е pa, V Е pc, V Е pa, V Е pc, V Е pa, V I Cu(L )2 1.09 0.13 0.61 1.50 1.12    0.71 0.12   II Cu(L )2 1.20 0.16 0.59 1.55 1.10    0.71 0.14 1.06 0.07 III Cu(L )2 0.53 0.20 0.37 1.30 1.13      1.55 0.13 IV Cu(L )2 1.38 0.18 0.78      0.82 0.15 1.83 0.20 V Cu(L )2 1.10 0.17 0.64 1.64 –    0.75  1.23 0.11 VI Cu(L )2 0.58 0.10 0.34 0.91  1.59 1.11  –0.2  -0.81 -0.12 VII Cu(L )2 0.60 0.18 0.39 1.07 0.40 1.66   –0.20 0.32 -0.75 –0.01 VIII Cu(L )2 1.3 1.0 1.15      –0.64 –0.30 –1.70 –0.34 IX Cu(L )2 0.89 0.28 0.59 1.50 1.10    –0.16 0.10 – – X Cu(L )2 0.76 0.01 0.39 1.56 1.12 1.73   –0.78 –0.05 – XI Cu(L )2 1.51 1.06 1.29      –0.63 –0.08 – – XII Cu(L )2 1.20 – – 1.87 –    –0.65 –0.07 –1.48 0.19 XIII Cu(L )2 0.75 – – 1.16 – 1.73   –0.66 –0.03 -1.08 –0.09 XIV Cu(L )2 1.24 – – 1.42 – 1.82   –0.66 –0.02 –0.99 0.20 XV Cu(L )2 0.78 – – 1.23 – 1.49  1.71 –0.66 –0.05 –1.50 –

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Because of the amorphous state of the complexes synthesized we used several spectroscopic methods to specify the coordination modes in the Cu(II) and Zn(II) complexes. Analysis of IR spectra of the compounds HLI–HLXV and their complexes made it possible to identify donor sites of binding of the ligands with Cu(II) and Zn(II) (Table 10). In the spectra of HLI–HLXV there are bands at 3536–3250 cm–1, indicating the presence of intermolecular hydrogen bonds involving phenolic hydroxyls [46]. The shift of these bands to the lower frequency region in the spectra of the metal(II) complexes suggests that phenolic hydroxyls participate in metal ion coordination. Besides, the frequencies of aromatic ring vibrations were found to be shifted due to the complexation. The change in the band intensity of ν(С–О) stretching vibrations and the appropriate bands being shifted to the lower frequency region in the spectra of the Cu(II) and Zn(II) complexes also are evidence in favour of the ligands being coordinated to the metal(II) ions via oxygen atoms of phenolic hydroxyls. On examination of the frequency range associated with ν(C–N) stretching vibrations of tertiary amines a blue shift of the bands and/or a change in their intensity can be observed in the spectra of the complexes. Hence the amino group of the compounds HLI–HLXV takes part in coordination to the metal ions. It should be noted that in the spectra of all these complexes there are novel bands in the region of 600–400 cm–1 which may be assigned to the stretching vibrations of M–O and M–N bonds and substantiate the participation of hydroxyl oxygen atoms and cycloaminomethyl nitrogen atoms in forming coordination cores [CuN2O2] and [ZnN2O2] [47]. Interpretation of IR spectra of the compounds HLIHLXV is in complete agreement with the composition and geometry of their coordination cores determined by the method of UV- Vis spectroscopy (Table 11). The UV-Vis absorption spectra of metal(II) complexes generally involve d-d transitions, the ligand-to-metal(II) charge transfer transitions (LMCT) as well as intraligand absorption (ILA) [48]. In the spectra of the Cu(II) complexes there are absorption maxima in UV region (220–300 nm) which belong to ILA. A band in the region 490–610 nm in the spectra of all the Cu(II) complexes under study may be indicative of the square planar shape (or very close to that type) of their chromophores [CuN2O2] [48]. The maxima in the regions 290–380 nm and 320–500 nm appearing in the spectra as a result of complexation with Cu(II) ions suggest the II II presence of LMCT transitions: respectively N()Cu and OphenCu [48]. In the UV-Vis spectra of the Zn(II) complexes there are just the ILA and LMCT bands (Table 11) [48]. Thus, the bands in the range of 300–380 nm and 350–550 nm belong to LMCT (respectively N(σ)M(II) and OphenM(II)), indicating the participation of the amino nitrogen atom and the hydroxyl oxygen atom of the ligands in complexation with Zn(II) ions [49]. The agreement of the results obtained with the literature for Zn(II) complexes of tetrahedral structure allows one to consider a tetrahedron as the most probable form of coordination cores [ZnO2N2] incorporated into the Zn(II) complexes synthesized [49–51].

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Table 10. Prominent IR absorption bands (ν, cm–1) of the compounds HLIHLXV and their metal complexes*

Compound ν(O–H) ν(C = C)arom ν(C–O) ν(C–N) ν(M–O, M–N)** HLI–V 3375–3410s 1588–1596w 1180–1293s 1063–1102m – 1489–1497s 1299–1307s 1327–1335m I–V * M(L )2 3300–3350m 1556–1557s 1098–1146s 1003–1037w 501–573m 1480–1493m 1273–1280s 1298–1310m HLVI–X 3392–3432s 1594–1596m 1380–1400m 1116–1186m – 1487–1490s 1263–1312m VI–X M(L )2 3300–3408m 1558–1564m 1325–1345m 1034–1084w 517–577m 1462–1472m 1259–1270m HLXI–XV 3437–3465m 1589–1592m 1386–1398s 1052–1069m – 1477–1481m 1353–1359m 1178–1184s XI–XV M(L )2 3300–3350m 1545–1558s 1357–1360s 1004–1033m 501–600m 1481–1483m 1245–1290m 1113–1117m *IR spectra of solids were recorded with a Nicolet 380 spectrometer at room temperature in the wavelength range 4000–400 cm–1, using «Smart Performer». **M = Cu(II), Zn(II).

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Table 11. UV-Vis spectral data for the compounds HLI–HLXV and their metal complexes*

Coordination Compound Chromophore Spectral assignments, λ , nm lg ε max max core HLI–V – 220–230 (ILA) 4.1–4.2 – 270–300 (ILA) 3.8–3.9 I–V Cu(L )2 CuO2N2 220–230 (ILA) 4.2–4.3 Square planar 270–300 (ILA) 3.9–4.0 290–320sh (N(σ)Cu(II) LMCT) 3.6–3.9

320–400 (OphenCu(II) LMCT) 3.3–3.6 490–580 (d–d) 2.5–2.8 I–V Zn(L )2 ZnO2N2 220–230 (ILA) 4.1–4.2 Tetrahedral 270–300 (ILA) 3.7–3.9 300–310sh (N(σ)  Zn(II) LMCT) 3.5–3.6

380–510sh (Ophen Zn(II) LMCT) 2.5–3.0 HLVI–X – 190–230 (ILA) 4.4–4.5 – 275–300 (ILA) 3.4–3.6 VI–X Cu(L )2 CuO2N2 190–220 (ILA) 4.0–4.2 Square planar 270–300 (ILA) 3.1–3.5 310–340 (N(σ)  Cu(II) LMCT) 2.9–3.1

370–450sh (Ophen Cu(II) LMCT) 2.6–3.0 490–600 (d–d) 2.1–2.3 VI–X Zn(L )2 ZnO2N2 225–235 (ILA) 4.1–4.3 Tetrahedral 270–300 (ILA) 3.4–3.7 305–340sh (N(σ)  Zn(II) LMCT) 2.7–3.2

350–410sh (Ophen Zn(II) LMCT) 2.0–2.5 HLXI–XV – 200–210 (ILA) 4.3–4.4 – 275–295 (ILA) 3.5–3.6 XI–XV Cu(L )2 CuO2N2 200–230 (ILA) 4.2–4.3 Square planar 250–300sh (ILA) 3.7–3.9 300–380 (N(σ)  Cu(II) LMCT) 3.3–3.5

440–530sh (Ophen Cu(II) LMCT) 2.6–3.0 560–610 (d–d) 2.0–2.3 XI–XV Zn(L )2 ZnO2N2 220–235 (ILA) 3.9–4.2 Tetrahedral 260–295sh (ILA) 3.5–3.6 300–370 (N(σ)  Zn(II) LMCT) 2.8–3.1

430–550sh (Ophen Zn(II) LMCT) 2.3–2.8 275–290 (ILA) 3.6 310–360 (N(σ)  Zn(II) LMCT) 3.0

470–550sh (Ophen Zn(II) LMCT) 2.0 *UV-Vis absorption spectra were recorded with a SPECORD S600 spectrophotometer.

To verify the composition of coordination cores of the Zn(II) complexes synthesized, 1H NMR spectra of the ligands and their complexes were investigated. Thus, in the spectra of HLI–HLV a strong singlet is observed at 1.26–1.34 ppm, corresponding to protons of tert- butyl groups, doublets of protons of aromatic ring at 6.50-7.00 ppm, singlets of hydroxyls at 7.28-8.19 ppm, singlets of methylene group between the aromatic ring and the nitrogen atom at 3.66-4.09 ppm as well as multiplets belonging to methylene groups of saturated heterocycles at 1.54-2.08, 2.58-3.01, and 3.64-3.99 ppm (Figure 3). In the spectra of the respective Zn(II) complexes the integral intensity of the signals assigned to hydroxyl protons corresponds to two hydrogen atoms, which is indicative of the ligand being coordinated in Complimentary Contributor Copy

160 N.V. Loginova, H.I. Harbatsevich, T.V. Koval’chuk et al. phenolate form. Besides, the hydroxyl proton signals of the complexes are shifted to the strong field region (7.25-7.40 ppm), thus substantiating the participation of hydroxyls in coordination with the metal ion [52]. The values of the chemical shifts of methylene groups (adjacent to the nitrogen atom taking part in coordination with Zn(II) ion) are also shifted in the spectra of the complexes. The signals from the protons of those groups which do not bind to Zn(II) (tert-butyl, aromatic ring, methylene groups remote from the nitrogen atom) virtually do not change their position in the spectra. 1H NMR spectra of the rest ligands and their complexes as a whole differ but little from those of the above compounds, excepting the positions of the signals for protons of some groups in the spectra of the ligands. The solid state ESR spectra of the Сu(II) complexes were recorded both at room temperature and at 77 К, and the ESR parameters are presented in Table 12. Unlike the Сu(II) complexes, the Zn(II) ones are diamagnetic. At room temperature the spectra of the Сu(II) complexes look virtually the same as at 77 К, and it is just the signal intensity that is significantly lower. Hyperfine structure is observed for all the complexes. The solid state ESR spectra of all these complexes at 77 K are quite similar and exhibit axially symmetric g-tensor parameters with g║>g┴>2.0023. The spin Hamiltonian parameters of the complexes do not differ much (Table 12), thus their coordination cores are similar. Because of the steric effects produced by bulky groups of cycloaminomethyl derivatives of sterically hindered diphenols in their Cu(II) complexes there is a significant distortion of their square planar coordination core. According to [53], g║ values less than 2.3 indicate a considerably covalent character of M–L bonds, and those greater than 2.3 are indicative of the ionic one. The geometric parameter G=(g║–2)/(g┴–2), which is a measure of the exchange interaction between copper centers in solid complexes, appears to be greater than 4 for all the compounds (Table 12) [54]. The calculated G values show that the exchange interaction between copper centers is negligible. Thus, all Cu(II) complexes with the trend g║ > g┴ > ge and G value falling within 2 2 the above-mentioned range are consistent with a dx –y ground state, and Cu(II) ion is in a distorted square planar environment formed by N,О-coordinating ligands, which agrees with the data obtained by other physico-chemical methods [55]. No signal of stabilized radicals present in ESR spectra of the Cu(II) and Zn(II) complexes in the range of g = 2.002 ÷ 2.006 confirms the phenolate character of the ligands [56]. To support the molecular structures deduced from the above-mentioned array of physico- chemical data, we performed quantum-chemical simulations. All calculations were carried out using the GAUSSIAN03 software package [57] and the theoretical approach described thoroughly in our previous publications [29, 30]. MO calculations were performed using the density functional theory (DFT) B3PW91 method together with the combined 6-31G(d) + LANL2DZ basis set (respectively for nonmetal and metal atoms) [58]. No symmetry constraints were implemented during the optimization process. In order to perform the stationary point characterization, harmonic vibrational frequencies were calculated at the same level of theory. The scaling factor of 0.951 was applied to the calculated values [59]. We used different initial guesses, regarding the most probable binding sites of free ligands. It was shown that the Cu(II) complexes should be square planar with a trans-form considerably more stable, whereas minima on the potential energy surface for Zn(II) derivatives correspond to the distorted tetrahedral arrangement. In the light of the physico-chemical characterization the general mode of the ligating atoms in the Cu(II) and Zn(II) complexes can be represented as shown in Figure 4.

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Selected three-dimensional structures of these complexes are given in Figure 5. Agreement between the calculated and experimental IR spectra was used as the main criterion for the calculated structure being veritable.

Figure 3. The 1H NMR spectra of the compound HLI and its Zn(II) complex.

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Table 12. Parameters of ESR spectra of the Cu(II) complexes*

Complex g║ g┴ G ΔH, Gs I Cu(L )2 2.290 2.062 4.82 175 II Cu(L )2 2.250 2.049 5.30 90 III Cu(L )2 2.250 2.049 5.30 175 IV Cu(L )2 2.276 2.062 4.58 125 V Cu(L )2 2.260 2.056 4.80 125 VI Cu(L )2 2.295 2.056 5.45 65 VII Cu(L )2 2.305 2.058 5.43 70 VIII Cu(L )2 2.300 2.059 5.25 70 IX Cu(L )2 2.280 2.058 4.99 75 X Cu(L )2 2.305 2.059 5.34 55 XI Cu(L )2 2.281 2.065 4.44 95 XII Cu(L )2 2.260 2.065 4.11 190 XIII Cu(L )2 2.270 2.063 4.41 120 XIV Cu(L )2 2.270 2.066 4.20 165 XV Cu(L )2 2.280 2.070 4.10 185 *ESR spectra of the Cu(II) complexes in the solid state were measured with ERS-220 X-band spectrometer (9.45 GHz) at room temperature and at 77 K, using 100-kHz field modulation; g factors were quoted relative to the standard marker 2,2-diphenyl-1-picrylhydrazyl (DPPH).

Figure 4. The plausible coordination modes of the Cu(II) and Zn(II) complexes.

I XIV Figure 5. DFT-optimized structures of the complexes Cu(L )2 and Zn(L )2.

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3. INTERACTION OF CYCLOAMINOMETHYL DERIVATIVES OF DIPHENOLS AND THEIR COPPER(II) AND ZINC(II) COMPLEXES WITH CYTOCHROME C

On the basis of our data for the properties of sterically hindered derivatives of diphenols and their transition metal complexes it may be suggested that biological properties of these compounds (including their antimicrobial activity) are due to their participation in redox cell processes [16]. Oxidoreductases may be possible macromolecular targets of the above- mentioned compounds. We carried out a spectrophotometrical investigation of reduction of bovine heart Cyt c (oxidized form) (Sigma) with the redox-active compounds synthesized, the results of which are presented in Table 13 and in Figures 6–8. As we demonstrated before [33], the complexes can interact with Cyt с both in molecular form and via anionic forms of the ligands and metal ions formed on their dissociation. Taking into account the findings presented in our previous publications [33] as well as the results of the electrochemical investigation of the compounds under study (Table 7), we may suggest a possible route of their oxidation on interaction with Cyt с: derivatives of diphenols transfer electrons in an outer-sphere process involving the exposed heme edge surrounded by positively charged amino acid residues; aromatic redox-active amino acid residues also can be potential participants of the electron transfer. Oxidation of o-diphenol derivatives with cycloaminomethyl substituents in vitro under anaerobic conditions can include two successive one-electron steps of oxidation of their ionic forms to yield o-benzoquinones on interaction with Cyt с via intermediate o-benzosemiquinone formation (Figure 6). Appearance of characteristic absorption bands at 550 and 520 nm when the ligands HLI– HLXV or their complexes are added to a solution of oxidized form of Cyt с indicates that Cyt с is reduced in vitro. The ligands HLI–HLV and their Zn(II) complexes were found to be the most active Cyt c reductants among the compounds under study (Table 13). Introduction of bulky substituents into HLVI–HLX molecules results in the reaction proceeding considerably slower, which is explicable by the interaction of these compounds with the hem edge of Cyt с being sterically hindered. Cycloaminomethyl derivatives of meta-diphenols HLXI–HLXV have no ability to reduce Cyt с (Table 13), because neither Zn(II) ion, nor resorcinol derivatives have pronounced reducing properties [62]. The Zn(II) complexes reduce Cyt с faster than the respective ligands (Table 13). It is likely that in this case a strong Lewis acid, Zn(II) ion, sterically orientates the hydroxyl groups participating in the process and provides for molecules of the complexes binding to the protein [63]. The reduction of Cyt с with the Cu(II) complexes proceeds noticeably slower than with the Zn(II) ones (Table 13). In this connection one should take into account that Cu(II) ions are able to catalyze the process of oxidation of phenols [64] and initiate the competing process resulting in oxidation of the reduced form of Cyt с. These processes can decrease the rate of the direct reaction involving the Cu(II) complexes, but they do not take place in the case of the Zn(II) complexes.

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Figure 6. Scheme of the reduction of Cyt c with the phenolate form of the ligand; Fe(III)-Cyt c and Fe(II)-Cyt c are respectively the oxidized and reduced forms of Cyt c.

Table 13. Rates of reduction of Cyt с (υ) with the compounds HLI–HLXV and their Zn(II) and Cu(II) complexes*

Compound υ, nmol· min–1 Compound υ, nmol· min–1 Compound υ, nmol· min–1 I I I HL 1.3 Zn(L )2 1.7 Cu(L )2 0.3 II II II HL 1.2 Zn(L )2 1.4 Cu(L )2 0.5 III III III HL 1.5 Zn(L )2 1.7 Cu(L )2 0.5 IV IV IV HL 1.3 Zn(L )2 1.8 Cu(L )2 0.8 V V V HL 1.0 Zn(L )2 1.1 Cu(L )2 0.6 VI VI VI HL 0.3 Zn(L )2 0.8 Cu(L )2 0.4 VII VII VII HL 0.5 Zn(L )2 0.5 Cu(L )2 0.4 VIII VIII VIII HL 0.4 Zn(L )2 0.3 Cu(L )2 0.5 IX IX IX HL 0.5 Zn(L )2 0.6 Cu(L )2 0.3 X X X HL 0.6 Zn(L )2 1.1 Cu(L )2 0.5 XI XI XI HL 0.1 Zn(L )2 0.1 Cu(L )2 0.2 XII XII XII HL 0.0 Zn(L )2 0.0 Cu(L )2 0.2 XIII XIII XIII HL 0.0 Zn(L )2 0.0 Cu(L )2 0.2 XIV XIV XIV HL 0.0 Zn(L )2 0.0 Cu(L )2 0.1 XV XV XV HL 0.0 Zn(L )2 0.1 Cu(L )2 0.1 *Cyt с concentration was determined on its interaction with excess sodium dithionite using the absorption -1 -1 coefficient ε550 = 21 mmol ·l·cm [60]. Ar-saturated acetonitrile solutions of the ligands and their complexes under study (3.5 µmol·l-1) and Cyt с (7.0 µmol·l-1) were used. Experiments were performed in 10 mmol·l-1 sodium phosphate buffer (pH 7.4) at 20 ±2 °С. Aliquots of the compounds under study were added to Cyt с solution up to the final concentration 35.0 µmol·l-1. The initial rate of Cyt с reduction (υ) was evaluated by the slope of the kinetic curve A550 vs. time according to [61]. The kinetic curves of Cyt c reduction were registered with the spectrophotometer SOLAR РВ2201В at the wave length 550 nm. The absolute error doesn’t exceed 0.1 nmol·min–1.

Thus, both the ligands and their metal complexes can reduce Cyt c; and it is the ligands and the metal complexes with the highest reducing ability (determined electrochemically) that exhibit the highest Cyt с reduction rate respectively among the ligands and metal complexes. IV IV For the compounds HL and Zn(L )2 reducing Cyt c at a high rate (Table 13) the products of their oxidation with this protein are identified. Figure 8 presents mass spectra for IV IV the extract of HL and Zn(L )2 oxidation products resulting from interaction with Cyt с as well as those for the extract from the negative control experiment (without adding Cyt с). In the spectra obtained in the mode of registering positive ions for control specimens there is a signal from [M+H]+ ions with m/z 266, while in the spectra of experimental specimens there is also a signal with m/z 264 from an ion which can be interpreted as [M+H]+ ion corresponding to the product of oxidation – 5-tert-butyl-(morpholinomethyl)cyclohexa-3,5- dien-1,2-dion (Figure 8, 2а, 4а). On the basis of comparing the levels of spectrophotometric Complimentary Contributor Copy

Redox-Active Metal Complexes with Cycloaminomethyl Derivatives … 165 signals from Cyt c reduced with excess sodium dithionite and with the above-mentioned compounds as well as of those from the oxidized and reduced forms of the ligand in the mass spectra of the extract it may be concluded that Cyt c oxidizes only 7±2 % of the compound HLIV (Figure 8, 2а) and of the Zn(II) complex (Figure 8, 4а). It is probable that the low degree of the redox transformation is due to the equilibrium character of the interaction of IV IV HL and Zn(L )2 with the protein as well as to the shift of the equilibrium towards formation of oxidized form of the protein Fe(III)-Cyt c and reduced forms of the compounds IV IV HL and Zn(L )2. The midpoint redox potential of Cyt c is about 250 mV, while that of the compound HLIV is equal to 440 mV and hence the reduction of the oxidized HLIV form with reduced Fe(II)- Cyt с is thermodynamically impossible. Besides, it should be taken into account that the IV + ligand [H2L ] with a protonated N-atom and the lysine residues of the active center of Cyt c are positively charged, which makes Cyt c interaction with the ligand more complicated IV because of the electrostatic repulsion [65]. The interaction of the complex Zn(L )2 with the protein may also be sterically hindered. Thus the peculiarities we have found of interaction of the compounds HLI–HLXV synthesized and their Cu(II) and Zn(II) complexes with Cyt c are in good agreement with the literature about the rate of interaction of this protein with redox-active compounds depending on steric factors, ionization/ protonation constants of compounds as well as on the ability of some metal ions to reduce Fe(III)-Cyt с or to oxidize Fe(II)-Cyt с [66–69].

Figure 7. Kinetic data for a change in absorbance of the Cyt c solution (at 550 nm) after adding the II II II II ligand HL and its complexes (1 – HL , 2 – Zn(L )2, 3 – Cu(L )2).

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Figure 8. Mass spectra (atmospheric pressure chemical ionization) of the extract obtained without II II adding (1, 3) and after adding (2, 4) Cyt c. HL (1, 2) and Zn(L )2 (3, 4) were selected as reductants. Registration of the spectra was performed in the modes of positive (a) and negative (b) ions.

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4. SOD-LIKE ACTIVITY OF CYCLOAMINOMETHYL DERIVATIVES OF DIPHENOLS AND THEIR COPPER(II) AND ZINC(II) COMPLEXES

The key problem in examining SOD activity in vitro is generation of superoxide followed by its detection. The following ways to generate superoxide are described in the literature: i) enzymatic oxidation of xanthine in the presence of xanthine oxidase [23, 70], ii) photoreduction of flavines [71], iii) autoxidation of epinephrine [72], iv) autoxidation of pyrogallol [73], v) autoxidation of 6-hydroxydopamine [74], vi) oxidation of NADH with phenazine methosulfate [75], vii) oxidation of alkaline dimethyl sulfoxide (DMSO) with atmospheric oxygen [76], viii) the use of potassium superoxide solution in DMSO [77], ix) electrochemical reduction of oxygen [78]. Superoxide formed is most commonly detected using Cyt c [70] or nitroblue tetrazolium (NBT) [79]. Previously we examined SOD-like activity of phenolic ligands and their metal complexes using enzymatic (xanthine/xanthine oxidase) and nonenzymatic (alkaline DMSO) models. To detect the superoxide formed, NBT was used, because the widely used alternative indicator Cyt с can be reduced with o-diphenol derivatives and their transition metal complexes. It was shown that the results obtained using the two above-mentioned procedures of generating superoxide virtually coincide. To investigate the compounds being discussed, the procedure of generating superoxide with the use of alkaline DMSO was selected, as it is well reproducible, economical and simple to perform [80]; the values of half maximal inhibitory concentration (IC50) obtained using it are given in Table 14. It was confirmed by ESR spectroscopy that alkaline DMSO contains superoxide: the corresponding signal (g║=2.087 and g┴=2.006) is present in ESR spectra at 77 K as described elsewhere [76]. The limited range of the inhibitor concentrations (≤100 µmol·l–1) is due to a low solubility of HLI–HLXV and their metal(II) complexes in water. The derivatives of o-diphenols (HLI–HLX) and m- diphenols (HLXI–HLXV) were found to be substantially different in their inhibiting action (Table 14). As noted above (Section 2), oxidation of the derivatives of o-diphenols includes two successive one-electron stages which cannot be realized with participation of the derivatives of m-diphenols, the reducing ability of the latter is low, and its level is insufficient to neutralize superoxide [62]. Complexation of the compounds HLI–HLXV with Zn(II) ions has generally no noticeable influence on their ability to neutralize superoxide, which suggests the ligand-centered redox interaction of Zn(II) complexes with the latter. SOD-like activity of the Cu(II) complexes is lower than that of the respective ligands (Table 14). Similarly to the case of Cyt c reduction with these compounds a competing reaction may proceed in the case of their redox interaction with superoxide. But the process of NBT reduction being irreversible, catalytic oxidation of formazane formed is unlikely [81]. According to [82], in the presence of redox-active HLI–HLXV ligands Cu(II) ions can be reduced to Cu(I) ones, which in their turn can react with atmospheric oxygen to give excessive superoxide able to decrease SOD-like effect of the compounds. Examination of SOD-like activity of HLX–HLXV (lacking a high reducing ability) and their Cu(II) complexes demonstrated that the activity of Cu(II) complexes is higher than that of the ligands, suggesting the metal-centered redox process. Similarly to the case of the process involving the eucariotic Cu/Zn SOD, superoxide is oxidized to molecular oxygen with the participation of Cu(II) ion which in its turn is reduced to Cu(I) and then, interacting Complimentary Contributor Copy

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∙– + with another superoxide, is transformed into the initial state again: Cu(I) + O2 + 2H  Cu(II) + H2O2 [83]. Note that the bulky substituents in HLVI–HLX sterically hinder interaction of their complexes with superoxide, which may be one of the reasons for the low SOD-like activity of these compounds and their complexes. Since one of the main requirements for synthetic SOD-mimics is their high ability to penetrate cellular walls and membranes, their lipophilicity should be also taken into account when searching for and developing these bioactive compounds. Zn(II) complexes were found to be the most lipophilic among the compounds under study (Table 6). Moreover, in parallel with lipophilicity the SOD-like activity of the complex can be substantially affected by the conformation of its active site, for it is known that the stable square planar structures exhibit no high SOD-like activity [84, 85], while the distortion of geometry of the active site results in enhancement of the latter, providing a higher accessibility of the active site for this substrate [86–88]. Thus the high SOD-like activity of the lipophilic Zn(II) complexes with the compounds HLI–HLV allows one to consider them as the most promising hit-compounds to produce new effective antioxidants – traps for superoxide.

Table 14. SOD-like activity of the compounds HLI–HLXV and their Zn(II) and Cu(II) complexes*

–1 –1 –1 Compound IC50, μmol·l Compound IC50, μmol·l Compound IC50, μmol·l I I I HL 1.1 Zn(L )2 1.4 Cu(L )2 9.1 II II II HL 1.2 Zn(L )2 1.8 Cu(L )2 10.0 III III III HL 2.4 Zn(L )2 2.0 Cu(L )2 3.9 IV IV IV HL 2.2 Zn(L )2 2.9 Cu(L )2 4.5 V V V HL 1.8 Zn(L )2 3.1 Cu(L )2 9.2 VI VI VI HL 14.1 Zn(L )2 18.3 Cu(L )2 116.6 VII VII VII HL 15.6 Zn(L )2 18.8 Cu(L )2 107.6 VIII VIII VIII HL 15.4 Zn(L )2 20.5 Cu(L )2 105.6 IX IX IX HL 17.0 Zn(L )2 18.7 Cu(L )2 109.8 X X X HL 15.5 Zn(L )2 16.1 Cu(L )2 123.6 XI XI XI HL >100 Zn(L )2 >100 Cu(L )2 16.3 XII XII XII HL >100 Zn(L )2 >100 Cu(L )2 26.7 XIII XIII XIII HL >100 Zn(L )2 >100 Cu(L )2 36.2 IVX XIV XIV HL >100 Zn(L )2 >100 Cu(L )2 23.9 VX XV XV HL >100 Zn(L )2 >100 Cu(L )2 36.2 * Ar-saturated acetonitrile solutions of the ligands and their metal(II) complexes under study, 0.5 mmol∙l-1 NBT solution (0.2 mol∙l-1 phosphate buffer pH 8.6) and alkaline DMSO (5 mmol∙l-1 , 1% water) were used. The experiments were performed at 20°C. Alkaline DMSO was added under stirring to the NBT solution containing aliquots of the compounds under study. After keeping for 5 min optical density A was measured (550 nm) of formazane formed from NBT. Similarly prepared mixtures without sodium hydroxide were used as reference solutions. The results were confirmed in three independent experiments. The blank experiment was performed using acetonitrile instead of solutions of the compounds under study [76]. The degree of inhibition (I %) of NBT reduction was calculated by the formula: I %=[(A–Aref)test/(A–Aref)blank]·100 %, A – optical density of solutions of the compounds under study, Aref – optical density of the reference solutions.

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5. ANTIBACTERIAL ACTIVITY OF CYCLOAMINOMETHYL DERIVATIVES OF DIPHENOLS AND THEIR COPPER(II) AND ZINC(II) COMPLEXES

Pharmacological screening of Cu(II) and Zn(II) complexes with the compounds HLI– HLXV was carried out in order to assess their antibacterial activity against test cultures of Gram-negative and Gram-positive bacteria; the data obtained are presented in Tables 15 and 16. The following test microorganisms (collection of Department of Microbiology, Belarusian State University) were used: Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Salmonella typhimurium) and Gram-positive bacteria (Bacillus subtilis, Sarcina lutea, Staphylococcus saprophyticus, Staphylococcus aureus, Mycobacterium smegmatis). The antimicrobial activity was determined as the lowest concentration of a compound which inhibits the visible microbial growth, i.e., the minimum inhibitory concentration (MIC, µmol·ml–1). The activity of the compounds under study was tested in vitro using the method of twofold serial dilutions in a liquid nutrient medium described elsewhere [89, 90]. The bacterial strains were subcultured for testing in Muller- Hinton broth (MHB, Merck). The compositions of these mediums provide favourable conditions for growing test strains, and they don’t contain any substances destroying the phenolic ligands and their metal complexes during testing in the incubation medium, as was shown spectrophotometrically in a preliminary examination. The cells were suspended, according to the McFarland protocol, in saline solution, to produce a suspension of about 104105 CFU ml–1 (colony-forming units per ml). All the compounds tested being insoluble in water, they were dissolved, according to standard recommendations [89], in a small volume of an organic solvent (DMSO or acetonitrile), which is well miscible with aqueous nutrient medium on dilution and doesn’t react with the compounds tested. This solvent was used for making stock solutions (3.2 mg·ml–1) of the test compounds. Stock solutions were further diluted with MHB medium. Serial dilutions of stock solutions to certain concentrations (from 200 to 1.56 g·ml–1) were carried out in test tubes. A specified volume of an inoculum was added to each tube. The organic solvent (DMSO or acetonitrile) at the final concentration (1%) in the nutrient medium did not affect the growth of the test microorganisms. The incubation was carried out at 37°C for 24 h, and optical density (OD 600) was determined for microbial cultures in the presence and absence of the compounds tested. The contents of the test tubes in which the concentration of these compounds was sufficient to suppress the microbial growth remained clear, while their turbidity was evidence for the presence of the microorganisms. The MIC was determined after an incubation period. MIC values are given in mol·ml–1 to reveal a correlation between the antimicrobial activity and reducing ability of the compounds. Tests using DMSO (or acetonitrile) as a negative control were carried out in parallel. Results were always verified in 4–5 separate experiments. The precision of this method is considered to be plus or minus one twofold concentration, in large part because of the practice of manually preparing serial twofold dilutions of the antibiotics [89, 90]. The compounds HLI–HLXV and their Cu(II) and Zn(II) complexes demonstrate a low antibacterial activity (MIC > 0.100 µmol·ml–1) against Gram-negative bacteria Pseudomonas aeruginosa, Serratia marcescens, Salmonella typhimurium, and Escherichia coli (Table 15). But in contrast to HLI–HLXV compounds their metal complexes effectively inhibit the growth of Gram-positive bacteria (Table 16), a high and moderate antibacterial activity being Complimentary Contributor Copy

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IV V VII demonstrated by the following complexes: Cu(L )2, Cu(L )2, and Cu(L )2 (Bacillus IV V VII VIII XIII XV X subtilis); Cu(L )2, Cu(L )2, Cu(L ), Cu(L )2, Cu(L )2, Cu(L )2, and Zn(L )2 (Sarcina V VII VIII I IV lutea); Cu(L )2, Cu(L )2, and Cu(L )2 (Staphylococcus saprophyticus); Cu(L )2, Cu(L )2, V VII VIII XIII I V Cu(L )2, Cu(L )2, Cu(L )2, and Cu(L )2 (Staphylococcus aureus); Cu(L )2–Cu(L )2, VII VIII XIII XV I V X XIII Cu(L )2, Cu(L )2, Cu(L )2, Cu(L )2, Zn(L )2–Zn(L )2, Zn(L )2, Zn(L )2, and XV Zn(L )2 (Mycobacterium smegmatis).

Table 15. MIC (µmol∙ml–1) of HLI–HLXV and their Zn(II) and Cu(II) complexes against Gram-negative bacteria

Pseudomonas Serratia Salmonella Escherichia Compound aeruginosa marcescens typhimurium coli HLI 0.401 0.401 0.401 0.401 I Cu(L )2 0.178 0.178 0.178 0.178 I Zn(L )2 0.178 0.178 0.178 0.178 HLII 0.380 0.380 0.380 0.380 II Cu(L )2 0.021 0.170 0.085 0.170 II Zn(L )2 0.169 0.169 0.169 0.169 HLIII 0.360 0.360 0.360 0.360 III Cu(L )2 0.162 0.162 0.162 0.162 III Zn(L )2 0.162 0.162 0.162 0.162 HLIV 0.754 0.754 0.754 0.754 IV Cu(L )2 0.042 0.169 0.169 0.042 IV Zn(L )2 0.168 0.168 0.168 0.168 HLV 0.359 0.359 0.359 0.359 V Cu(L )2 0.162 0.162 0.162 0.162 V Zn(L )2 0.161 0.161 0.161 0.161 HLVI 0.230 0.230 0.230 0.230 VI Cu(L )2 0.107 0.107 0.107 0.107 VI Zn(L )2 0.107 0.107 0.107 0.107 HLVII 0.222 0.222 0.222 0.222 VII Cu(L )2 0.104 0.104 0.026 0.052 VII Zn(L )2 0.104 0.104 0.052 0.104 HLVIII 0.216 0.216 0.216 0.216 VIII Cu(L )2 0.101 0.101 0.101 0.101 VIII Zn(L )2 0.101 0.101 0.101 0.101 HLIX 0.221 0.221 0.221 0.221 IX Cu(L )2 0.104 0.104 0.104 0.104 IX Zn(L )2 0.103 0.103 0.103 0.103 HLX 0.215 0.215 0.215 0.215 X Cu(L )2 0.101 0.101 0.101 0.101 X Zn(L )2 0.101 0.101 0.101 0.101 HLXI 0.327 0.327 0.327 0.327 XI Cu(L )2 0.074 0.149 0.149 0.074 XI Zn(L )2 0.148 0.148 0.148 0.148

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Pseudomonas Serratia Salmonella Escherichia Compound aeruginosa marcescens typhimurium coli HLXII 0.313 0.313 0.313 0.313 XII Cu(L )2 0.143 0.143 0.071 0.143 XII Zn(L )2 0.142 0.142 0.142 0.142 HLXIII 0.300 0.300 0.300 0.300 XIII Cu(L )2 0.137 0.137 0.137 0.137 XIII Zn(L )2 0.137 0.137 0.137 0.137 HLXIV 0.311 0.311 0.311 0.311 XIV Cu(L )2 0.142 0.142 0.142 0.142 XIV Zn(L )2 0.142 0.142 0.142 0.142 HLXV 0.299 0.299 0.299 0.299 XV Cu(L )2 0.137 0.137 0.137 0.137 XV Zn(L )2 0.137 0.137 0.137 0.137 Streptomycin 0.172 0.011 0.021 0.005 Tetracycline – – 0.014 0.007 Chloramphenicol 0.039 – 0.019 0.019

Table 16. MIC (µmol∙ml–1) of HLI–HLXV and their Zn(II) and Cu(II) complexes against Gram-positive bacteria

Bacillus Sarcina Staphylococcus Staphylococcus Mycobacterium Compound subtilis lutea saprophyticus aureus smegmatis HLI 0.201 0.201 0.201 0.201 0.201 I Cu(L )2 0.045 0.045 0.045 0.022 <0.011 I Zn(L )2 0.044 0.089 0.089 0.089 <0.011 HLII 0.190 0.190 0.190 0.095 0.095 II Cu(L )2 0.042 0.042 0.042 0.042 0.021 II Zn(L )2 0.085 0.085 0.085 0.042 <0.011 HLIII 0.180 0.180 0.180 0.180 0.180 III Cu(L )2 0.081 0.081 0.081 0.081 0.020 III Zn(L )2 0.081 0.081 0.040 0.040 0.010 HLIV 0.377 0.377 0.377 0.377 0.188 IV Cu(L )2 0.021 0.021 0.021 0.021 0.011 IV Zn(L )2 0.084 0.084 0.084 0.084 0.021 HLV 0.359 0.180 0.180 0.180 0.090 V Cu(L )2 0.020 0.020 0.010 0.010 <0.010 V Zn(L )2 0.040 0.040 0.040 0.081 <0.010 HLVI 0.230 0.230 0.115 0.115 0.230 VI Cu(L )2 0.054 0.054 0.054 0.054 0.054 VI Zn(L )2 0.054 0.027 0.054 0.054 0.027 HLVII 0.222 0.222 0.222 0.222 0.222 VII Cu(L )2 0.013 0.007 0.003 0.007 0.003 VII Zn(L )2 0.026 0.026 0.052 0.052 0.026 HLVIII 0.216 0.216 0.216 0.216 0.216

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Table 16. (Continued)

Bacillus Sarcina Staphylococcus Staphylococcus Mycobacterium Compound subtilis lutea saprophyticus aureus smegmatis VIII Cu(L )2 0.025 0.013 0.013 0.013 0.013 VIII Zn(L )2 0.025 0.025 0.025 0.025 0.025 HLIX 0.221 0.221 0.221 0.221 0.221 IX Cu(L )2 0.052 0.052 0.052 0.052 0.052 IX Zn(L )2 0.052 0.052 0.052 0.052 0.052 HLX 0.215 0.215 0.215 0.215 0.215 X Cu(L )2 0.050 0.025 0.025 0.025 0.025 X Zn(L )2 0.025 0.013 0.025 0.025 0.013 HLXI 0.327 0.164 0.164 0.164 0.164 XI Cu(L )2 0.037 0.037 0.037 0.037 0.037 XI Zn(L )2 0.074 0.074 0.074 0.037 0.074 HLXII 0.313 0.313 0.313 0.157 0.313 XII Cu(L )2 0.036 0.036 0.036 0.036 0.036 XII Zn(L )2 0.071 0.071 0.071 0.071 0.036 HLXIII 0.300 0.300 0.300 0.300 0.300 XIII Cu(L )2 0.034 0.017 0.034 0.017 0.017 XIII Zn(L )2 0.068 0.068 0.068 0.068 <0.009 HLXIV 0.622 0.622 0.622 0.622 0.622 XIV Cu(L )2 0.071 0.071 0.071 0.071 0.071 XIV Zn(L )2 0.071 0.071 0.071 0.071 0.071 HLXV 0.299 0.299 0.299 0.299 0.299 XV Cu(L )2 0.068 0.017 0.034 0.068 0.017 XV Zn(L )2 0.068 0.034 0.068 0.034 0.017 Streptomycin 0.011 0.021 0.011 0.011 0.005 Tetracycline 0.014 0.014 0.014 0.007 – Chloramphenicol 0.010 – 0.019 0.019 0.039

IV V VII Based on the results of the screening, the complexes Cu(L )2, Cu(L )2, Cu(L )2, and VIII Cu(L )2 are considered as hit-compounds, their activity against the test cultures of Gram- positive bacteria being comparable to or higher than that of the standard antibiotics. Besides, IV VII Cu(L )2 and Cu(L )2 complexes are moderately active against some Gram-negative bacteria (Table 15). It was found that the level of activity of the compounds under study against Gram-positive bacteria depended on their lipophilicity: the compounds HLI–HLV and their metal complexes with low lipophilicity were also characterized by a lower level of antibacterial activity against Staphylococcus saprophiticus, Staphylococcus aureus, Bacillus subtilis, Sarcina lutea and Mycobacterium smegmatis as compared to more lipophilic compounds HLVI–HLXV and their complexes (Tables 6, 16). It should be noted that the degree VII VIII of lipophilicity of the hit-compounds Cu(L )2 and Cu(L )2 (logPow=2.9–3.1) corresponds to the optimal range of drug-like properties (logPow=1–3) [41]. For Cu(II) and Zn(II) complexes a correlation was noted between the reducing ability and antibacterial activity. It was shown that o-diphenol derivatives HLI–HLX and their complexes manifesting reducing properties were generally more active against Gram-positive bacteria than m-diphenol derivatives HLXI–HLXV and their complexes (Tables 7–9, 15, 16). Complimentary Contributor Copy

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Among the compounds investigated, those which are characterized by a high or moderate SOD-like activity are also effective antibacterial agents with a broad spectrum of action, IV V IV specifically Cu(L )2 and Cu(L )2 (Table 14). It should be emphasized that Cu(L )2 complex, which interacts actively with superoxide, displays also a moderate inhibiting effect on Escherichia coli (Table 15) capable of producing superoxide under certain conditions [22].

CONCLUSION

The results of investigations presented in this chapter show that Cu(II) and Zn(II) chelation plays an important role in the antibacterial and SOD-like activity of the novel redox-active cycloaminomethyl derivatives of ortho- and meta-diphenols as well as in Cyt c reduction with these substances. Their Cu(II) and Zn(II) complexes were found to have a low inhibition activity against Gram-negative bacteria, while Gram-positive ones in vitro are more sensitive to these compounds. The lowest MIC value (0.003–0.012 µmol∙ml–1) comparable to those of commonly used antibiotics (tetracycline, streptomycin, chloramphenicol) was achieved by structural modification of the ligands and complexation with metal ions which purposefully change the hydrophilic-lipophilic balance, acid-base and redox properties of these substances. All the metal complexes are more lipophilic and more active against the test microorganisms than the diphenols forming them. Both the cycloaminomethyl derivatives of ortho-diphenols and their Cu(II) and Zn(II) complexes exhibited the capability for the Cyt c reduction and displayed also SOD-like activity. The correlation between the antimicrobial activity of these compounds and their reducing ability deserves particular attention since they possess both antioxidant and antimicrobial activity.

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Chapter 7

A 21ST CENTURY CONTRIBUTION TO THE ANTIBACTERIAL ARMAMENTARIUM: A MEDICINAL CHEMIST’S PERSPECTIVE

1, ,‡ 2, Stefano Biondi * and Mauro Panunzio †‡ 1Allecra Therapeutics SAS, 13, St-Louis, France 2Dipartimento di Chimica “G. Ciamician,” ISOF-CNR, Bologna, Italy

ABSTRACT

Bacterial infections are a serious and growing problem. Some antibacterial agents (AAs), successfully used for decades, are no longer effective due to the emergence and spread of resistance. Multidrug-resistant strains (MDR), and extremely drug-resistant strains (XDR) are non-susceptible to all classes of antimicrobials, have compromised the clinical utility of currently available antibiotics and underscore the need for new compounds. To ensure that the supply of new antibiotics keeps pace with evolving pathogens, it is necessary to build a robust, sustainable pipeline of new drugs and innovative therapeutic approaches. Since 2000, 27 new antibiotics, i.e., less than 2 per year have been launched, covering five new drug classes for combating bacterial diseases. In this review we analyze recent progress in the discovery and development of novel antibiotics during the first 15 years of the 21st century.

Keywords: antibacterial agents, oxazolidinones, ketolides, carbapenems, quinolones, lipopeptides, tetracyclines, cephalosporins, lipoglycopeptides, polyene macrocycle, diarylquinoline

* Correspondence should be addressed: MP: Tel.: +39 3204313396; Fax: +39-0512099508; SB: Tel.: +33 389 689876; [email protected]. † Correspondence should be addressed: MP: Tel.: +39 3204313396; Fax: +39-0512099508; SB: Tel.: +33 389 689876; [email protected]. ‡ Author Contributions: The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Complimentary Contributor Copy 182 Stefano Biondi and Mauro Panunzio

ABBREVIATIONS

AA Antbacterial Agents AECB Acute Exacerbations Chronic Bronchitis AMS Acute Maxillary Sinusitis AUC Area under Concentration-time Curve Bid twice a day CAP Community-Acquired Pneumonia CDC Centers Disease Control Prevention Cl Clearance Cmax maximal plasma Concentration CONS Coagulase Negative Staphylococci CRE Carbapenem Resistant Enterobacteriaceae EMA European Medicines Agency ESKAPE Enterococcus faecium, S taphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter spp. FDA Food and Drug Administration ICU Intensive Care Unit MDR MultiDrug-Resistant; MDR-TB Multi Drug Resistant Tubercolosis MIC Minimal Inhibitory Concentration MLSB Macrolide Lincosamide Streptogramin mRNA messenger RiboNucleic Acid MRSA Methicillin Resistant Staphylococcus Aureus MSSA Methicillin Susceptible Staphylococcus Aureus MRSE Methicillin Resistant Staphylococcus Epidermidis MSSE Methicillin Susceptible Staphylococcus Epidermidis OD One a Day PO Per Os PRSP Penicillin Resistant Streptococcus pneumoniae PSSP Penicillin Susceptible Streptococcus pneumoniae t1/2 half-life tmax time at which Cmax occurs Vd distribution volume VR Vancomycin Resistant VRE Vancomycin Resistant Enterococci VS Vancomycin Susceptible tRNA transfer RiboNucleic Acid XDR Extremely Drug-Resistant

1. INTRODUCTION

Bacterial infections are a serious and growing problem that has attracted the attention of health authorities and prompted the FDA and EMA to take initiatives aimed at supporting Complimentary Contributor Copy

A 21st Century Contribution to the Antibacterial Armamentarium 183 research activities directed towards the identification and development of novel antibiotics [1]. Some antibacterial agents (AAs), successfully used for decades, no longer are effective due to emergence and spread of resistance. During the past 20 years there has been a significant increase in morbidity and mortality due to bacterial infections in both community and hospital settings that experienced clinical failure by currently available antibiotics, underscoring the need for new compounds:

 multidrug-resistant strains (MDR), which are non-susceptible to one or more drugs belonging to ≥3 antimicrobial classes;  extremely drug-resistant strains (XDR), which are non-susceptible to nearly all classes of antimicrobials [2].

The bacteria that constitute the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter spp.) are responsible for about 30-35% of nosocomial infections, including the vast majority of MDR and XDR strains, leaving physicians with limited therapeutic options [3]. According to a report by the Centers for Disease Control and Prevention (CDC-USA) [4], 2 million Americans acquire serious infections caused by antibiotic-resistant bacteria each year, and 23,000 of them die as a result. To ensure that the supply of new antibiotics keeps pace with these evolving pathogens, it is necessary to build a robust, sustainable pipeline of new drugs and innovative therapeutic approaches. Despite the alarm warning launched in the last few years by many Agencies and single researchers on the real pandemic risk, the “Big Pharma” are far away from a real confront with the problem of “Super Bugs.” Financial return is basis of this decision, mainly because there is poor return on investment for the antibacterial drugs compared to drugs for other indications such as cancer, diabetes and cardiovascular diseases. This review aims to analyze and describe recent progress in the discovery and development of novel antibiotics.

2. ANTIBACTERIAL AGENTS ENTERED IN THE CLINICAL PRACTICE IN THE 21ST CENTURY

Since 2000, 27 new antibiotics, i.e., less than 2 per year, have been launched, covering five new drug- classes for fighting bacterial diseases. These five new classes include: oxazolidinones, linezolid, being the most important representative active against Gram- positive cocci, approved in 2000; lipopeptides, exemplified by daptomycin, active against Gram-positive cocci, approved in 2003; pleuromutilins, retapamulin, a topical antibacterial agent, active against Gram-positive cocci, approved in 2007; fidaxomicin, an oral macrocycle active against Clostridium difﬁcile, approved in 2010, and bedaquiline, a diarylquinoline, active against Mycobacterium tuberculosis, approved in 2012. Table 1 lists new antibiotics approved since the start of the second millennium, and Chart 1summarizes the number of antibacterial approvals per year.

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Table 1. Antibiotics approved since 2000

Year Drug name Chemical structure Class Bacterial Special features approved profile 2000 Linezolid Oxazolidinone G+ MRSA

2001 Telithromycin macrolide (ketolide) G+/some G- Safety concerns

2002 Biapenem Carbapenem G+, G- Broad spectrum, including many - lactamase producers

2002 Ertapenem Carbapenem G+, G- Broad spectrum, including many - lactamase producers

2002 Prulifloxacin fluoroquinolone G+, G- Broad spectrum

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Year Drug name Chemical structure Class Bacterial Special features approved profile 2002 Pazufloxacin fluoroquinolone G+, G- Broad spectrum

2002 Balofloxacin fluoroquinolone G+, G- Broad spectrum

2003 Daptomycin Lipopeptide G+ MRSA, VRE

2004 Gemifloxacin fluoroquinolone G+, G- Broad spectrum

2005 Doripenem Carbapenem G+, G- Broad spectrum, including many - lactamase producers

2005 Tigecycline tetracycline G+, G- Broad spectrum (glycylglycine)

2007 Retapamulin Pleuromutilin G+ MRSA

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Table 1. (Continued)

Year Drug name Chemical structure Class Bacterial Special features approved profile 2007 Garenoxacin Quinolone G+, G- Broad spectrum,

2008 Ceftobiprole Cephalosporin G+, G- Broad spectrum, medocaril MRSA

2008 Sitafloxacin fluoroquinolone G+, G- Broad spectrum

2009 Tebipenem Carbapenem G+, G- Broad spectrum, pivoxil including many - lactamase producers

2009 Telavancin lipoglycopeptide G+ MRSA

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Year Drug name Chemical structure Class Bacterial Special features approved profile 2009 Antofloxacin fluoroquinolone G+, G- Broad spectrum

2009 Besifloxacin fluoroquinolone G+, G- Broad spectrum

2010 Ceftaroline Cephalosporin G+, G- Broad spectrum fosaminyl

2011 Fidaxomicin Macrocycle G+ C.difficile

2012 Bedaquiline Diarylquinoline acid-fast Mycobacterium bacteria tuberculosis

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Table 1. (Continued)

Year Drug name Chemical structure Class Bacterial Special features approved profile 2014 Dalbavancin lipoglycopeptide G+ MRSA, VRE

2014 Tedizolid Oxazolidinone G+ MRSA

2014 Ceftolozane + cephalosporin + G+, G- Broad spectrum Tazobactam lactamase inhibitor

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Year Drug name Chemical structure Class Bacterial Special features approved profile 2014 Oritavancin Glycopeptide G+ MRSA

2015 Ceftazidime + Cephalosporin + G+, G- Broad spectrum avibactam lactamase inhibitor

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Chart 1. Novel antibacterial agents approved in the last 15 years.

2.1. Oxazolidinones

Oxazolidinones have a unique mechanism of action which, involving inhibition of the formation of N-formylmethionyl-tRNA-mRNA 70S ribosomal ternary complex, precludes protein synthesis. Oxazolidinones bind to the 23S peptidyltransferase center of the 50S ribosomal subunit, preventing binding of tRNA and mRNA to the 50S and 30S subunits that would lead to the formation of the initiation complex. The key elements in chemical structure of oxazolidinones, described in Figure 1, are an aromatic or heteroaromatic group attached to the nitrogen of the oxazolidinone ring that affects the antibacterial activity, and a hydrophilic moiety (morpholine in linezolid) that affects pharmacokinetic and solubility. Though the (5S)- acetamidomethyl substitutent was believed essential for antibacterial activity, it was replaced by a hydroxyl group in tedizolid. Oxazolidinones are used mainly for treatment of bacterial infection were methicillin resistant Staphylococcus aureus (MRSA) is suspected, and offer the advantage of being both injectable and oral.

2.1.1. Linezolid Linezolid, the most representative oxazolidinone, shows an excellent in vitro activity against Gram-positive bacteria including methicillin- and vancomycin-resistant strains (Table 2) [5].

Table 2. Linezolid MIC50/MIC90 in g/ml

MSSA MRSA S. epidermidis E. faecalis E. faecium S. pneumoniae Goup A Group B streptococci streptococci 1/2 1/1 0.5/1 1/2 ½ 1/1 1/1 1/1 MSSA = methicillin susceptible Staphylococcus aureus; MRSA = methicillin resistant Staphylococcus aureus.

Linezolid is available as injectable and oral drug with a bioavailability of 95-100% and has a half-life ranging from 4.8 to 5.4 hours depending on the dosage form (Table 4). The pharmacokinetic/pharmacodynamics parameters, that correlate with the clinical outcome, are AUC/MIC and T>MIC [6]. It exhibits good penetration in tissues, including lung, skin and skin structures. The daily dosage varies from 400 to 600 mg bid, and does not require Complimentary Contributor Copy

A 21st Century Contribution to the Antibacterial Armamentarium 191 adjustment in renal or hepatic dysfunction. Linezolid manifests duration-dependent thrombocytopenia and its use has been associated with cases of myelosuppression, anemia, leukopenia, pancytopenia, and thrombocytopenia [7]. Other potential side effects of prolonged linezolid use include lactic acidosis, optic and peripheral neuropathies and inhibition of monoamine oxidase [8]. The (S)-configuration at C-5 has been correlated with inhibition of mitochondrial protein synthesis and monoamine oxidase. Figure 1 shows a representation of oxazolidinone’s structure properties relationship.

Figure 1. Schematic representation of structure properties relationship for oxazolidinones.

Resistance has been documented in surveillance studies like Zyvox® Annual Appraisal of Potency and Spectrum (ZAAPS) [7, 9] and Linezolid Experience and Accurate Determination of Resistance (LEADER) Program [10]. The incidence of non susceptibility is rather low, between 0.03 and 1.83% and is mainly associated with alterations in the 23S target [11]. Additional resistance mechanisms include alterations of L3 and L4 proteins and acquisition of the cfr gene, which is plasmid-encoded and catalyzes the post-transcriptional methylation of the C8 atom of A2503 in the 23S subunit [12]. This modification confers a MDR phenotype, affecting susceptibility to phenicols, licosamides, oxazolidinones, pleuromutilins, and streptogramin A [13]. After the first outbreak of cfr-carrying S.aureus in 2008 in an ICU of Hospital Clínico San Carlos [14], the incidence of linezolid-resistant S. epidermidis has reached 20% or even more and has remained steady despite implementation of infection control interventions. Fortunately, overall resistance to linezolid remains low, but an increase due to the mobility of the genetic element is reasonably expected.

2.1.2. Tedizolid Fourteen years after linezolid, tedizolid phosphate (Sivextro®) reached the market. Tedizolid phosphate (TR-701) is a prodrug that is transformed in the serum into the active drug tedizolid (TR-700). It has very good oral bioavailability (91%) and is used once daily at lower recommended doses (200 mg/day vs 400 or 600mg bid) and for a shorter period (6 vs. 10 to 14 days) than linezolid. Tedizolid has broad activity against Gram-positive pathogens, including strains that are resistant to linezolid (Table 3) [15]. The pharmacodynamic index for tedizolid most closely linked with efficacy in animals, is AUC/MIC [16]. The major improvements achieved by tedizolid relate mainly to its pharmacokinetic profile and improved tissue penetration. From a medicinal chemistry perspective, tedizolid phosphate presents some important structural features. The morpholine ring of linezolid is replaced by a 2-tetrazolyl, 5-pyridyl group, but most importantly the acetamido moiety Complimentary Contributor Copy

192 Stefano Biondi and Mauro Panunzio attached at C5 on the oxazolidinone ring is replaced by a hydroxyl group. This structural change was believed detrimental for the antibacterial activity of oxazolidinones but clearly this is not the case. Table 4 compares the pharmacokinetic profile of the two drugs following oral administration. Clinical studies described higher concentrations in the epithelial lining fluid and alveolar macrophages compared to free plasma concentrations, indicating extensive penetration into both extracellular and intracellular pulmonary compartments [17].

Table 3. Susceptibilities of Gram-positive organisms to tedizolid and linezolid

Linezolid Tedizolid Strain MIC range (g/ml) MSSA 1-4 0.25-1 MRSA 1-4 0.25-1 CA-MRSA 1-4 0.25-1 MSSE 0.5-4 0.12-1 MRSE 0.5-4 0.12-1 VS Enterococcus faecalis 1-4 0.25-1 VR E. faecalis 1-4 0.25-1 VS Enterococcus faecium 1-4 0.25-2 VR E. faecium 1-4 0.12-1 PSSP 0.25-1 0.06-0.5 PRSP 0.25-2 0.06-0.5 Streptococcus pyogenes 0.06-2 0.06-0.5 Streptococcus agalactiae 1-2 0.06-1 CONS* 8->128 0.06-16 S.aureus* 8-16 0.5 MSSA = methicillin susceptible Staphylococcus aureus; MRSA = methicillin resistant Staphylococcus aureus; CA-MRSA = Community aquired methicillin resistant Staphylococcus aureus; MSSE = methicillin susceptible Staphylococcus epidermidis; MRSE = methicillin resistant Staphylococcus epidermidis; VS = vancomycin susceptible; VR = vancomycin resistant; PSSP = Penicillin-susceptible Streptococcus pneumoniae; PRSP = Penicillin-resistant Streptococcus pneumoniae;CONS = Coagulase negative staphylococci. * Linezolid resistant isolates.

Table 4. Pharmacokinetic profile of currently marketed oxazolidinones

Parameter Linezolid Tedizolid phosphate (600mg PO bid) (200mg PO OD) Cmax (g/ml) 21.2±5.78 2.2±0.6 Tmax (hrs) 1.03±0.62 3.5±2.5 t1/2 (hrs) 5.4±2.06 12 Cl (l/hr) 4.8±1.74 8.4±2.1 Vd (L) 40-50 67-80 AUC (g*hr/ml) 138±42.1 25.6±8.4 Oral bioavailability (%) 100 91 Plasma protein binding (%) 31 70-90

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A 21st Century Contribution to the Antibacterial Armamentarium 193

3. KETOLIDES

Ketolides (Figure 2) are semisynthetic derivatives of 14 membered macrolide erythomycin, that have increased potency and are active against many macrolide resistant strains. They have a keto group at position 3 instead of the cladinose sugar moiety, a chemical modification that is believed critical for activity against inducible resistant strains (MLSB) [18]. They are characterized by the presence of a cyclic carbamate at position C11-C12, that reduces the flexibility of the molecules and increase antibacterial potency. The C11C12 carbamate also appears to confer the ability to overcome resistance caused by efflux encoded by mef genes. There is also a heteroaromatic group tethered to the ketolide structure, either attached to the carbamate (telithromycin, solithromycin) or at the oxygen at position 6 (cethromycin) that is believed to interact with ribosomal RNA ina manner mimicking the interactions between nucleotides.

3.1. Telithromycin

Telithromycin (HMR3647, ketek®) is the first-in-class ketolide, approved in 2001 for treatment of adults with community-acquired pneumonia (CAP), acute exacerbations of chronic bronchitis (AECB), and acute maxillary sinusitis (AMS) in adults. Telithromycin retains good activity against streptococci harboring mef genes and it is a poor substrate for the efﬂux pumps that are effective against erythromycin and other macrolides [19].

Figure 2. Chemical structure of representative ketolides.

The mechanism of antimicrobial activity of ketolides and macrolides tends to be bacteriostatic and acts through inhibition of protein synthesis. Ketolides interact with the 50S ribosomal subunit near the peptidyl-transferase site to inhibit translation and prevent elongation during protein synthesis [20]. They also interfere with protein synthesis earlier in the process by interacting with partially assembled 50S subunit precursors to block formation of a functional 50S subunit. Telithromycin, like other ketolides, binds to domains II and V of the 50S subunit on the 23S rRNA [21]. Binding to domain II, due to the cyclic carbamate moiety, increases the affinity of telithromycin for the ribosomal subunit of about 10 fold, compared to erythromycin [22]. The pharmacokinetic parameters of telithromycin suggest that it can be administered once-daily (Table 5). The recommended dose is 800 mg (2 tablets Complimentary Contributor Copy

194 Stefano Biondi and Mauro Panunzio of 400 mg) taken orally once a day, for 7–10 days. Ketek® (telithromycin) tablets can be administered with or without food and does not require dose adjustment for hepatic impairment. In the presence of severe renal impairment (CLCR <30 ml/min), including patients who need dialysis, the dose should be reduced to 600 mg once daily. Whereas for severe renal impairment (CLCR <30 ml/min), with coexisting hepatic impairment, the dose should be reduced to Ketek®400 mg once daily. The oral bioavailability of telithromycin is 57% in both young and elderly subjects [23] and is not affected by food. Maximum serum concentrations are reached 1 hr after a single oral dose of telithromycin, and the elimination half-life (t½) ranges from 7.16 to 13 hrs [24]. Telithromycin achieves concentrations in lower respiratory tract infection sites that are higher than the MICs of common respiratory pathogens. Table 6 shows the concentration of clarithromycin, azithromycin and telithromycin in lower respiratory tract infection sites. In particular, the concentration of telithromycin in alveolar macrophages is significantly higher than the concentration observed for macrolides and seems to accumulate over time.

Table 5. Comparative pharmacokinetics of the ketolide telithromycin and macrolide antibiotics [22]

Erythromycin Azithromycin Clarithromycin Telithromycin Bioavailability (%) 25 37 55 57 Cmax(g/ml) 0.3-0.9 0.4 2.1-2.4 1.9-2.0 tmax (hrs) 3-4 2 2 1 t1/2 (hrs) 2-3 40-68 3-5 7.16-13 AUC (mg*hr/L) 8 3.4 19 7.9-8.25 Protein binding (%) 75-95 7-51 70 60-70 *Mean values after a single 500-mg oral dose (azithromycin or clarithromycin) or 800-mg dose (telithromycin).

Table 6. Drug concentration in lower respiratory tract infection sites of macrolides and telithromycin after multiple oral doses

Clarithromycin Azithromycin Telithromycin 4hrs 24hrs 4hrs 24hrs 2hrs 24hrs bronchial mucosa (mg/kg) 3.88 0.78 epithelial lining ﬂuid (mg/L) 34.5 4.6 1.01 1.22 14.89 0.97 alveolar macrophages (mg/L) 480 99.4 42.7 41.7 69.32 161.57

At 2 hours after multiple oral doses, the concentration of telithromycin was 3.88 mg/kg in bronchial mucosa, 14.89 g/ml in epithelial lining ﬂuid (ELF), and 69.32 g/ml in alveolar macrophages (AMs). Corresponding concentrations at 24 hours were 0.78 mg/kg, 0.97 g/ml, and 161.57 g/ml, respectively [25]. Telithromycin is active against common Gram-positive and some Gram-negative organisms, as well as atypical and intracellular respiratory pathogens, including MLSB-resistant strains (Table 7) [26]. It is active against S. pneumoniae isolates expressing erm or mef genes [27], and it is more active than clarithromycin against erythromycin-susceptible Gram-positive cocci. Telithromycin essentially is inactive against Enterobacteriaceae species; nonfermentative, Gram-negative bacilli; and constitutively, MLSB-resistant S aureus [27]. Complimentary Contributor Copy

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Table 7. MIC50/90 of the ketolide telithromycin and macrolide antibiotics [28]

Organism (nr. of strains) MIC50/90 (g/ml) Erythromycin Azithromycin Clarithromycin Telithromycin S.pneumoniae PenS (100) 0.015/0.03 0.06/0.12 0.015/0.03 0.008/0.008 S.pneumoniae EryR, ClinR, ermB >128/>128 >128/>128 >128/>128 0.06/0.5 (150) S.pneumoniae EryR, ClinS, mef(E) 4/8 8/8 4/8 0.12/0.25 (50) S.pyogenes PenS, EryS, ClinS, 0.06/0.06 0.12/0.12 0.06/0.06 0.03/0.03 levoS (60) S.pyogenes EryR, ClinS, mef(A) 8/8 8/8 16/16 0.06/0.25 (10) S.pyogenes EryR, ClinS, erm(A) 16/32 8/16 16/16 0.12/0.5 inducible (10) Moraxella catarrhalis -lactamase 0.12/0.25 0.06/0.06 0.12/0.25 0.06/0.12 positive (428) Moraxella catarrhalis -lactamase 0.12/0.25 0.06/0.06 0.12/0.5 0.06/0.12 negative (50) Haemophilus influenzae - 4/8 1/2 4/16 2/4 lactamase positive (370) Haemophilus influenzae - 4/8 1/2 4/16 2/4 lactamase negative (213)

The safety profile of telithromycin is similar to that of other antibacterial agents used in community-aquired respiratory tract infections (CA-RTI), but it is not recommended for patients with myastenia gravis as it may worsen the conditions [28]. Telithromycin has the potential to prolong the QTc interval in some subjects, a feature that is also observed in macrolides. Telithromycin is a competitive inhibitor of the CYP3A4 pathway and, to a lesser extent, the CYP2D6 pathway and thus has the potential for drug-drug interactions with other inhibitors and substrates [29]. Overall, telithromycin represent a major improvement with respect to macrolides, particularly because of its ability to overcome some resistance mechanisms, although it lacks efficacy against constitutive ermB S.aureus. An important feature of macrolides and ketolides is their activity against atypical pathogens (Chlamydia, Legionella, Mycoplasma).

4. CARBAPENEMS

There are four carbapenems that have been approved for clinical use in the US (imipenem, meropenem, ertapenem, doripenem), and three others (biapenem, panipenem, tebipenem), approved for use in Japan. Carbapenems are characterized by a 1- azabicyclo[3.2.0]hept-2-ene ring system and differ from penicillin ring system (4-thia-1- azabicyclo[3.2.0]heptane) in two main aspects: the sulfur atom has been replaced by a carbon atom, and there is an unsaturated double bond. These structural changes increase the strain in the molecule, making it more reactive and also modifying the relative position of the carboxy group and the carbonyl groups. Other important features of carbapenems are the presence of a hydroxyethyl side chain, which most likely is responsible for resistance to most class A, C Complimentary Contributor Copy

196 Stefano Biondi and Mauro Panunzio and D -lactamases, and a trans configuration on the -lactam ring (Figure 3). The trans configuration is unique to carbapenems and provides protection from nucleophilic attack by serine -lactamases by hindering the lower face of the -lactam ring through the formation of an intramolecular hydrogen bond. Synthetic or natural-occurring carbapenems lacking the trans configuration are susceptible to hydrolysis by -lactamases [30]. The comparative antibacterial activities of imipenem, meropenem, doripenem, and ertapenem are shown in Table 8.

Table 8. Comparative activity of imipenem, meropenem, doripenem and ertapenem against selected aerobic and anaerobic pathogens

Organism MIC90 (g/ml) Imipenem Meropenem Doripenem Ertapenem Gram-positive organisms MSSA 0.12 0.12 0.06 0.25 MRSA >16 >16 >16 >16 Coagulase negative staphylococci MSS 0.12 0.12 0.03 0.25 Coagulase negative staphylococci MRS >16 >16 >16 >16 Streptococcus pneumoniae 0.06-0.25 0.06-1 0.06-0.5 0.06-0.5 -haemolytic streptococci 0.06 0.06 0.06 0.06 viridans group streptococci 0.03 0.03 0.06 0.12 Enterococcus faecalis 1 8 4 8 Enterococcus faecium >8 >8 >8 >8 Bacillus anthracis 0.12 0.05 - - Gram-negative organisms Listeria monocytogenes 0.06 0.12 0.12 0.25 Haemophilus influenzae 0.25 0.06 0.12 0.03 Moraxella catarrhalis 0.25 0.03 0.03 0.03 Neisseria gonorrhoeae 0.25 0.03 0.05 0.06 Neisseria meningitidis 0.03 0.03 0.06 0.03 Escherichia coli 0.5 0.03 0.03 0.06 Escherichia coli ESBL-producing 0.5 0.06 0.06 0.06 Salmonella spp. ≤0.5 0.03 0.06 0.06 Shigella spp. ≤0.5 0.03 0.06 0.06 Klebsiella pneumoniae 0.5 0.12 0.12 0.12 Enterobacter cloacae 0.5 0.12 0.12 0.06 Enterobacter aerogenes 0.5 0.12 0.12 0.06 Morganella morganii 8 0.12 0.5 0.06 Citrobacter spp. 0.5 0.12 0.12 0.06 Serratia marcescens 0.5 0.12 0.12 0.06 Proteus mirabilis 1 0.12 0.12 0.06 Aeromonas spp. 0.5 0.12 0.5 0.25 Pseudomonas aeruginosa 1->8 0.5->8 0.25->8 >8 Acinetobacter baumanii 0.5->8 0.5->8 0.25->8 4->8 Stenotrophomonas maltophilia >8 >8 >8 >8 Burkolderia cepacia >8 4 8 >8 Anaerobes Peptostreptococcus spp. 0.25 0.125 0.125 0.125 Fusobacterium spp. 0.12 0.03 0.03 0.03 Bacteroides fragilis 0.5 0.25 0.5 0.5 Clostridium perfringens 0.5 0.06 0.06 0.06 Clostridium difficile 2 2 2 4

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Figure 3. Key structural differences between carbapenems and penicillins.

4.1. Biapenem

Biapenem is a parenteral carbapenem antibiotic possessing antibacterial activities against a wide range of Gram-positive and Gram-negative bacteria [31] and anaerobes. It is more stable than imipenem, meropenem or panipenem to hydrolysis by human renal dihydropeptidase-I (DHP-I) [32]. The in vitro antibacterial activity of biapenem has been evaluated in several studies and is comparable to that of meropenem (see Table 9). Like other carbapenems it is inactivated by KPC and metallo -lactamases. Biapenem exhibited a marked bactericidal effect against clinically relevant bacteria (e.g., P. aeruginosa, S. aureus, E. coli and K. pneumoniae) [33], as well as a significant post- antibiotic effect against Gram-negative and Gram-positive bacteria [34]. The recommended dose of biapenem for adults has not been totally determinated but depends on the clinical condition. In studies conducted in Japan biapenem, 300 mg twice daily given for chronic respiratory infection, bacterial pneumonia and complicated urinary tract infections, was effective in eradicating bacteria and showed clinical efﬁcacy [35]. In a Swedish study, biapenem, 500 mg, was administered every 8 hours to patients with complicated intra- abdominal infection [36]. Dosage adjustment is not necessary for patients with renal impairment. The pharmacokinetic properties of biapenem, administered as single or multiple doses, have been investigated in healthy adult volunteers [37], individuals with renal impairment [38], and elderly volunteers [39]. Biapenem was administered as an intravenous infusion over 30 or 60 minutes in all of the investigations. Table 10 summarizes the pharmacokinetic properties of biapenem in comparison with meropenem. Although there are slight differences in the activity profile between meropenem and biapenem, it is difficult to discern a real clinical advantage. Moreover, the growth of resistance, in particular KPC and metallo -lactamases, are undermining the efficacy of all marketed carbapenems.

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198 Stefano Biondi and Mauro Panunzio

Table 9. Minimum inhibitory concentration (MIC, g/ml) of biapenem in comparison to meropenem

Organism Biapenem Meropenem MIC50 MIC90 n. isolates MIC50 MIC90 n. isolates S. aureus MSSA ≤0.063 0.125 85 0.06 0.06 20 S. aureus MRSA 32 64 25 8 >8 10 S. epidermidis MSSE ≤0.063 ≤0.063 17 S. epidermidis MRSE 4 >32 93 S. haemolyticus 0.25 >8 10 0.5 >8 10 Coagulase-negative ≤0.063 ≤0.063 119 staphylococci CONS Streptococcus pyogenes ≤0.063 ≤0.063 63 ≤0.015 ≤0.015 20 Streptococcus agalactiae 0.25 0.25 56 0.03 0.06 20 S. pneumoniae PenS ≤0.06 ≤0.06 36 S. pneumoniae PenI 0.25 0.25 41 S. pneumoniae PenR 0.25 0.5 39 Other streptococci ≤0.063 ≤0.125 51 ≤0.015 0.06 10 Enterococcus faecalis 4 16 114 4 4 20 Enterococcus faecium >64 >64 69 >8 >8 10 Escherichia coli ≤0.063 ≤0.063 148 ≤0.016 0.03 491 Klebsiella pneumoniae 0.25 0.5 75 0.06 0.06 20 Klebsiella pneumoniae ESBL 0.5 0.5 12 Klebsiella oxytoca 0.25 0.5 60 0.06 0.06 10 Klebsiella spp. ESBL 0.25 1 220 Klebsiella spp. 0.03 0.03 450 Proteus mirabilis 2 4 53 0.06 0.06 147 Proteus vulgaris 2 4 46 0.06 0.12 10 Yersinia enterocolitica 0.12 0.12 21 0.03 0.06 10 Salmonella enteritidis 0.06 0.12 10 0.03 0.03 10 Salmonella spp. 0.03 0.06 15 Shigella spp. 0.25 0.25 15 0.03 0.03 10 Providencia rettgeri 0.5 1 10 0.06 0.12 10 Providencia stuartii 0.5 1 10 0.12 0.25 10 Providencia spp. 1 2 35 Morganella morganii 2 2 55 0.12 0.12 10 Citrobacter freundii 0.125 0.25 106 0.03 0.06 20 Citrobacter diversus 0.03 0.06 10 0.03 0.03 10 Citrobacter spp. 0.03 0.06 146 Enterobacter cloacae 0.125 0.5 80 0.06 0.06 20 Enterobacter aerogenes 0.06 0.25 21 0.06 0.06 21 Enterobacter agglomerans 0.12 0.25 11 0.06 0.06 11 Enterobacter spp. 0.03 0.06 160 Serratia marcescens 1 2 87 0.06 0.12 20 Serratia spp. 0.03 0.06 134 Haemophilus influenzae 1 8 200 Haemophilus influenzae 0.5 1 20 0.06 0.12 20 AmpS Haemophilus influenzae 1 2 25 0.06 0.25 25 AmpR Haemophilus parainfluenzae 1 2 28 Complimentary Contributor Copy

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Organism Biapenem Meropenem MIC50 MIC90 n. isolates MIC50 MIC90 n. isolates Campylobacter jejuni 0.015 0.03 22 Neisseria meningitidis 0.06 0.06 49 Neisseria gonorrhoeae 0.06 0.12 65 Pseudomonas aeruginosa 0.5 16 90 0.5 1 30 Moraxella catarrhalis ≤0.015 0.03 30 ≤0.015 ≤0.015 30 Burkholderia cepacia 8 8 21 Burkholderia pseudomallei 0.25 0.25 100 Acinetobacter anitratus 0.12 0.25 10 0.5 1 10 Acinetobacter spp. 0.125 0.5 43 Stenotrophomonas >8 >8 61 maltophilia Aeromonas hydrophila 0.12 1 11 Xanthomonas maltophilia >8 >8 10 >8 >8 10 Peptostreptococcus spp. 0.25 1 50 C. difficile 8 16 23 Bacteroides fragilis 0.25 4 71 Prevotella spp. 0.125 0.125 36

Table 10. Pharmacokinetic properties of biapenem

Parameter Meropenem Biapenem t1/2 (hrs) 1.01 ± 0.19 1.6 ±0.2 (1.42-3.4) [40] AUC (mg*hr/L) 70.5 ±10.3 30 (300mg) 50 (500mg) Vdss (L/kg) 0.2-0.3 0.18-0.23 Cl (L/h/1.73m2) 9.59±0.80 9.1±0.66 (6.66-9.90) [41] Fraction excreted unchanged (%) 75 49±10 Protein binding (%) 9 7

4.2. Ertapenem

Ertapenem is a once-daily parenteral carbapenem active against Enterobacteriaceae producing extended-spectrum -lactamases (ESBLs) and AmpC -lactamases [42]. It belongs to group I carbapenems, which have limited activity against non-fermentative enterobacteriaceae (P.aeruginosa and Acinetobacter species), in contrast to group II that is active against these bacteria (imipenem, meropenem, doripenem, biapenem). The main structural difference between meropenem and ertapenem is the introduction of the meta- aminobenzoic group (see Figure 4); this modification improves the pharmacokinetic profile, but restricts the antibacterial spectrum. The meta-aminobenzoic group also imparts an overall negative charge to the molecule due to ionization of the carboxylic acid on the benzene ring at physiologic pH [43]. This ionization is responsible for the extensive protein binding (85-95%) of ertapenem, which increases its half-life, thereby allowing once-daily dosing. Ertapenem MICs for Gram-positive bacteria are generally comparable to those of meropenem and it shows good activity against the most common pathogens; exceptions Complimentary Contributor Copy

200 Stefano Biondi and Mauro Panunzio include enterococci, methicillin-resistant Staphylococcus aureus (MRSA), oxacillin-resistant coagulase-negative staphylococci (CONS), S. haemolyticus, and some Streptococcus viridans (see Table 8). Although ertapenem MICs for Gram-negative bacilli are generally lower than those for imipenem, its clinical relevance is arguable [44]. The increased activity against Gram-negative organisms by meropenem and ertapenem with respect to imipenem has been attributed to their different PBP binding profiles [45]. Meropenem and ertapenem bind with highest affinity to PBP2, followed by PBP3, whereas imipenem bind preferentially to PBP2 and then PBP1a and 1b. This difference in PBP binding also results in different bactericidality. Gram-negative bacterial cells treated with imipenem form small spheres or elipsoids with rapid lysis, while ertapenem- or meropenem-treated cells form elongated filaments accompanied by diminished endotoxin release [46]. Mean plasma concentration proﬁles of total ertapenem were very similar on day 1 and on day 8 following multiple dosing. The ratio of day 8 AUC0-24 over day 1 AUC0-24 for each dose was close to or slightly lower than 1, indicating that ertapenem does not accumulate following once-daily dosing over 8 days. Ertapenem distributes well into interstitial fluids [47] with AUC0–24 blister fluid/AUC0– 24plasma = 0.61. Penetration into the cerebrospinal fluid (CSF) of rabbits was 2.4% into non- inflamed and 7.1% into inflamed meninges [48]. Ertapenem is mainly eliminated through the kidneys by glomerular filtration and secretory processes [49], with almost 80% of a 1 gram dose of 14C-labeled ertapenem collected in the urine as approximately equal amounts of unchanged ertapenem and its major metabolite, the open ring -lactam derivative formed through hydrolysis by DHP-1 [50]. These two compounds constituted 95% of the total radioactivity measured in the urine. Radioactivity in the feces was less than 10% of the dose.

Figure 4. Chemical structure of meropenem and ertapenem.

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A 21st Century Contribution to the Antibacterial Armamentarium 201

Table 11. Pharmacokinetic parameters of ertapenem at different intravenous doses in healthy volunteers (n=16) [51]

Mean value (SD) at indicated dose Parameter 0.5g 1g 2g 3g

AUC0-∞ (g/ml) 305.6 (36.8) 572.1 (68.6) 1011.4 (118.0) 1407.2 (230.1)

ClP (ml/min) 27.6 (3.2) 29.5 (3.4) 33.4 (4.1) 36.3 (5.2)

ClR (ml/min) 12.7 (3.4) 12.9 (4.3 14.9 (4.6) 16.1 (4.9

ClNR (ml/min) 15.0 (4.0) 16.1 (5.4) 18.5 (5.5) 20.3 (5.9) Vss (L) 7.8 (1.3) 8.2 (1.5) 9.2 (1.4) 9.4 (1.7)

Cmax ((g/ml) 83.0 (12.1) 154.9 (22.0) 282.9 (41.4) 274.3 (41.3)

C24 ((g/ml) 0.6 (0.3) 1.2 (0.6) 2.0 (1.0) 3.0 (1.9) fe (%) 45.6 (12.1) 44.4 (14.8) 44.3 (13.3) 44.1 (12.5) t1/2 (h) 3.8 3.8 3.8 3.6

ClP = plasma clearance; ClR = renal clearance; ClNR = non renal clearance; Vss = distribution volume at stedy state; Cmax = concentration at end of infusion. The length of infusion was 0.5 h except for the 3-g dose, which was administered over 2 h; fe = fraction excreted unchanged in the urines; t = terminal half life.

Following a 1 gram i.v. dose of ertapenem, the total and free drug concentrations remained above the MIC90 of MSSA, Streptococci, Enterobacteriaceae, Moraxella catarrhalis, Haemophilus influenza, and most anaerobes (= 1 g/ml) for 24 and 8 h, respectively, which corresponds to 100 and 33% of the dosing interval [51-52]. Results from in vivo efficacy studies in neutropenic mice support that maximal bactericidal activity is observed when the T> MIC exceeds 30% of the dosing interval [53]. Overall ertapenem has a unique pharmacokinetic profile that supports once-daily administration, though this is not a requirement for an injectable drug used in the hospital, and the weaker antibacterial profile compared to other carbapenems outweighs its pharmacokinetic advantages. However, its use could offer advantages in therapies were non-fermenters are not involved and were ESBL, class C and the majority of class D -lactamases, are involved or suspected. Like other carbapenems, ertapenem is inactivated by carbapenemases (KPC and metallo -lactamases).

4.3. Doripenem

Doripenem, (formerly S-4661), is a synthetic 1--methylcarbapenem [54] similar to meropenem but with a sulfamoylaminomethyl group in place of the N,N- dimethylcarboxamide [55]. This substituent is considered important for the activity of doripenem against non-fermentative Gram-negative bacteria [56]. The 1--methyl group confer stability to human dehydropeptidases. The antibacterial spectrum of doripenem is similar to that of meropenem [56] and includes a broad range of Gram-positive, Gram- negative, and anaerobic pathogens. The US Food and Drug Administration (FDA) approved doripenem in October 2007 for treatment of complicated intra-abdominal infection and complicated urinary tract infections. The in vitro activity of doripenem is similar to that of imipenem and better than that of meropenem and ertapenem against Gram-positive organisms [57]. Like other carbapenems, doripenem has no activity against MRSA, Enterococcus Complimentary Contributor Copy

202 Stefano Biondi and Mauro Panunzio faecium, Stenotrophomonas maltophilia, Chryseobacterium indologenes, Elizabethkingia meningoseptica, or mycobacteria [58]. Table 12 shows the antibacterial activity of doripenem against common pathogenic bacteria. The issue of emerging resistance to doripenem is quite similar to that for imipenem and meropenem. In Gram-negative organisms, the most important mechanism of resistance is production of -lactamases capable of signiﬁcant carbapenem hydrolysis [58b, 59]. The most relevant examples are the KPC -lactamases, metallo -lactamases and some OXA -lactamases produced by enterobacteriaceae and Acinetobacter baumannii [60]. Pharmacokinetic models using Monte Carlo simulations were used to predict the appropriate dosing of doripenem [60b]. The recommended dose for doripenem is 500 mg every 8 hours (administered over a 1-hour infusion), in patients with creatinine clearance >50 ml/min [60a]. The pharmacokinetic parameters derived from multiple dosing in healthy subjects [61] are reported in Table 13. Plasma protein binding is about 8% and independent of doripenem concentration. The volume of distribution at steady state is similar to that of the extracellular volume with a median (range) of 16.8 L (8.09–55.5 L) [61b]. There is no difference in doripenem t1/2 between short or prolonged infusion, though, plasma clearance (range: 15-36 L) and total body Vdss (range: 19-56 L) trended larger for longer infusion time [62]. The elimination half-lives of doripenem and its primary metabolite (-lactam ring opening product), are 1.1 and 2.5 hours, respectively [63]. The mean urinary excretion accounts for 97.2% of the administered dose. Doripenem is not a substrate for cytochrome P450 enzymes, and is not metabolized by the liver.

Table 12. In Vitro Activity of Doripenem Against Common Pathogenic Microorganisms

Organism MIC50 (g/ml) MIC90 (g/ml) Gram-positive Staphylococcus aureus Methicillin susceptible 0.06 0.06 Methicillin resistant 16 16 Streptococcus pneumoniae Penicillin susceptible ≤0.008 ≤0.008 Penicillin intermediate 0.12 Penicillin resistant 0.5 1 E. faecalis 4 8 E. faecium >16 >16 Gram negative Fermentative Gram-negative Escherichia coli 0.03 0.3 ESBL E. coli 0.03 0.06 Klebsiella pneumoniae 0.03 0.06 ESBL K. Pneumoniae 0.06 0.12 Enterobacter cloacae 0.03 0.06 Proteus mirabilis 0.12 0.25 Non fermentative Gram-negative Acinetobacter baumanii 0.25 4 Pseudomonas aeruginosa 0.5 8 Stenotrophomonas maltophila >16 >16 Anaerobes Bacteroides fragilis 0.5 1

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Table 13. Pharmacokinetic parameters of doripenem

Parameter 500 mg/tid 500 mg/tid 1000 mg/tid 1000 mg/tid (n = 6) (n = 6) (n = 6) (n = 6) 30 min. infusion 4 hrs infusion 1 hr infusion 4 hrs infusion Cmax (g/ml) 31.8 7.4 48.0 10.6 t1/2 (hrs) 1.0 1.1 1.0 0.9 Cl (L/h) 13.9 18.0 13.1 25.1 Vdss (L/kg) 14.8 25.0 16.4 34.9 AUC0-∞ (g/ml*h) 36.1 28.5 76.5 40.6 Protein binding (%) 8

The most important pharmacodynamic parameter predicting bacteriologic and clinical efficacy of carbapenems is the time that the free (unbound) plasma concentration of drug exceeds the MIC (fT>MIC [62, 64]; an fT>MIC of approximately 20–30% is required for bacteriostasis) of the infecting pathogen, whereas fT>MIC of approximately 40–50% is bactericidal. Administration of doripenem in a neutropenic murine thigh infection model mimicking human exposure was studied [65]. The experiment used 24 P. aeruginosa isolates with a range of MICs (0.125–16 g/ml). Simulated human dose of 500 mg doripenem infused over 1 and 4 hours resulted bactericidal for isolates with MICs of ≤2 mg/ml. The stability of reconstituted doripenem solution supports the use of prolonged infusions of doripenem to increase fT>MIC in order to achieve efficacy against bacteria with higher MICs, and minimize emergenge/amplification of antimicrobial resistance. This is perhaps the major advantage of doripenem over other carbapenems, however does not resolve the problem of resistant strains, particularly KPC and metallo -lactamase producers.

4.4. Tebipenem Pivoxil

Tebipenem-pivoxil (TBM-PI) is an oral carbapenem agent whose active metabolite, tebipenem, shows broad-spectrum activity against Gram-positive and Gram-negative bacteria, except for P. aeruginosa [66]. Unlike other oral -lactam antibiotics, tebipenem displays excellent activity against penicillin-resistant S. pneumoniae. TBM-PI is indicated for treatment of community-acquired pediatric infections such as pneumonia, acute otitis media (AOM), and sinusitis. It is currently marketed in Japan. Tebipenem antibacterial activity has been compared to reference antibiotics and is reported in Table 14. The pharmacokinetic profile of tebipenem was determined in several animal species after oral administration of tebipenem pivoxil [67]. In mouse, rat, dog, and monkey, TBPM-PI was absorbed quickly, and the bioavailability was 71.4%, 59.1%, 34.8% and 44.9%, respectively. TBPM-PI was rapidly converted to tebipenem, and through blood circulation, distributed into the kidney at a high concentration and quickly eliminated. Tebipenem was found in high concentration and for a prolongued period of time only in kidneys. The penetration ratio of tebipenem to ELF was 21.8 +/- 14.7%. Serum protein binding of TBPM was 90.4-98.3% for mouse, 78.5-90.0% for rat, 15.7-18.7% for dog, 35.3-39.3% for monkey and 59.7-73.9% for human. Table 15 reports the primary pharmacokinetic parameters obtained in a phase II clinical trial [68]. Complimentary Contributor Copy

204 Stefano Biondi and Mauro Panunzio

Table 14. Comparative in vitro activities of tebipenem and reference drugs against clinical isolates (g/ml)

Tebipenem Imipenem Levofloxacin Organism Range MIC50 MIC90 Range MIC50 MIC90 Range MIC50 MIC90 (nr. of strains) S. aureus ≤0.063 ≤0.063 ≤0.063 ≤0.063 ≤0.063 ≤0.063 0.125–1 0.25 0.5 MSSA (43) S. aureus 0.125– ≤0.063-8 4 8 0.063–64 32 64 8 128 MRSA (39) >128 S. epidermidis ≤0.063– ≤0.063 ≤0.063 0.125 ≤0.063 0.125 0.125–8 0.25 4 MSSE (34) 0.125 S. epidermidis 0.5–8 2 8 0.25–64 4 64 0.25–128 4 16 MRSE (30) S. pneumoniae 0.0005– 0.002– 0.002 0.032 0.008 0.125 0.25–2 0.5 1 PenS (66) 0.032 0.25 S. pneumoniae 0.032–0.5 0.063 0.125 0.25–2 0.25 1 0.5–1 1 1 PenR (32) E. faecalis 0.25–2 0.5 2 8–>128 128 >128 1–64 1 64 (35) E. faecium 2–>128 64 >128 8–>128 128 >128 1–128 16 64 (45) E. coli ≤0.063- ≤0.063– ≤0.063 ≤0.063 ≤0.063 0.125 0.125 ≤0.063 8 (42) 0.25 64 K. pneumoniae 0.125– ≤0.063– ≤0.063–0.5 ≤0.063 ≤0.063 0.25 0.25 ≤0.063 0.25 (34) 0.5 4 H. influenzae ≤0.063–0.5 ≤0.063 0.25 0.25–16 1 4 ≤0.063 ≤0.063 ≤0.063 (38) Legionella pneumophila ≤0.063 ≤0.063 ≤0.063 ≤0.063 ≤0.063 ≤0.063 ≤0.063 ≤0.063 ≤0.063 (11) Moraxella ≤0.063- ≤0.063 ≤0.063 ≤0.063 ≤0.063 ≤0.063 ≤0.063 ≤0.063 ≤0.063 catarrhalis (34) 0.125

Changes in the regimen and dosage did not influence the pharmacokinetic properties of TBPM-PI. As the use of carbapenems in Europe and US is restricted to hospitals, it is unlikely that tebipenem could reach the market in these countries, where it would compete with generic -lactams.

Table 15. Pharmacokinetic parameter of tebipenem pivoxil

Parameter 150 mg (tid) 250 mg (bid) 300 mg (tid) -1 ka (hr ) 5.64 +/- 2.76 5.11 +/- 3.06 2.51 +/- 1.13 -1 Kel (hr ) 1.75 +/- 0.25, 2.03 +/- 0.10 1.34 +/- 0.27 Vd/F (L) 17.62 +/- 5.09 15.83 +/- 6.14 19.34 +/- 8.80 tmax (hr) 0.85 +/- 0.29 0.81 +/- 0.33 1.18 +/- 1.53 Cmax (g/ml) 5.08 +/- 2.05 7.92 +/- 4.02 8.69 +/- 4.01 t1/2 (hr) 0.40 +/- 0.06 0.34 +/- 0.01 0.54 +/- 0.10 AUC0-8 (g•hr/ml)* 5.22 +/- 1.90 7.93 +/- 4.04 13.62 +/- 6.29 AUC0-24 (g•hr/ml)* 15.65 +/- 5.70 15.85 +/- 8.08 40.87+/- 18.87 ka = absorption rate constant; Kel = elimination rate constant from the central compartment. * For 250mg (bid) AUC0-12.

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5. QUINOLONES

The quinolone antibiotics target two essential bacterial enzymes: DNA gyrase (topoisomerase II) and topoisomerase IV. While most of the compounds in this class have the chemical structure of quinolones, it also includes naphthyridines (nalidixic acid, gemifloxacin) and quinazolinediones. Figure 5 shows the typical structures of “quinolone antibiotics.”

O O O O O O F F OH OH OH

N N N N N N HN N O

Nalidixic acid Ciprofloxacin Levofloxacin

O O O O F OH OH H N N N N F HN O H2N H F F F Trovafloxacin Garenoxacin

Figure 5. Chemical structures of quinolone antibiotics.

The quinolone saga begun in 1962 with the discovery of nalidixic acid, which was used for oral treatment of uncomplicated urinary tract infections. Unfortunately, the drug is active only against a few Gram-negative pathogens, has very low serum and tissue concentrations, and has an half-life so short that it had to be given four times daily. Before the new millennium, quinolones were classified into four generations, mainly on the basis of their spectrum of activity (see Table 16) [69]. First and second generation fluoroquinolones selectively inhibit the topoisomerase II ligase domain, leaving the two nuclease domains intact. This modification, coupled with the constant action of the topoisomerase II in the bacterial cell, leads to DNA fragmentation via the nucleasic activity of the intact enzyme domains. Third and fourth generation fluoroquinolones are more selective for the topoisomerase IV ligase domain, and have enhanced Gram-positive coverage. For many Gram-negative bacteria, DNA gyrase is the target, whereas topoisomerase IV is the target for many Gram-positive bacteria. The main structural difference between first and second generation quinolones is the presence of a fluorine atom at C6, which increases significantly the activity. The third generation consists of molecules characterized by an alkyl-substituted piperazine or pyrrolidine at position 7, and of a methoxy at position 8, while the fourth generation has a fluoro-naphthyridine ring and the fifth generation is defined a C6 desfluoro quinolones. There are four known mechanisms of resistance to ﬂuoroquinolones: altered target site due to point mutations in DNA gyrase Complimentary Contributor Copy

206 Stefano Biondi and Mauro Panunzio and/or topoisomerase IV; up-regulation of efﬂux pumps with or without decreased expression of outer membrane porins; plasmid-mediated gyrase inhibitor binding site protection (Qnr proteins); enzymatic degradation [70]. Adverse events of quinolones include, but are not limited to, anaphylaxis, hypersensitivity reactions, tendinopathy arthropathy, central nervous system effects, peripheral neuropathy, C. difﬁcile-associated diarrhea, and teratogenic effects in animals. The most important differentiation among recently approved quinolones is in the breadth of spectrum, activity against multidrug-resistant strains, including quinolone resistant ones, possibility to perform intravenous-to-oral switch and to a lesser extent safety profile.

Table 16. Classification of Quinolone Antibiotics

Classification Agents Antimicrobial spectrum General clinical indications First generation Nalidixic acid Gram-negatives Uncomplicated urinary tract Cinoxacin (but not Pseudomonas spp.) infections Second Norfloxacin Gram-negatives (including Uncomplicated and generation Lomefloxacin Pseudomonas species), complicated urinary tract Enoxacin Ofloxacin some Gram-positives infections and pyelonephritis, Ciprofloxacin (including S. aureus but not sexually transmitted diseases, S. pneumoniae) and some prostatitis, skin and soft atypicals tissue infections Third generation Levofloxacin Expanded Gram-positive Acute exacerbations of Sparfloxacin coverage (penicillin- chronic bronchitis, Gatifloxacin sensitive and penicillin- community-acquired resistant S. pneumoniae) and pneumonia expanded activity against atypicals Fourth Trovafloxacin Includes broad anaerobic Intra- abdominal infections, generation Moxifloxacin coverage nosocomial pneumonia, pelvic infections

5.1. PRULIFLOXACIN (ULIFLOXACIN)

Prulifloxacin is the orally available prodrug of ulifloxacin [71], a fluoroquinolone with a broad spectrum of activity against various Gram-negative and Gram-positive bacteria (Figure 6). Like other fluoroquinolones, prulifloxacin prevents bacterial DNA replication, transcription, repair and recombination through inhibition of bacterial DNA gyrase [72]. Prulifloxacin has a long elimination half-life and is administered once daily.

Figure 6. Schematic representation of prulifloxacin conversion into the active fluoroquinolone ulifloxacin.

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Table 17. In vitro activity of ulifloxacin and ciprofloxacin against Gram-negative and Gram-positive bacteria (MIC and MBC values in g/ml)

Species (nr. of isolates) Ulifloxacin Ciprofloxacin MIC50 MIC90 MBC90 MIC50 MIC90 MBC90 Gram-negative bacteria Escherichia coli Italian nosocomial (41) ≤0.015 4 16 ≤0.015 16 32 Italian community (37) ≤0.015 0.12 0.5 ≤0.015 0.5 1 Spanish [nalidixic acid-resistant] (26) ≤0.015 ≤0.015 NT ≤0.015 ≤0.015 NT Haemophilus spp. Italian community (24) ≤0.015 ≤0.015 0.03 ≤0.015 ≤0.015 0.03 H. influenzae, Spanish (20) ≤0.015 ≤0.015 NT ≤0.015 0.03 NT Klebsiella spp. Italian nosocomial (33) ≤0.015 2 4 0.03 8 16 Italian community (15) ≤0.015 0.12 0.12 0.03 0.25 0.25 K. pneumoniae, Spanish [nalidixic acid-resistant] (23) 0.03 0.12 NT 0.03 0.25 NT Moraxella catarrhalis Italian community (16) ≤0.015 0.03 0.03 ≤0.015 ≤0.015 0.03 Spanish (8) 0.03 0.06 NT 0.12 0.12 NT Proteus, Providencia, Morganella spp. Italian nosocomial (44) ≤0.015 0.5 0.5 0.03 2 2 Italian community (23) ≤0.015 0.5 1 0.03 1 4 P. mirabilis Spanish [nalidixic acid-resistant] (22) ≤0.015 ≤0.015 NT 0.03 0.03 NT Pseudomonas aeruginosa Italian nosocomial (45) 2 64 64 8 128 128 Italian community (16) 1 32 128 2 64 128 Spanish [ciprofloxacin-sensitive] (75) 0.25 1 NT 0.5 1 NT Gram-positive bacteria Enterococcus spp. Italian community (19) 0.5 1 4 0.5 1 2 E. faecalis Italian nosocomial (26) 0.5 2 16 1 2 8 Spanish [vancomycin-resistant] (21) 2 4 NT 1 1 NT Staphylococcus aureus Italian nosocomial, oxacillin-susceptible (30) 0.25 0.25 0.5 0.25 0.5 1 Italian community, oxacillin-susceptible (26) 0.25 0.25 0.5 0.25 0.5 0.5 Spanish [meticillin-resistant] (20) 0.5 0.5 NT 0.5 0.5 NT Streptococcus pneumoniae Italian community (36) 0.5 1 2 0.5 1 2 Spanish penicillin-susceptible, -intermediate 1–2 2–4 NT 1–2 2–4 NT or -resistant (58) S. pyogenes Italian community (21) 0.25 1 2 0.25 1 4 Spanish (17) 0.25 0.25 NT 0.5 1 NT

Ulifloxacin has in vitro activity against a variety of clinical isolates of Gram-negative or Gram-positive bacteria commonly associated with chronic lower respiratory tract infections or lower UTIs (Table 1). It has limited activity against Chlamydia pneumoniae, an atypical respiratory tract pathogen (MIC90 = 4 g/ml), and against anaerobic bacteria. The in vitro activity of ulifloxacin is generally greater than that of ciprofloxacin against most Gram- Complimentary Contributor Copy

208 Stefano Biondi and Mauro Panunzio negative bacteria such as Escherichia coli, Klebsiella spp., Proteus, Providencia and Morganella spp., Pseudomonas aeruginosa, Moraxella catarrhalis and Haemophilus spp. Emerging bacterial resistance in Gram-negative pathogens after repeated exposure to ulifloxacin is similar to that observed with ciprofloxacin. After seven passages in media containing subinhibitory concentrations of either agents, the MIC of ulifloxacin against E. coli was unchanged whilst that of ciprofloxacin was increased 2-fold. Against P. aeruginosa, the MIC of ulifloxacin increased 4-fold, whereas that of ciprofloxacin increased 16-fold [73]. The in vitro activity of ulifloxacin against Gram-positive bacteria is similar to ciprofloxacin [74]. Prulifloxacin has similar or greater activity than ciprofloxacin in mouse models of systemic or respiratory tract infection or UTI against Gram-negative (including E. coli, K. pneumoniae and P. aeruginosa) or Gram-positive organisms (including S. aureus and S. pneumoniae) [72, 75]. Ulifloxacin is unlikely to prolong the QTc interval during therapy, as shown both in vitro and in vivo [76]. The pharmacokinetic parameters of ulifloxacin, following a single 600 mg dose of prulifloxacin, are reported in table 18.

Table 18. Pharmacokinetic parameters of ulifloxacin after single/ multiple oral administration of prulifloxacin [77]

Parameter Value Parameter Value Dose (single) 600 mg Dose (12 days) 600mg Cmax (g/ml) 1.6 Cmax ss (g/ml) 2 tmax (hr) 1 tmax ss (hr) 0.75 AUC0-∞ (g•hr/ml) 7.3 AUC0-∞ ss (g•hr/ml) 7.6 Vd (L) 1231 t1/2 (hr) 12.1 t1/2 ss (hr) 7.6 ClR (ml/min) 170 ClR ss (ml/min) 193 Protein binding (%) 45 Urinary recovery (%) 18

Protein binding of ulifloxacin is 45% and urinary recovery is about 18% of the administered dose, sufficient to reach a therapeutically viable concentration (3g/ml) for many pathogens but not P. aeruginosa. Co-administration of probenecid and oral prulifloxacin increased systemic exposure to ulifloxacin by 46%, prolonged the ulifloxacin t1/2 by 60% and reduced the apparent total clearance and urinary excretion of ulifloxacin by 30% and 57%, indicating that renal excretion of ulifloxacin occurs by active tubular secretion as well as glomerular filtration. Prulifloxacin is a once daily orally available fluoroquinolone with antibacterial activity comparable or slightly superior to ciprofloxacin. It does not address the problem of multidrug resistance and is ideally positioned as an antibacterial agent for community-acquired infections.

5.2. Pazufloxacin

Pazufloxacin is a fused tricyclic fluoroquinolone derivative with a 1-aminocyclopropyl substituent at position C10 position. The drug shows potent broad spectrum activity against Gram-positive, Gram-negative, and anaerobic bacteria [78]. It has particularly good activity

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against P.aeruginosa (MIC90 = 0.78g/ml). The activity of pazufloxacin versus comparator drugs is reported in Table 19 and 20.

Table 19. Antibacterial activity (g/ml) of pazufloxacin and comparators

Organism Pazufloxacin Ceftazidime Imipenem Gentamicin Ciprofloxacin S. aureus FDA209P 0.2 12.5 0.025 0.05 0.2 S. epidermidis IID866 0.2 3.13 0.0125 0.1 0.2 S. pyogenes S-8 0.78 0.78 0.0125 0.39 0.2 S. pneumoniae IID552 3.13 0.39 0.0125 25 0.78 E. faecalis IID682 0.78 >100 12.5 6.25 0.39 B. subtilis ATCC6633 0.1 12.5 0.025 0.1 0.05 Escherichia coli NIHJ 0.0125 0.05 0.39 0.2 0.00625 Salmonella paratyphy 0.025 0.2 0.39 0.2 0.0125 IID605 Shigella sonnei EW33 0.0125 0.05 0.39 0.78 0.00625 Citrobater freundi 0.0125 0.39 0.1 0.1 0.00313 IFO12681 Klebsiella pneumoniae 0.025 0.025 0.78 0.39 0.0625 ATCC10031 Enterobacter aerogenes 0.0125 0.2 1.56 0.39 0.00313 IID972 Enterobacter cloacae 0.0125 0.78 1.56 0.78 0.00625 IID977 Serratia marcescens 0.05 0.05 0.78 0.39 0.05 IID620 Proteus mirabilis 0.05 0.39 6.25 3.13 0.1 IFO3849 Proteus vulgaris 0.0125 0.025 1.56 0.2 0.0125 IFO3851 Morganella morganii 0.025 0.1 3.13 0.39 0.025 IID602 Providencia rettgeri 0.0125 0.0125 3.13 0.2 0.00313 IFO13501 Pseudomonas 0.2 1.56 1.56 1.56 0.2 aeruginosa IFO3445 Peptostreptococcus 1.56 3.13 0.05 100 0.2 magnus WAL2508 Clostridium perfringens 0.2 0.1 0.1 100 0.2 WAL3503 Bacteroides fragilis 3.13 12.5 0.1 >100 1.56 ATCC25285

The recommended dose of pazufloxacin for most infections is 500 mg twice a day administered as an intravenous infusion over 30-60 minutes. Depending on age, symptoms, and severity of infection, the dose of the drug may be reduced to 300 mg twice a day. The total duration of therapy with the drug should not exceed 14 days. The dose of the drug needs to be modified in patients with moderate to severe renal failure. The maximum recommended dose of the drug should be 300 mg twice a day in such patients. Table 21 shows the main pharmacokinetic parameters for pazufloxacin. Complimentary Contributor Copy

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Pazufloxacin may be used in community-acquired infections in respiratory tract, abdominal and urinary tract infections. It is suitable for empirical therapy for unknown infections specially hospital acquired till the culture sensitivity reports are made available thanks to its broad coverage against Gram-positive organisms including MRSA and Gram- negative organisms including Pseudomonas, Enterobacter and Klebsiella.

Table 20. Antibacterial activity (g/ml) of pazufloxacin against some respiratory clinical isolates

Organism Drug MIC50 MIC90 Range Moraxella catarrhalis Pazufloxacin ≤0.05 0.1 ≤0.05-0.2 Ceftazidime 0.1 0.2 ≤0.05-0.39 Imipenem 0.1 0.2 ≤0.05-0.2 Gentamicin 0.39 0.39 ≤0.05-0.78 Ciprofloxacin ≤0.05 0.1 ≤0.05-0.1 Bacteroides fragilis Pazufloxacin 6.25 25 1.56->100 Ceftazidime 25 >100 6.25->100 Imipenem 0.2 0.78 ≤0.05-12.5 Gentamicin >100 >100 >100 Ciprofloxacin 6.25 50 1.56->100 Prevotella spp. Pazufloxacin 1.56 50 0.39->100 Ceftazidime 1.56 >100 ≤0.05->100 Imipenem ≤0.05 0.2 ≤0.05-25 Gentamicin >100 >100 25->100 Ciprofloxacin 1.56 50 0.39->100 Mycobacterium Pazufloxacin 4 4 1-8 tubeculosis Ciprofloxacin 0.5 1 0.125-2 Sparfloxacin 0.125 0.25 ≤0.063-1 Rifampicin ≤0.063 0.25 ≤0.063-1 Mycobacterium avium Pazufloxacin 16 64 2->128 Ciprofloxacin 2 8 0.25-32 Sparfloxacin 2 8 0.125-16 Rifampicin 64 128 4->128

5.3. Balofloxacin

Balofloxacin is a fluoroquinolone derivative, is prescribed for the treatment of bacterial infections including uncomplicated urinary tract infections, infective ophthalmitis, community-acquired pneumonia, acute exacerbation of chronic bronchitis, sinusitis, and skin infections. Balofloxacin is effective against Gram-negative and Gram-positive microbes including MRSA. The comparative antibacterial activity of balofloxacin and other representative fluoroquinolones against MRSA and S.epidermidis is reported in Table 22. Balofloxacin showed equal or superior activity against MRSA and S.epidermidis. The antibacterial activity of balofloxacin and reference fluoroquinolones against clinical isolates is shown in Table 23 [79].

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Table 21. Pharmacokinetic parameters of pazufloxacin

Parameter Value Dose (mg) 500 Cmax (g/ml) 11. 0 ± 2. 4 Tmax (hrs) 0.5 t1/2 (hrs) 1.65 -1.88 Urinary excretion (%) 89.5-93.8

Table 22. Antibacterial activities of balofloxacin and reference quinolones against MRSA and S. epidermidis

Strain gyrA MIC (g/ml) mutation Balofloxacin Ofloxacin Ciprofloxacin Tosufloxacin Sparfloxacin MRSA ATJ-8 - 0.39 3.13 12.5 0.39 0.05 MRSA ATJ-17 - 0.39 3.13 12.5 0.78 0.05 MRSA ATJ-29 - 0.78 3.13 12.5 0.78 0.05 MRSA ATJ-34 - 0.05 0.39 0.39 0.05 0.05 MRSA QA 389 - 0.2 0.78 3.13 0.2 0.05 MRSA ATJ-2 - 0.78 3.13 12.5 0.78 0.2 MRSA ATJ-10 + 3.13 25 100 12.5 6.25 MRSA ATJ-18 + 6.25 25 100 6.25 6.25 MRSA ATJ-26 + 6.25 25 100 6.25 6.25 MRSA ATJ-27 + 1.56 6.25 12.5 6.25 6.25 MRSA ATJ-43 + 1.56 12.5 12.5 3.13 6.25 MRSA QA 266-1 + 6.25 25 100 12.5 6.25 MRSA ATJ-4 + 3.13 25 50 6.25 12.5 MRSA ATJ-22 + 6.25 50 100 3.13 12.5 MRSA ATJ-38 + 6.25 25 100 3.13 12.5 MRSA ATJ-42 + 6.25 50 100 3.13 12.5 MRSA QA 217 + 3.13 25 100 12.5 12.5 MRSA ATJ-49 + 12.5 50 100 6.25 25 S. epidermidis QA 184-2 NT 0.2 0.78 0.39 0.2 0.1 S. epidermidis QA 129 NT 1.56 12.5 50 12.5 6.25 S. epidermidis QA 244-1 NT 6.25 100 12.5 12.5 12.5

Table 23. Antibacterial activities of balofloxacin and other quinolones against clinical isolates

Organism Drug Range (g/ml) MIC50 MIC90 (nr. of strains) (g/ml) (g/ml) Methicillin-susceptible Balofloxacin 0.05-6.25 0.1 0.2 S. aureus (91) Ofloxacin 0.2-50 0.39 0.78 Ciprofloxacin 0.2-50 0.39 3.13 Tosufloxacin 0.012-50 0.05 0.1 Lomefloxacin 0.39-50 0.78 3.13 Sparfloxacin 0.025-12.5 0.1 0.2

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Table 23. (Continued)

Organism Drug Range (g/ml) MIC50 MIC90 (nr. of strains) (g/ml) (g/ml) Methicillin-resistant Balofloxacin 0.05-25 0.2 6.25 S. aureus (132) Ofloxacin 0.2-100 0.78 50 Ciprofloxacin 0.2->100 1.56 50 Tosufloxacin 0.025->100 0.05 25 Lomefloxacin 0.78->100 3.13 100 Sparfloxacin 0.05-25 0.1 12.5 S. epidermidis (104) Balofloxacin 0.012-1.56 0.1 0.2 Ofloxacin 0.1-25 0.39 1.56 Ciprofloxacin 0.012-25 0.2 1.56 Tosufloxacin 0.025-6.25 0.05 0.2 Lomefloxacin 0.05-100 0.78 3.13 Sparfloxacin 0.05-12.5 0.1 0.2 Ciprofloxacin-susceptible Balofloxacin 0.012-0.78 0.1 0.2 staphylococci (245) Ofloxacin 0.1-1.56 0.39 0.78 Ciprofloxacin 0.012-1.56 0.39 0.78 Tosufloxacin 0.012-0.39 0.05 0.1 Lomefloxacin 0.05-12.5 0.78 1.56 Sparfloxacin 0.025-3.13 0.1 0.1 Ciprofloxacin-resistant Balofloxacin 0.2-25 1.56 6.25 staphylococci (82) Ofloxacin 0.78-100 12.5 50 Ciprofloxacin 3.13->100 12.5 100 Tosufloxacin 0.05->100 1.56 50 Lomefloxacin 3.13->100 25 100 Sparfloxacin 0.05-25 3.13 12.5 S. pneumoniae (33) Balofloxacin 0.1-6.25 0.39 0.39 Ofloxacin 0.39-100 1.56 3.13 Ciprofloxacin 0.2-50 1.56 3.13 Tosufloxacin 0.05-12.5 0.2 0.39 Lomefloxacin 0.39-100 6.25 12.5 Sparfloxacin 0.1-50 0.39 0.39 S. pyogenes (34) Balofloxacin 0.1-0.39 0.2 0.39 Ofloxacin 0.78-1.56 0.78 1.56 Ciprofloxacin 0.39-0.78 0.39 0.78 Tosufloxacin 0.1-0.2 0.1 0.2 Lomefloxacin 1.56-6.25 3.13 6.25 Sparfloxacin 0.2-0.78 0.2 0.39 Enterococcus faecalis (80) Balofloxacin 0.2-25 0.78 12.5 [102, 79b] Norfloxacin 1.56->100 3.13 100 Ofloxacin 1.56->100 3.13 100 Ciprofloxacin 0.39->100 0.78 25 Sparfloxacin 0.2-50 0.78 50 E. faecalis (40) Balofloxacin 0.2-0.78 0.39 0.78 Ofloxacin 0.78-3.13 1.56 3.13 Ciprofloxacin 0.2-1.56 0.78 1.56 Tosufloxacin 0.1-0.78 0.39 0.39 Lomefloxacin 3.13-12.5 6.25 6.25 Sparfloxacin 0.2-0.78 0.39 0.78

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Organism Drug Range (g/ml) MIC50 MIC90 (nr. of strains) (g/ml) (g/ml) E. faecium (60) Balofloxacin 0.2-3.13 0.78 1.56 Ofloxacin 0.78-12.5 3.13 3.13 Ciprofloxacin 0.2-12.5 0.78 1.56 Tosufloxacin 0.1-6.25 0.39 0.78 Lomefloxacin 1.56-50 3.13 6.25 Sparfloxacin 0.1-3.13 0.39 0.78 E. coli (40) Balofloxacin 0.1-0.78 0.2 0.2 Ofloxacin 0.05-0.78 0.1 0.1 Ciprofloxacin 0.012-0.2 0.025 0.025 Tosufloxacin 0.025-0.2 0.025 0.05 Lomefloxacin 0.1-1.56 0.2 0.2 Sparfloxacin 0.012-0.39 0.025 0.05 C. freundii (34) Balofloxacin 0.1-6.25 0.78 1.56 Ofloxacin 0.05-3.13 0.2 0.39 Ciprofloxacin 0.012-0.39 0.05 0.2 Tosufloxacin 0.025-1.56 0.2 0.39 Lomefloxacin 0.2-3.13 0.39 0.78 Sparfloxacin 0.025-3.13 0.2 0.78 K pneumoniae (40) Balofloxacin 0.1-3.13 0.39 1.56 Ofloxacin 0.05-1.56 0.1 0.78 Ciprofloxacin 0.025-0.39 0.05 0.2 Tosufloxacin 0.012-0.39 0.05 0.2 Lomefloxacin 0.1-1.56 0.2 1.56 Sparfloxacin 0.025-0.78 0.1 0.39 E. cloacae (39) Balofloxacin 0.2-6.25 0.39 0.78 Ofloxacin 0.05-0.78 0.1 0.39 Ciprofloxacin 0.012-0.39 0.025 0.1 Tosufloxacin 0.025-0.78 0.05 0.1 Lomefloxacin 0.1-1.56 0.2 0.39 Sparfloxacin 0.025-3.13 0.05 0.2 E. aerogenes (39) Balofloxacin 0.2-3.13 0.39 0.78 Ofloxacin 0.05-0.78 0.2 0.2 Ciprofloxacin <0.006-0.39 0.05 0.1 Tosufloxacin 0.025-0.2 0.05 0.1 Lomefloxacin 0.1-1.56 0.2 0.39 Sparfloxacin 0.025-0.39 0.1 0.2 P. vulgaris (40) Balofloxacin 0.2-3.13 0.78 1.56 Ofloxacin 0.05-0.39 0.1 0.39 Ciprofloxacin 0.012-0.2 0.05 0.1 Tosufloxacin 0.025-0.39 0.1 0.2 Lomefloxacin 0.1-0.78 0.2 0.39 Sparfloxacin 0.05-0.78 0.39 0.78 P. mirabilis (38) Balofloxacin 0.39-50 0.78 3.13 Ofloxacin 0.1-50 0.2 0.78 Ciprofloxacin 0.025-12.5 0.05 0.2 Tosufloxacin 0.05-50 0.1 0.78 Lomefloxacin 0.2-50 0.39 1.56 Sparfloxacin 0.2-25 0.39 1.56

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Table 23. (Continued)

Organism Drug Range (g/ml) MIC50 MIC90 (nr. of strains) (g/ml) (g/ml) M. morganii (40) Balofloxacin 0.012-25 0.39 0.78 Ofloxacin 0.012-12.5 0.1 0.2 Ciprofloxacin <0.006-6.25 0.025 0.05 Tosufloxacin <0.006-12.5 0.05 0.1 Lomefloxacin 0.025-12.5 0.1 0.2 Sparfloxacin <0.006-6.25 0.1 0.2 P. rettgeri (36) Balofloxacin 0.2->100 6.25 100 Ofloxacin 0.1-> 100 3.13 50 Ciprofloxacin 0.025-> 100 0.78 100 Tosufloxacin 0.025->100 0.39 25 Lomefloxacin 0.2-> 100 6.25 50 Sparfloxacin 0.05-50 1.56 12.5 S. marcescens (39) Balofloxacin 0.39-100 6.25 50 Ofloxacin 0.1-100 1.56 25 Ciprofloxacin 0.05-25 0.78 12.5 Tosufloxacin 0.05->100 0.78 12.5 Lomefloxacin 0.2-50 3.13 25 Sparfloxacin 0.05-50 1.56 12.5 P. aeruginosa (50) Balofloxacin 1.56-50 6.25 12.5 Ofloxacin 0.78-12.5 1.56 3.13 Ciprofloxacin 0.1-0.78 0.39 0.78 Tosufloxacin 0.1-1.56 0.39 0.78 Lomefloxacin 1.56-6.25 1.56 6.25 Sparfloxacin 0.39-3.13 1.56 3.13 A. calcoaceticus (32) Balofloxacin 0.2-6.25 0.39 0.78 Ofloxacin 0.1-3.13 0.2 0.78 Ciprofloxacin 0.1-3.13 0.2 0.78 Tosufloxacin 0.025-0.39 0.05 0.1 Lomefloxacin 0.39-12.5 0.78 1.56 Sparfloxacin 0.012-0.78 0.05 0.1 H. influenzae (52) Balofloxacin 0.2-0.025 0.05 0.05 Ofloxacin 0.025-0.2 0.05 0.05 Ciprofloxacin <0.006-0.05 0.025 0.025 Tosufloxacin <0.006-0.05 0.012 0.012 Lomefloxacin 0.05-0.39 0.1 0.1 Sparfloxacin 0.012-0.1 0.025 0.05 N. gonorrhoeae (31) Balofloxacin <0.006-0.2 0.025 0.05 Ofloxacin <0.006-0.39 0.012 0.025 Ciprofloxacin <0.006-0.1 <0.006 <0.006 Tosufloxacin <0.006-0.1 0.012 0.025 Lomefloxacin 0.012-0.78 0.025 0.05 Sparfloxacin <0.006-0.05 <0.006 <0.006 M. catarrhalis (52) Balofloxacin 0.05-3.13 0.2 0.2 Ofloxacin 0.05-6.25 0.1 0.1 Ciprofloxacin 0.025-1.56 0.05 0.05 Tosufloxacin 0.012-0.78 0.025 0.025 Lomefloxacin 0.1-12.5 0.2 0.2 Sparfloxacin <0.006-0.78 0.025 0.025

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After oral administration, balofloxacin is well absorbed and distributed, and is not primarily metabolized by hepatic enzymes. Table 24 reports the main pharmacokinetic parameter of balofloxacin in healthy volunteers [80]. Balofloxacin is administered at a dose of 100 mg twice daily. It has improved efficacy, broader spectrum and better safety profile than other fluoroquinolones, however it has been registered only in some Asian countries.

Table 24. Pharmacokinetic parameters of balofloxacin

Parameter Value Dose (mg) 100

Cmax (g/ml) 2.17 ± 0.36

Tmax (hrs) 1.1 ± 0.5

AUC0-∞ (g•hr/ml) 17.1 ± 1.8

t1/2 (hrs) 7.8 ± 1.2 Vdss/F (L) 38.3 ± 21.1 Cl/F (L/h) 11.80 ± 1.16

ClR/F (L/h) 9.47 ± 1.78 Urinary excretion (%) 70-80

5.4. Gemifloxacin

Gemifloxacin is a 6-fluoronaphthyridone and is usually considered related member of fluoroquinolones. It was originally designated LB20304 (later also termed SB-265805) by LG Chemical Ltd in Korea, the pharmaceutical company that first synthesized it [81]. Gemifloxacin was approved by the FDA in 2003 to treat AECB and mild-to-moderate CAP caused by several pathogens, including multidrug-resistant S. pneumoniae. Gemifloxacin shows enhanced activity against pneumococci including most ciprofloxacin-resistant strains, improved activity against atypical pathogens [82], activity against Gram-negatives (excluding Pseudomonas aeruginosa), a favorable pharmacokinetic profile suitable for once daily dosing, limited drug-drug interactions. It is well tolerated with only a few discontinuations [83]. The antibacterial activity of gemifloxacin is summarized in Table 25. The recommended oral dose of gemifloxacin for adults with normal renal function is 320 mg once daily. The usual duration of therapy is 5 days for acute bacterial exacerbations of chronic bronchitis and mild to-moderate community-acquired pneumonia caused by pneumococci, H. influenzae, M. pneumoniae, or C. pneumoniae. The course for community- acquired pneumonia caused by multidrug-resistant pneumococci, K. pneumoniae, or M. catarrhalis is extended to 7 days. For patients with a creatinine clearance <40 ml/min the recommended dose is 160 mg every 24 h. The pharmacokinetic parameters for gemifloxacin are reported in Table 26 [84].

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Table 25. Antibacterial activity of gemifloxacin

Organism MIC50 (g/ml) MIC90 (g/ml) MIC range (g/ml) Gram-positive Staphylococcus aureus Methicillin susceptible 0.016–0.03 0.03–0.12 ≤0.004–16 Methicillin resistant 0.016–4 1–8 ≤0.008–128 Fluoroquinolone susceptible 0.016–0.03 0.03–0.06 ≤0.004–0.5 Fluoroquinolone resistant 0.5–4 8–64 0.06 - >64 Staphylococcus epidermidis 0.06–0.5 1–2 0.008–64 Methicillin susceptible 0.016 0.016–0.12 ≤0.004–2 Methicillin resistant 0.008–0.5 0.25–2 ≤0.004–2 Streptococcus pneumoniae 0.015 0.03 ≤0.004–4 Fluoroquinolone resistant 0.06–0.25 0.06–1 0.03–2 Streptococcus pyogenes 0.015–0.03 0.015–0.25 ≤0.004–2 Streptococcus agalactiae 0.015–0.06 0.03–0.125 0.008–1 Group C or G Streptococci 0.008–0.03 0.015–0.06 ≤0.004–0.5 Viridans group Streptococci ≤0.015–0.06 0.06–0.125 0.004–4 Enterococcus faecalis 0.06–32 0.25–32 0.008–64 Enterococcus faecium 0.25–64 2 - >128 0.016 - >128 Gram-negative Haemophilus inﬂuenzae 0.03 ≤0.004–0.03 Haemophilus parainﬂuenzae 0.03–0.06 Moraxella catarrhalis 0.015 ≤0.004–0.016 Citrobacter freundii 0.125–8 Citrobacter koseri 0.03–0.12 Enterobacter aerogenes 0.25–2 Enterobacter cloacae 0.25 0.25–2 Escherichia coli 0.25 0.03–128 Hafnia alvei 0.03 Klebsiella oxytoca 0.06–0.5 Klebsiella pneumoniae 0.5 0.12–32 Morganella morganii 0.06–4 Proteus mirabilis 0.12 - >32 Proteus vulgaris 0.06–0.25 Providencia rettgeri 0.06–8 Providencia stuartii 0.25 - >16 Neisseria gonorrhoeae 0.125 Salmonella spp. 0.015–0.5 Serratia marcescens 1-4 0.25–8 Shigella spp. 0.008–0.016 Acinetobacter baumannii >4 - >16 Burkholderia cepacia 8–32 Pseudomonas aeruginosa >8 2 - >128 Stenotrophomonas maltophilia 1–8 Anaerobes Bacteroides fragilis 0.5 – 1 0.5 – 16 Other Bacteroides fragilis group spp. >4 B. gracilis 1 B. stercoralis 0.5 B. tectum 0.25 B. urealyticus 2 Prevotella spp. 0.5–16 Veillonella spp. 8 Porphyromonas spp ≤0.25 -≤1 Fusobacterium spp. ≥2 Complimentary Contributor Copy

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Organism MIC50 (g/ml) MIC90 (g/ml) MIC range (g/ml) Clostridium perfringens 0.06-0.12 Clostridium difﬁcile 2 - >16 Peptostreptococcus spp. 0.5 - 4 Atypical Mycoplasma pneumoniae 0.12-0.25 Mycoplasma homini ≤0.008-0.03 Mycobacterium tuberculosis 8 Mycobacterium marinum >32 Legionella pneumophila 0.015 Chlamydia pneumoniae 0.25 Ureaplasma urealyticum 0.25 Helicobacter pylori 0.12

Table 26. Pharmacokinetic parameters of gemifloxacin

Parameter Value Dose (mg) 320 Cmax (g/ml) 1.48 ± 0.39 Tmax (hrs) 1.00 AUC0-∞ (g•hr/ml) 9.82 ± 2.70 t1/2 (hrs) 6.65 ± 1.25 Vd/F (L/kg) 4.97 ± 0.96 ClR/F (ml/min) 151 ± 8.2 Urinary excretion (%) 27.5 ± 6.4 Protein binding (%) 61 F (%) 70

Gemifloxacin is well distributed and accumulates in human polymorphonuclear leukocytes to levels seven times the extracellular concentration in which cells are incubated in vitro [85]. Approximately 60% of the drug is excreted into the feces and 20-30% into the urine. A limited extent is metabolized in the liver forming minor metabolites [83]. The pharmacodynamics driver that correlates with clinical efficacy in models of pneumococcal infection is fAUC0-24/MIC ≥ 30 hrs [86]. Monte Carlo simulation predicted a high probability of reaching such a value using a daily dose of gemifloxacin of 320 mg [87]. Gemifloxacin has been developed for treatment of respiratory tract infection and its potency and spectrum of activity make it suitable for use in cases of infection where resistance to other classes of antibiotics is suspected.

5.5. Garenoxacin

Garenoxacin is a 6-desﬂuoro quinolone bearing a chiral group at C-7, namely 7-[(1R)(1- methyl-2,3-dihydro-1H-5-isoindolyl) and a diﬂuoromethoxy substitutent at C-8 [88]. Similar to fluoroquinolones, garenoxacin is active against many Gram-positive and Gram-negative bacteria and intracellular respiratory pathogens. In particular it is active against penicillin susceptible and -resistant Streptococcus pneumoniae, methicillin-susceptible Staphylococcus aureus (MSSA) and some anaerobes, however it lacks significant activity against Pseudomonas aeruginosa. Garenoxacin has been registered in Japan, but not in USA and

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Europe following withdrawal of the application by Schering-Plough. The antibacterial activity of garenoxacin is reported in Table 27.

Table 27. Antibacterial activity of garenoxacin

Organism MIC50 (g/ml) MIC90 (g/ml) MIC range (g/ml) Gram-negative Escherichia coli ≤0.03 0.12–>4 ≤0.03–>4 Enterobacter spp. 0.12 4 Enterobacter aerogenes 0.12 1–>4 Enterobacter cloacae 0.12 >4 Klebsiella spp. 0.12 1–>4 ≤0.03–>4 Proteus mirabilis 0.5 >4 Morganella morganii 1 >4 Serratia marcescens 1 >4 Citrobacter spp. 0.12 4 Salmonella spp. 0.06 0.25 Nalidixic acid susceptible 0.06 0.12 ≤0.03–4 Nalidixic acid resistant 0.5 1 0.12–>4 Acinetobacter spp. 0.06 0.12–>4 Haemophilus inﬂuenzae ≤0.03 ≤0.03 ≤0.03–2 Moraxella catarrhalis ≤0.03 ≤0.03 ≤0.03–1 Pseudomonas aeruginosa 2–4 >4 0.25–>4 Stenotrophomonas maltophilia 2 >4 Gram-positive Staphylococcus aureus MSSA ≤0.03 0.03–0.06 MRSA 1–2 4 Coagulase negative methicillin sensitive ≤0.03–0.03 0.06–0. 12 Coagulase negative methicillin resistant 1 4 Streptococcus pneumoniae 0.06 0.06 ≤0.03–0.5 Penicillin sensitive 0.03–0.06 0.03–0.06 ≤0.015–4 Penicillin intermediate 0.03–0.06 0.06 ≤0.015–4 Penicillin resistant 0.03–0.06 0.06–0.12 ≤0.015–>4 Streptococcus pyogenes 0.06 0.12 Beta-hemolytic streptococci 0.06 0.12 Viridans streptococci 0.06 0.12 Enterococcus faecalis 0.25 4 0.06–8 Vancomycin susceptible 0.25 >4 0.12–>4 Vancomycin resistant 2 4 2-4 Enterococcus faecium >4 >4 0.03-128 Vancomycin susceptible 4 >4 0.5->4 Vancomycin resistant >4 >4 4->4 Clostridium perfringens 0.25–0.5 0.25–2 0.03–4.0 Peptostreptococcus spp. 0.06–0.5 0.125–8 ≤0.03–8 Atypical Mycoplasma pneumoniae 0.016–0.031 0.031–0.06 0.008–0.12 Mycoplasma hominis ≤0.008–0.03 ≤0.008–0.03 ≤0.008–0.25 Chlamydophila pneumoniae 0.015 0.015–0.06 0.015–0.06 Ureaplasma urealyticum 0.063–0.12 0.25 0.016–1 Legionella pneumophila spp. ≤0.004–016 0.03 ≤0.004–0.25 Bacteroides fragilis 0.03–2 0.12–8 0.06–64 Mycobacterium tuberculosis 2 2–4 0.03–16 Mycobacterium fortuitum 0.13 0.25 0.06–0.5 Mycobacterium chelonae 4 16 0.5–>16

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Garenoxacin prevents the development of resistant strains with double-point mutations and has a lower potential to select mutant for resistant strains in vitro than levofloxacin and moxifloxacin [89]. Similarly garenoxacin gave complete bacteriological eradication rates against quinolone-resistant (100%), -lactam resistant (97.7%) and macrolide resistant (98.7%) S. pneumonia [90]. The pharmacokinetic properties of garenoxacin are reported in Table 28 [91]. A high fat meal delays the tmax approximately 1 hour [94] but there is no detrimental effect on bioavailability Garenoxacin undergoes both hepatic metabolism and renal elimination, with 40% of the drug being excreted unchanged in the urine [95]. Renal elimination is by a combination of ﬁltration and active tubular secretion. Clearance is inversely proportional to the dose (range 27–56 ml/min). There are two major metabolites, the sulfate (M1) and the glucuronide (M6), both of which are excreted in the bile [96]. The pharmacokinetic parameters of garenoxacin are dose-proportional and time-independent in the recommended dose range (400–600 mg once daily orally) [97]. The pharmacodynamic parameter that best predicts efﬁcacy for garenoxacin against Gram-positive and Gram- negative bacteria is AUC/MIC [98]. Mean PAEs (at ten times MIC) for pneumococci, staphylococci, and enterococci were 0.3-2.2 hours. For E. coli and P. aeruginosa PAEs were in the range 0.9-1.6 hours.

Table 28. Pharmacokinetic properties of garenoxacin

Parameter Value Value Dose (mg) 400 600 Cmax (g/ml) 7.43 9.3-15.3 Tmax (hrs) 1.5 1-2 AUC0-∞ (g•hr/ml) 100.7 96.7–136 t1/2 (hrs) 12.36 9.8-15 Vd/F (L/kg) 67.1 ClR/F (ml/min) 56 27 Urinary recovery (%) 40 Protein binding (%) 80-90 [92] F (%) 92 [93]

5.6. Sitafloxacin

Sitafloxacin is a N-1-fluorocyclopropyl quinolone with very potent bactericidal activity against MRSA, VRE, Pseudomonas aeruginosa and Bacteroides spp. The compound contains a cis-oriented (1R,2S)-2-fluorocyclopropylamine moiety which significantly contributes to the overall biological profile. Sitafloxacin is available as 50-mg tablets, and has a 10% fine granular preparation. The usual recommended dose is 50 or 100 mg twice daily. Sitafloxacin is approved in Japan for the treatment of urinary sepsis and community-acquired pneumonia [99]. Occurrence of photosensitivity adverse effect has been reported. With respect to ciprofloxacin, sitafloxacin is characterized by improved in vitro activity [100] against Gram- positive bacteria, anaerobes, and some Gram-negative non-fermenters [101]. It is also effective against methicillin-resistant S. aureus and vancomycin-resistant enterococci. The antibacterial activity of sitafloxacin is shown in Table 29. Complimentary Contributor Copy

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Table 29. Antibacterial activity of sitafloxacin

Organism MIC90 (g/ml) Organism MIC90 (g/ml) Gram-negative bacteria Gram-positive bacteria Escherichia coli 1 Stapylococcus aureus (MSSA) 0.025 Enterobacter aerogenes 1 Staphylococcus aureus (MRSA) 0.5 Enterobacter cloacae 0.25 Staphylococcus epidermidis (MSSE) 0.12 Klebsiella pneumoniae 0.12 Staphylococcus epidermidis (MRSE) 0.25 Proteus mirabilis 0.5 Streptococcus pneumoniae 0.06 Proteus vulgaris 0.12 Streptococcus pyogenes 0.03 Morganella morganii 0.25 Enterococcus faecalis 2 Providentia rettgeri 2 Enterococcus faecium 4–8 Providentia stuartii 2 Listeria monocytogenes – Serratia marcescens 0.5 Clostridium perfringens 0.05–0.125 Citrobacter freundii 0.5 Clostridum difﬁcile 0.12–0.25 Salmonella spp. 0.015 Peptostreptococcus spp. 0.008–0.125 Shigella spp. 0.015 Other bacteria Yersinia enterocolitica 0.015 Mycoplasma pneumoniae – Campylobacter jejuni 1 Mycoplasma hominis – Acinetobacter calcoaceticus 2 Chlamydia pneumoniae 0.06 Haemophilus inﬂuenzae ≤0.008 Chlamydia trachomatis 0.06 Moraxella catarrhalis ≤0.008 Mycobacterium tuberculosis 0.2 Neisseria meningitidis ≤0.008 Mycobacterium avium complex 6.2 Neisseria gonorrhoeae ≤0.008 Pseudomonas aeruginosa 4 Burkholderia cepacia 4 Stenotrophomonas 0.25 maltophilia Bacteroides fragilis 0.25–1

Sitafloxacin inhibited the growth of ciprofloxacin-susceptible and ciprofloxacin-resistant S. aureus isolates at 0.015-0.5 g/mL and produced rapid killing at twice their MIC values. No regrowth occurred by 24h at eight times the MIC of sitafloxacin [102]. In one study a low rate of spontaneous emergence of single-step resistant subclones was observed. In the case of levofloxacin-resistant strains of S. pneumoniae due to DNA gyrase and topoisomerase IV mutations, sitafloxacin remains potent and may be clinically useful [103]. Similar results were obtained for strains with efflux-mediated resistance [104]. Resistance to sitafloxacin and other fluoroquinolones appears to be uncommon among strains of M. tuberculosis, but when it occurs it is generally associated with multidrug resistance to first-line anti-tuberculosis agents. The pharmacokinetic parameters of sitafloxacin [105] are reported in Table 30. Absorption is slightly delayed if the drug is given with food rather than in the fasting state. Sitafloxacin is only slightly metabolized; some of the metabolites identified in serum, urine, and feces are the glucuronide, 7'-oxo, 7'S-hydroxy, glucuronide of 7'S-hydroxy and N- acetyl conjugate. An in vitro study, performed using human tissue specimens, showed that sitafloxacin modestly inhibited CYP1A1 and CYP1A2, but did not cause any inhibition of other CYP isoforms.

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Table 30. Pharmacokinetic properties of sitafloxacin

Parameter Value Value Value Value Route Oral Oral oral i.v. (1hr infusion) Dose (mg) 50 100 500 400

Cmax (g/ml) 0.51±0.14 1±0.14 4.65 5.53 Tmax (hrs) 1.2±0.5 1.2±0.5 1.25 1

AUC0-∞ (g•hr/ml) 2..62±0.52 5.55±1.22 28.1 25.4 t1/2 (hrs) 6.2±0.4 5.7±0.7 7.0 6.6 Vd/Fss (L/kg) 2.8±0.5 2.5±0.7 1.46–1.96 Cl/F (ml/min) 296 263 ClR/F (ml/min) 181 198 Urinary recovery (%) 26-86 42-101 Protein binding (%) 50 F (%) 70-94

5.7. Antofloxacin

Antofloxacin is a new fluoroquinolone agent developed jointly by the Shanghai Institute of Materia Medica, the Chinese Academy of Sciences, and Anhui Huanqiu Pharmaceutical Co., Ltd [106]. Antofloxacin has antibacterial activity in vitro against both Gram-positive and Gram-negative organisms, similar to levofloxacin [107]. The pharmacokinetic parameters of antofloxacin are reported in Table 31.

Table 31. Pharmacokinetic parameters of antofloxacin after single oral dose

Parameter 300 mg 400 mg 500 mg t1/2 (hrs) 7.46±3.44 7.49±1.91 9.77±4.6 Tmax (hrs) 1.09±0.58 1.4±0.48 1.62±0.58

Cmax (g/ml) 2.91±0.13 3.53±0.52 4.32±0.1

AUC0-∞ (gh/ml) 51.2±7.28 66.6±11.61 85.9±16.62 Vd/F (l/kg) 2.88±0.56 3.00±1.07 2.75±0.86 Cl/F (L*kg/h) 0.1±0.02 0.1±0.03 0.09±0.02

t1/2 (hrs) 20.30±4.35 20.22±3.33 20.61±4.58 Urinary recovery (mean, %) 45.63 43.60 40.03

5.8. Besifloxacin

Besifloxacin is a new fluoroquinolone anti-infective developed for ophthalmic use. A 0.6% suspension (Besivance™) of this fluoroquinolone is indicated for treatment of bacterial conjunctivitis, one of the most common ocular infections encountered in a primary care setting. Besifloxacin ophthalmic suspension 0.6% is formulated in a mucoadhesive polymer (DuraSite®, InSite Vision., Inc., Alameda, CA), designed to prolong the drug’s residence time on the ocular surface [108]. Besifloxacin has a chlorine atom at position 8 that is believed to increase potency against DNA gyrase and topoisomerase IV enzymes [109] and a C-7 amino- azepinyl substituent that is thought to contribute to the spectrum of activity and improved Complimentary Contributor Copy 222 Stefano Biondi and Mauro Panunzio potency against Gram-positive bacteria [110]. In vitro studies of spontaneous resistance development at a concentration 4XMIC resulted in resistance frequiencies < 3.3 × 10-10 for S. aureus and < 4.7 × 10-10 for S. pneumonia [110]. The in vitro activities of besifloxacin and six other topical ophthalmic antibacterial agents, including towards multidrug-resistant strains, are reported in Table 32 [111].

Table 32. Antibacterial activity of besifloxacin and comparators against Gram-positive and Gram-negative bacteria

MIC90 Organism Pheno- N BES CIP LEV TOB GAT MXF AZI type Gram-positive All 30 4 > 8 > 8 > 32 > 8 > 8 > 8 Meths 19 0.25 8 4 1 2 1 > 8 MethR 11 4 > 8 > 8 > 32 > 8 > 8 > 8 S. aureus CipS 14 0.12 0.5 0.25 1 0.25 0.06 > 8 CipR 16 4 > 8 > 8 > 32 > 8 > 8 > 8 All 15 4 > 8 > 8 8 > 8 > 8 > 8 Meths 6 0.03* 0.12* 0.12* 0.03* 0.06* 0.06* 0.5* S. epidermidis MethR 9 0.25* 2* 2* 4* 1* 1* > 8* CipS 9 0.03* 0.12* 0.12* 0.03* 0.06* 0.06* > 8* CipR 6 0.25* > 8* 8* 2* 1* 2* > 8* S. haemolyticus 101 1 > 8 > 8 32 8 8 > 8 S. hominis 50 1 > 8 > 8 32 4 4 > 8 S. lugdunensis 15 0.5 > 8 > 8 32 2 2 > 8 S. saprophyticus 101 0.12 0.5 0.5 0.06 0.25 0.12 > 8 S. warneri 50 1 > 8 > 8 8 4 4 > 8 S. agalactiae 100 0.06 1 1 64 0.25 0.25 > 8 All 35 0.06 1 1 32 0.25 0.12 > 8 S. pneumoniae PSSP 31 0.06 1 1 32 0.25 0.12 > 8 S. pyogenes 101 0.06 0.5 0.5 16 0.25 0.25 8 group C, F, G 50 0.06 0.5 0.5 16 0.25 0.12 > 8 streptococci Viridans streptococi 156 0.12 4 1 32 0.5 0.25 > 8 Gram-negative Citrobacter koseri All 100 0.25 0.06 0.12 1 0.12 0.25 > 8 All 40 0.015 0.008 0.015 4 0.008 0.03 1 Haemophilus Bla- 24 0.015 0.015 0.015 4 0.008 0.03 2 influenzae Bla* 16 0.03 0.008 0.015 2 0.008 0.03 1 Klebsiella oxytoca All 50 1 0.5 0.5 1 0.5 2 > 8 Legionella All 50 0.03 0.03 0.03 2 0.06 0.06 1 pneumophila Moraxella catarrhalis All 101 0.03 0.015 0.03 0.25 0.015 0.03 0.03 Morganella morganii All 51 4 >8 8 4 >8 >8 >8 R *MIC50 value is given. AZI: Azithromycin; BES: Besifloxacin; Bla: -lactamase; CIP: Ciprofloxacin; Cip : Ciprofloxacin-resistant; CipS: Ciprofloxacin-susceptible; GAT: Gatifloxacin; LEV: Levofloxacin; MethR: Methicillin-resistant; MethS: Methicillin-susceptible; MXF: Moxifloxacin; PSSP: Penicillin- susceptible S. pneumoniae; TOB: Tobramycin.

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Besifloxacin showed bactericidal activity against S. aureus, S. epidermidis, S. pneumoniae and H. influenzae, with a similar or greater potency than those of comparator fluoroquinolones [112]. After a single topical ocular administration [113] to healthy subjects, the maximum besifloxacin concentration in tears was 610 g/g with concentrations decreasing to approximately 1.6 g/g by 24 h. Total exposure of besifloxacin in tears was 1232 g h/g. Elimination from tears occurred with an estimated half-life of 3.4 h. Based on these pharmacokinetic parameters, the Cmax/MIC90 ratio for besifloxacin in tears was determined to be ≥ 1220, while the AUC0 – 24/MIC90 ratios were determined to be ≥ 2500 against S. pneumoniae, S. aureus, S. epidermidis, and H. influenza [114]. The approved dosage is one drop in the affected eye(s) t.i.d., at 4-12 hour intervals for 7 days.

6. LIPOPEPTIDES

Lipopeptides are natural or semisynthetic compounds characterized by the presence of a lipophilic, fatty acid attached to an amino sugar of the glycopeptide core structure [192]. Lipoglycopeptide antibiotics are more effective than vancomycin against MRSA, and may be active against vancomycin resistant strains (VRSA, VRE).

6.1. Daptomycin

Daptomycin (Cubicin®) is an injectable, high molecular weight cyclic lipopeptide containing a hydrophilic core and a hydrophobic tail produced by Streptomyces roseosporus. It is more active in vitro than glycopeptides against a wide range of Gram-positive aerobic and anaerobic organisms, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE); it is not active against Gram-negative pathogens. It is inactivated by pulmonary surfactant and therefore has no role in treatment of pneumonia [115]. It also does not reliably cross the blood-brain barrier, and may not prove to be a useful agent for treatment of meningitis. The initial development program started in the 1980s was terminated due to failures of treatment of endocarditis with 2 mg/kg and the occurrence of potential drug-induced myopathy. Clinical development was restarted by Cubist in 1997 by evaluating once-daily dosing regimens [116]. Daptomycin was approved for use in the USA in 2003 and in Europe in 2006. The mechanism of action of daptomycin involves calcium- dependent bacterial membrane depolarization and consequent rapid concentration-dependent bactericidality [117] In the presence of physiologic levels of calcium, the hydrophobic tail of the molecule inserts irreversibly into the cell membrane of Gram-positive bacteria,. followed by formation of pores, which leads to loss of K+ and membrane depolarization. The observation that daptomycin is bactericidal even against stationary-phase S. aureus supports this hypothesis [118]. In addition to K+, also Mg2+ and ATP loss may also play a role [119]. The antibacterial activity of daptomycin and comparator drugs is reported in Table 33 [120].

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224 Stefano Biondi and Mauro Panunzio

Table 33. Antibacterial activity of daptomycin and comparators against Gram-positive organisms

Organism Drug MIC50 MIC90 Range (g/ml) (g/ml) (g/ml) S. aureus MSSA Daptomycin 0.25 0.5 ≤0.12-2 Clindamycin ≤0.25 ≤0.25 ≤0.25- >2 Erythromycin ≤0.25 > 2 ≤0.25->2 Levofloxacin ≤0.5 ≤0.5 ≤0.5->4 Linezolid 2 2 0.25-4 Tetracycline ≤4 ≤4 ≤4->8 TMP/SMX* ≤0.5 ≤0.5 ≤0.5->2 Vancomycin 1 1 ≤0.12-2 MRSA Daptomycin 0.25 0.5 ≤0.12-4 Clindamycin ≤0.25 > 2 ≤0.25->2 Erythromycin >2 >2 ≤0.25->2 Levofloxacin >4 >4 ≤0.5->4 Linezolid 2 2 ≤0.25->8 Tetracycline ≤4 ≤4 ≤4->8 TMP/SMX ≤0.5 ≤0.5 ≤0.5->2 Vancomycin 1 1 0.25-4 Coagulase-negative staphylococci Daptomycin 0.25 0.5 ≤0.12-4 Oxacillin > 2 > 2 ≤0.25->2 Clindamycin ≤0.25 > 2 ≤0.25->2 Erythromycin > 2 > 2 ≤0.25->2 Levofloxacin 4 > 4 ≤0.5->4 Linezolid 1 1 ≤0.25->8 Tetracycline ≤4 >8 ≤4->8 TMP/SMX ≤0.5 > 2 ≤0.5->2 Vancomycin 1 2 ≤0.12-8 Vancomycin-resistant E. faecalis Daptomycin 0.5 1 ≤0.06-2 Ampicillin ≤2 ≤2 ≤2-> 16 Gentamicin > 1000 > 1000 ≤500->1000 Linezolid 1 2 0.5->8 Teicoplanin >16 > 16 ≤2->16 Vancomycin-resistant E. faecium Daptomycin 2 4 ≤0.06-8 Ampicillin >16 >16 ≤1->16 Gentamicin ≤500 >1000 ≤500->1000 Linezolid 1 2 ≤0.06->8 Q/D** 0.5 1 ≤0.25->2 Teicoplanin >16 >16 ≤2->16 *Trimethoprim–sulfamethoxazole. **Quinopristin-Dalfopristin.

The approved dose of daptomycin is 4-6 mg/kg every 24 hrs, although many clinicians now use up to 8 or 10 mg/kg for serious infections [121]. Resistance mechanisms to daptomycin include mutations in mprF (encoding lysylphosphatidylglycerol synthetase), yycG (encoding histidine kinase) and rpoB and rpoC (encoding RNA polymerase subunits) [122]. Increases in glycopeptide MICs are often accompanied by increases in the MICs of daptomycin, suggesting a common mechanism [123]. The pharmacokinetic profile of daptomycin after single and repeated dose in healthy subjects are reported in Table 34 [124]. Complimentary Contributor Copy

A 21st Century Contribution to the Antibacterial Armamentarium 225

Table 34. Pharmacokinetic properties of daptomycin

Parameter Value Dose (mg/kg) 4 6 8 4 6 8 Day 1 7 Cmax (g/ml) 54.6±5.4 86.4±7.1 116.3±10.1 57.8±3.0 98.6±12.0 133.0±13.5 AUC0-∞ 425±58 705±67 1127±161 494±75 747±91 1130±117 (g•hr/ml) t1/2 (hrs) 7.4±0.9 7.8±1.0 9.6±1.1 8.1±1.0 8.9±1.3 9.0±1.2 Vd (l/kg) 0.101±0.013 0.096±0.009 0.099±0.014 0.096±0.009 0.104±0.013 0.092±0.012 ClT (ml/min) 9.6±1.3 8.6±0.8 7.2±1.1 8.3±1.3 8.1±1.0 7.1±0.8 ClR,unbound 84.6±33.6 59.5±16.7 50.0±5.2 62.9±18.5 59.8±13.8 40.9±6.28 (ml/min) Urinary 53±10.8 47.4±11.5 52.1±5.2 recovery (%) Protein binding 92 93 91 92 92 91 (%) Ra* 1.4±0.2 1.2±0.1 1.2±0.1 *Ra = accumulation index

Daptomycin has linear pharmacokinetics, with a half-life of 9 hrs. For all three doses of daptomycin studied, the Cmaxss, AUCs were about 20% greater at steady state (day 7) than following a single dose (day 1). Urinary excretion of unchanged drug varies among studies, between 37% and 68% and about 6% is eliminated in the feces. The pharmacodynamics indexes that best correlate in vivo activity with clinical efficacy are AUC0-24/MIC and Cmax/MIC [125]. Daptomycin has a post-antibiotic effect (PAE) of 5 hrs against S. aureus in an animal model [125a]. An in vitro study showed a dose-dependent PAE of 1-6.3 hrs against S. aureus and 0.6-6.7 hrs against E. faecalis [126]. Overall, daptomycin is the first in class lipopeptide showing antibacterial activity against Gram-positive organisms. It has rapid bactericidal activity and a unique mechanism of action. It has favorable pharmacokinetic properties allowing once-daily dosing. Daptomycin has been approved for treatment of complicated skin and skin-structure infections, bacteremia, and right-sided endocarditis. Toxicity is rare with currently recommended dosing, though there have been case reports of rhabdomyolysis [127]. As reduced susceptibility to vancomycin has been often accompanied by reduced susceptibility to daptomycin, its clinical utility might be limited with this type of bacteria. It is worth noting that in a large study of daptomycin efﬁcacy in patients with S. aureus bacteremia with or without right-side endocarditis, emergence of daptomycin resistance appeared to occur relatively commonly [128]. About one-third (6/19) of daptomycin-treated patients who suffered microbiologic failure were found to have isolates that had developed resistance to daptomycin (MIC ≥2 mg/ml) during treatment.

7. TETRACYCLINES

Tetracyclines are an old class of broad-spectrum antibiotics including Gram-positive, Gram-negative, intracellular bacteria and protozoan pathogens [129]. Tetracyclines

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226 Stefano Biondi and Mauro Panunzio commonly are divided in short- and long-acting, and more recently it has been expanded to include “glycylcyclines” represented by the recently approved tigecycline.

7.1. Tigecycline

Tigecycline is the first glycylcycline to become available for clinical use. Structurally, it is related to minocycline, but has a glycylamido moiety attached to the 9-position of minocycline [130] which confers a broader spectrum of activity and overcome some tetracycline resistance mechanisms. Like other tetracyclines, it inhibits bacterial protein synthesis, but is able to bind to target sites with higher affinity, and overcomes the two major tetracyclines resistance mechanisms drug-specific efflux and ribosomal protection [131]. Tigecycline is active against many Gram-positive and Gram-negative organisms, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci [132] and - lactamase-producing enterobacteriaceae. It is also active against many anaerobic bacteria, as well as atypical pathogens, including rapidly growing, non-tuberculous mycobacteria [133]. However, tigecycline has no activity against Pseudomonas aeruginosa [134], and decreased activity to some members of the Enterobacteriaceae, in particular Proteus spp., Providencia spp., and Morganella spp. The antibacterial activity [134-135] of tigecycline is reported in Table 35. Tigecycline binds reversibly to the 30S ribosomal subunit, inhibiting protein translation by preventing the amino-acyl tRNA entry into the A site of the ribosome [136]. Tigecycline has higher binding affinity to the ribosomal target sites than tetracyclines [137]. The post- antibiotic effects of tigecycline for S. pneumoniae and E. coli in a murine thigh infection model were 8.9 and 4.9 hours respectively, following an intravenous administration of a 3 mg/kg dose [138]. Overexpression of mepA, a multidrug and toxic compound efflux pump, may contribute to decreased susceptibility of tigecycline in S. aureus [139]. Tigecycline’s activity may be compromised in certain Gram-negative organisms, especially those with a multidrug efflux pump, e.g., AcrAB, AcrEF, or AdeABC [140]. A specific enzymatic resistance mechanism to tigecycline has been identified [141]. Tigecycline is a substrate for TetX, generating 11-hydroxy tigecycline, which is a weak inhibitor of protein translation. Tigecycline is administered by intravenous infusion of 30-60 minutes at the recommended loading dose of 100 mg, followed by 50 mg every 12 hours for 5-14 days. The pharmacokinetic profile of tigecycline is reported in Table 36. Radiolabeling experiments in rats have shown that elimination of tigecycline was slower from tissue than from plasma, yielding high tissue to plasma concentrations. In a study performed in healthy subjects who received 100 mg followed by 50 mg every 12 hours the tigecycline AUC0–12 (134 g/ml*h) in alveolar cells was approximately 78-fold higher than the AUC0–12 in the serum, and the AUC0–12 (2.28 g/ml) in epithelial lining fluid was approximately 32% higher than the AUC0–12 in serum. Concentrations in patients undergoing surgery at 4 hours after administration of 100 mg of tigecycline were higher in gallbladder (38-fold, n =6), lung (8.6-fold, n =1), and colon (2.1-fold, n =5) relative to serum [142]. Tigecycline is eliminated primarily by the liver (59%) via biliary excretion of the unchanged drug in the feces [143] Glucuronidation and excretion of tigecycline, inactive epimer and N- actetyl-9-aminominocycline are secondary elimination routes [144]. Tigecycline is not a Complimentary Contributor Copy

A 21st Century Contribution to the Antibacterial Armamentarium 227 substrate, inhibitor, or inducer of common cytochrome P450 enzymes [133], therefore drug- drug interactions are unlikely. The best pharmacodynamics driver that correlates with tigecylcine probability of clinical cure is AUC/MIC [144]. Tigecyline is a modified tetracycline that is able to overcome common tetracycline resistance mechanisms including efflux pump and ribosomal modification of the binding site. It has broad spectrum activity but it lacks efficacy against P. aeruginosa. The safety profile is similar to other tetracyclines and its use requires careful clinical monitoring.

Table 35. Antibacterial activity of tigecycline

Organism MIC50 (g/ml) MIC90 Range (g/ml) (g/ml) Gram-positive S.aureus methicillin resistant 0.12–0.25 0.25–0.5 0.06–1 S.aureus methicillin sensitive 0.12 0.12–0.5 ≤0.06–1 S.epidermidis methicillin resistant 0.12–0.25 0.25–0.5 ≤0.06–2 S.epidermidis methicillin sensitive 0.12–0.25 0.25–0.5 0.06–2 Enterococcus faecalis, vancomycin susceptible ≤0.06–0.12 0.12–0.25 ≤0.06–2 Enterococcus faecium, vancomycin susceptible ≤0.06 ≤0.06–0.25 ≤0.06–0.5 Enterococcus faecium, vancomycin resistant ≤0.06 ≤0.06–0.12 Streptococcus pyogenes 0.06 0.06–0.12 0.06–0.5 Streptococcus agalactiae 0.03–0.06 0.12–0.25 0.03–1 Streptococcus pneumonia ≤0.03–0.12 0.06–0.5 ≤0.06–1 Gram-negative Enterobacter spp. 0.5 1–2 0.06–8 Enterobacter cloacae 0.5–1 1–2 0.5 –16 Enterobacter aerogenes 0.5–1 1–2 0.06–16 Escherichia coli 0.12–0.25 0.25–0.5 0.03–4 Haemophilus inﬂuenzae 0.12–0.5 0.25–1 ≤0.008–2 Klebsiella spp. 0.5 1–2 0.5–8 Klebsiella pneumoniae ESBL+ 1 2 0.12–8 Moraxella catarrhalis 0.06–0.12 0.12–0.25 ≤0.03–0.5 Morganella morganii 2 4 1–8 Proteus spp. 4 4–8 1–16 Serratia spp. 1–2 1–4 0.5–16 Stenotrophomonas maltophilia 0.38–1 1.5–4 0.25–8 Pseudomonas aeruginosa 8 –16 16–≥32 0.12–64 Acinetobacter spp. 0.5–3 1–6 0.03–8 Anaerobes Bacteroides fragilis group 0.5–1 0.5–8 0.06–32 Clostridium difﬁcile 0.06–0.12 0.06–0.25 ≤0.06–2 Clostridium perfringens 0.12 1 0.06–2 Fusobacterium spp. 0.03–0.12 0.06–0.12 ≤0.015–0.25 Peptostreptococcus spp. 0.06 0.06–0.125 0.06 – 0.12

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228 Stefano Biondi and Mauro Panunzio

Table 36. Pharmacokinetic properties of tigecycline

Parameter Single dose 100 mg Repeated dose 50 mg Cmax (g/ml) 1.45 0.87 Tmax (min) 30 30 AUC0-∞ (g*h/ml) 5.19 AUC0-24 (g*h/ml) 4.70 Vd L) 568 639 Cl (L*kg/h) 21.8 23.8 ClR (L*kg/h) 38.0 51.0 t1/2 (hrs) 27.1 42.4 Urinary recovery (%) <30 <30 Protein binding (%) 71-89

8. PLEUROMUTILINS

Pleuromutilin is a natural product obtained from the fungi Pleurotus mutilus that was first reported several decades ago and has modest antibacterial activity, mainly against Gram- positive bacteria [145].

Table 37. Antibacterial activity of retapamulin and comparator drugs versus clinical isolates [151]

Organism Substance MIC50 (g/ml) MIC90 (g/ml) Range (g/ml) S. aureus Retapamulin 0.06 0.12 0.004-0.5 Bacitracin 128 >128 ≤0.006->128 Clindamycin 0.12 >32 0.03->32 Fusidic acid 0.12 2 0.03->32 Gentamycin 0.25 4 ≤0.03->64 Mupirocin 0.25 4 0.06->256 S. aureus MRSA Retapamulin 0.12 Bacitracin >128 Clindamycin >32 Fusidic acid 2 Gentamycin 32 Mupirocin 16 S. pyogenes Retapamulin 0.03 0.03 0.004-0.25 Bacitracin 2 32 0.25->128 Clindamycin 0.03 0.12 ≤0.015->32 Fusidic acid 4 8 0.5-32 Gentamycin 4 16 0.5->64 Mupirocin 0.12 0.5 ≤0.03->256 Propionibacterium Retapamulin 0.03 0.03 ≤0.015-0.03 acnes Bacteroides fragilis Retapamulin 0.25 64 0.03-64 Clostridium perfringens Retapamulin 0.125 1 0.03-1 Other Clostridium Retapamulin 1 16 ≤0.015->64 Prevotella spp. Retapamulin ≤0.015 0.06 ≤0.015-0.125 Porphyromonas spp. Retapamulin ≤0.015 0.03 ≤0.015-0.03 Fusobacterium spp. Retapamulin 0.125 1 ≤0.015-1 Complimentary Contributor Copy

A 21st Century Contribution to the Antibacterial Armamentarium 229

8.1. Retapamulin

Retapamulin is a semisynthetic pleuromutilin that has been approved in the USA for topical treatment of impetigo [146], a skin infection caused by bacteria, and approved in Europe [147] (where it is marketed as Altargo) for treatment of impetigo and infected small lacerations, abrasions, or sutured wounds. Retapamulin inhibits bacterial protein synthesis by binding to a site on the 50S subunit of the bacterial ribosome through an interaction that is different from that of other ribosomotropic antibiotics [148]. Resistance to retapamulin has been observed in strain with mutations in the rplC gene encoding the ribosomal protein L3 [149]. The cfr gene, which encodes a rRNA methyltransferase, confers resistance to retapamulin as well as to phenicols, lincosamides, streptogramin A [150]. Efflux mechanisms have also been reported to reduce susceptibility to retapamulin. The antibacterial activity of retapamulin and comparators is reported in Table 37. Systemic exposure following topical application of retapamulin 1% ointment through intact skin is very low. The geometric mean 2 Cmax value in plasma after application to 200 cm of abraded skin is 9.75 ng/ml on day 1 and 8.79 ng/ml on day 7. In clinical studies of secondarily infected open wounds, the efficacy of retapamulin was inadequate in patients with infections caused by MRSA. The reason for the reduced clinical efficacy observed in these patients is unknown. Two pleuromutilins, tiamulin and valnemulin, have been approved for veterinary uses, and retapamulin is the first to be approved for use in humans.

9. CEPHALOSPORINS: CEFTOBIPROLE MEDOCARIL, CEFTAROLINE FOSAMIL, CEFTOLOZANE + TAZOBACTAM, CEFTAZIDIME + AVIBACTAM

Nearly all recently developed -lactam antibiotics have been studied in combination with -lactamase inhibitors, because bacteria have evolved -lactamases that hydrolyze (inactivate) all -lactam classes, including carbapenems. The antibacterial pipeline contains several combinations of novel -lactamase inhibitors with “old” -lactam antibiotics and “old” -lactamase inhibitors with new -lactam products.

9.1. Ceftobiprole Medocaril

Ceftobiprole (BAL 9141, RO 63-9141) and its water-soluble prodrug, ceftobiprole medocaril (BAL 5788, RO-5788), are cephalosporin that were developed to combat methicillin-resistant Staphylococcus aureus (MRSA) [152] and vancomycin-resistant S. aureus (VRSA) [153]. Ceftobiprole inhibits the activity of a modified penicillin-binding proteins (PBP) of MRSA (PBP2a) and penicillin-resistant Streptococcus pneumoniae (PRSP) (PBP1A, 1B, and PBP2X) and is relatively stable to hydrolysis from AmpC -lactamases and narrow-spectrum-acquired cephalosporinases [154]. It shows bactericidal activity typical of -lactam antibiotics [155]. Ceftobiprole has activity against a wide range of aerobic,

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230 Stefano Biondi and Mauro Panunzio facultative, and some anaerobic Gram-positive and Gram-negative bacteria as shown in Table 38.

Table 38. Antibacterial activity of ceftobiprole

Organism MIC50 MIC90 (g/ml) (g/ml) Gram-positive Staphylococcus aureus methicillin resistant (MRSA) 1 2 Staphylococcus aureus methicillin sensitive (MSSA) 0.25 0.5 Staphylococcus coagulase negative methicillin susceptible ≤0.12 0.25 Staphylococcus coagulase negative methicillin resistant 1 2 Staphylococcus epidermidis methicillin susceptible 0.25 1 Staphylococcus epidermidis methicillin resistant 1 2 S.pneumoniae penicillin susceptible ≤0.006 ≤0.006 S.pneumoniae penicillin intermediate 0.06 0.25 S.pneumoniae penicillin resitant 0.25 0.5 Viridans streptococci penicillin susceptible ≤0.015 0.06 Viridans streptococci penicillin intermediate 0.03 0.25 Viridans streptococci penicillin resistant 0.5 1 -Hemolytic streptococci (groups A, B, C, F, G) ≤0.015 ≤0.015 Enterococcus faecalis 0.5 4 Enterococcus faecium >32 >32 Enterococcus spp. 0.5 >32 Gram-negative Citrobacter freundii 0.03 0. Citrobacter koseri ≤2 ≤2 Citrobacter spp. (derepressed AmpC) 2 >32 Enterobacter cloacae 0.06 0.12 Enterobacter cloacae (derepressed AmpC) 8 >32 Enterobacter aerogenes 0.03 >32 Escherichia coli ≤0.06 0.12 Escherichia coli ESBL producing >32 >32 Escherichia coli AmpC 0.12 0.5 Klebsiella pneumonia 0.03 0.06 Klebsiella pneumoniae ESBL producing 64 128 Klebsiella oxytoca 0.06 0.5 Proteus mirabilis ≤0.06 0.12 Proteus mirabilis ESBL producing >32 >32 Indole-positive proteaceae ≤0.015 >32 Serratia spp. 0.06 8 Serratia marcescens ceftazidime.resistant >32 >32 Salmonella spp. 0.03 0.03 Shigella spp. 0.03 0.03 Haemophilus influenza 0.03 0.25 Haemophilus influenzae -lac + 0.06 0.25 Moraxella catarrhalis 0.06 0.5 Moraxella catarrhalis -lac + 0.12 0.5 Neisseria meningitides ≤0.002 0.004 Neisseria gonorrhoeae 0.03 0.06 Complimentary Contributor Copy

A 21st Century Contribution to the Antibacterial Armamentarium 231

Organism MIC50 MIC90 (g/ml) (g/ml) Pseudomonas aeruginosa 4 16 Pseudomonas aeruginosa ceftazidime-resistant 16 >32 Stenotrophomonas maltophila >64 >64 Acinetobacter spp. imipenem-susceptible 0.5 >32 Acinetobacter spp. imipenem-resistant 32 >32 Acinetobacter baumanii 2 16 Burkholderia cepacia 8 64 Chriseobacterium indologenes 16 64 Chryseobacterium meningosepticum 32 64 Comamonas Acidovorans 4 16 Ochrobactrum anthropic 1 2 Anaerobes Actinomyces spp. 0.06 8 Bacteroides fragilis 16 >128 Bacteroides thetaiotaomicron 16 32 Bacteroides distasonis 1 >64 Bacteroides vulgatus 4 >64 Porphyromonas spp. ≤0.03 16 Prevotella bivia 4 64 Prevotella buccae 0.06 32 Fusobacterium spp. 0.12 8 Fusobacterium nucleatum ≤0.016 ≤0.016 Lactobacillus spp. 1 >128 Clostridium spp. 2 64 Clostrium difficile 4 8 Clostridium perfringens ≤0.016 ≤0.016 Propionibacterium acnes 0.06 0.125 Anaerococcus prevoti 0.03 0.125 Micromonas micros 0.06 0.25 Finegoldia magna 0.25 0.5 Peptostreptococcus anaerobius 2 32

Ceftobiprole has a low potential to select for resistance [156]. The highest MIC achieved after 50 passages in 1 of 10 strains was 8 g/ml, which represented a 4-fold increase in the initial MIC. PBP mutations in ceftobiprol- resistant MRSA strains have been observed by in vitro selection [157]. Ceftobiprole medocaril is a water-soluble prodrug that is rapidly converted in plasma by esterases [158], into ceftobiprole [159]. The pharmacokinetic properties of ceftobiprole medocarril are reported in Table 39. The concentration of ceftobiprole in lungs has been evaluated in a murine model where epithelial lining fluid concentrations were in the range 60-94% of serum concentration [160]. Ceftobiprole does not interact with cytochrome P450 or p-glycoproteins and is eliminated by the kidneys via glomerular filtration. The pharmacodynamics parameter that best predict clinical success rate for ceftobiprole is fT>MIC. At 500 mg i.v. every 8h, the probabilities of achieving 40–60% fT>MIC exceeded 90% for MICs ≤4 and ≤2 g/ml, respectively. Ceftobiprole is approved in thirteen European countries for the treatment of community- acquired pneumonia (CAP) and hospital-acquired pneumonia (HAP) in adults, excluding ventilator-associated pneumonia (VAP). FDA has requested additional phase 3 studies. Complimentary Contributor Copy

232 Stefano Biondi and Mauro Panunzio

Table 39. Pharmacokinetic properties of ceftobiprole medocaril following multiple 2 hr intravenous infusion of ceftobiprole 500 mg every 8h in healthy adults

Parameter Value Cmax (g/ml) 33.0 ± 4.83 Tmax (min) 2 AUC0-8 (g*h/ml) 102 ± 11.9 Vdss (L) 15.5 ± 2.33 Cl (L*kg/h) 4.98 ± 0.58 ClR (L*kg/h) 4.28 ± 0.57 t1/2 (hrs) 3.3 ± 0.3 Urinary recovery (%) >80 Protein binding (%) 16

Table 40. In vitro antibacterial activity of ceftaroline against selected pathogens

Organism MIC50 MIC90 Range (g/ml) (g/ml) (g/ml) Gram-positive S.aureus methicillin susceptible 0.25 0.5 0.13–0.5 S.aureus methicillin resistant 1 2 0.012–2 S.aureus vancomycin intermediate 1 2 0.25–4 Staphylococcus coagulase negative, methicillin- 0.06 0.25 ≤0.016–0.5 susceptible Staphylococcus coagulase negative, methicillin- 0.25 0.5 ≤0.016–2 resistant Staphylococcus coagulase negative, vancomycin- 1 2 ≤0.016–2 intermediate S. epidermidis, methicillin-susceptible 0.13 0.13 0.06–0.13 S. epidermidis, methicillin-resistant 0.5 1 0.25–1 Streptococcus pneumoniae, penicillin-susceptible ≤0.008 0.06 ≤0.008–0.13 S. pneumoniae, penicillin-intermediate 0.13 0.13 0.03–0.25 S. pneumoniae, penicillin-resistant 0.13 0.25 0.06–0.5 S. pyogenes ≤0.016 ≤0.016 ≤0.016 S. agalactiae 0.016 0.03 0.016-0.06 Viridans streptococci, penicillin-susceptible ≤0.016 0.03 ≤0.016–1 Viridans streptococci, penicillin-intermediate 0.03 0.12 ≤0.016–0.5 Viridans streptococci, penicillin-resistant 0.25 1 0.03–8 Other beta-hemolytic streptococci ≤0.016 ≤0.016 ≤0.016–0.03 E. faecalis, vancomycin-susceptible 2 16 0.5–>32 E. faecalis, vancomycin-resistant 4 8 1–16 E. faecium, vancomycin-susceptible 16 >32 0.5–>32 E. faecium, vancomycin-resistant (vanA) >32 >32 16->32 Bacillus spp. 4 8 0.06-32 Gram negative Acinetobacter spp. 4 8 1-32 Acinetobacter baumannii 16 >32 2->32 Alcaligenes spp. >32 >32 16->32 Citrobacter freundii 0.25 64 0.13–128 E. cloacae, wild type 0.12 32 0.03–>32 Complimentary Contributor Copy

A 21st Century Contribution to the Antibacterial Armamentarium 233

Organism MIC50 MIC90 Range (g/ml) (g/ml) (g/ml) E. cloacae, ESBL-producing >32 >32 4->32 E. coli, wild type 0.06 0.12 ≤0.016–0.25 E. coli, ESBL-producing >32 >32 0.5->32 H. influenzae -lac - ≤0.016 ≤0.016 ≤0.016 H. influenzae, -lac + ≤0.016 ≤0.016 ≤0.016-0.25 H. influenzae, -lac -- ampicillin resistant ≤0.016 0.03 ≤0.016–0.03 Klebsiella oxytoca 0.25 2 0.03–>128 K. pneumoniae, wild type 0.06 0.5 0.03–4 K. pneumoniae, ESBL-producing >32 >32 32->32 Moraxella catarrhalis 0.25 0.5 ≤0.008–0.5 Morganella morganii 0.13 8 0.03–16 Neisseria meningitidis ≤0.016 ≤0.016 ≤0.016 P. mirabilis, wild type 0.12 0.12 0.03–4 P. mirabilis, ESBL-producing >3 >32 4->32 Proteus vulgaris 8 64 0.25-64 Pseudomonas aeruginosa 16 128 1–>128 Salmonella spp. 0.13 0.25 0.13–2 Serratia marcescens 1 32 0.5->128 Stenotrophomonas maltophilia >32 >32 32->32 Anaerobes Bacteroides fragilis 32 >32 4->32 Clostridium difficile 2 4 0.06-8 Clostridium spp. 0.06 1 ≤0.06-1 Prevotella spp. 8 >32 ≤0.03->32

9.2. Ceftaroline Fosamil

Ceftaroline is the active metabolite of ceftaroline fosamil, formerly PPI-0903 or TAK- 599, an N-phosphonoamino water-soluble cephalosporin prodrug [161]. Similar to ceftobiprole, it shows potent activity against MRSA, while maintaining the broad Gram- negative activity and favorable safety proﬁle of cephalosporins [162]. Ceftaroline was approved by the FDA and is indicated for acute bacterial skin and skin structure infections (ABSSSI) and community-acquired bacterial pneumonia (CABP). Ceftaroline has a high binding affinity for common PBP targets for S. pneumoniae (PBP-1a, -2b, and -2x) and S. aureus (PBP-1, -2, -2a, and -3) [163]. Notably, ceftaroline demonstrates high afﬁnity for PBP-2a, a peptidoglycan trans-peptidase that is responsible for -lactam resistance in MRSA. This accounts for its strong antibacterial activity against MRSA [164]. The antibacterial activity of ceftaroline is reported in Table 40. Like many old generation cephalosporins ceftaroline is susceptible to -lactamases and has been studies in combination with avibactam, a novel -lactamase inhibitor (vide infra). The pharmacokinetic profile of ceftaroline following intravenous administration of ceftaroline fosamil is shown in Table 41 [165]. Ceftaroline is primarily excreted in the urine as the active compound, together with its inactive metabolite M1. About 50.7% of active ceftaroline and 7.4% of the metabolite M1 is recovered in the urine. Ceftaroline shows little cytochrome P450-dependent metabolism in vitro. Complimentary Contributor Copy

234 Stefano Biondi and Mauro Panunzio

Table 41. Ceftaroline pharmacokinetics following multiple intravenous infusions of the prodrug in healthy males

Parameter Value Value Dose (mg) 600 600 Dosing interval (hrs) 8 12

Cmax (g/ml) 21 21.33 Tmax (min) 60 55

AUC0-∞ (g*h/ml) 56 56.25 Vdss (L) 35.3 Cl (L*kg/h) 9.6

t1/2 (hrs) 2.6 2.66 Urinary recovery (%) 50 Protein binding (%) <20

Ceftaroline has time-dependent, bactericidal activity in vitro and in vivo [166]. Murine thigh and lung infection models were used to demonstrate that the best PK/PD index that correlated with efﬁcacy of ceftaroline is the percentage of time that the free drug concentration remains above the MIC (% fT>MIC) [214]. Ceftaroline fosamil is a cephalosporin developed speciﬁcally for its enhanced activity against Gram-positive pathogens [166-167]. It has demonstrated good in vitro activity against most Gram-positive isolates including drug resistant strains of S. aureus and S. pneumoniae. The Gram-negative coverage includes common respiratory pathogens [168] and members of the Enterobacteriaceae. Ceftaroline has poor anaerobe and atypical coverage, and does not have useful activity against P. aeruginosa, ESBL producers, or AmpC-producers, restricting its utility as a monotherapy agent for healthcare–associated infections to drug resistant Gram- positives. Overall, ceftaroline could offer a good alternative to currently used drugs for critically ill patients with MRSA [169].

9.3. Ceftolozane + Tazobactam

CXA-101 (ceftolozane) is a cephalosporin [170] discovered in 2004 by the Fujisawa Pharmaceutical Co., Ltd. (now Astellas) and originally named FR264205 [171]. The compound is particularly active against MDR P. aeruginosa, including isolates from chronically-infected cystic fibrosis patients [172], due to enhanced affinity of the β-lactam for the PBPs of this species, reported stability to many -lactamases including AmpCs (but not ESBLs, KPC carbapenemases, or metallo-β-lactamases), and indifference to efflux pumps [173]. However, it also has relatively poor activity towards other Gram-negative pathogens, as well as towards multidrug-resistant Gram-positive cocci and anaerobes. Ceftolozane MICs against clinical isolates of P. aeruginosa are 8- to 16-fold lower than those of ceftazidime [157-158] and are little affected by MexAB-OprM overexpression and/or OprD deletion. Susceptibility of ceftolozane to ESBLs is evidenced by the 4- to 128-fold increase in MIC by producers of this β-lactamase [174]. CXA-201 (Zebraxa™) is a 2:1 combination of ceftolozane and tazobactam, a sulfone penam β-lactamase inhibitor, that has been developed

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A 21st Century Contribution to the Antibacterial Armamentarium 235 by Cubist. Zerbaxa is indicated for treatment of the following infections caused by susceptible microorganisms:

1) complicated intra-abdominal infections, used in combination with metronidazole 2) complicated urinary tract infections, including Pyelonephritis.

Addition of tazobactam at a fixed concentration of 8 µg/ml restored the in vitro susceptibility of 93% of ESBL producers and 95% of the AmpC overproducers examined. However, tazobactam was unable to lower MICs, for Enterobacteriaceae producing KPCs [175], Cubist recently filed a patent covering use of ceftolozane/tazobactam (2:1) for treating pulmonary infections [176]. Ceftolozane/tazobactam is relatively stable against P. aeruginosa resistance mechanisms, such as hydrolysis by AmpC, efflux, and porins deficiencies [177]. Addition of tazobactam has no significant impact on the antipseudomonal activity of ceftolozane [178]. The spectrum of microbiological activity in comparison with other - lactam antibiotics is shown in Table 42 [179].

Table 42. In vitro activity of ceftolozane/tazobactam and selected comparators

Ceftolozane/ Piperacillin/ Ceftazidime tazobactam tazobactam Isolates MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 (g/ml) (g/ml) (g/ml) (g/ml) (g/ml) (g/ml) S. aureus 16 32 ≤1 ≤1 16 16 S. salivarius 1 2 S. anginosus 1 2 S. constellatus 0.5 2 S. pneumoniae ≤0.12 1 ≤1 ≤1 H. influenzae ≤0.12 ≤0.12 ≤1 ≤1 E. coli 0.2 0.5 2 16 0.12 4 E. coli ESBL 0.5 32 16 >64 16 >32 K. pneumoniae 0.25 32 4 >64 0.25 >32 K. pneumoniae ESBL 4 >32 64 >64 32 >32 K. oxytoca 0.25 1 Enterobacter spp. 0.5 8 4 >64 0.5 >32 Enterobacter cloacae 0.25 8 Enterobacter aerogenes 0.25 2 4 32 0.5 >32 Serratia marcescens 0.5 1 2 16 0.12 1 Citrobacter spp. 0.25 32 2 32 0.25 >32 C. freundii ≤0.12 ≤0.12 2 16 0.5 >32 Indole-positive Proteae 0.25 1 ≤0.5 1 0.12 8 Proteus mirabilis 0.5 0.5 M. morganii ≤0.12 0.25 ≤1 ≤1 ≤0.5 4 P. aeruginosa 0.5 32 8 >64 2 >32 P. aeruginosa 1 8 32 256 16 128 carbapenem-resistant P. aeruginosa MDR 4 >32 >64 >64 32 >32 S. maltophilia 32 >64 256 >512 >32 >32 A. baumanii 0.5 2 2 128 8 32 B. fragilis 1

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236 Stefano Biondi and Mauro Panunzio

The antipseudomonal activity of ceftolozane/tazobactam is the most potent among all currently available -lactams and has been shown to be active against carbapenem-resistant P. aeruginosa including the majority of MDR isolates [179b]. Tazobactam broadens the activity of ceftolozane to include some important anaerobic pathogens such as B. fragilis and is active against the anaerobes Fusobacterium spp. and Prevotella spp. [180]. The pharmacodynamic parameter best predicting in vivo bacteriological efficacy is the time t > MIC, as was shown in a neutropenic murine thigh infection model using ESBL- producing enterobacteria or P. aeruginosa [181]. In a humanized pharmacokinetic model of murine thigh infection by ESBL-producing E. coli and K. pneumoniae simulating % t>MIC for ceftolozane/tazobactam in humans, 1 to 3 log reductions were achieved with 37.5% t > MIC, showing improved efficacy versus piperacillin/tazobactam [182]. The pharmacokinetic profile of ceftolozane after single and multiple doses is shown in Table 43 [183].

Table 43. Ceftolozane pharmacokinetic parameters following 1 hr infusion of ceftolozane/tazobactam

Mean (%CV) PK value Parameter 500 mg 500/250 mg 2000 mg 2000/1000 mg 1000/500 mg 1000/500 mg C C/T C C/T C/T day 1a C/T day 10 a Cmax (g/ml) 42.6 (13.5) 40.2 (12.6) 153 (10.8) 140 (14.9) 69.1 (11.3) 74.4 (13.6) Tmax (min) 1 1 1 1 1 1 AUC0-∞ 98.6 (16.3) 97.3 (15.0) 375 (16.4) 353 (18.0) 172.0 (13.8) 197 (16.6)* (g*h/ml) t1/2 (hrs) 2.48 (8.2) 2.43 (18.9) 2.62 (16.9) 2.62 (18.2) 2.77 (30.0) 3.12 (21.9) Cl (L*kg/h) 5.86 (13.7) 5.58 (12.6) ClR (L*kg/h) 5.58 (24.0) 6.80 (49.4) Vdss (L) 14.6 (16.0) 14.2 (16.6) Accumulation 1.14 (5.7) index Urinary recovery 100 (%) C: ceftolozane; T: tazobactam. a. 10 day repeated dose every 8 hrs. * AUC0-8 (g*h/ml)

PK parameters of ceftolozane were not affected by coadministration of tazobactam. Ceftolozane exhibits rapid tissue distribution, low protein binding (20%) [179a], and concentrates well at extracellular sites of infection [242, 184]. Monte Carlo simulation using the standard 1000/500 mg every 8 h dosing regimen estimated that >90% of simulated patients could achieve free-drug t > MIC ≥32.2% for P. aeruginosa with MIC = 8 g/ml supporting the proposed in vitro susceptibility breakpoint of 8 g/ml [185].

9.4. Ceftazidime + Avibactam

Ceftazidime is a parenteral extended-spectrum antipseudomonal cephalosporin with Gram-negative activity and marginal Gram-positive activity. Avibactam is a non--lactam - lactamase inhibitor which inactivates susceptible -lactamases by covalent acylation of the - lactamases active site serine residue [184, 186]. Avibactam inhibits class A and class C - Complimentary Contributor Copy

A 21st Century Contribution to the Antibacterial Armamentarium 237 lactamases as well as some class D enzymes. Avibactam binds covalently to -lactamases through the formation of a carbamate bond at the same active-site serine that reacts with - lactam substrates. The covalent nature of the bond has been conﬁrmed by x-ray analysis of the crystal structure of avibactam bound to a variety of -lactamases [187], representing all three molecular classes of serine -lactamases. The potential for avibactam to select for resistance is little documented; it has been reported to not induce chromosomal ampC expression in Enterobacter cloacae [188]. The antibacterial activity of ceftazidime/avibactam (Avycaz) is shown in Table 44. The pharmacodynamic (PD) parameter that best predict the clinical efficacy of ceftazidime is free drug time above the MIC (fT>MIC) [65]. This has been demonstrated clinically using nosocomial pneumonia clinical trial data where ceftazidime was used as a comparator [189] showing that a fT>MIC required to result in a likely favorable outcome was >45% of the dosing interval. A second study, of ceftazidime and cefepime in the treatment of ventilator-associated pneumonia, found that achieving a > 53% fT>MIC was a predictor of success [190]. The pharmacokinetic parameters for ceftazidime and avibactam in healthy adult male subjects with normal renal function after single and multiple 2 hour intravenous infusions of Avycaz (2 g ceftazidime, 0.5 g avibactam) administered every 8 hours is shown in Table 45.

Table 44. Antibacterial activity of ceftazidime/avibactam

Ceftazidime Ceftazidime/avibactam Cefepime Isolates MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 (g/ml) (g/ml) (g/m-l) (g/ml) (g/ml) (g/ml) Gram-negative Citrobacter freundii 0.5 >32 0.25 0.5 ≤1 ≤1 Citrobacter spp. Ceftazidime I/R 0.25 >32 0.12 0.5 ≤0.12 4 32 >32 0.25 1 1 >16 Enterobacter aerogenes ≤0.5 >32 0.25 0.5 ≤1 ≤1 Enterobacter cloacae 0.5 >32 0.25 1 ≤1 1 Enterobacter spp. Ceftazidime R 0.25 >32 0.25 1 ≤0.12 2 AmpC + porin loss 32 >32 0.5 2 2 >16 256 256 1 1 NA NA Escherichia coli 0.25 2 0.12 0.25 ≤0.12 1 ESBL producing 16 32 0.12 0.25 8 32 AmpC hyper-producing ESBL 16 64 0.12 0.5 0.25 0.5 producing + AmpC hyper- 32 >64 0.12 0.12 16 32 producing Klebsiella oxytoca ≤0.25 0.5 0.12 0.5 ≤1 ≤1 Klebsiella pneumoniae ≤0.25 1 0.12 0.5 ≤1 ≤1 ESBL producing 64 >64 0.5 1 8 64 OXA-48 producing 256 512 0.25 0.5 32 512 KPC-producing ≥512 ≥512 0.25 1 32 128 ESBL-+ porin loss 256 512 1 1 NA NA Klebsiella spp. 0.12 32 0.12 0.5 ≤0.12 16 ESBL >32 >32 0.5 2 >16 >16 Carbapenem I/R >32 >32 0.5 2 >16 >16 Morganella morganii 0.2 8 0.06 0.12 ≤0.12 0.25 Complimentary Contributor Copy

238 Stefano Biondi and Mauro Panunzio

Table 44. (Continued)

Ceftazidime Ceftazidime/avibactam Cefepime Isolates MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 (g/ml) (g/ml) (g/m-l) (g/ml) (g/ml) (g/ml) Proteus mirabilis 0.06 0.12 0.06 0.12 ≤0.12 0.25 Indole + Proteus spp. 0.12 8 0.06 0.25 ≤0.12 0.25 Salmonella spp. 0.25 0.5 0.25 0.5 ≤0.12 0.25 Serratia marcescens 0.12 2 0.25 0.5 ≤0.12 2 Serratia spp. 0.25 0.5 0.25 0.5 ≤0.12 0.5 Burkholderia cepacia 64 >128 8 >128 NA NA Pseudomonas aeruginosa MDR 4 32 2 8 4 16 AmpC-derepressed NA NA 8 32 NA NA Intrinsic MexA/OprM 64 >128 4 8 NA NA 4 8 4 8 NA NA Acinetobacter baumannii OXA + 8 32 8 >16 64 128 carbapenemase 128 >128 8 >128 NA NA Acinetobacter spp. Imipenem- >32 >32 16 >32 >16 >16 resistant >32 >32 32 >32 >16 >16 Anaerobes Bacteroides caccae >128 >128 32 >128 Bacteroides fragilis 64 >128 4 32 Bacteroides ovatus >128 >128 128 >128 Bacteroides thetaiotaomicron >128 >128 128 >128 Bacteroides vulgatus >128 >128 32 128 Bacteroides spp. 128 >128 8 64 Parabacteroides spp. >128 >128 16 64 Clostridium difﬁcile 128 >128 32 64 Clostridium perfringens 64 >128 ≤0.06 2 Prevotella/Porphyromonas spp. 32 >128 2 4 Gram-positive S. aureus 8 >32 8 >32

Table 45. Pharmacokinetic parameters (geometric mean (%CV)) of ceftazidime and avibactam in healthy subjects

Ceftazidime Avibactam Parameter Single dose Multiple dose Avycaz Single dose Multiple dose Avycaz Avycaz (11days) Avycaz (11days) Cmax (g/ml) 88.1 (14) 90.4 (16) 15.2 (14) 14.6 (17) t1/2 (min) 3.27 (33) 2.76 (7) 2.22 (31) 2.71 (25) AUC0-∞ (g*h/ml) 289 (15) 291 (15) 42.1 (16) 38.2 (19) Vdss (L) 18.1 (20) 17 (16) 23.2 (23) 22.2 (18) Cl L/h) 6.93 (15) 6.86 (15) 11.9 (16) 13.1 (19) Protein binding (%) <10 5.7-8-2

No significant accumulation of ceftazidime or avibactam was observed following multiple intravenous infusions in healthy adult subjects with normal renal function. Both ceftazidime and avibactam are excreted mainly by the kidneys. The addition of avibactam restores the activity of ceftazidime against Gram-negative bacilli that express class A ESBLs, Complimentary Contributor Copy

A 21st Century Contribution to the Antibacterial Armamentarium 239 chromosomal or mobile class C -lactamases, serine carbapenemases, and some class D - lactamases. According to the Centers for Disease Control and Prevention, carbapenem resistant enterobacteriaceae (CRE), are the most urgent unmet medical need that can potentially be addressed by ceftazidime-avibactam [191]. Through potential improvements in clinical efficacy and spared use of more toxic treatment alternatives, such as polymyxin- containing regimens, ceftazidime-avibactam may have a profound impact on the outcomes of patients with CRE infections. The antimicrobial stewardship implications associated with introduction of ceftazidime-avibactam clinical use also are of crucial consideration.

10. LIPOGLYCOPEPTIDES: TELAVANCIN, ORITAVANCIN, DALBAVANCIN

Lipoglycopeptides represent a subgroup of the glycopeptide antibiotic class [192]. Natural product lipoglycopeptides are identified by presence of a lipophilic, often long chain fatty acid, attached to an amino sugar on the ring 4 amino acid [193]. Lipoglycopeptide antibiotics are more effective than vancomycin against MRSA as they carry an extra aliphatic acyl side chain on glucosamine. The spectrum of activity of lipoglycopeptides covers most Gram-positive organisms and some anaerobes, and their activity against Gram-negative organisms is marginal. The most striking difference between vancomycin and the lipoglycopeptides is their capacity to bind serum proteins and is in accordance with their prolonged half-life. However, high protein binding also needs to be taken into account pharmacodynamics predictions, since only the free serum fraction is microbiologically active.

10.1. Telavancin

Telavancin (TD-6424, is a semisynthetic derivate of vancomycin featuring a lipophilic side chain (decylaminoethyl), and a hydrophilic moiety (phosphonomethyl aminomethyl) on the 4’-position of amino acid 7, which is attached to a vancosamine sugar [194] (Figure 7). The decylaminoethyl side chain provides improved binding afﬁnity to D-Ala-D-Ala– containing peptidoglycan intermediates, and the negatively charged phosphonic acid moiety increases the urinary excretion of this compound [195]. The drug has high protein binding, a serum half-life of approximately 7-9 hours and usually is administrated intravenously at 10 mg/kg once a day [195b]. Telavancin is excreted by the kidneys, and dosage adjustments is required in cases of renal failure. Clinical trials have demonstrated non-inferiority to vancomycin, in the treatment of complicated skin and skin-structure infections and pneumonia. The antibacterial activity of telavancin covers a spectrum similar to that of vancomycin, but additionally has activity against some multidrug- resistant Gram-positive organisms [196] like MRSA, VISA, VRSA, and VRE [197]. Telavancin’s activity against Gram-positive anaerobes is approximately two to four times greater than that of vancomycin (see Table 46). Telavancin potency is negatively affected by the VanA resistance phenotype; however, the MICs increase less for telavancin than for vancomycin and dalbavancin [198]. It has a low potential in vitro to select for resistant mutations among S. aureus and enterococci [199] and has not shown cross-resistance with the Complimentary Contributor Copy

240 Stefano Biondi and Mauro Panunzio other antimicrobials (vancomycin, teicoplanin, daptomycin, linezolid, oxacillin, erythromycin) [200].

Table 46. In vitro activity of Telavancin against Gram-positive bacteria

MIC50 Organism (nr. of isolates) MIC90 (g/ml) Range (g/ml) (g/ml) Staphylococcus aureus Methicillin susceptible (1077) 0.25 0.5 0.03–1 Methicillin resistant (885) 0.25 0.5 0.06–1 Vancomycin intermediate (50) 0.5 1.0 0.125–1 Coagulase negative staphylococci Methicillin susceptible (30) NA 0.5 0.125–1 Methicillin resistant (30) NA 1.0 0.25–2 Streptococcus pneumoniae (412) NA 0 .015–0.03 0.002–0.06 Beta-hemolytic streptococci (218) NA 0.015–0.125 0.06–0.125 Viridans-group streptococci (102) NA 0.12 0.001–1 Enterococcus faecalis Vancomycin susceptible (345) 0.5 1.0 0.12–2 Vancomycin resistant (184) 8.0 16 0.5–16 Enterococcus faecium Vancomycin susceptible (76) 0.12 0.25 <0.015–0.5 Vancomycin resistant (184) 4.0 8.0 <0.015–16 Actinomyces israelii (13) 0.25 0.25 0.125–0.25 Actinomyces meyeri–Actinomyces turicensis 0.125 0.25 0.125–0.25 group (12) Actinomyces odontolyticus (10) 0.25 0.25 0.125–0.25 Actinomyces viscosus (10) 0.125 0.25 0.125–0.25 Anaerococcus (Peptostreptococcus) prevotii (11) 0.03 0.06 <0.015 – 0.5 Bacillius anthracis (15) 0.12 0.12 <0.03 – 0.5 Clostridium clostridioforme (15) 1.0 8.0 0.25–8 Clostridium difﬁcile (14) 0.25 0.25 0.125–0.5 Clostridium innocuum (15) 4.0 4.0 2–4 Clostridium perfringens (12) 0.06 0.125 0.006–0.125 Clostridium ramosum (16) 0.5 1.0 0.25–8 Corynebacterium amycolatum (10) 0.03 0.06 0.03–0.06 Corynebacterium jeikeium (11) 0.06 0.06 0.03–0.06 Lactobacillus casei (6) 32 NA 32–64 Lactobacillus plantarum (10) 0.25 0.25 0.125–0.25 Peptostreptococcus anaerobius (10) 0.06 0.25 0.06–0.25

Inhibition of peptidoglycan synthesis and transglycosylase activity is more than 10 times greater with telavancin than that of vancomycin The comparative antibacterial activity of telavancin, vancomycin, linezolid, and daptomycin for Gram-Positive organisms is reported in Table 47. The in vitro post-antibiotic effect (PAE) of telavancin is 4-6 hours [201], which is approximately four times longer than that of vancomycin against staphylococci.

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A 21st Century Contribution to the Antibacterial Armamentarium 241

Table 47. Comparative in vitro Minimum Inhibitory Concentrations (MICs) of Telavancin, Vancomycin, Linezolid, and Daptomycin for Gram-Positive Organisms

MIC90 (g/ml) Organism (nr. of isolates) Telavancin Vancomycin Linezolid Daptomycin Staphylococcus aureus MSSA (1217) 0.5 1 2 0.5 MRSA (1082) 0.25 1 2 0.5 VISA (23) 1 8 2 4 DNSSA (7) 0.5 2 2 4 Coagulase-negative staphylococci Methicillin susceptible (100) 0.5 2 1 1 Methicillin resistant (272) 0.5 2 1 1 Enterococcus faecalis Vancomycin susceptible (429) 1 2 2 1 Enterococcus faecium Vancomycin susceptible (92) 0.25 1 2 4 Van A (223) 8 512 2 4 Van B (17) 2 512 2 4 Streptococcus pyogenes (68) 0.06 0.5 1 0.06 Streptococcus agalactiae (45) 0.06 0.5 1 0.25 Streptococcus pneumoniae (204) 0.03 0.5 1 - Actinomyces israelii (13) 0.25 1 16 4 Clostridium difﬁcile (14) 0.25 1 8 2 Clostridium perfringens (12) 0.125 0.5 2 1 Clostridium ramosum (16) 1 4 8 32 Peptococcus anaerobius (10) 0.25 0.5 8 0.5

Figure 7. Chemical structure of Telavancin and metabolites.

Telavancin inhibits cell wall synthesis by binding to D-alanyl-D-alanine terminal residues involved in peptidoglycan synthesis. It also binds no-covalently to the lipophilic moiety of the cell membrane disrupting [200a] the membrane integrity, leading to depolarization of the cell membrane and increased membrane permeability accompanied by leakage of cytoplasmic Complimentary Contributor Copy

242 Stefano Biondi and Mauro Panunzio adenosine triphosphate and potassium ions, this is associated with the rapid bacterial killing proﬁle of telavancin [256]. Telavancin is metabolized by cytochrome P450 via hydroxylation of the lipophilic side chain at positions 7, 8, and 9 (see Figure 7). The pharmacokinetic and mass balance of telavancin has been evaluated in healthy volunteers using [14C]- telavancin (0.68 Ci/kg) [200b]. Telavancin accounted for >95% and 83% of total radioactivity in plasma at 12 hrs and 24 hrs, respectively. By 216 hrs, approximately 76% of the total administered dose was recovered in urine, while only 1% was collected in feces. Unchanged telavancin accounted for most (83%) of the eliminated dose. Telavancin metabolite THRX- 651540 (M3), along with two other hydroxylated metabolites (designated M1 and M2), accounted for the remaining radioactivity recovered from urine. The pharmacokinetic parameters of telavancin [195b, 202] at different doses following repeated administration are summarized in Table 48.

Table 48. Pharmacokinetic parameters of Telavancin

Parameter Value 7.5 mg/kg/day 12.5 mg/kg/day 15 mg/kg/day Dose (60 min infusion) (30 min infusion) (60 min infusion) Protein binding (%) 93 t1/2 (hrs) 6.0±0.6 9.11±2.33 7.5±1.3 Vss (ml/kg) 111±32 119±18 119±14 Cmax (g/ml) 87.5±12.8 151±17 186±27 AUCss (g•h/ml) 599±96 1033±9.1 1282±201 Clss (ml/h/kg) 13.0±3.6 12.2±1.1 12.0±1.9 Clr (ml/h/kg) 8.3±3.9 NA 8.7±4.3 Urine with >80% as unchanged drug and <20% as hydroxylated metabolites Elimination route (with dose of 10mg/kg); Feces (<1%) Cmax, maximum plasma concentration; AUCss, area under the curve from time 0 to 24 h at steady state; t1/2, terminal elimination half-life; Clss, clearance at steady state; Vss, steady-state volume of distribution; Clr, renal clearance. NA, not available.

Telavancin shows good penetration into pulmonary epithelial lining ﬂuid and alveolar macrophages [203] reaching concentrations comparable to calculated free (unbound) plasma levels [204] (i.e AUCELF/AUCplasma penetration ratio of 10%). However, the mean concentration of telavancin in ELF was between two and eight times higher than the MIC90 of MRSA (0.5 g/ml). In the alveolar macrophages the concentration of telavancin was on average 40 to 85 times above the MIC90 of MRSA [204a]. The dosage of telavancin is 10 mg/kg every 24 h if the estimated creatinine clearance is >50 ml/min. It is reduced to 7.5 mg/kg if the creatinine clearance is 30–50 ml/min, and the dose is further reduced to 10 mg/kg every 48 h if the clearance is <30 ml/min. In contrast to vancomycin, telavancin produces rapid and concentration-dependent killing against both extracellular and intracellular S. aureus [201, 205]. Pharmacodynamic studies in mice have indicated that the pharmacodynamic parameter that best correlates with the antimicrobial effect of telavancin is AUC/MIC ratio [206]. In an in vitro kinetic model, the minimum AUC/MIC ratio resulting in >3-log killing without regrowth was 50 (corresponding to a dose of 10 mg/kg) [207]. Telavancin has been approved for treatment of complicated skin and skin structure infections due to susceptible Gram-positive bacteria and hospital-acquired/ventilator-associated bacterial pneumonia due to Staphylococcus aureus when other alternatives are unsuitable. Complimentary Contributor Copy

A 21st Century Contribution to the Antibacterial Armamentarium 243

Telavancin has proven to be efficacious in multiple animal models of soft tissue, cardiac, systemic, lung, bone, brain and device-associated infections involving clinically relevant Gram-positive pathogens, including MRSA, GISA, heterogeneous vancomycin-intermediate S. aureus and daptomycin non-susceptible MRSA.

10.2. Dalbavancin

Dalbavancin (Dalvance) is a lipoglycopeptide with an extended half-life (8.5 days) that enables once-weekly dosing. Dalvance is indicated for acute bacterial skin and skin structure infections caused by designated susceptible strains of Gram-positive microorganisms. It is more potent in vitro against most Gram-positive organisms than vancomycin, oritavancin, and telavancin. It is active against MRSA and VRE, except for strains exhibiting vanA resistance. Dalbavancin is a semisynthetic derivative of the naturally occurring A-40926, a member of teicoplanin family (Figure 8) [208] obtained by fermentation of Nonomuraea spp. It has several structural changes with respect to A-40926, which include a 3,3- dimethylaminopropylamide in place of a carboxy group, that potentiate antistaphylococcal activity, particularly against coagulase-negative staphylococci [209]. The lipophilic side chain increases protein binding and provides the long half-life typical of teicoplanin-type derivatives. The side chain also enhanced the interaction of dalbavancin with its target binding site [210]. Dalbavancin is a mixture of five closely related active homologues (A0, A1, B0, B1, and B2); B0 is the major component of dalbavancin. The homologues share the same core structure and differ in the fatty acid side chain of the N-acylaminoglucuronic acid moiety (R1) and/or the presence of an additional methyl group (R2) on the terminal amino group (Figure 8). The antibacterial activity of dalbavancin is restricted to Gram-positive organisms including MRSA and VRE, with the exception of vanA enterococci [211] (Table 49) [210a]. Table 50 shows comparative in vitro inhibitory activity for dalbavancin and the other lipoglycopeptides. In order to get reproducible MIC values, it was necessary to add 0.002% polysorbate-80 to assay broths [210b]. Dalbavancin has comparable activity to vancomycin, linezolid, daptomycin and quinupristin–dalfopristin towards less common Gram-positive aerobes such as Bacillus spp., Corynebacterium spp., Listeria spp., and Micrococcus spp., with MIC values (0.016–0.25 g/ml) [210a, 212]. It is generally 2- to 4-fold more potent than vancomycin against Gram-positive anaerobes such as Actinomyces spp., Eubacterium spp., Propionibacterium spp. and Peptostreptococcus spp. [213] and 8 times more potent than vancomycin and daptomycin against C.difficile. Dalbavancin inhibits the ﬁnal stages of peptidoglycan synthesis by binding to the D- Alanyl-D-Alanine terminus of peptidoglycan precursors [210, 214]. The complex created between the heptapeptide backbone of dalbavancin and the D-Alanyl-D-Alanine dipeptide hampers binding of transglycosylases and transpeptidases, preventing the elongation and cross-linking of the peptidoglycan. The lipophilic side chain seems to enhance the interaction with D-Alanyl-D-Alanine peptides [210b, 215]. Dalbavancin is believed to form dimers with a high binding afﬁnity to the target site locking the binding pocket into a prime position to facilitate cooperative binding [210]. These features are considered to contribute to the potent antimicrobial activity of dalbavancin. Of the marketed lipoglycopeptides, only telavancin is capable of overcoming vanA resistance by virtue of a dual mechanism of action, membrane Complimentary Contributor Copy

244 Stefano Biondi and Mauro Panunzio depolarization and permeabilization, which is not shown by dalbavancin [197]. Dalbavancin is available in a two-dose regimen: 1000 mg followed one week later by 500 mg. Dosage adjustment for patients with creatinine clearance less than 30 ml/min and not receiving regularly scheduled hemodialysis: 750 mg followed one week later by 375 mg. It is administer by intravenous infusion over 30 minutes. The antibacterial activity of dalbavancin best correlates with AUC/MIC for S. aureus, based on animal models of infection. An exposure-response analysis in patients with complicated skin and skin structure infections supports the two-dose regimen. Dalbavancin pharmacokinetic parameters have been characterized in healthy subjects, patients, and specific populations. Pharmacokinetic parameters following administration of a single i.v. 1000 mg dose are shown in Table 51. The pharmacokinetics of dalbavancin is best described using a three-compartment model.

Table 49. Antimicrobial activity of dalbavancin against Gram-positive bacteria

Organism (n.of isolates) MIC50 (g/ml) MIC90 (g/ml) MIC range (g/ml) S.aureus Methicillin susceptible (1815) 0.06 0.06 ≤0.015-0.25 Methicillin resistant (1177) 0.06 0.06 ≤0.015-0.5 Coagulase negative staphylococci Methicillin susceptible (157) 0.03 0.06 ≤0.015-0.25 Methicillin resistant (617) 0.03 0.06 ≤0.015-0.5 S.pneumoniae Penicillin susceptible (996) ≤0.015 0.03 ≤0.015-0.06 Penicillin resistant (400) ≤0.015 0.03 ≤0.015-0.25 Viridans group streptococci Penicillin susceptible (104) ≤0.015 0.03 ≤0.015-0.06 Penicillin non susceptible (30) ≤0.015 0.03 ≤0.015-0.03 -Hemolytic (234) ≤0.015 0.03 ≤0.015-0.25 E.faecalis Vancomycin susceptible (586) 0.03 0.06 ≤0.015-4 Vancomycin resistant (20) 4 0.06 ≤0.015-32 E.faecium Vancomycin susceptible (77) 0.06 0.12 ≤0.015-4 Vancomycin resistant (51) 8 32 0.03-32

O OH OH O OH OH HO OH O HO OH O HO O O O O N H N N O R H Cl 1 Cl O O H OH Cl O O O O O O HO OH O O O OH HO OH O H H O H H HO O N O H H O H O O H O H O N N H H N N N R O N N NH2 O N N N H H 2 HO NH N N N N N N H O O H O H O H H H O H HN H O O O O O HN OH HN OH HO H O HO H O O HO O OH OH OH OH OH OH N HO O O HO O O HO O O OH OH OH HO OH HO OH HO OH OH OH OH Dalbavancin A40926 Teicoplanin A2

R Mol. formula Dalbavancin R1 2 Mol. weight

A0 CH(CH3)2 H C87H 98N10OO28Cl2 1802.7

A1 CH2CH2CH3 H C87H 98N10OO28Cl2 1802.7

B0 CH2CH(CH3)2 H C88H100N10OO28Cl2 1816.7

B1 CH2CH2CH2CH3 H C88H100N10OO28Cl2 1816.7

B 2 CH2CH(CH3)2 CH3 C89H102N10OO28Cl2 1830.7

Figure 8. Chemical structures of teicoplanin A2, A40926 and dalbavancin. Complimentary Contributor Copy

A 21st Century Contribution to the Antibacterial Armamentarium 245

Table 50. Comparative antimicrobial activity of semisynthetic lipoglycopeptides and vancomycin against Gram-positive bacteria [213]

MIC range (g/ml) Organism Dalbavancin Oritavancin Telavancin Vancomycin Staphylococcus aureus methicillin-susceptible 0.06–0.5 0.12-2 0.12-2 0.25-2 methicillin-resistant 0.06–1 0.12-4 ≤0.06-2 0.5-4 Coagulase negative Staphylococci methicillin-susceptible ≤0.03-0.25 0.25-1 0.12-2 0.12-1 methicillin-resistant 0.06-1 0.25-4 0.12-2 1-4 Streptococcus spp. S.pneumoniae 0.008-0.12 ≤0.002-0.06 0.004-0.03 0.25-2 -Hemolytic streptococci ≤0.03-0.12 0.016-0.12 0.03-0.12 0.5 Enterococcus spp. 0.25-4 vancomycin susceptible ≤0.03-1 0.06-0.25 0.06-1 8-128 vancomycin-resistant (vanB) 0.02-2 0.12-2 0.12-2 >128 vancomycin-resistant (vanA) 0.5->128 1-4 0.12-8

Table 51. Dalbavancin Pharmacokinetic Parameters in Healthy Subjects

Parameter Single 1000 mg Dose Mean (% coefficient of variation) Cmax (g/ml) 287 (13.9) AUC0-24 (mg•h/L) 3185 (12.8) AUC0-Day7 (mg•h/L) 11160 (41.1) AUC0-inf (mg•h/L) 23443 (40.9) Terminal t½ (hrs) 346 (16.5) Cl (L/h) 0.0513 (46.8)

In healthy subjects, dalbavancin AUC0-24h and Cmax show a dose proportional increase following single intravenous administration ranging from 140 mg to 1500 mg, indicating linear pharmacokinetics. Dalbavancin is reversibly bound to human plasma proteins (93%), primarily to albumin. The mean concentrations of dalbavancin achieved in skin blister fluid remain above 30 g/ml up to 7 days following intravenous dosing of 1000 mg. The mean ratio of the AUC0-144 hrs skin blister fluid/AUC0-144 hrs plasma is 0.60 (range 0.44-0.64). Dalbavancin is not a substrate, inhibitor, or inducer of CYP450 isoenzymes. Analogous to what observed for telavancin, a minor metabolite of dalbavancin (hydroxy-dalbavancin) has been observed in the urine of healthy subjects. Following administration of a single 1000 mg dose in healthy subjects, 20% of the dose was excreted in feces through 70 days post dose. An average of 33% of the administered dose was excreted in urine as unchanged dalbavancin and approximately 12% was excreted in urine as hydroxy-dalbavancin metabolite through 42 days post dose.

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246 Stefano Biondi and Mauro Panunzio

10.3. Oritavancin

Oritavancin (LY333328) is a second-generation semisynthetic lipoglycopeptide antibiotic obtained by modiﬁcations of chloroeremomycin, a naturally occurring glycopeptides [216] isolated from Kibdelosporangium orienticin. The chemical structure of oritavancin is similar to that of vancomycin (Figure 9), the only difference being introduction of an aromatic lipophilic side chain and an unsubstituted sugar in oritavancin. Compared to vancomycin, oritavancin possesses additional hydrophilic and hydrophobic regions, which confer an increased spectrum of activity and an increased half-life. Oritavancin is effective against Gram-positive aerobes, including vancomycin-resistant organisms, such as vancomycin- resistant Enterococci (VRE) and Staphylococcus aureus (VRSA), or vancomycin intermediate-resistant S. aureus (VISA). It also possesses activity against a number of Gram- positive anaerobic bacteria. The antibacterial activity of Oritavancin is shown in Table 52 [217].

Table 52. In vitro activity of Oritavancin against Gram-positive aerobic and anaerobic bacteria

Organism MIC50 MIC90 MIC range (g/ml) (g/ml) (g/ml) S. aureus Methicillin susceptible 0.5-1 1-2 0.06-4 Methicillin resistant 1 2 0.03-4 Vancomycin resistant ND ND 0.25-1 Coagulase negative staphylococci Methicillin susceptible 0.5-1 2 0.06-2 Methicillin resistant 1 2 0.015-4 S. pneumoniae Penicillin susceptible 0.04 0.008 0.0002-0.12 Penicillin intermediate 0.04 0.015 0.0002-0.06 Penicillin resistant 0.04 0.015 0.0002-0.06 Viridans group streptococci Penicillin susceptible ≤0.01 ≤0.01 ≤0.01 Penicillin non susceptible ≤0.01 ≤0.01 ≤0.01-0.25 -Hemolytic ≤0.01 0.06 ≤0.01-0.06 Group A Streptococci ≤0.01 0.06 ≤0.01-0.06 E. faecalis Vancomycin susceptible 0.5 1 0.008-4 Vancomycin resistant 1 2 0.03-2 E. faecium Vancomycin susceptible 0.06 0.12-0.5 ≤0.015-4 Vancomycin resistant 1 1-2 0.03-32 Clostridium perfringens 0.5 1 0.016-2 Clostridium difficile ND ND 0.016-2 Peptostreptococcus spp. 0.125 0.25 0.016-1 Propionibacterium acnes 0.125 0.25 0.125-0.25

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A 21st Century Contribution to the Antibacterial Armamentarium 247

Figure 9. Chemical structures of vancomycin, chloroeremomycin and oritavancin.

Similar to telavancin, the antibacterial activity of oritavancin is attributed to a dual mechanism of action. The first is similar to other lipoglycopeptides, involving the inhibition of cell wall biosynthesis [218] by forming a complex with the D-Ala-D-Ala terminus of the developing peptidoglycan chain. Afﬁnity for the peptidoglycan terminus shown by oritavancin far exceeds that of vancomycin, especially for VRE and resistant S. aureus [219]. It has been postulated that oritavancin form a homodimer prior to binding, this dimer interacts with two developing peptidoglycan precursors and this in turn an extra binding site to the cytoplasmic membrane. Moreover, the p-chlorophenylbenzyl side chains of the dimer molecules also form a bond with the membrane [295]. Alternatively, it has been proposed that the activity of oritavancin is the result of a secondary binding action [220]. Oritavancin’s second mechanism of action is due to the side chains binding to the cell membrane and causing rapid changes in membrane potential and permeability [221]. This has been attributed to interactions of oritavancin with phospholipids bilayers and thereby changes in lipid organization [222]. As a result of this dual mechanism of action oritavancin, unlike vancomycin, displays rapid bactericidal activity. This mechanism has also been postulated to explain the activity of oritavancin against S. aureus in several phases of growth, including in stationary-phase and bioﬁlms [223]. Resistance to oritavancin has been described in vitro either via current glycopeptide resistance mechanisms (Van operons), and the VISA-type thickened cell wall phenotype. MIC distributions for staphylococcal and enterococcal strains indicate an absence of cross-resistance with resistant phenotypes such as VanA, VanB, VanC, or VISA [224]. Oritavancin demonstrates concentration-dependent activity [142, 225], and a long post-antibiotic effect both in vitro and in vivo [226]. In vitro [227] and Phase I [228] studies have shown accumulation of oritavancin in the lysosomes of macrophages, revealing a 300-fold higher intracellular concentration than extracellular. Once inside the cells, oritavancin efflux to the extracellular space is slow. This accounts for the long half-life of oritavancin and its intraphagocytic activity against some pathogens that survive lysosomes, such as S. aureus. Orbactiv™ (Oritavancin) for injection is indicated for treatment of adults with acute bacterial skin and skin structure infections (ABSSSI) caused by susceptible isolates of the following Gram-positive microorganisms: Staphylococcus aureus (including methicillin-susceptible and methicillin–resistant isolates), Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus anginosus group, and Enterococcus faecalis (vancomycin-susceptible isolates only). The recommended dosing for Orbactiv is a single 1200 mg dose administered by intravenous infusion over 3 hours. The population PK analysis was derived using data from the two Phase 3 ABSSSI clinical trials in Complimentary Contributor Copy

248 Stefano Biondi and Mauro Panunzio

297 patients. The mean pharmacokinetic parameters of Oritavancin in patients following a single 1200 mg dose are presented in Table 53 together with other parameters derived from different studies.

Table 53. Mean PK parameters for Oritavancin 1200 mg dose

Parameter Mean (CV%) Cmax (g/ml) 138 (23.0%) AUC0-24 (g•h/ml) 1110 (33.9%) AUC0-∞ (g•h/ml) 2800 (28.6%) t½,α (hrs) 2.29 (49.8%) t½,β (hrs) 13.4 (10.5%) t½,γ (hrs) 245 (14.9%) Vd (L) 87.6 Cl (L/hr) 0.445 Protein binding (%) 86-90 Excretion (%) Feces (1), urines (5)* *Data collected for two weeks.

Oritavancin exhibits linear pharmacokinetics at a dose up to 1200 mg. The mean, population-predicted oritavancin concentration-time profile displays a multi-exponential decline with a long terminal plasma half-life. Unlike vancomycin, oritavancin demonstrates a high degree of protein binding (86-90%) [229]. Based on population PK analysis, the population mean total volume of distribution is estimated to be approximately 87.6l, indicating that oritavancin is extensively distributed into the tissues. Exposures of oritavancin in skin blister fluid were approximately 20% of those in plasma (AUC0-24) after single 800 mg dose in healthy subjects. Non-clinical studies including in vitro human liver microsome studies indicated that oritavancin is not metabolized. No mass balance study has been conducted in humans. In humans, oritavancin is slowly excreted unchanged in feces and urine, with less than 1% and 5% of the dose recovered in feces and urine, respectively, after 2 weeks of collection. Oritavancin has a terminal half-life of approximately 245 hrs and a clearance of 0.445 L/hr based on population pharmacokinetic analyses. Tissue distribution studies in mice, rats, and beagle dogs reveal the greatest uptake to occur in the liver, with approximately 59–64% of the administered dose found. Oritavancin seems to be an effective one-dose treatment for ABSSSI and a single dose of oritavancin was non-inferior to vancomycin given twice daily for a period of 7 to 10 days. The long half-life, multiple mechanisms of action, possibility for outpatient therapy, and favorable safety profile are all desirable features of this new lipoglycopeptide. One point that still remains unclear is the elimination from the body, as the studies performed so far in humans do not completely describe this pharmacokinetic aspect.

11. P OLYENE MACROCYCLE: FIDAXOMYCIN

Fidaxomicin, previously known as OPT-80, difimicin and PAR-101, is a first-in-class 18- membered macrocyclic antibiotic [230]. Other related agents, tiacumicin and lipiarmycin, are naturally occurring products, composed of a mixture of isomers related to tiacumicin B, derived from fermentation of a strain of the soil-borne actinomycete Dactylosporangium Complimentary Contributor Copy

A 21st Century Contribution to the Antibacterial Armamentarium 249 aurantiacum subspecies hamdenensis (strain 718C-41). Fidaxomicin (Dificid®) was approved for C. difficile-associated diarrhea in 2011 by the US FDA becoming the first new antimicrobial approved for this infection in 30 years. Fidaxomicin is supplied as 200 mg tablets administered twice-daily for a 10-day course of therapy. Although data from Phase III indicate its superiority for the outcomes of recurrence and global cure or sustained clinical response, use of this new antimicrobial has been limited due to the high costs of therapy compared to alternative therapies [231]. Fidaxomicin is active against most clostridial spp., including C. difficile, and it has moderate activity against other Gram-positive organisms, such as staphylococci and enterococci [232]. It is inactive against Gram-negative bacteria, fungi and protozoa. Fidaxomicin has bactericidal activity against C. difficile. The mechanism of action of fidaxomicin involves inhibition of RNA polymerase. Unlike RNA polymerase initiation inhibitors (rifamycins), or elongation inhibitors, fidaxomicin inhibits initial DNA strand separation within the promoter complex of the enzyme [232-233]. This represents a unique mechanism that precludes cross-resistance to other agents targeting RNA polymerase. The MIC90 for fidaxomicin for C. difficile is typically 0.125 g/ml, with reported ranges between ≤0.016 and 0.25 g/ml [234]. In these studies, the MIC of fidaxomicin was compared with that of vancomycin and metronidazole, and it was found that fidaxomicin was two- to eight-fold more potent against C. difficile. Fidaxomicin shows minimal systemic absorption [234]. Serum concentrations of fidaxomicin are generally in the low ng/ml range and are not affected by fed or fasted states. The main metabolite, OP-1118, is derived from hydrolysis of the O-isobutyryl ester at 4´ position of the sugar attached at C11 of the macrolactone. Mean plasma concentrations of fidaxomicin and OP-1118 are two- to six-times higher in individuals with CDI than in healthy volunteers. The majority of administered fidaxomicin (approximately 92%) is excreted in feces, with ≥66% excreted as the metabolite OP-1118. Less than 1% of fidaxomicin is recovered in urine. Fidaxomicin shows minimal CYP450 inhibition and no propensity to induce or inhibit expression of CYP450 enzymes. Fidaxomicin and OP-1118 are both P-glycoprotein (P-gp) substrates and inhibitors. Following successful treatment with oral metronidazole or vancomycin, 30 to 50% of the patients continue to harbor C. difficile in their stool. Current CDI treatment options are effective in treating the acute symptoms; however, relapse of CDI symptoms can occur in up to 40% of cases. None of the available treatments is without significant problems. Currently available antimicrobial treatments for CDI show the typical recurrence rate of approximately 20–30% [235]. Vancomycin use is associated with recurrence rates of 25% and, in addition, there is concern for a risk of acquiring resistant organisms, especially VRE with its use as an oral agent in treating this infection. Fidaxomicin has been shown to treat CDI as effectively as vancomycin; however, compared with vancomycin, fidaxomicin reduces rates of recurrence by 47%. Spontaneous development of resistance has been shown to be low.

12. DIARYLQUINOLINE: BEDAQUILINE

Bedaquiline, also known as TMC207, R207910 and compound J, currently marketed under the brand name Sirturo, is a diarylquinoline, with a novel mechanism of action and activity against M. tuberculosis [236]. The drug is indicated as part of combination therapy in adults (≥ 18 years) with pulmonary multi-drug resistant tuberculosis (MDR-TB). Sirturo is Complimentary Contributor Copy

250 Stefano Biondi and Mauro Panunzio reserved for use when an effective treatment regimen cannot otherwise be provided. The recommended dosage is 400 mg once daily for 2 weeks followed by 200 mg 3 times per week for 22 weeks with food. The structure is formed by a quinoline heterocyclic nucleus and side chains with a tertiary alcohol and a tertiary amine group, which are responsible for the antitubercular action [237]. The protonated basic amino group is likely to interact with the carboxyl group of glutamate 61 in subunit c of the active site of ATP synthase [238]. Important factors in the binding of bedaquiline to its active site are electrostatic interactions and aromatic rings binding by hydrophobic and stacking interactions [239]. The selectivity of bedaquiline for mycobacterial ATP synthase might be due to a different amino acid in the proximity of the binding side of subunit c (position 63 for mycobacteria: alanine and for humans: methionine) [240]. The gene encoding subunit c of ATP synthase is atpE, has a highly conserved amino acid sequence in nonrelated M. tuberculosis isolates [241]. The mechanism of resistance of M. tuberculosis to bedaquiline is due to the presence of mutations at position 63 (with a proline substituting alanine) or at position 66 (with a methionine substituting a leucine) of the atpE gene reducing the binding affinity to the c subunit of ATP synthase [242]. These mutations are inside the binding pocket of bedaquiline and confer either natural (e.g., in M. xenopi) or acquired (e.g., in M. tuberculosis mutants) resistance. The effect of bedaquiline on bacteria has two phases: during the first week of treatment it is bacteriostatic, during which time mycobacteria reduce their ATP consumption and remodel the ATP production pathways; afterwards it becomes bactericidal [239, 243]. Dormant Mycobacterium tuberculosis cells require a 10-times lower ATP level than replicating bacilli, but are successfully killed by bedaquiline when ATP levels drop even further [243b]. Bedaquiline is active against drug-susceptible and drug-resistant TB and it shows also activity against latent MDR-TB in animal models [244]. The in vitro antibacterial activity of bedaquiline is reported in Table 54. The pharmacokinetic properties of bedaquiline after single and repeated doses [245] are reported in Table 55. Food intake increases the oral bioavailability of bedaquiline by 2.0- to 2.4-fold relative to fasted conditions. Human protein binding of bedaquiline is greater than 99.9% at a concentration of 5 mg/L and also its active metabolite M2 (see below), is 99.7% bound to human plasma protein at concentrations of 10 and 30 mg/L. The volume of distribution in the central compartment is estimated to be approximately 164 L. In mice, bedaquiline is extensively distributed to tissues, including the lungs and spleen. Brain uptake was low. In a Phase II study, administration of bedaquiline at 400 mg once daily for 7 days, gave a Cmax in sputum of 5 mg/L, comparable to the mean concentration in plasma for the same dose and time-point (see Table 54) [248]. No clinical data on the distribution of bedaquiline into cerebrospinal fluid, bone or other tissues are available. TMC-207 is metabolized by CYP3A4 to an active N-desmethyl metabolite, M2 [249] and one minor metabolite M3, formed by the N-demethylation of M2. M2 is 4- to 6-fold less active against M. tuberculosis than bedaquiline, and M3 is inactive. Coadministration with rifampin resuts in a 50% reduction in bedaquiline concentrations [250]. Approximately 75%85% of the drug-related material is eliminated in 24 h in the feces, whereas M2 represented 3.7%–7.2%. Bedaquiline has a black- box warning for QT interval prolongation and an increase in all-cause mortality. Bedaquiline is the first novel antitubercular agent approved by the FDA since 1971 [251]. As MDR and extensively drug-resistant TB are increasing worldwide, this is an important advancement [252]. However, access to bedaquiline is still limited for many patients by infrastructural or Complimentary Contributor Copy

A 21st Century Contribution to the Antibacterial Armamentarium 251 regulatory obstacles, or by restrictions due to insufficient data, as in the case of children [253].

Table 54. Antibacterial activity of bedaquiline

Organism MIC range (g/ml) MIC99 (g/ml) Drug sensitive M. tuberculosis 0.03-0.12 0.06 MDR M. tuberculosis 0.03-0.03 0.03 M. avium 0.007-0.01 0.01 M. intracellulare 0.007-0.01 0.01 M. chelonae 0.06-0.5 M. fortuitum 0.007-0.01 M. kansasii 0.003-0.03 M. malmoense 0.5 m. gordonae 0.03 M scrofulaceum 0.03 M. marinum 0.003 M. xenopi 4-8 M. shimoidei 8 M. novocastrense 8 Helicobacter pylorii 2-4 4 Nocardia asteroides >16 Nocardia farcinca >16 Escherichia coli >32 Streptococcus pneumoniae 16-24 >32 Staphylococcus aureus >32

Table 55. Pharmacokinetic properties of bedaquiline after single and repeated dose

Dose Regimen Tmax (hrs) Cmax (g/ml) AUC0-144 (mg*h/L) Cmin (g/ml) (range) 10 single 6.0 0.0686±0.0148 1.248±0.233 (6.0–8.0) 30 single 5.0 0.276±0.064 4.418±1.424 (5.0–5.0) 100 single 5.0 0.854±0.283 13.604±5.115 (2.0–6.0) 300 single 5.0 2.547±1.305 38.737±14.584 (2.0–6.0) 450 Single 5.0 3.755±1.165 64.530±26.927 (2.0–5.0) 700 Single 5.0 6.747±2.210 97.816±38.074 (5.0–6.0) 25 Once daily 3.9 0.319±0.0977 0.0983±0.0368 Week one (2.0–6.2) 100 Once daily 4.0 1.208±0.395 0.380±0.165 Week one (2.0–8.0) 400 Once daily 4.0 5.502±2.965 1.448±0.437 Week one (2.1–6.0) 400 Once daily 2.763±1.185 0.728±0.257 (+ background Week two regimen) The effective half-life of bedaquiline is 24-30 hours [246] and the terminal half-life is approximately 5 months [247]. Complimentary Contributor Copy

252 Stefano Biondi and Mauro Panunzio

CONCLUSION

During the first 15 years of the new millennium the interest and need for new antibiotics has stimulated the pharmaceutical industry to return researching and developing new antibiotics to solve the growing problem of bacterial resistance. Overall, twelve classes and 27 new antibacterial agents have been introduced in the market but, of these, only five classes were newly introduced while the other were already present. In particular -lactam antibiotics constitute a very numerous group, with new cephalosporins, carbapenems and -lactamase inhibitors, including avibactam, the first inhibitor devoid of -lactam structure, but acting through acylation of the enzyme in the active site similarly to classical inhibitors. The main difference in the mechanism is related to the fact that the slow hydrolysis of the covalently bound inhibitor regenerates avibactam that could in theory be available for inhibiting another -lactamase molecule. The practical implication of this in not clear, as other more complex factors are implicated in determining the efficacy of the combination of a specific -lactam antibiotic with a -lactamase inhibitor. The five new classes are oxazolidinones represented by linezolid, lipopeptides (daptomycin), pleuromutilins (retapamulin, macrocyclic lactones (fidaxomicin), and diarylquinolines (bedaquiline). The first three classes remain still object of intensive research and more drugs of these classes are avaited in the near future. As a matter of fact in the coming years it is highly desirable that more antibiotics will reach the market, particularly focusing on high unmet medical needs, such as MDR Gram-negative pathogens, and pathogens that could be used for bioterrorism. In addition the globalization, facilitate mobility of travelers and environmental changes such as the global warming are increasing the spread of pathogen that historically were highly localized. Research activities should try to address all these problems by identifying new antibiotics, in particular the pharmaceutical companies should have two main options: identifying compounds with new antibacterial mechanisms of action, or identifying new chemical classes with a know mechanism of action that could evade existing esistance mechanisms. An example of the first strategy is POL7080, a protein epitope mimetic (PEM) under development for treatment of P. aeruginosa infections. The cellular target identified as a homolog of the beta-barrel protein LptD (Imp/OstA), which functions in outer membrane biogenesis [254]. If the new antibiotic is highly specific for a limited number of pathogens like POL7080, there are two options to adequately develop the drug: using the drug as an add-on therapy, or develop the drug in conjunction with a diagnostic companion. Two examples of the second strategy are avibactam, a non -lactam -lactamase inhibitor, or the boronic acid RPX7009 [255]. Other options to develop new antibacterial therapy include vaccines, monoclonal antibodies, biofilm inhibitors, quorum sensing, to mention but a few.

ACKNOWLEDGMENT

The authors would like to thank Dr. Stuart Shapiro (Allecra Therapeutics) for unvaluable support during the preparation of the manuscript and for revising the same.

Funding Sources: No funds were used to support the preparation of the manuscript.

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BIOGRAPHICAL SKETCHES

Dr. Stefano Biondi Head of Chemistry, Mfg and Controls

Dr., Univ. Bologna, Italy; MRSC CChem, UK. Dr. Stefano Biondi is a medicinal chemist with experience in both major pharma and biotech companies. For 12 years in GSK, he covered several positions, as Antibacterials Laboratory Head, he contributed to the discovery of sanfetrinem and other tricyclic carbapenems, invented and developed a number of novel ketolides. Previous roles include Director of Chemistry at Neuro 3D S.A. Director Research Laboratories, NicOx. Contributed to the identification and development of Epivir a marketed HIV/AIDS drug, VESNEO (latanoprostene bunod) an anti glaucoma drug, and numerous clinical compounds Inventor on over 40 patents dealing with antibiotics (macrolides, β- lactams) and CNS, cardiovascular, ophthalmic, and anti-inflammatory drugs.

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A 21st Century Contribution to the Antibacterial Armamentarium 267

Dr. Mauro Panunzio Prof. Organic Chemistry Associate Director of Research - National Research Council ISOF-CNR Institute of Organic Synthesis and Photoreactivity (ISOF); Department of Chemistry University of Bologna Via Selmi, 2 - I-40126, Italy Email: [email protected]

Activities: Co-author of some 150 papers and Patents concerning the synthesis of biologically active compounds (Biological active amines and aminols, Indoles, Prostaglandins, Steroids, -Lactam Antibiotics, Amino-acids, NCS-drugs) and new synthetic methodologies including Combinatorial Chemistry by Solid Phase or Parallel Solution Techniques and use of Microwaves in Organic Synthesis (MAOS). Visiting Scholar joining the Prof. B. K. Sharpless’ (Nobel Laureate) group/Stanford University, California, USA (1/10/’77-31/9/’78) Visiting Professor joining the Prof. Bijorn Akermark's group/ Department of Organic Chemistry/ Royal Institute of Technology/Stockholm, Sweden (1/10/’79-30/6/’80) Visiting Professor joining the Prof. L.S.Hegedus' group/ Department of Chemistry/Colorado State University/Fort Collins/ Colorado, USA. (1/1/’86-10/12/’86) Visiting Professor joining the Prof. L.S.Liebeskin's group/ Department of Chemistry/Emory University/Atlanta/ Georgia, USA. (1/8/’86-10/12/’86) Recipient of a fellowship grant by JSPS (Japan Society Promotion of Science) of Japan from 1/05/2000 to 1/06/2000 has been a visinting professor in Japan delivering lectures in several Japan Universities. Chairman of the ICCA-1 (1st International Conference on Chemistry of Antibiotics and Relate Microbial Compounds, formerly 6th ICSA, Bologna August 30 - September 4 1998). Visiting Professor of some Indian Universities India from October 2th 2002 to Novembre 2th 2002. Visiting Professor of Universities of Pais Basques (Spain) (May 2006). Visiting Professor of some Mexican Universities (March 2009). Italian Coordinator of the Project Fu Sc 14 “New Synthetic Processes for the preparation of non classical beta-lactam antiniotics” (Chinese Counterpartner Prof. Xia, Zhining, University of Chongqing. R.P. China) within the “Executive Protocol of the 12th Joint

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268 Stefano Biondi and Mauro Panunzio

Commission on S&T Cooperation between the People’s Republic of China (MOST) and the Italian Republic (Ministero Affari Esteri) for the Years 2006-2009 extended to 20010-2012. Dr. Panunzio has delivered a number of Plenary and Invited Lectures at different Internationals Meetings Conferences and Public as well as Private Scientific Institutions.

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INDEX

biocompatible, 2, 28, 30, 31, 37, 81 # biocompatible polymers, 28, 31, 37 biocorrosion, 80 21st Century, vi, vii, 181, 183 biofilms, 79, 81, 85 biomass antibacterials, vii, 1, 2, 3, 12, 13, 23 A biomedical applications, vii, 1, 60

Aeromonas, 78, 80, 196, 199 C affinity chromatography, 72, 87 AFM, 31, 32, 33, 34 Ca+2-dependent, 77 agglutination, viii, 69, 74, 77 calreticulin, 76, 85, 88 AgX, 19, 20, 26 CAPES, 82, 134 algae, 78, 81 carbohydrate specificity, 70, 72 anaerobes, 196, 197, 201, 202, 216, 217, 219, 227, carbohydrate-binding proteins, 70 231, 233, 234, 236, 238, 239, 243 carbohydrate-recognizing domain, 70, 71 animal lectins, 75, 76 cariogenic, 81 anionic matrices, 72 cationic matrices, 72 antibacterial armamentarium, vi, 181 cell permeabilization, 74 antibacterials materials, 2 cell wall, viii, 35, 69, 73, 74, 81, 241, 247 antibiofilm, 78, 79, 81, 82, 85 cellulose/AgCl/Ag hybrids, 21 antimicrobial lectins, 72, 73 cellulose-AgBr hybrid, 19, 21 antimicrobial properties, 28, 29, 53, 71, 84, 116, 120, cellulose-AgCl hybrid, 20 145 cellulose-silver nanocomposites, 13, 15, 25 autophagy, 77, 88 chemical structure, ix, 31, 190, 205, 246 chitin-binding lectin, 77, 81, 85 B chitosan coatings, 39 CNPq, 82, 134 Bacillus cereus, 78, 79 complement, viii, 69, 76 Bacillus megaterium, 79 corrosive bacteria, 79 Bacillus pumillus, 79 Corynebacterium callunae, 77, 79 Bacillus subtilis, x, 74, 77, 78, 143, 169, 170, 171, C-type lectins, 76 172 Cu nanoparticles biomass antibacterials, 6 bacterial inhibition, v, 27, 30, 37 CuO nanoparticles biomass antibacterials, 12 bacterial membrane, 50, 74, 223 bactericide, 78, 79, 80, 145 D bacteriocins, 77, 86 bacteriostatic, 78, 79, 80 defense, viii, 69, 75, 76, 81, 85, 86, 118 bedaquiline, 183, 187, 249, 250, 251, 252 depolarization, 223, 241, 244 Complimentary Contributor Copy

270 Index device-associated infections, 81, 243 interaction mechanism, 23 dialysis, 71, 194 intracellular responses, 74 dissacharides, 70 invading pathogen, 75 doripenem, 185, 195, 196, 199, 201, 202, 203, 255 invertebrate, 77, 89 ion exchange chromatography, 72 E K Enterococcus faecalis, 78, 80, 174, 192, 196, 198, 212, 216, 218, 220, 227, 230, 240, 241, 247 Klebsiella pneumoniae, 77, 78, 182, 183, 196, 198, Escherichia coli, viii, ix, x, 27, 31, 39, 74, 77, 78, 85, 202, 209, 216, 220, 227, 230, 237 116, 125, 127, 135, 136, 143, 169, 170, 171, 173, 174, 196, 198, 202, 207, 208, 209, 216, 218, 220, 227, 230, 237, 251 L eutrophic water, 79 lactamases, 118, 196, 197, 199, 201, 202, 229, 233, 234, 236, 239 F lectin purification, 71, 72 lignocelluloses/silver hybrids, 22 fabrication, 3, 4, 14, 23, 29 lipopolysaccharides, viii, 70, 73, 77, 80, 88 FACEPE, 82, 134 FINEP, 134 fish, 77, 83 M FUNCAP, 134 future perspectives, vii, 1, 2 mannose-specific lectins, 77, 88 marine sponge, 80, 86 mechanisms, v, vii, viii, 30, 69, 73, 74, 76, 78, 79, G 81, 86, 117, 118, 119, 121, 133, 138, 144, 145, 173, 174, 177, 191, 195, 205, 224, 226, 227, 229, galectins, 77, 84 235, 247, 248, 252, 253 gel filtration, 72 melanization, viii, 69, 76 glucose/mannose specific lectin, 81, 84 metal nanoparticles, viii, 2, 3, 28, 29, 30, 48, 49, 61 glycolipids, 70 metal nanoparticles biomass antibacterials, 2, 3 glycoproteins, 70, 73, 231 metal oxides nanoparticles biomass antibacterials, 2, Gold and silver nanoparticles biomass antibacterials, 7 6 Micrococcus, 78, 79, 80, 243 gram-negative, 31, 34, 35, 43, 53, 60, 118 microwave-assisted ionic liquids method, 13, 19 gram-negative bacteria, 43, 53, 60, 118 microwave-assisted method, 13, 15 gram-positive, 31, 34, 35, 43, 53, 57, 60, 240, 241 monosaccharides, 70, 71, 72 growth inhibition, viii, 53, 69, 74, 80, 81 N H nanoparticle, 3, 10, 12, 25, 28, 29, 47, 58, 59, 62, 65 hemolymph, 77, 78, 89 N-demethylation, 250 N-desmethyl, 250 I O infection(s), vii, x, 5, 27, 28, 34, 38, 58, 75, 76, 77, 81, 83, 84, 87, 116, 118, 119, 133, 137, 138, 181, opsonization, 76 182, 183, 190, 191, 194, 195, 197, 201, 203, 205, organic biomass antibacterials, 2 206, 207, 208, 209, 210, 217, 221, 224, 225, 226, others biomass antibacterials, 12 229, 233, 234, 235, 236, 239, 242, 243, 244, 247, oxidative burst, 76 249, 252 Complimentary Contributor Copy

Index 271

P S

P. aeruginosa, 52, 53, 77, 79, 80, 81, 118, 119, 132, Salmonella enteritidis, 77, 78, 79, 198 197, 203, 208, 214, 219, 227, 234, 235, 236, 252 salt fractionation, 71 pathogen recognition, 75 Serratia marcescens, x, 79, 80, 143, 169, 170, 171, pathogens, viii, x, 28, 69, 75, 76, 81, 84, 86, 123, 196, 198, 209, 216, 218, 220, 230, 233, 235, 238 139, 181, 183, 191, 194, 195, 196, 199, 201, 205, silver chloride, viii, 4, 19, 24, 28, 38, 60, 62 208, 215, 217, 223, 225, 226, 232, 234, 236, 243, skin mucus, 80, 86 247, 252 S-Lac-type lectins, 76, 77 peptidoglycans, 73 snake venom, 80, 86 permeabilization, viii, 70, 244 Staphylococcus aureus, viii, ix, x, 27, 74, 77, 78, 87, pH responsive materials, 55 116, 118, 125, 127, 135, 137, 139, 140, 143, 169, phagocytosis, viii, 69, 76, 89 170, 171, 172, 182, 183, 190, 192, 200, 202, 207, phytopathogenic bacteria, 80 216, 217, 218, 220, 223, 226, 229, 230, 240, 241, plants, viii, ix, 69, 70, 73, 75, 76, 77, 78, 81, 85, 87, 242, 245, 246, 247, 251 88, 115, 116, 119, 120, 132, 133, 136, 140 Staphylococcus epidermidis, viii, 27, 31, 39, 78, 81, pneumonia, 2, 84, 193, 197, 203, 206, 210, 215, 219, 192, 216, 220, 230 222, 223, 227, 230, 231, 233, 237, 239, 242 Streptococcus, 77, 78, 79, 81, 84, 85, 123, 182, 192, polymer nanofibers, 53, 54, 55 196, 198, 200, 202, 207, 216, 217, 218, 220, 227, polymer thin films, 51 229, 232, 240, 241, 245, 247, 251 polysaccharides, vii, 1, 2, 44, 70, 72, 81 Streptococcus faecalis, 77, 79 pores, viii, 5, 70, 72, 74, 119, 223 surface morphology, 29, 31 problems, vii, 1, 2, 117, 177, 249, 252 protein extraction, 71 protein synthesis, 117, 190, 191, 193, 226, 229 T Proteus mirabilis, 77, 79, 196, 198, 202, 209, 216, 218, 220, 230, 235, 238 TiO2 nanoparticles biomass antibacterials, 10 prulifloxacin, 184, 206, 208 transmission electron microscopy, 47 Pseudomonas, viii, ix, x, 27, 76, 77, 78, 79, 80, 84, 86, 116, 119, 125, 137, 138, 139, 143, 169, 170, U 171, 182, 183, 196, 199, 202, 206, 207, 208, 209, 210, 215, 216, 217, 218, 219, 220, 226, 227, 231, ultrasound agitation, 13, 21 233, 238 Universidade Regional do Cariri, 115, 137 Pseudomonas aeruginosa, viii, ix, x, 27, 77, 78, 116, URCA, 115 119, 125, 137, 138, 139, 143, 169, 170, 171, 182, 183, 196, 199, 202, 207, 208, 209, 215, 216, 217, 218, 219, 220, 226, 227, 231, 233, 238 W Pseudomonas fluorescens, 79, 80 Pseudomonas stutzeri, 79, 80 washability, 23 wound healing, 5, 23, 38, 44, 60 Q X quorum sensing signaling, 79 Xanthomonas campestris, 78, 80 XPS, 31, 33, 34, 35, 37, 39, 40, 42, 47, 48, 52 R

recognition, 70, 75, 76, 77, 84, 86, 87 Z reducing mechanism, 16 reducing reagents, 15, 22, 25 ZnO nanoparticles biomass antibacterials, 7 ZnS-cellulose nanocomposite, 12, 25

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