Protein and in Drug Targeting and its Therapeutic Approach Las proteínas y péptidos en la orientación de fármacos y su abordaje terapéutico Raj K. Keservani1 · Anil K. Sharma2 · Urmila Jarouliya3 1. School of Pharmaceutical Sciences, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, India-462036. 2. Department of Pharmaceutics, Delhi Institute of Pharmaceutical Sciences and Research, New Delhi, India-110017. 3. School of Studies in Biotechnology, Jiwaji University, Gwalior (M.P), 474011, India.

Artículo de revisión RESUMEN Review Article Objetivo. El objetivo principal de este artículo de revisión es proporcionar información sobre las ventajas de las proteínas y péptidos a través de diferentes vías de administración de fármacos, dirigidos a un sitio Correspondencia Correspondence en particular y su implicación en el sistema de administración de fármacos.

Raj K. Keservani Métodos. Con ese objetivo, los sitios web de PubMed, HCAplus, Thomson, se utilizaron como las prin- School of Pharmaceutical Sciences, cipales fuentes para realizar la búsqueda de los artículos de investigación más importantes publicados Rajiv Gandhi Proudyogiki Vishwavidyala- ya, Bhopal, India-462036 sobre el tema. La información fue luego cuidadosamente analizada, destacando los resultados más im- Mobile: +917897803904 portantes en el desarrollo de proteína y péptido de direccionamiento de drogas, así como su actividad [email protected] terapéutica.

Financiación Resultados. En los últimos años muchos investigadores utilizan las proteínas y los péptidos como un Fundings sitio diana de fármaco por un sistema de administración diferente. Las proteínas y los péptidos se utili- No funding agency for the research zan como agentes terapéuticos específicos y eficaces, debido a la inestabilidad y los efectos secundarios work. de su uso es complicado. Las proteínas quinasas son reguladores importantes de la mayoría, si no todos, los procesos biológicos. La actividad anormal de proteínas y péptidos se ha implicado en muchas en- Conflicto de interés fermedades humanas, tales como diabetes, cáncer y trastornos neurodegenerativos. Competing interest Author declares no conflict of interest. Conclusión. Finalmente concluyó que la proteína y el péptido se utilizaron en fármaco que se dirige al sitio específico y también se utiliza en diferentes estados de enfermedad como el cáncer, la diabetes, Agradecimientos como sustancias inmunomoduladora, efectos neurodegenerativos y actividad antimicrobiana. Acknowledgements Authors are thankful to the Mr. Adi Dev Palabras clave: proteína, péptido, liberación controlada, polímero bioerosionable. Bharti, for valuable suggestion. ABSTRACT Received: 19.04.2015 Accepted: 26.06.2015 Aim. The main aim of this review article is to provide information like advantages of protein and pep- tides via different routes of drug administration, targeted to a particular site and its implication in drug delivery system.

Methods. To that aim, from the web sites of PubMed, HCAplus, Thomson, and Registry were used as the main sources to perform the search for the most significant research articles published on the subject. The information was then carefully analyzed, highlighting the most important results in the development of protein and peptide drug targeting as well as its therapeutic activity.

Results. In recent years many researchers use protein and peptide as a target site of drug by a different delivery system. Proteins and are used as specific and effective therapeutic agents, due to in- stability and side effects their use is complicated. Protein kinases are important regulators of most, if not all, biological processes. Abnormal activity of proteins and peptides has been implicated in many human diseases, such as diabetes, cancer and neurodegenerative disorders.

Conclusions. It is concluded that the protein and peptide were used in drug targeting to specific site and also used in different diseased states like cancer, diabetes, immunomodulating, neurodegenerative effects and antimicrobial activity.

LICENSE 3.0 UNPORTED. Keywords: Protein, Peptide, Controlled release, Bioerodible polymer.

Ars Pharm. 2015; 56(3): 165-177 165 Keservani, R.; Sharma, A. & Jarouliya, U.

INTRODUCTION mostly used in medical therapy. The rapid development of biotechnology and progress in peptide and protein chemis- Protein and peptide enhance the successfully delivery of the try allowed the mass production of many compounds and drug to the desired site of action. In past twenty years many made their broad introduction into medical therapy pos- biopharmaceutical process development aspects have been sible 8. studied by researchers. For the production of many phar- maceutical proteins that have been well characterized and In biological processes protein and peptides play important overcome the problem associated with culture, purifi- role such as regulator in most cases not all. Their abnormal cation, recovery and fermentation 1. Thus, the successful activity has been implicated as causal factors in many hu- development of protein and peptide formulations is totally man diseases, including cancer, diabetes and neurodegen- depending on the study of in vitro, in vivo drug characteri- erative disorders 9-11. Pharmaceutical carriers were used to zation and its intended application 1. The recently protein increase the stability, improve their efficacy and decrease and peptide show great progress to understand the erosion undesired side effects and to assist intracellular delivery of mechanism of biodegradable polymer and the preparation the drugs in most of the cases 8,12. of controlled release devices. The results provide useful in- STRUCTURE OF PROTEIN AND PEPTIDES formation on the microstructure and chemical environment inside these polymers during erosion. Many protein and The Protein are complex of large molecules, consist of one peptide cannot be administered orally because it degraded or more amino acids in specific order. The protein and pep- inside the gastrointestinal tract (GI tract) 2 due to their short tide acquire three dimensional conformations and protein half life in the body fluids. structure is directly related to its function. It means that if the structure or shape of protein disrupted the function will These limitations forced the preparation of controlled re- also disrupted. There are four types of protein structures13 lease dosage form and enhance the protein and peptide (Figure 1), namely, (a) primary structure (b) secondary stability and drug activity for long period of time after its structure (c) tertiary structure (d) quaternary structure. application. Processing of this substance into dosage forms at all times not easily achieved. Many of them have limited A target peptide (Table 1) is short peptide chain of amino chemical and physical stability. Common instabilities are acids (usually 3-70) that directs the transport of protein irreversible aggregation (J) 3, oxidation 4 or conformational and peptide to specific region in the cell, such as includ- changes 5 all of which may affect activity. The controlled re- ing mitochondria, nucleus, (ER), lease devices for buccal, oral, nasal, rectal, parenteral, vagi- apolpast, , and plasma membrane. nal and transdermal routes have been prepared 6,7. In the enzyme cleaved the target peptide from last few decades number of bioactive protein and peptide protein after those proteins were transported14. The amino have discovered due to the progress in the field of biology acids joined together by amide linkage15 known as peptide and biochemistry. Insulin is a classical example, which is bond (Figure 2).

Figure 1. Four type of protein structure. Figure 2. Structure of peptide bond

166 Ars Pharm. 2015; 56(3): 165-177 Protein and Peptide in Drug Targeting and its Therapeutic Approach

Table 1. Examples of target peptides

S. N. Cell Peptide Chain

1. Transported to the nucleus (NLS) -Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-

H2N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val- Transported to the secretory pathway (plasma membrane of 2. Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-Glu-Gln- and endoplasmic reticulum of ) Leu-Thr-Lys-Cys-Glu-Val-Phe-Gln-

3. Retention to the endoplasmic reticulum -Lys-Asp-Glu-Leu-COOH

H2N-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe- 4. Transported to the mitochondrial matrix Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu-Cys-Ser-Ser- Arg-Tyr-Leu-Leu-

5. Transported to the peroxisome (PTS1) -Ser-Lys-Leu-COOH

6. Transported to the peroxisome (PTS2) H2N-----Arg-Leu-X5-His-Leu-

ADVANTAGES OF PROTEIN AND PEPTIDES IN DIF- trointestinal breakdown of drugs. Quick abrupt termina- FERENT ROUTES tions of drug effect by easily remove the delivery system from the skin surface 20-23. Protein and peptides delivery of drugs provides several ad- vantages when drug delivered by various routes, some of PEPTIDE AND PROTEIN DRUGS – BRIEF OVER- the mentioned below: VIEW AND DELIVERY PROBLEMS

Buccal route Many proteins and peptides acquire biological activity that makes them potent therapeutics. The enzymes represent When the drug delivered via buccal routes it provides an important and, probably, the best investigated group of much less pain, irritation in case of long term of treatment. protein drugs 24-26. The insulin peptide hormones used most It can be easily attached and removed without any discom- likely than that of other hormone. To the treatment of pitui- 16 fort and easily accessible and acceptable by patients . tary and gastrointestinal (GI) tumors, somatostatin analogs peptides such as lanreotide, octreotide and vapreotide be- Nasal route come available in the clinics 27. Antibodies against certain Nasal route is simple, convenient and provide rapid onset cancer-specific ligands can also be considered as protein of drug action and also avoid the first pass metabolism13,17 . anti cancer drugs 28,29. Still, the use of proteins and peptides as therapeutic agents is hampered by the whole set of their Rectal route intrinsic properties associated with their nature as complex A large dose of drug can be administered by rectal route, macromolecules, which are, as a rule, foreign to the recipi- drugs can be target to lymphic system, it avoids presys- ent organism. temic or first pass metabolism and suitable for drugs that Stability of protein may vary at different condition of phys- cause nausea/vomiting and irritation in GI tract on oral iological pH and temperature, most of the peptide espe- administration 16. cially protein show low stability at normal physiological Pulmonary route pH and temperature. Due to receptor interaction most of the protein and peptide and antibodies exert their action Pulmonary route of drug administration provides direct extracellularly. Many others, however, have their targets route to the circulation, safe route for drug entry even in inside the cell. In the latter case, low permeability of cell patient with lung disease. Less dose required, less pain or membranes to macromolecules often represents an addi- discomfort and improve patient compliance 13,18,19. tional obstacle for the development of peptide-based and protein-based drug formulations 30. Transdermal route PEPTIDES AS COMPETITIVE INHIBITORS Transdermal route provides controlled and sustained re- lease of drugs. Self administered and improve patient com- The sequences responsible for the protein ki- pliance due to its convenience and ease of use. Reduced nases specificity to substrate phosphorylation which leads side effects and avoid the hepatic first pass effect and gas- to phosphorylation called as consensus sequences. The

Ars Pharm. 2015; 56(3): 165-177 167 Keservani, R.; Sharma, A. & Jarouliya, U. numbers and types of interactions with residues surround- properties of the protein such as the isoelectric point, mo- ing the phosphorylation site vary considerably among ki- lecular weight, glycosylation or other post-translational nases, reflecting differences in sequence specificity. By vir- modification, and overall amino acid composition. These tue of that consensus sequence can serve as a template for a properties along with any known behavior of the drug in peptide that will acts as a competitive inhibitor 31,32. different solutions (e.g. different buffers, cofactors, etc.) as well as its in vivo behavior should guide the choice of PEPTIDES COMPETING WITH ANCHORING AND formulation components for testing in the initial screen of TARGETING PROTEINS candidate formulations. The potential candidate formula- Modular binding partners target some Ser/Thr kinases to tions are composed of U. S. Food and Drug Administration their specific substrates via changes in cellular localization. (FDA) approved buffer components, excipients, and any Binding of a peptide to this targeting domain may prevent required cofactors (e.g. metal ions). Often, the first choice of the interaction of the kinase with its substrates. The Protein candidate formulations is based upon the previous experi- kinase C isoforms are known to translocate from cytosol to ence of the formulation scientist with other proteins or pep- particulate fractions in response to various stimuli. A fam- tides and, in many cases; a simple phosphate buffered sa- ily of adaptor molecules, termed RACKs (Figure 3) (from line solution may be one of the initial candidates. The pH of Receptors for Activated C-Kinase), interact with the vari- the solution impacts major degradation pathway. Thus, the ous Protein kinase C isozymes and facilitate their cellular initial formulations also assess the pH dependence of the translocation 33,34. The RACK binding domain is a short degradation reactions and the mechanism for degradation 38 sequence located in the Protein kinase C’s N terminal-sub can often be determined from the pH dependence . The domain and is different in each isoform. Thus, the trans- stability of each solution quickly analyzed by the formula- location of Protein kinase C isoforms could be disrupted tion scientist. Rapid screening methods usually involve the selectively using peptides derived from the specific RACK use of accelerated stability at elevated temperatures (e.g. docking region 35,36. The use of such peptides has enabled 40°C) 39-42. analysis of the functions of individual Protein kinase C Many proteins and peptides shows extensive degradation isozymes. For example, peptides derived from the C-2 re- without affecting their safety and efficacy, such as recom- gion of PKC-β II prevented hormone-induced membrane binant human growth hormone (rhGH) is fully bioactive translocation of PKC β II and inhibited insulin-induced and nonimmunogenic but 70 % deamidated, this value of Xenopusoocyte maturation 37. degradation not acceptable by regulatory agency standard 43 Figure 3. Structure of Receptors for Activated C-Kinase for therapeutic protein . (RACK) Scientists must consider potential conditions and all the major degradation route of optimization to fulfill the regu- latory requirements for a stable formulation. Surfactant is added in case of aggregation or sugar can prevent denatur- ation process, which leads to irreversible aggregation. The use of amine buffers (like Tris, ammonium, or imidazole) may slow the deamidation rate when degradation rate dominant 44. In another degradation pathway, for example, by adding surfactants or other polymers to prevent aggre- gation, the residual peroxide in the surfactant may cause a more rapid oxidation 45.

To decrease the deamidation rate pH of the formulation must be reduced in some cases. Reduction of pH may also alter the protein solubility, since many proteins have iso- FORMULATION DEVELOPMENT CONSIDERA- electric points at or near the optimal pH (pH 5-6) for mini- TIONS mizing the deamidation rate. For each protein formulation, Novel delivery system includes the complete characteri- all the degradation pathways must be evaluated and often zation of the drug properties and drug stability in differ- a balance must be achieved between the different degra- ent formulations, is the main key for development of any dation pathways. The researchers have options to develop protein and peptide formulation. Typically, a formulation solid formulation like lyophilized powder. The removal of scientist will begin by considering the physicochemical excess water from the formulations decreases rate of the

168 Ars Pharm. 2015; 56(3): 165-177 Protein and Peptide in Drug Targeting and its Therapeutic Approach degradation in process of deamidation and hydrolysis. The without excipients required some residual water for stabil- residual moisture in a solid protein or peptide formulation ity purpose 46. Different routes of protein and peptide deliv- can contribute to the physical stability of the protein by pre- ery and polylactide applications presented in Table 2 and venting its denaturation and subsequent aggregation upon Table 3 respectively. reconstitution. Recent studies on lyophilization of proteins

Table 2. Routes of delivery for proteins and peptides 45,46

Formulation and Device Commercial Prod- S.N. Delivery Routes Manufacturer Requirements ucts

1. Invasive

Liquids or reconstituted solids, Direct Injection syringes

intravenous (i.v.) Activase® Genentech, Inc.

subcutaneous (s.q.) Nutropin® Genentech, Inc.

intramuscular (i.m.) RecombiVax® Merck & co.

intracerebral vein (i.c.v.)

Biodegradable polymers, li- posomespermeable polymers Depot system (not degradable),microspheres, implants

Takeda Pharmaceu- Lupron Depot® ticals. LHRH analogs (s.q. or Zoladex® Imperial Chemical i.m.) Industries, Ltd. Decapeptyl® Debiopharm

2. Noninvasive

Liquids or powders formulations, Pulmonary nebulizers, metered dose inhalers, Pulmozyme® Genentech, Inc. dry powder inhaler

Solids, emulsions, microparticulate, Oral absorption enhancer

Liquids, usually requires permea- Nasal tion Synarel® Syntex Corporation enhancer

Emulsions, creams or pastes Topical (liposomes)

Electrophoretic (iontophoresis), electroporation, chemical permea- Transdermal tion enhancer, prodrug, ultrasonic

Gel, suppositories, bioadhesives, Buccal, Rectal, Vaginal particles

Ars Pharm. 2015; 56(3): 165-177 169 Keservani, R.; Sharma, A. & Jarouliya, U.

Table 3. List of polylactide for controlled release of proteins or peptides and its application 47-60

S.N. Protein or Peptide Polymer (*) Application

Bone morphogenetic PLA-PEG copolymer (650 Da PLA - 200 1. Bone formation protein Da PEG)

Transforming 50:50 PLGA (40-100 kDa) (+ demineral- 2. Bone formation growth factor-βι ized bone matrix)

Central nervous 3. Thyrotropin 75:25 PLGA (11 kDa) system dysfunction

Growth hormone Growth hormone 4. 75:25 PLGA (91 kDa) releasing factor Deficiency

5. Somatostatin analogue 55:45 PLGA (23-76 kDa) Acromegaly, tumors

6. Neurotensin Analogue PLA (2-6 kDa) Psychotropic

50:50 PLGA 7. Cyclosporin A Immunosuppression (0.44 & 0.80 dL/g)

Colonizing factor 8. PLGA (0.73 dL/g) Oral vaccine antigen (E. coli)

Cholera toxin Β 9. PLA (2 kDa ) Oral vaccine subunit

Diphtheria toxoid 10. PLA (49 kDa) Vaccine formilin treated

50:50 PLGA (22 kDa) 11. Ovalbumin Vaccine 85:15 PLGA (53 kDa)

12. Tetanus toxoid 50:50 PLGA (100 kDa) Vaccine

50:50 PLGA 13. LHRH antagonists Tumor suppression 75:25 PLGA

Horse radish Marker proteins 14. peroxidase 75:25 PLGA (10 kDa) Mechanistic studies Bovine serum albumin

(*) X: Y PLGA indicates the mole fraction (%) of lactide (X) and glycolide (Y) in the copolymer. The polymer size is reported as either molecular weight in kilo Daltons (kDa) or intrinsic viscosity in decaliters/gram. Some references did not provide complete descrip- tions of the polymer.

THE BASIC CONCEPT FOR DRUG DELIVERY FROM CONTROLLED RELEASE DEVICES FOR THE DELIV- BIOERODIBLE POLYMERS ERY OF PROTEINS AND PEPTIDES FROM POLY- MERS Polymer matrix was represented by two dimensional com- putational grids, and each of grid point represents either Biodegradable and non degradable polymers have been used in controlled release delivery of proteins and pep- an amorphous or crystalline part of the polymer matrix. tides. Direct diffusion through a non degradable polymer The erosion of polymer mainly depends on two features matrix is not possible due to the high molecular weight (a) contact with the degradation medium (b) crystalline or of proteins 63. By the introduction of a network of pores amorphous nature of matrix. Polymer that’s not contact in manufacturing, however, the release of such large com- with water will not erode. Pixels on the surface of the poly- pounds does occur 64. Another possibility is the creation of mer matrix or next to an eroded neighbor have contact to hydrophilic pathways using swellable polymers, or em- water. The amorphous parts of polymer erode faster than bedding the compounds into gels. In the case of biodegrad- the crystalline matrix 62. able polymers, pores are created upon erosion of the poly-

170 Ars Pharm. 2015; 56(3): 165-177 Protein and Peptide in Drug Targeting and its Therapeutic Approach mer matrix enabling the release of proteins from the dosage polypeptide (ELP), which can be synthesized in various se- form. All of these options have certain advantages and quences and sizes to stabilize the therapeutic peptide and disadvantages. Embedding suspended compounds into a avoid crossing the placental interface. This prevents fetal non-degradable matrix prevents some of the protein from exposure and potential developmental effects 82. being released 65, or might cause some instability due to the Targeted delivery by cell-targeting peptides (CTPs) with intense contact with organic solvents 66. Their disadvantage the ability to recognize cancer cells is particularly attractive with respect to parenteral application, however, is the need for cancer therapy; the use of these peptides has increased for removing such systems after therapy. By using gels as the specificity and efficacy of drug delivery while reducing a carrier, the protein may be released very quickly if not side effects in a model 83. PEGA, a homing peptide used in combined with some other sort of material67,68. Degradable the diagnosis of breast cancer, this peptide conjugated to polymers change their properties substantially during ero- the cell-penetrating peptide pVEC was taken up by differ- sion, a characteristic which may or may not be beneficial for ent breast cancer cells 84. Peptide D2A21 as therapeutic for the stability of proteins and peptides. In general, decisions several types of cancer and has been developing this pep- about the suitability of a release device for specific proteins tide gel formulation as a wound healing product to treat in- or peptides and appropriate manufacturing technology fected burns and wounds. A TAT peptide derived from the must be made on a case by case basis. In the following sec- N-terminus of p53 has been used in the application of sev- tion, they provide a number of examples from the progress eral tumor suppressor and apoptotic genes, TAT was fused they made in the delivery of proteins from polymers. to a peptide derived from the VHL tumor suppressor gene BIODEGRADABLE POLYMERS FOR IMMUNIZA- that inhibits insulin-like growth factor-I receptor (IGF-IR) TION signaling in renal cell carcinomas 85. Another study done by Su et al., observed that naturally existing anti-cancer In seventies, the idea of polymers were using as antigens bioactive peptide obtained from liver (ACBP-L) alone or in 69 releasing carriers for the simulation of response . Due combination with the low dose Cisplatin improves quality to their progress and ease of application, potential effi- of life in the xenograft tumor model having human gastric cient carriers (microspheres) emerged to enhance immune cancer 86. The bioactive peptide RRM-MV obtained by the 64 response . They have so far been used for a number of resonant recognition model and Myxoma virus (RRM-MV), 70-72 vaccines . These microspheres were made from poly- mimicks the bioactivity of specific MV proteins and evalu- mers based on poly-lactic acid (PLA) and its copolymers ated the suitability of RRM-MV for human skin cancer with glycolic acid (PLGA). For these applications, loading therapy 87. a polymer with a high molecular weight protein requires adjusting the desired release rate and preventing the pro- Diabetes therapy tein’s loss of activity. In an attempt to develop a controlled In the study by Kaspar et al. (2013), recent successes in the release system for vaccination against tetanus, Sluzky et al. development of peptides as therapeutics, most notably investigated the design of microspheres with a desired re- glucagon-like peptide 1 receptor (GLP-1R) agonists such as lease rate and methods to maintain the imunogenicity of Lixisenatide (Lyxumia1), a GLP-1R agonist, was approved the processed tetanus toxoid 73. as a treatment for type 2 diabetes88. It competes with two APPLICATION OF PROTEIN AND PEPTIDES IN DIS- other GLP-1R agonist peptides, exena-tide and liraglutide, EASES for type 2 diabetes, they are administered subcutaneous- ly. The GLP-1R agonist administered s.c. weekly is being Protein and peptides widely used in the management compared to other diabetes drugs (i.e. exenatide, insulin and treatment of various diseases such as cancer manage- glargine, metformin, sitagliptin, liraglutide) or placebo in 74 75 76 ments , cancer therapy , diabetes therapy , Antimicrobial type 2 diabetes patients. It acts (e.g. cell-targeting peptides, 77,78 activity , Peptides and peptidomimetics can function as cell-penetrating peptides) by linking the peptides to vari- immunomodulating agents by blocking the immune re- ous molecular formats (e.g. peptides linked to small mol- 79,80 81 sponse , neurodegenerative diseases etc. ecules, lipids, carbohydrates, biopolymers, polyethylene glycol or proteins) and GLP-1R is able to normalize the Cancer therapy and managements levels of blood glucose in Type 2 diabetes through several Gene et al., describes a number of peptide based therapies mechanisms: the release of insulin is stimulated and the we have developed to target theses pathways, and which release of glucagon is suppressed89. In the another study are currently being tested in preclinical models. Therapeu- by Wahren et al., C-peptide activates insulin receptor tyros- tics are based on a synthetic polymeric carrier elastin-like ine kinase phosphorylation, and glycogen synthase kinase

Ars Pharm. 2015; 56(3): 165-177 171 Keservani, R.; Sharma, A. & Jarouliya, U.

3 phosphorylation , with downstream effects leading to within neutrophil granules and play an important microbi- GLUT mobilization, promotion of amino acid uptake, and cidal role in phagolysosomes. glycogen synthesis, suggesting that C-peptide signaling Neurodegenerative diseases may cross-talk with the insulin pathway at the level of the insulin receptor 90. Several clinical studies, with C-peptide There are currently no effective drugs for the treatment of replacement in patients with type 1 diabetes, show benefi- neurodegenerative diseases; potential therapeutic targets cial effects on somatic and autonomic Diabetic peripheral for symptomatic treatments of neurodegenerative dis- neuropathy (DPN), also the C-peptide mediates reduction eases may include neuroprotective factors, neurotrophins of glomerular hyperfiltration and decrease of urinary al- and neuroprotective peptides 102,103. Peptides like NAP bumin excretion in the diabetic state 91. Another protein (NAPVSIPQ) derived from activity of neuroprotective peptide in diabetic study is G-protein-coupled receptors protein(NAP) and ADNF-9 peptide derived from activity- (peptide-binding GPCRs) that play an important role in the dependent neurotrophic factor (ADNF) in the treatment of pathophysiology of vascular dysfunction during diabetes92. Alzheimer’s disease (AD) and Amyotrophic lateral scle- rosis (ALS) disease. ADNF-9 has been found to protect Immunomodulating agents against Aβ, apolipoprotein E deficiencies, and oxidative stress, as well as increasing synapse formation. Colivelin The immunomodulatory action of biopeptides is related another protein peptide also has potential neuroprotecrive to the stimulation of proliferation of human lymphocytes effect in neurodegenerative diseases such as AD 104. NAP and macrophages phagocytic activity 93. Lactoferrin (milk mimics the neuroprotective activity of ADNP in its abil- protein peptide) and its derivatives affect the production of ity to cross then blood-brain barrier, interact with tubulin, cytokines involved in immune reactions of the organism94. enhance assembly of microtubules, and promote neuronal LF-derived peptides have clinical applications due to their outgrowth in AD and ALS disease 104. In the another discov- chemopreventive and immunomodulatory properties 95. ery of poly Q binding protein (QBP1) peptide, these QBP1 α-lactalbumin (whey protein fraction of bovine milk) has specifically binds to the expanded polyQ stretch and inhib- immunomodulatory properties, whereas products of its its its misfolding and aggregation, resulting in suppression degradation by trypsin and chemotrypsin (f1 – 5, f17 – 31- of neurodegeneration in cell culture and animal models of SS-f109 –114 and f61 – 68-SS-f75 – 80) or pepsin exhibit both the polyQ diseases (Huntington’s disease, spinocerebellar immnunomodulatory and antimicrobial properties against ataxia different types, dentatorubral pallidoluysian atro- 96 bacteria, viruses and fungi . Acidic peptides derived from phy, and spinobulbar muscular atrophy (SBMA)] 106. β-Lactoglobulin under the action of peptidases of Lactobacil- lus paracasei decreased the stimulation of lymphocytes and Antimicrobial activity regulated production of IL-10, IFN-γ and IL-4 this indicates Antimicrobial peptides (AMPs) are widely distributed eliminating allergic reaction in cow milk 97. Casein fraction in nature, are found in species ranging from bacteria and and its derived peptide (isracidin) of milk total protein, is insects to mammals 107. Most of these peptides are synthe- a rich source of bioactive peptides that stimulate and aid sized as a prepropeptide consisting of an N-terminal signal 98 the immune system . In the study by Rodrigo and Albani, sequence, a pro segment and a C-terminal cationic peptide Peptides derived from the E. coli (hsp) that demonstrates antimicrobial activity after it is cleaved dnaJ are antigenic in human autoimmune disorder rheu- from the rest of the protein 108. Compared to conventional 99 matic arthritis . T cell recognition of these peptides is as- anti-infective agents, some AMP may kill bacteria but also sociated with TH-I type and pro-inflammatory responses, simultaneously neutralize released pathogenic factors, like including production of TNF-α and IFN-g in the pathogen- lipopolysaccharide (LPS) or lipoprotein (LP). AMPs have esis of autoimmune inflammation. Another studied pep- anti-Gram-positive and -negative effects 109 as well as anti- tide LL-37 is a human antimicrobial peptide derived from viral and anti-yeast effects. Peptides have shown potential cathelicidin has anti-endotoxic activity, induces chemokine and desirable therapeutic properties like antimicrobial, an- production, promotes IL-1b , but inhibits inflam- tiviral, anticancer and contraceptive activities. Members of matory responses to certain TLR ligands 100,101. Neutrophils the major groups of antimicrobial peptides have been clas- LL-37 and defensins are produced by neutrophils, stored sified mainly on the basis of their biochemical (net charge)

172 Ars Pharm. 2015; 56(3): 165-177 Protein and Peptide in Drug Targeting and its Therapeutic Approach and/or structural features (linear/circular/amino acid tives show the antibacterial activity in vitro against various composition). pathogens, e.g. Clostridium perfringens, Candida albicans, Haemophilus influenzae, Helicobacter pylori, Pseudomonas aer- Cationic peptides uginosa, Staphylococcus aureus, Streptoccccus mutans, Vibrio This is the largest group and the first to be reported, being cholerae as well as antiviral activity against hepatitis C and widely distributed in animals and plants. On the basis of B virus; HIV-1; poliovirus; rotavirus; and herpes simplex 115 their structural features, cationic peptides can be divided virus . Hydrolysis of αs2-casein (by chymosin acting) re- as well into three different classes: (1) linear peptides form- sults in releasing casocidin, this peptide shows antibacterial ing-helical structures (such as Cecropins) ; (2) cysteine-rich properties. It was also reported that antibacterial peptides open ended peptides containing single or several disulfide obtained from αs2-casein (i.e. f183-207 and f164-179) inhibit bridges (such as Defensins); and (3) molecules rich in spe- growth of both Gram-positive and Gram-negative bacteria. cific amino acids such as proline, glycine, histidine and In the study by Rivas et al., resulted that Penetratin, the tryptophan (such as amino-acid enriched class) 110. Some peptide derived from the drosophila transcription factor other cationic peptides include, Thionins (plant peptides) antennapedia and the HIV1-Tat derived nonapeptide, be- 111 and histone derived compounds, beta-hairpin, cathelici- long to the arginine-class of peptides. These peptides ex- dins and thrombocidins. hibit antimicrobial or antifungal properties 116.

Anionic peptides CONCLUSIONS

This is a smaller group of antimicrobial peptides which in- Several variables play important role in the design, devel- cludes; Neuropeptide derived molecules,Oxygen-binding opment and delivery methods of protein and peptides. To proteins, Aromatic dipeptides and Aspartic-acid-rich mol- make successful protein and peptide drugs, the relation- ecules. Some other AMPs Bacterial ribosomes synthesize ship between the routes of administration, pharmacoki- antimicrobial peptides which are generally called as bac- netics, toxicity and clinical indication should be carefully teriocins (in bacteria), cecropins, Drosomycin and metch- balanced. Many novel systems like liposome and polymer nikowin (in insect). These peptides however, have certain offers smart alternative to solid and liquid dosage form. properties in common. They all have an affinity for mem- New delivery often requires invasive method but noninva- brane lipids and their specificity for microbial membranes sive delivery routes such as transdermal or pulmonary de- in many cases has been shown to be related to the positive livery may give up capable results for protein and peptides charge on the peptide favouring interaction with the ex- in future. The delivery of DNA and RNA in gene therapy posed anionic lipids of microorganisms. The peptides may and antisense therapy respectively is addressed by formu- form pores in the membrane allowing for leakage of ions lations scientist. A new type of system such as biodegrad- and other materials from the cell. The activity of the pep- able nanosphere is available in near future because pres- tide is explained by mechanisms like carpet, barrel stave, ently they are the focus for research by scientists. Finally toroidal along with these mechanisms, it shows an intracel- we concluded that the protein and peptide have versatile lular killing activity which affects the nucleic acid of the applications in the field of drug delivery. microorganism 112. REFERENCES Numerous studies focus on multipotential activity of lacto- 1. Cleland J, et al.. In Formulation and Delivery of Proteins and ferricin (Lfcin) that is a product of hydrolytic degradation Peptides; ACS Symposium Series; Amer. Chem. Societ. Wash- of lactoferrin (LF). Lactoferrin itself exhibits a strong bacte- ington, DC. 1994; 1: 1-2. ricidal activity through its ability to bind iron. This peptide shows a considerably higher antimicrobial activity than 2. Luft FC, Lang GR, Aronoff H, Ruskoaho M, Toth M, Ganten the native protein 113. Lactoferrampin (Lfampin) is another D, Sterzel RB, Unger T. Atriopeptin III kinetics and pharma- peptide derived from lactoferrin. It has a wide spectrum codynamics in norma and anephric rats. J. Pharmacol. Exp. of antifungal and antibacterial properties. The peptide ex- Ther. 1986; 236: 416-428. erted antifungal (against Candida) activity higher than LF 3. Zhang F, Liu CL, Hu BR. Irreversible aggregation of protein and was also active against Bacillus subtilis, Escherichia coli synthesis machinery after focal brain ischemia. J. Neurochem. and Pseudomonas aeruginosa 114. Lactoferrin and its deriva- 2006; 98(1):102-112.

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