Copyright by Natalie Kay Boyd 2020 The Dissertation Committee for Natalie Kay Boyd Certifies that this is the approved version of the following dissertation:

THE IN VITRO ANTIBIOTIC ACTIVITY OF NON-ANTIBIOTIC DRUGS AGAINST S. AUREUS CLINICAL STRAINS

Committee:

Christopher R. Frei, Supervisor

Kelly R. Reveles

Jim M. Koeller

Jose L. Lopez-Ribot

Manjunath P. Pai THE IN VITRO ANTIBIOTIC ACTIVITY OF NON-ANTIBIOTIC DRUGS AGAINST S. AUREUS CLINICAL STRAINS

by

Natalie Kay Boyd

Dissertation

Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

The University of Texas at Austin August 2020 Dedication

I dedicated this dissertation to my family, friends, and colleagues, who have supported and loved me unconditionally. Abstract

THE IN VITRO ANTIBIOTIC ACTIVITY OF NON-ANTIBIOTIC DRUGS AGAINST S. AUREUS CLINICAL STRAINS

Natalie Kay Boyd, Ph.D. The University of Texas at Austin, 2020

Supervisor: Christopher R. Frei

Drug repurposing, or identifying new uses for existing drugs, has emerged as an alternative to traditional drug discovery processes involving de novo synthesis. Drugs that are currently approved or under development for non- antibiotic indications may possess antibiotic properties, and therefore may have repurposing potential, either alone or in combination with an antibiotic. They might also serve as “antibiotic adjuvants” to enhance the activity of certain antibiotics. The objective of the proposed research is to utilize novel screening tools to characterize antibiotic effects of select non-antibiotic drugs against

Staphylococcus aureus clinical isolates. We hypothesized that one or more of the non-antibiotic candidates selected for this study would demonstrate antibiotic activity against Staphylococcus aureus, either directly (as monotherapy) or indirectly (in combination with antibiotic drugs). We determined minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) for non-antibiotic drugs (amlodipine,

v azelastine, ebselen, and sertraline) against five clinical Staphylococcus aureus isolates and one quality control strain using microplate alamar blue assays. Clinical isolates were selected from previous clinical and epidemiological studies by our research group. These isolates were obtained from wound swab cultures of patients with skin and soft tissue infections (SSTIs) who were seen at primary care clinics in the South Texas Ambulatory Research Network (STARNet) from 2010 to 2013. Our group had previously identified resistance genes and determined susceptibilities in these isolates using whole genome sequencing and Vitek 2 automated testing system (bioMerieux, Durham, NC), respectively. Each clinical isolate was genetically and phenotypically distinct from one another. Amlodipine, azelastine, and sertraline MICs were 64 µg/ml (112 µM), 200 µg/ml (480 µM), and 20 µg/ml (60 µM), respectively, while ebselen ranged from 0.25 - 1 µg/ml (1 - 4µM). MBCs for amlodipine, azelastine, sertraline, were within one dilution of their MICs, indicating bactericidal activity for all test isolates. Ebselen MICs were 1 - 2 dilutions higher in isolates carrying two or more resistant determinants. Though the microplate reader has the capability of continuous monitoring of fluorometric data, this functionality was limited in this case due to significant evaporation, likely from absence of the microplate lid during incubation. Evaporation was immediately evident upon visual inspection, as the volume loss created a more pale or colorless appearance. Not surprisingly, volume loss from evaporation also affected antibiotic concentrations, which gave the appearance of increased potency. We observed this with the control strain after measuring fluorescence activity over 16 hours against non-antibiotics. Azelastine 100 µg/ml and sertraline 10 µg/ml appeared to inhibit growth by more than 60% compared to vi the control. However, azelastine and sertraline MICs, determined prior to this experiment, were shown to be one dilution higher at 200 µg/ml and 20 µg/ml, respectively. Therefore, it less plausible that azelastine and sertraline inhibited growth to this extent at sub-MIC concentrations. The overall level of fluorescence intensity, when tested as part of continuous monitoring, was also at least 30% lower than expected compared to initial testing for signal to background ratio. These inconsistencies can easily be explained by evaporation. Dose-response curves for non-antibiotics were plotted by drug concentration (log [µg/ml]) and % inhibition. Calculated IC50 ranges were 37.9 - 47.2 µg/ml (66.3 - 82.6 µM) for amlodipine, 116 - 119 µg/ml (278 - 286 µM) for azelastine, 11.7 - 18.3 µg/ml (35.1 - 54.9 µM) for sertraline, and 0.17 - 0.62 µg/ml (0.67 to 2.5 µM) for ebselen. In summary, all four non-antibiotics demonstrated in vitro activity to varying degrees against S. aureus clinical isolates. Ebselen was the most potent of the four non-antibiotics tested, with MICs between 0.25 to 1 µg/ml, and MBC/MIC ratios of 1-2. This range is consistent with what prior investigations have observed against S. aureus. In fact, ebselen has demonstrated bactericidal activity various multidrug resistant strains of S. aureus, including vancomycin-intermediate and vancomycin-resistant strains. Additionally, ebselen has been shown to inhibit toxin production as well as biofilm formation. When viewed in the context of available literature, these data suggest that the non-antibiotic candidates selected for this study might have future potential as antibiotics or antibiotic adjuvants.

vii Table of Contents

Chapter One. Drug repurposing for antibiotic development...... 1 Drug discovery: Overview of process ...... 1 Antibiotic drug development: A historical perspective ...... 3 Penicillin: A landmark 'bench to bedside' breakthrough ...... 3 The antibiotic era ...... 5 Antibiotic innovation gap ...... 7 Emergence of resistance ...... 17 Staphylococcus aureus - an urgent threat ...... 19 Addressing the unmet clinical need ...... 21 Drug repurposing ...... 24 Overview ...... 24 Non-antibiotics ...... 28 Antibiotic adjuvants ...... 31 References ...... 33

Chapter Two. The in vitro activity of non-antibiotic drugs against Staphylococcus aureus clinical strains ...... 44 Introduction ...... 44 Central Hypothesis, Specific Aim, and Objectives ...... 45 Central Hypothesis ...... 45 Specific Aim ...... 46 Methods and rationale ...... 46 Staphylococcus aureus isolates ...... 46 Selection of non-antibiotic drug candidates ...... 47 Non-antibiotic drugs: Solubility and storage ...... 49 Selection of non-antibiotic drug concentrations ...... 50 Antibiotic susceptibility testing ...... 51 Data analysis ...... 54 Assay optimization ...... 54 viii Results ...... 56 Assay optimization ...... 56 Non-antibiotic susceptibilities ...... 56 Fluorometric analysis ...... 57 Discussion and Conclusion ...... 61 Limitations ...... 65 References ...... 66

Chapter Three. Translational application ...... 72 References ...... 76

Appendices ...... 77 Appendix A ...... 77 Appendix B ...... 78 Appendix C ...... 79 Appendix D ...... 80

Bibliography ...... 81

ix CHAPTER 1

Drug repurposing for antibiotic development

DRUG DISCOVERY: OVERVIEW OF PROCESS

Traditional drug discovery strategies aim to identify the next new chemical or molecular entity that possesses a novel mechanism of action. For any promising new compound, the path from initial discovery to market launch is sluggish, costly, and fraught with a multitude of barriers. Moving a new drug from pre-clinical phases to market generally requires a minimum timeframe of 10-12 years and over

$2 billion in resources.1,2 Additionally, the probability of success is low, with only 1-2 drugs from an initial 10,000 compounds reaching Federal Drug Administration (FDA) approval. An overview of this lengthy process is illustrated in Figure 1.1. Following target identification and validation, high-throughput screening (HTS) assays are developed and run against compound libraries to generate ‘hits’, which are the compounds that demonstrate the desired activity or interaction with the target of interest. Each hit series undergoes additional screening and/or chemical modifications to become more ‘druggable’ lead compounds before in vitro and in vivo pharmacokinetic testing is performed in animal models (preclinical). Generally, only a handful of drug candidates from the initial 10,000 compounds

enter clinical trials. Success rates for drugs entering Phase I clinical trials have approximately 10% chance of gaining FDA approval for the desired indication.3

1 Figure 1.1 Pharmaceutical research and development process4

2 ANTIBIOTIC DRUG DEVELOPMENT: A HISTORICAL PERSPECTIVE

Milestones of antibiotic discovery and development can offer insights to future solutions. The pre-antibiotic era bears striking resemblance to circumstances of today, regarding a need for: 1) novel, effective antibiotics, 2) large scale collaboration, and 2) efficient processes/timelines for antibiotic approvals.

Penicillin: A landmark ‘bench to bedside’ breakthrough

The discovery of penicillin in 1928 is regarded as one of the most significant

medical and scientific breakthroughs in history.5-8 It represents one of history’s earliest examples of translating a scientific discovery into medicine. The story of how penicillin was developed is as important as the discovery of the drug itself. Overcoming the major barriers during that time helped establish methods that led to next-generation penicillins and development of other antibiotic classes.6,8 When a fungal contaminant (Penicillium notatum) on a petri dish was found to produce a potent substance that inhibited growth of Staphylococcus aureus, Alexander Fleming had unknowingly discovered a life-saving antibiotic. He published his findings the following year,9 but surprisingly, the world did not take notice right away. Furthermore, there were problems with the drug itself. Penicillin was chemically unstable and difficult to isolate from the mold, raising serious doubts about its potential as a therapeutic agent. More than ten years later, in 1939, a group of scientists at Oxford University (Howard Florey, Ernest Chain, Norman Heatley, and Edward Abraham) took on this challenge, and successfully developed a procedure for isolating and purifying penicillin,10 thereby enabling extraction of sufficient material to conduct in vivo 3 efficacy studies.11 Clinical trials began in 1941, demonstrating drug stability and efficacy against Streptococcus pyogenes and Staphylococcus aureus, with no signs of toxicity.5,8 With World War II underway, there was an established need for penicillin use in wounded soldiers and civilians5,12; thus, creating an intense sense of urgency for large-scale production. Funding, however, was limited in England for a task of this size, leading the Oxford team to relocate to the United States for assistance. Collaborative efforts soon began at an unprecedented level between the United States and United Kingdom that included academic and government entities teaming up with multiple pharmaceutical companies (including Merck, Squibb, Lilly, and Pfizer to start). Through their combined resources and expertise, they developed new procedures for the purification and mass production of penicillin - and just in time for D-Day (1944).12,13 These were ground-breaking techniques (i.e. submerged fermentation, microbial strain production, mutational strain improvement) representing a combination of scientific disciplines including microbiology, biochemistry, and chemical engineering, that quickly became integral for the development of subsequent antibiotics.14 Shortly after the war in 1946, penicillin became widely available by

prescription, which revolutionized medicine. Providers were able to, for the first time, effectively treat previously incurable diseases such as rheumatic fever, scarlet fever, syphilis, severe wounds, and infections with aggressive pathogens such as Staphylococcus or Streptococcus spp.6,15-17 Alexander Fleming’s serendipitous discovery of penicillin was the breakthrough of the century; however, it took an international collaboration composed of government, academia, and

4 industry scientists to translate this discovery into one of the most important medical treatments in history.

The antibiotic era

The drug discovery landscape was forever changed after the arrival of penicillin. Not only did it save thousands of lives, it also ushered in an era of natural

products discovery.18,19 Building on the work of Fleming, microbiologist Selman Waksman sought to find more sources of antibiotic-producing microbes from soil. His approach involved the screening of soil-derived bacteria (mostly Actinomycetes spp.) against susceptible test organisms and evaluating zones of

inhibited growth on an overlay plate.20 This method is similar to Fleming’s discovery of penicillin; however, Waksman applied a more systematic, deliberate screening approach, while Fleming’s discovery of an antibiotic-producing mold was accidental. This new screening approach, otherwise known as the ‘Waksman platform’ led to the discovery of an important antibiotic streptomycin, which exhibited in vitro activity against gram-positive and gram-negative bacteria.21 Though penicillin was highly effective and in frequent use at the time, its antibacterial activity was primarily limited to gram-positive bacteria. Streptomycin, the first of the aminoglycoside antibiotic class, was also the first drug with activity against Mycobacterium tuberculosis. After the successful launch of streptomycin, the Waksman platform quickly became the quintessential tool for antibiotic discovery at the time, and ultimately the most successful and widely adopted antibiotic discovery platform to date. Discovery of other antibiotics occurred shortly thereafter, and continued over the

5 next 20 years, famously referred to as the ‘golden age’ of antibiotics.22,23 In fact, the bulk of antibiotics in use today are from natural products or their semisynthetic derivatives, that were discovered by this method of mining through soil-derived compounds.18,24,25 Vancomycin, clindamycin, rifampin, tetracycline, and daptomycin are among a few important natural product antibiotics discovered during this era that remain in use today (Table 1.1).

Table 1.1 Antibiotics derived from natural products19,22

Antibiotic class Example of clinically used drugs Biological target

Penicillins: amoxicillin, ampicillin, piperacillin, cephalosporins: cephalexin, β‐lactam Peptidoglycan synthesis; transpeptidases cefaclor, ceftazidime, Carbapenems: imipenem, meropenem Peptidoglycan synthesis; binding to acyl‐D‐Ala‐D‐ Glycopeptide Vancomycin Ala Erythromycin, clarithromycin, Ribosome; blocks peptide exit tunnel in the large Macrolide azithromycin subunit Ribosome; blocks peptide exit tunnel in the large Lincosamide Clindamycin subunit Ribosome; impairs cognate aminoacyl‐tRNA Aminoglycoside Gentamicin, tobramycin, amikacin recognition Ribosome: inhibits peptidyl transfer, blocks Streptogramin Synercid (quinupristin + dalfopristin) peptide exit tunnel in large subunit Ribosome: inhibits aminoacyl‐tRNA transfer, blocks Tetracycline Doxycycline, minocycline peptide exit tunnel in small subunit

Rifamycin Rifampin RNA polymerase

Lipopeptide Daptomycin Cell membrane

Cationic peptide Colistin Cell membrane

6 Antibiotic innovation gap

During the golden age of antibiotics, from 1940 through 1960s, the antibiotic development pipeline flourished.26 In fact, the rapidity of new antibiotics discovered at the time appeared to be outpacing the spread of antibiotic resistance. Though the majority of antibiotics developed during this period were through natural product discovery, a few synthetic antibiotic classes or “scaffolds” were also developed with success and remain in used today: fluoroquinolones (ciprofloxacin, levofloxacin), sulfonamides (sulfamethoxazole), oxazolidinones (linezolid), nitroimidazole (metronidazole).19,27 Rapid advances in biotechnology gave rise to HTS in the early 1990s.28 Plus, with the advancements in medicinal chemistry, molecular biology, and arrival of genomic tools, the pharmaceutical industry was seemingly more equipped than ever to discover the next wave of novel antibiotic compounds. Expectations were high for productivity as the industry moved away from laboriously mining soil for naturally-occurring compounds, opting instead for target-based HTS of synthetic compounds.22,29 However, these high-tech platforms yielded only disappointing returns. What occurred instead was nearly a 40-year innovation gap, as depicted in Figure 1.2. After the introduction of nalidixic acid in 1962, no new structural classes of antibiotics were developed again until linezolid in 2000.30,31

7 Figure 1.2 Timeline of antibiotic discovery30

A combination of several important factors is likely to blame for the antibiotic innovation gap or discover void lasting several decades: Collapse of the Waksman platform. This drug discovery platform was a success for approximately 20 years. Unfortunately, mining through soil microbes eventually led to frequent re-isolation or rediscovery of known compounds.25 After yielding diminished returns, the platform was abandoned. Still, many experts now advocate for a revival of this platform, as synthetic approaches have been unable to replace the success of natural product drug discovery. Furthermore, soil and marine environments may still be promising untapped sources for antibiotic compounds. Metagenomic analyses have shown that 99% of bacteria from soil and marine samples are “uncultured,” meaning they do not grow under normal laboratory conditions.32,33 Recently, investigators unveiled the discovery of a new antibiotic, teixobactin,34,35 using a method similar to the Waksman platform, but

8 with a modified technique for isolating and growing uncultured bacteria (Appendix

A).36 Golden age of medicinal chemistry.26 Much of the focus in the pharmaceutical industry during the 1960s and 1970s shifted from novel discovery of compounds to the chemical tailoring of existing antibiotics to create successive generations of antibiotics (2nd, 3rd, 4th, etc.). This has been important for improving the efficacy and/or pharmacological properties of antibiotics but has not lead to any new molecular entities or novel antibiotic scaffolds.17,27,31 Adherence to Lipinski ‘rule of five.’ Poor absorption was a major source of attrition during drug discovery in the early 1990s.37 Renowned medicinal chemist, Christopher Lipinski, along with his team, sought to determine the physiochemical properties of compounds that best predict absorption and advancement to clinical stages of development. Lipinski analyzed properties of compounds that had emerged from Phase I and entered Phase II clinical studies, accessed through the World Drug Index (WDI) and United States Adopted Name (USAN) databases.38 More than 90% of compounds that reached Phase II status had the following physiochemical parameters: i) molecular weight < 500, ii) number of hydrogen- bond donors < 5, iii) number of hydrogen-bond acceptors < 10, and iv) calculated octanol-water partition coefficient < 5. These characteristics, which became known as Lipinski’s rule of five are associated with solubility and permeability, and therefore increased absorption.39 This rule-based approach for synthesizing or screening new compounds was widely adopted by the pharmaceutical industry. Antibiotics, however, are unique molecules, and have always been an exception to the Lipinski rules. Unlike drugs developed for other therapeutic areas, antibiotic drug candidates must be able to penetrate bacterial cells, and not just human cells. 9 Lipinski’s rules do not account for this critical physiochemical property.22,40 In fact, the widespread use of Lipinski’s ‘rule of five’ may have inadvertently selected against discovery of new antibiotic compounds. Phenotypic versus target-based screens. Antibiotics during the Golden Age were discovered empirically using in vitro growth inhibition assays, in which phenotypic endpoints were recorded as bacterial ‘growth’ or ‘no growth’.5,18,25,41 Mechanisms of action were usually determined later, often many years after approval - a significant downside to using traditional whole-cell phenotypic assays. Following the arrival of genomics, bioinformatics, and high throughput screening, drug screening strategies shifted from phenotypic to molecular target-based platforms, thereby enabling target identification and validation of important disease-related targets.22,42 A target-based method involves the in vitro interaction between a drug candidate and a defined/validated target (e.g. enzyme or receptor) in a cell-free system. Other distinguishing characteristics between phenotypic and target-based screening is described in Table 1.2.

10 Table 1.2 Comparing target-based and phenotypic-based screens43-47

Target-based Phenotypic-based

 Often recombinant proteins (cell- free system) or cells over-  Whole cells (e.g. cell lines) or expressing the target of interest organisms (e.g. bacteria) are used

Assay format/  High throughput  Low to medium throughput

characteristics  Screen measures drug effect on  Screen measures biological effect target of interest on cells, tissue, or organism

 Hypothesis-driven (target known  Does not rely on hypothesis at starting point)

 Screen hits are biologically active Advantages  Target or mechanism identified  Screen activity may translate better to human disease

 Additional study needed to  Screen hits may not be determine target or mechanism of Disadvantages biologically active action

Perceived at the time as the more sophisticated and more promising screening platform for anti-infective research and development, target-based screens soon became widely favored in industry. However, this highly anticipated method was met with disappointing results - no new antibiotics emerged from these platforms. Payne et al. published a highly influential article, in which the authors provided candid insight on experiences with target-based screening platforms at GlaxoSmithKline (GSK).48 Between 1995 and 2001, a total of 67 HTS campaigns using genomic-derived antibiotic targets were run against a large compound collection, consisting of 260,000 to 530,000 compounds. This was an unprecedented amount of effort and resources for screening only one single

11 therapeutic area. Only 16 ‘hits’ were identified, five of which became ‘lead’ compounds; but ultimately none of them reached clinical trial phases. Payne et al. concluded that whole-cell phenotypic assays, rather than target-based, are more likely to produce successful leads.48 The authors discussed several possibilities for the poor performing results, one of which is an inability to translate in vitro activity observed from target-based assays to activity that occurs with live bacterial cells. Target-based screening can produce many ‘hits.’ However, if these compounds cannot overcome the permeability barriers and tendencies for efflux pump activity in bacteria, then none of them, not one single hit, will progress to a lead compound.17,27,45,49,50 According to Dr. Kim Lewis, Ph.D., Distinguished Professor of Biology and Director of Antimicrobial Discovery Center at Northeastern University, “…simply doing more high-throughput screening or adding yet another target to the long list of potential ones will not do.”27 Although the cell permeability hurdle was specific to bacterial cells, the lower productivity from target-based screens does not appear to be limited to the development of antibiotics. An analysis of FDA drug approvals between 1999 and 2008 revealed a higher number of first-in-class compounds (i.e. new molecular entities) discovered through phenotypic screening compared to molecular target-

based approach.43 From a total of 50 new in-class drugs, 28 (56%) were discovered using a phenotypic approach, while 17 (34%) were from target-based methods. One area in which target screening appears to be more successful, however, is in the field of cancer. Between 1999 and 2013, 31 of the 48 first in- class oncology drugs were discovered through target-based screens, 21 of which were kinase inhibitors.51

12 Despite having fallen out of favor more than two decades ago, and replaced with molecular target-based platforms, phenotypic screening has been undergoing

a resurgence.45-47 The ideal screening strategy to improve productivity in antibiotic drug discovery, however, is one that combines advantages of both phenotypic and target screening, while circumventing their limitations (Figure 1.3).46,52,53 This can be accomplished through a number of ways. One approach is through the use of a parent wildtype bacterial strain paired with a mutant or modified strain, in which a specific target or mechanism of interest has been altered.53 Comparing the in vitro response of a wildtype/mutant pair to drug candidates can preferentially reveal compounds that inhibit a target or pathway. This method allows for a more hypothesis-driven phenotypic approach, for which the screen hits are biologically active, and the mechanism of action can also be deduced. There are increasing reports of using this integrated strategy, some referring to it as target-, pathway-, or mechanism-based whole cell screens.52,54 55,56

13 Figure 1.3 Drug discovery screening strategies53

A

B

C

(A) Target-based screens identify compounds active against a purified target in a cell-free system. (B) Phenotypic screens identify compounds that impact activity of a complex biological system (i.e. growth versus no growth), without knowing the target or mechanism. (C) Integrated target- based whole-cell screens can circumvent limitations of target-based or phenotype-based screens.

14 High-risk investments. Antimicrobial research and development (R&D)

programs were becoming unattractive investments. After the ‘high-tech’ target- based platforms failed to produce new antibiotics, the anti-infective divisions within the pharmaceutical industry began shutting down.27,48,57 Turning a profit may take years, possibly not until final years of the patent life, depending on the circumstances (Appendix B).58 Furthermore, antibiotics in general tend to have short durations of therapy (≤ 14 days). In contrast, chronic conditions, such as diabetes or hypertension may require daily treatment for many years, if not for life. Pharmaceutical companies estimate risk/benefit and profitability of a developing product using a metric known as net present value (NPV). This is a summation of Research and Development (R&D) costs and expected value of future revenues.59 A threshold target NPV of $200 million is recommended for an antibiotic to be an attractive investment and comparable to other therapeutic classes.60 However, the predicted NPV of an antibiotic is estimated at negative $50 million, meaning the developmental costs would exceed projected earnings.60 Among the more recently launched antibiotics, Avycaz (ceftazidime/avibactam) and Teflaro (ceftaroline fosamil) had the highest sales at ~ $80 and ~$50 million, respectively, two years post-launch (Figure 1.4A). Meanwhile, other popular non- antibiotic drugs had sales ranging from $500 million to over $1 billion (Figure 1.4B).61 Thus, even for antibiotics that bring in revenues, the return of investment is low compared to other popular ‘blockbuster’ treatments for other conditions.

15 Figure 1.4 Sales of recently launched antibiotics

A B

First and 2nd year post-launch sales of (A) newer antibiotics and (B) top selling non-antibiotic brands, relative to Teflaro.

16 Emergence of resistance

Antibiotic resistance is an extremely complicated problem. The urgency to

develop new antibiotics is almost entirely driven by escalating resistance rates.62 The first sign of penicillin resistance was observed in 1940, several years before penicillin was available for widespread use (1945), when a penicillin-inactivating enzyme (penicillinase) was discovered in an E. coli strain.63 In 1942, penicillin resistance was noted in four clinical strains of S. aureus, also by a penicillinase.64 Unfortunately, this was only the beginning. As each new antibiotic was launched into market, reports of resistance followed shortly thereafter (Figure 1.5). Over time, this pattern began occurring in a variety of bacterial pathogens, spanning several decades. Today, there is no shortage of antibiotic resistant bacteria, but there is a shortage of effective treatment options. Until better control measures are in place and more novel antibiotics are available, the threat of resistance will loom, putting an expiration date on each and every antibiotic in use.

17 Figure 1.5 Timeline of antibiotic deployment and evolution of resistance65

Antibiotic deployment

Antibiotic resistance

The year each antibiotic was introduced is displayed above the line, while the year resistance developed for each antibiotic is shown below the timeline.

18 Staphylococcus aureus - an urgent threat

Antibiotic resistance rates have reached an all-time high.66-68 Currently there are several high priority pathogens (e.g. carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa),69-71 but none with the disease burden quite like Staphylococcus aureus. As a frequent source of infections and a known colonizer of the skin and nares,72-78 S. aureus has a more ubiquitous presence than other bacterial pathogens. S. aureus continues to be one of the most common antimicrobial-resistant pathogens causing invasive infections, both in health care settings and in the community.76,79 For these reasons, we chose S. aureus as the pathogen of study for this dissertation. Initially, S. aureus was fully susceptible to penicillin, but this was short-lived. By the early 1950s, just a few years after penicillin was launched, it was no longer a reliable treatment choice for S. aureus infections due to production of penicillinase.64 The development and use of semi-synthetic penicillins, specifically designed to overcome resistance in S. aureus, not only failed to prevent emergence of resistance, but gave rise to methicillin-resistant S. aureus (MRSA) strains (Figure 1.6).

19 Figure 1.6 Evolution of S. aureus antibiotic resistance in nosocomial and community-acquired infections

Black squares = nosocomial infections; grey squares = community-acquired

20 ADDRESSING THE UNMET CLINICAL NEED

The emergence and spread of resistant bacteria, coupled with the paucity

of new antibiotics, has evolved into a global health crisis.66,67,80-82 The Centers for Disease Control and Prevention (CDC) estimated that two million patients per year in the U.S. have infections associated with drug-resistant bacteria and 23,000 die annually as a result.83 If antimicrobial resistance continues its current trajectory, an estimated 10 million deaths worldwide are predicted by 2050 (surpassing cancer).84 Industry sponsors, regulatory agencies, and organizations at the national and international level are taking action to overcome hurdles that led to a dry antibiotic pipeline. Just as the Oxford group discovered during their wartime efforts of mass-producing penicillin, public-private collaborations are critical to successfully revive the antibiotic pipeline and bring new antibiotics to patients in need.85,86 In an effort to improve regulatory processes for drug approvals, the FDA developed four expedited drug review pathways (Table 1.3) for the treatment of life-threatening/serious or rare conditions: i) accelerated approval, ii) priority review, iii) fast track, and iv) breakthrough therapy.87,88 Bacterial infections were not specifically addressed in these pathways. That changed, however, in 2012 when the Generating Antibiotic Incentives Now (GAIN) act was signed into law. Under the GAIN act, industry sponsors can petition the FDA for a Qualified Infectious Disease Product (QIDP) designation, defined as an antibacterial or antifungal drug for human use intended to treat serious or life-threatening infections. Antibiotics with QIDP designation can receive both fast track and priority review status.89 The FDA is expected to have frequent meetings and written communications with the sponsor and provide guidance along the way on 21 developing pathogen-focused antibiotics. QIDP antibiotics are also eligible for five additional years of exclusivity, which allows the sponsor a longer timeframe to recoup development costs.

Table 1.3 FDA expedited regulatory pathways90-92

Year Program Criteria or Data required Key characteristics initiated

 FDA approval based on surrogate Accelerated  Treatment of serious condition endpoint 1992  Early evidence showing advantage  Approval granted on conditional approval over existing therapies basis, post-approval trial required to confirm clinical benefit

 Treatment of serious condition or drug Priority is designated as QIDP  Shorter FDA review timeline (six vs. 1992 review  Improvement in safety or ten months) effectiveness over existing therapies

 Treatment of serious condition or drug is designated as QIDP  Rolling NDA review 1997; Fast track  More frequent written 2012*  Preclinical or clinical evidence demonstrating potential to address communication from FDA unmet medical need.

 Treatment of serious condition  Intensive FDA guidance throughout development to generate additional Breakthrough  Demonstrates substantial 2012 improvement over existing therapies safety and efficacy data therapy on one or more clinically important  Largely oncology and orphan endpoints diseases

*Fast track designation was amended by the FDA Safety and Innovation Act (FDASIA) in 2012 to include the GAIN Act. NDA = new drug application, QIDP = Qualified Infectious Disease Product, FDA = Food and Drug Administration

22 In addition to expedited approval programs, another pathway, the Limited Population Antimicrobial Drug (LPAD) pathway, was signed into law in 2016 as a

provision to the 21st Century Cures Act.92,93 Established with the intention to streamline antibiotic development, LPAD allows faster access to antibiotics for patients with serious or life-threatening bacterial infections in which no appropriate treatment options exist. The drug’s safety and effectiveness can be studied in significantly smaller, more rapid, and less expensive clinical trials using this mechanism, which is similar to the orphan drug approval process.94,95 Data considered acceptable for drug approval using this pathway can include a combination of non-clinical, in vitro susceptibility, pharmacokinetic- pharmacodynamic, and phase II data (Appendix C).96 An antibiotic approved by this pathway must have “Limited Population” in the labeling of the drug. The GAIN act and LPAD pathway are important milestones for revitalizing the antibiotic pipeline. However, some remain skeptical about the benefits of these expedited programs and have concern that speed is being favored over safety. Critics argue that expedited approval has been granted for drugs that did not meet qualifying criteria (i.e. life-threatening/serious, urgent unmet medical need, breakthrough) and have also pointed out a lack of oversight (or enforcement) of

post-marketing surveillance studies for approvals based on surrogate markers.90,97-102 Others have questioned how safety and efficacy can adequately be assessed from such limited data.103 Streamlining drug approval processes is an important strategy to help bring new antibiotics to market in a shorter timeline, but this is only one of several measures needed to combat antibiotic resistance. Several national and international initiatives aimed at incentivizing antibiotic R&D are currently 23 underway, including tax credits, market exclusivity extension, public-private

partnerships, and reimbursements.59,85,104 Still, despite an expanding antibiotic pipeline, experts remain concerned that these measures will simply not be enough and that we will be outmatched by worsening antibiotic resistance rates.105-107

DRUG REPURPOSING

Overview

As of September 2017, 48 new antibiotics were in Phase I to Phase III

development.108 While this news was initially encouraging, further investigation revealed a more sobering outlook. First, only approximately 20-30% will translate to a marketable product, given the success rates of an antibiotic moving through development.48,109 Second, most of these antibiotics do not have a novel mechanism of action but are instead modifications of existing antibiotic classes.106 Third, only 38% of the antibiotics in development are expected to be active against

ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella

pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter), which have been regarded as high priority for more than a decade.110 These factors, combined with the efficacy and safety uncertainties of expedited/LPAD pathway approvals leave little optimism for the future of antibiotics. Perhaps an alternative solution to the scientific, regulatory, and safety barriers may be to seek new therapeutic uses from existing drugs, also known as

drug repurposing or drug repositioning.111 This is becoming an increasingly attractive translational strategy to expedite therapies into the clinic, circumventing

24 much of the early phases of drug development. Drug repurposing is, in itself, an expedited process. Drug repurposing provides a way for pharmaceutical companies to reduce

costs, increase efficiency, and minimize investment and safety risks.112-114 Developing a repurposed drug, as opposed to a newly discovered compound, has the potential to save more than $1 billion and reduce the time to FDA approval by 50%.111,115 Using this strategy can also bring a failed drug back to life, often in the form of a different indication, thereby adding value back to a lost investment. There is no ‘standardized’ definition for drug repurposing. However, the term does have many other names, which has caused some confusion: “repositioning”, “reprofiling”, “redirecting”, “rediscovery”, and “redeployment”, to name a few.116 These terms are generally considered interchangeable, with the exception of one -- “drug rescue.” This is specifically applied to drugs that failed their intended indication and have successfully been developed for an unrelated indication.117,118 There is also some disagreement as to whether the terms “drug repurposing” and “drug repositioning” are synonymous. The source of some of this confusion is that the wording does not indicate whether a drug failed, was withdrawn, or was abandoned (i.e., early versus late development stages). Even the wording “existing drugs” is not very clear, since this could be interpreted as drug candidates in development or FDA approved drugs. Clearly, the terminology is a work in progress. Dr. Hermann Mucke, PhD, editor of Drug Repurposing, Rescue, and Repositioning, suggested that the term “drug repurposing” be used as a ‘catch-all’ phrase to describe the general concept of “developing an active pharmaceutical ingredient, at any stage of the life cycle and regardless of the success or misfortune it has encountered so far; to serve a therapeutic purpose 25 that is significantly different from the originally intended one.”119 For the sake of simplicity, we will follow this suggestion and use the wording “drug repurposing” as an all-encompassing term. Drug repurposing is not a new concept. In fact, it accounts for approximately 30% of all FDA approved drugs in recent years.120 Repurposing is a rapidly emerging field, galvanizing interest from both industry sponsors and government agencies. In 2012, the National Center for Advancing Translational Sciences (NCATS) launched a drug repurposing program, entitled “Discovering New Therapeutic Uses for Existing Molecules.” 121 Recognizing the value of drug repurposing, as well as the value of public-private collaborations, NCATS established a three-way partnership between academia, industry, and government with the goal to identify new therapeutic uses of propriety assets (drugs/biologics) across a range of human diseases in areas of unmet clinical need. Drug repurposing partnerships are established through the use of template agreements. AstraZeneca, Janssen Research, Pfizer, and Sanofi are a few of the companies making their assets available to researchers. Essentially, NCATS acts as the ‘matchmaker’ between the ideas from academia and experimental assets from pharmaceutical companies.

Successful drug repurposing may lead to three possible outcomes: (1) new indications for shelved candidates, (2) line extension for existing drugs, and (3) new targets and new indications for existing drugs.111,114 Shelved drugs may have failed due to efficacy or safety reasons or were discontinued by the sponsor for strategic reasons. ‘Existing drugs’ refers to those that are FDA approved and currently available. Drugs for which there is pre-existing knowledge of safety and toxicity data (i.e. cleared Phase I trials) are generally the most ideal candidates for 26 repurposing, as this significantly de-risks the clinical development phases (Appendix D). Furthermore, sponsors can leverage pre-existing safety and/or efficacy data to streamline the regulatory approval process of their drug through another expedited pathway, known as the 505(b)(2) pathway. Established in 1984, this pathway is used when changes have been made to previously approved drugs. Ceftazidime-avibactam, which is a combination of an approved cephalosporin (ceftazidime) and a novel β-lactamase inhibitor (avibactam) was

approved in 2015 using the 505(b)(2) pathway.122 Ceftazidime-avibactam was also a QIDP designated drug, therefore receiving priority review, fast-track designation, and an additional five years of exclusivity. Repurposing has led to a number of successful drug launches (Table 1.4).111,123-129 Many of these success stories were the result of serendipitous re- discoveries, having once been abandoned or shelved after failing during early development for the intended indication. Zidovudine, for example, was originally developed as an anticancer agent, but development came to a halt after testing in animal models was unsuccessful. Years later, zidovudine was found to have potent in vitro activity against HIV, and after a rigorous clinical trial, was fast-tracked to FDA approval in 1987.130 This marked the beginning of the antiretroviral era,

paving the way for discovery of additional life-saving antiretroviral drugs.131 Probably the most notable example of reviving a discontinued drug is the case of thalidomide. Originally marketed in 1957 to pregnant women for treatment of morning sickness, thalidomide was found to have devastating teratogenic effects, causing more than 10,000 birth defects,132,133 and was withdrawn in 1961. Thalidomide resurfaced in the late 1990s due to growing interest in the drug for treatment of erythema nodosum leprosum, an indication for which it received FDA 27 approval in 1998 (pregnant women were excluded from study population). This launched an aggressive effort by the sponsor (Celgene) to pursue additional indications and to develop analogues that lacked the teratogenic side effects. In 2003, thalidomide was also approved for treatment of multiple myeloma and

quickly became Celgene’s blockbuster drug.134 The thalidomide case, a redeployment of a once considered disastrous drug, demonstrates that repurposing possibilities can arise even from the unlikeliest of sources. This fuels hope that similar repurposing successes will be possible with antimicrobials.

Table 1.4 Examples of drugs successfully repurposed or repositioned Drug Original Indication New Indication Amantadine Influenza Parkinson’s Amphotericin B Fungal infections Leishmaniosis Aspirin Inflammation, pain Antiplatelet Duloxetine Depression Fibromyalgia Finasteride Prostate hyperplasia Hair loss Gabapentin Epilepsy Neuropathic pain Minoxidil Hypertension Hair loss Thalidomide Morning sickness Leprosy, multiple myeloma Sildenafil Angina Pulmonary hypertension, erectile dysfunction Zidovudine Cancer HIV/AIDS

Non-antibiotics

No drugs to date have been repurposed as antibiotics. However, a number of existing drugs have demonstrated in vitro activity against bacterial pathogens and are therefore known as “non-antibiotics.” They exist among a range of drug classes (Table 1.5),127,135-141 and their microbiological activity is highly variable

28 depending on the specific drug. In many cases, non-antibiotic concentrations used during in vitro testing exceed human plasma levels.142 In those instances, it is unlikely a non-antibiotic would be pursued (alone) as a therapeutic agent, given the lack of potent in vitro activity. That being said, plasma concentration is not the only metric for drug exposure. Drugs with a large volume of distribution have more drug distributed into the tissues than in plasma. Tigecycline, for example, a broad- spectrum antibiotic given intravenously, has an average peak serum concentration of 0.6 mg/L, which falls below minimum concentrations needed to inhibit growth of many bacterial pathogens in vitro.143 However, tissue penetration, expressed as a ratio of tissue to serum concentrations were 537 for bile, 23 for gall bladder, 2 for lung.144 Thus, despite subtherapeutic serum concentrations (compared to those tested in vitro), and a general recommendation to avoid use for treatment of bloodstream infections,145 tigecycline is considered an effective treatment for other types of infections such as intra-abdominal and community-acquired bacterial pneumonia.

29 Table 1.5 Example drug classes containing non-antibiotics

Drug Classes

Antihistamines Antidepressants Antihypertensives Antimalarial agents Antineoplastic Calcium channel blockers Proton pump inhibitors Statins Selective serotonin reuptake inhibitors Non-steroidal anti-inflammatory agents

30 Antibiotic adjuvants

Whether or not a non-antibiotic demonstrates antibacterial activity, directly, as monotherapy, is only one part of the story. Antibacterial properties of a drug can also be apparent when in combination with an antibiotic. When presence of a non- antibiotic drug enhances the in vitro activity of an antibiotic, that drug is referred to

as an “antibiotic adjuvant” or “antibiotic potentiator.” 146 Some investigators refer to them as “antibiotic helper compounds,” “resistance modulators,” or “resistance breakers.” 141,147-151 Adjuvants not only potentiate antibiotic activity, but they can also minimize or even prevent antibiotic resistance.152,153 Use of combination regimens for the prevention or reversal of antibiotic resistance is already a common strategy, but is typically performed with the combination of two antibiotics, rather than an antibiotic plus antibiotic adjuvant. The goal with co-administration of two antibiotics is to achieve synergistic activity, or when the sum of in vitro activity of two drugs is greater than with either agent alone.154 The ideal scenario is to administer reduced doses of both antibiotics to decrease the risk of toxicity. However, given the increasing presence of multi-drug resistant pathogens (e.g., Pseudomonas aeruginosa), this is not a realistic treatment approach. Though it is possible that one of the combined antibiotics could be administered at a reduced dose, this applies only to certain types of infections and/or specific bacterial pathogens, such as the use of ampicillin with low-dose gentamicin for enterococcal endocarditis.155 An antibiotic plus antibiotic adjuvant combination differs from antibiotic- antibiotic combination in that the adjuvant itself may have little to no in vitro activity against bacteria, especially at clinically relevant doses.152,153 The primary action of the adjuvant is to enhance antibiotic activity. Gill et al. has characterized adjuvant 31 mechanisms by dividing them into anti-resistance and anti-virulence, as described

in Figure 1.7.152 Currently, the β-lactamase inhibitor category is the only one for which drugs have successfully been developed and FDA approved. Clavulanic acid, for example, paired with amoxicillin, as Augmentin®, has remained in use for over 30 years.156,157 Quorum sensing inhibitors have demonstrated some success in vitro and in murine models for treatment and prevention of biofilm formation.158,159 Meanwhile several anti-toxin antibodies are undergoing clinical trials. Efflux pumps have become a source of considerable research, particularly the NorA efflux pump that confers a multidrug resistant phenotype of S. aureus.160- 163

Figure 1.7 Anti-resistance and anti-virulence mechanisms of adjuvants

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Padhy BM, Gupta YK. Drug repositioning: re-investigating existing drugs for new therapeutic indications. J Postgrad Med. 2011;57(2):153-160. 130. Yarchoan R, Broder S. Development of antiretroviral therapy for the acquired immunodeficiency syndrome and related disorders. A progress report. N Engl J Med. 1987;316(9):557-564. 131. Broder S. The development of antiretroviral therapy and its impact on the HIV-1/AIDS pandemic. Antiviral Res. 2010;85(1):1-18. 132. Vargesson N. Thalidomide-induced teratogenesis: history and mechanisms. Birth Defects Res C Embryo Today. 2015;105(2):140-156. 133. McBride WG. Studies of the etiology of thalidomide dysmorphogenesis. Teratology. 1976;14(1):71-87. 134. Novac N. Challenges and opportunities of drug repositioning. Trends Pharmacol Sci. 2013;34(5):267-272. 135. Thangamani S, Mohammad H, Younis W, Seleem MN. Drug repurposing for the treatment of staphylococcal infections. Curr Pharm Des. 2015;21(16):2089-2100. 136. Enserink M. Infectious diseases. 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Drug combination therapy increases successful drug repositioning. Drug Discov Today. 2016;21(7):1189-1195. 41 143. Agwuh KN, MacGowan A. Pharmacokinetics and pharmacodynamics of the tetracyclines including glycylcyclines. J Antimicrob Chemother. 2006;58(2):256-265. 144. Rodvold KA, Gotfried MH, Cwik M, Korth-Bradley JM, Dukart G, Ellis- Grosse EJ. Serum, tissue and body fluid concentrations of tigecycline after a single 100 mg dose. J Antimicrob Chemother. 2006;58(6):1221-1229. 145. Wang J, Pan Y, Shen J, Xu Y. The efficacy and safety of tigecycline for the treatment of bloodstream infections: a systematic review and meta- analysis. Ann Clin Microbiol Antimicrob. 2017;16(1):24. 146. Ejim L, Farha MA, Falconer SB, et al. Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial efficacy. Nat Chem Biol. 2011;7(6):348-350. 147. Stenger M, Hendel K, Bollen P, Licht PB, Kolmos HJ, Klitgaard JK. Assessments of Thioridazine as a Helper Compound to Dicloxacillin against Methicillin-Resistant Staphylococcus aureus: In Vivo Trials in a Mouse Peritonitis Model. PLoS One. 2015;10(8):e0135571. 148. Gibbons S, Udo EE. The effect of reserpine, a modulator of multidrug efflux pumps, on the in vitro activity of tetracycline against clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) possessing the tet(K) determinant. Phytother Res. 2000;14(2):139-140. 149. de Araujo RS, Barbosa-Filho JM, Scotti MT, et al. Modulation of Drug Resistance in Staphylococcus aureus with Coumarin Derivatives. Scientifica (Cairo). 2016;2016:6894758. 150. Brown D. Antibiotic resistance breakers: can repurposed drugs fill the antibiotic discovery void? Nat Rev Drug Discov. 2015;14(12):821-832. 151. Bernal P, Molina-Santiago C, Daddaoua A, Llamas MA. Antibiotic adjuvants: identification and clinical use. Microb Biotechnol. 2013;6(5):445-449. 152. Gill EE, Franco OL, Hancock RE. Antibiotic adjuvants: diverse strategies for controlling drug-resistant pathogens. Chem Biol Drug Des. 2015;85(1):56-78. 153. Kalan L, Wright GD. Antibiotic adjuvants: multicomponent anti-infective strategies. Expert Rev Mol Med. 2011;13:e5. 154. Pillai SK, Moellering RC, Eliopoulos GM. Antimicrobial combinations. In: Lorian V, ed. Antibiotics in laboratory medicine. Vol 5th. Philadelphia, PA: Lippincott Williams & Wilkins; 2005:365 - 440. 155. Le T, Bayer AS. Combination antibiotic therapy for infective endocarditis. Clin Infect Dis. 2003;36(5):615-621. 156. Neu HC, Fu KP. Clavulanic acid, a novel inhibitor of beta-lactamases. Antimicrob Agents Chemother. 1978;14(5):650-655. 157. Bush K. A resurgence of beta-lactamase inhibitor combinations effective against multidrug-resistant Gram-negative pathogens. Int J Antimicrob Agents. 2015;46(5):483-493. 42 158. Chow JY, Yang Y, Tay SB, Chua KL, Yew WS. Disruption of biofilm formation by the human pathogen Acinetobacter baumannii using engineered quorum-quenching lactonases. Antimicrob Agents Chemother. 2014;58(3):1802-1805. 159. Sully EK, Malachowa N, Elmore BO, et al. Selective chemical inhibition of agr quorum sensing in Staphylococcus aureus promotes host defense with minimal impact on resistance. PLoS Pathog. 2014;10(6):e1004174. 160. Couto I, Costa SS, Viveiros M, Martins M, Amaral L. Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide. J Antimicrob Chemother. 2008;62(3):504-513. 161. Gibbons S, Oluwatuyi M, Kaatz GW. A novel inhibitor of multidrug efflux pumps in Staphylococcus aureus. J Antimicrob Chemother. 2003;51(1):13-17. 162. Zimmermann S, Tuchscherr L, Rodel J, Loffler B, Bohnert JA. Optimized efflux assay for the NorA multidrug efflux pump in Staphylococcus aureus. J Microbiol Methods. 2017;142:39-40. 163. Felicetti T, Cannalire R, Burali MS, et al. 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43 CHAPTER TWO

The in vitro antibiotic activity of non-antibiotic drugs against Staphylococcus aureus clinical strains

INTRODUCTION

For decades, drug discovery and development was largely guided by a “one drug-one target” approach, also known as a “lock and key” model, which focused on the identification of a single drug that selectively binds to one single target, while avoiding any “off-targets” that could produce undesirable or adverse effects. Until recently, this was the prevailing principle of drug design, but has since shifted from “one drug-one target” model to “one drug-multiple targets” model, otherwise known

as “polypharmacology.” 1,2 While it is true that the interaction of a drug with its off- targets can lead to adverse effects in certain cases, it is also this very concept that creates opportunities to find new treatments. Drug repurposing is the discovery of unknown off-targets of existing drugs.3 Many of the repurposed success stories occurred through the serendipitous discovery of these off-targets. Now, however, the same success can be achieved through a more deliberate, systematic approach.

After a near 40-year innovation gap in antibiotic drug discovery, and antibiotic resistance rates persistently on the rise,4,5 it is no surprise that drug repurposing is being pursued as another option for developing antimicrobials.6-10 Staphylococcus aureus, for example, remains one of the most burdensome antibiotic resistant infectious pathogens in hospital and community settings.11-13 Large library collections of FDA approved non-antibiotic drugs and other small molecules (proven safe but not approved) are now available for investigators to 44 perform high-throughput screening (HTS).9,14,15 Several drug classes have, in fact, shown evidence of possessing antibacterial properties. These include calcium channel blockers,16 antihistamines,17,18 and psychotropic medications such as selective serotonin reuptake inhibitors (SSRIs).19,20 Additionally, another group of drugs, which possesses a unique mechanism of disrupting the thioredoxin system of microbes (auranofin, ebselen, PX-12), has been making headlines recently21-23 due to increasing potential to be used as antibacterial agents. Interestingly, each of the drugs discussed here are either approved or under development for non- anti-infective indications. Because of our interest in drug repurposing for the development of antibiotics, we chose one drug from each of these classes to investigate against S. aureus. We aimed to assess the in vitro activity of four non- antibiotic drugs, amlodipine, azelastine, ebselen, and sertraline, against S. aureus using microplate alamar blue assays.

CENTRAL HYPOTHESIS, SPECIFIC AIM, AND OBJECTIVES

Central hypothesis

Drug repurposing, or identifying new uses for existing drugs, has emerged as an alternative to traditional drug discovery processes involving de novo

synthesis. Drugs that are currently approved or under development for non- antibiotic indications may possess antibiotic properties, and therefore may have repurposing potential, either alone or in combination with an antibiotic. They might also serve as “antibiotic adjuvants” to enhance the activity of certain antibiotics. The objective of the proposed research is to utilize novel screening tools to

45 characterize antibiotic effects of select non-antibiotic drugs against Staphylococcus aureus clinical isolates. We hypothesize that one or more of the non-antibiotic candidates selected for this study will demonstrate antibiotic activity against S. aureus, either directly (as monotherapy) or indirectly (in combination with antibiotic drugs).

Specific aim

Quantify the in vitro antibiotic activity of each non-antibiotic drug against S. aureus clinical strains with known resistance genes. Objective 1.1: Determine minimum inhibitory concentrations (MICs) for non- antibiotic drugs (amlodipine, azelastine, ebselen, and sertraline) against S. aureus using microplate alamar blue assays. Objective 1.2: Determine minimum bactericidal concentrations (MBCs) for non-antibiotic drugs (amlodipine, azelastine, ebselen, and sertraline) against S. aureus using microplate alamar blue assays.

METHODS AND RATIONALE

Staphylococcus aureus isolates

Non-antibiotic minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for five clinical isolates (Table 3.1) and one quality control strain (S. aureus 29213). Clinical isolates were selected from previous clinical and epidemiological studies by our research

group.12,24,25 These isolates were obtained from wound swab cultures of patients with skin and soft tissue infections (SSTIs) who were seen at primary care clinics 46 in the South Texas Ambulatory Research Network (STARNet) from 2010 to 2013. Our group had previously identified resistance genes and determined susceptibilities (Table 3.1) in these isolates using whole genome sequencing and

Vitek 2 automated testing system (bioMerieux, Durham, NC), respectively.12 Each clinical isolate is genetically and phenotypically distinct from one another.

Table 3.1. Whole genomic sequencing and antibiotic susceptibilities† for clinical isolates Resistance TMP- Isolate OX CLIN ERY GENT DOX TET VAN CIP Genes SMX A1019 mecA ≥ 4 ≤ 0.25 ≤ 0.5 ≤ 0.5 ≤ 10 ≤ 2 ≤ 4 ≤ 0.5 ≤ 0.5 mecA, ermC, A32 ≥ 4 ≥ 4 ≥ 8 ≤ 0.5 20 ≤ 2 ≥ 16 1 ≥ 8 tetK, gyrA(S84L) gyrA(S84L), ≤ B60 ≤ 0.25 ≤ 0.5 ≤ 0.5 ≤ 10 ≤ 2 ≤ 4 ≤ 0.5 ≥ 8 norA 0.5 ≤ B72 ermC, tetK ≥ 4 ≥ 8 ≤ 0.5 > 320 4 ≥ 16 1 ≤ 0.5 0.5 ≤ J1019 norA ≤ 0.25 ≤ 0.5 ≤ 0.5 ≤ 10 ≤ 2 ≤ 4 ≤ 0.5 ≤ 0.5 0.5 †From VITEK® 2 automated system; shaded areas indicate resistance; OX = oxacillin, CLIN = clindamycin, ERY = erythromycin, GENT = gentamicin, TMP-SMX = trimethoprim- sulfamethoxazole, DOX = doxycycline, TET = tetracycline, VAN = vancomycin, CIP = ciprofloxacin

Selection of non-antibiotic drug candidates

Several drugs were initially considered for this study. Candidates in each drug class from Table 1.5 were evaluated and selected based on several criteria.

 Safety and tolerability. Drug candidates had to have proven safety and tolerability data in humans. FDA approval was also preferred.

 Dosage forms/routes. We also considered dosage forms and routes of administration, with oral, inhalation, or intranasal routes being preferred over parenteral medications.

47  Different drug classes. We selected drugs that represented various therapeutic classes (one from each class, rather than all from one class).

 Antibiotic activity. We sought drugs for which antibiotic activity has been demonstrated previously, but with limited data. If this activity appeared among several drugs within a class or a generalized “class effect”, the drug that demonstrated the greatest potency. Efflux pump inhibition. We also considered if a drug had previously been shown to inhibit p- glycoprotein efflux pumps, since this has previously been correlated with intrinsic antibiotic activity.26

After consideration of all of the above factors, we chose one drug from each of the following classes: calcium channel blockers (amlodipine), selective serotonin reuptake inhibitors (sertraline), and antihistamines (azelastine). In addition to the three non-antibiotics, we included ebselen, a unique organoselenium compound with antioxidant and anti-inflammatory properties that has demonstrated biological activity for a variety of targets (Figure 3.1).27-31 Though not yet FDA-approved, ebselen has been shown to be safe in humans, and is being evaluated in phase II trials for bipolar disorder, as well as for the treatment and prevention of hearing loss.27,32 Recent in vitro studies have also observed antimicrobial activity for a wide range of infectious pathogens, including S. aureus.33-36

48 Figure 3.1 Schematic representation of biological activities of ebselen27

Non-antibiotic drugs: solubility and storage

Standard laboratory grade powders were obtained for the following non- antibiotic drugs: amlodipine besylate (Tocris, Minneapolis, MN), ebselen (Cayman Chemical, Ann Arbor, MI), azelastine (Selleck Chemicals, Houston, TX), and sertraline (Selleck Chemicals, Houston, TX). Stock solutions were prepared according to manufacturer instructions and stored at -80°C. Azelastine was solubilized in distilled water (up to 35mg/ml), while amlodipine, ebselen, and sertraline were solubilized in dimethyl sulfoxide (DMSO). Each drug was further diluted with cation-adjusted Mueller-Hinton broth in 96-well microtiter plates for the microplate alamar blue assays. The highest concentration of DMSO remaining in microtiter wells did not exceed 2% (v/v).

49 Selection of non-antibiotic drug concentrations

Non-antibiotic drug concentrations used for in vitro testing are shown in Table 3.2. These were similar to concentrations that were tested in a small number

of prior reports that evaluated in vitro antibacterial effects of non-antibiotics.21,37-39 While the in vitro drug concentrations used in this study generally exceed those observed clinically (Table 3.2), they may still be clinically useful. Drug concentrations are most commonly assessed from samples collected from plasma; however, plasma concentrations are not the only indicators of drug exposure. Additional factors such as route of administration, site of infection, and drug concentration at tissue sites must also considered. Sertraline concentrations in brain tissue, for example, can be 20 to 40-fold higher than in plasma.40,41 Azelastine is an antihistamine that is available for use as a nasal spray. As one may expect, the drug concentrates in the nasal passages--not in the plasma--after intranasal administration.42 If azelastine has antibiotic activity, it is possible that the drug may impact S. aureus nasal colonization or potentially other important bacterial flora. Furthermore, several alternative mechanisms of drug delivery currently exist, in which high concentrations of antibiotics are required for effectiveness. Examples include antibiotic lock therapy, aerosolized antibiotics, antibiotic-impregnated catheters, and topical antibiotics.43,44 Note that ebselen is not yet FDA approved. Peak plasma concentrations shown in Table 3.2 are from a phase I trial using oral doses up to 1600mg per day. Additional trials are currently underway using similar oral doses (Appendix E), but it is unknown at this time whether this indication or dosing range will gain FDA approval.

50 Table 3.2 Non-antibiotic drugs32,39,40,42,45-48

Drug class or Concentrations for Generic drug FDA Mean peak plasma primary MIC determination name approved concentrations indication (µg/ml)

Ca+ channel Amlodipine Yes 6‐14 ng/ml 12.5 ‐ 256 blocker Azelastine Antihistamine Yes 200 pg/ml 12.5 ‐ 400

Ebselen Antioxidant No 30 ‐ 83 ng/ml† 0.12 ‐ 4 Sertraline SSRI Yes 100 ‐ 500 ng/ml 2.5 ‐ 160 †Based on a 200mg - 1600mg orally per day during phase I clinical trial48 SSRI = Selective serotonin reuptake in inhibitor

Antibiotic susceptibility testing

MIC and MBC determinations. Standard broth microdilution procedures

were followed according to Clinical Laboratory and Standards Institute (CLSI) methods, but with the addition of alamar blue dye (Invitrogen, Carlsbad, CA). Bacterial cultures were inoculated in 96-well, flat bottom, microtiter plates containing serially diluted drugs at a final concentration of ~105 colony forming units/ml (CFU/ml). To prevent evaporation, 200-µl volumes of sterile water was added to outer-perimeter wells.27 Control wells included Mueller-Hinton broth with alamar blue (background) and Mueller-Hinton broth with alamar blue plus bacteria (growth control). Alamar blue was added at a volume equal to 10% of the total volume, while protecting from light exposure, prior to incubating 16 to 20 hours at 35 ± 2°C. MICs were defined as the lowest drug concentration that prevents a change in color. Additionally, 10µl samples were withdrawn, primarily from growth control wells, and plated for colony counting. MICs were recorded and contents from wells for which there was no growth were plated onto Mueller-Hinton agar 51 plates and incubated 18-24 hours at 35 ± 2°C. The MBC was defined as the

minimum drug concentration resulting in a ≥ 99.9% reduction (≥ 3-log10) in CFU/ml from initial inoculum. Fluorescence measurements. Fluorescence was included as an additional measure of antibiotic activity. Fluorescence intensity, expressed arbitrarily as relative fluorescence units, was measured with Synergy HT Microplate Reader (excitation, 530 nm; emission 590 nm). This microplate reader is temperature controlled and can also be used for incubation of microplates if continuous monitoring is desired. This particular plate reader model reads fluorescence from the top of the microplate wells to the bottom, and therefore, microplate lids must be removed during any fluorescence measurements or incubations in the plate reader. Growth control wells represented 100% fluorescence, while Mueller-Hinton broth with alamar blue represented the negative controls. The percent of bacterial growth inhibited by non-antibiotic drugs was calculated with respect to the growth control. Percent (%) inhibition for a given drug concentration was calculated as: ୊୪୳୭୰ୣୱୡୣ୬ୡୣ ൬1 െ ౪౛౩౪ ౚ౨౫ౝ ൰ X 100. ୊୪୳୭୰ୣୱୡୣ୬ୡୣౝ౨౥౭౪౞ ౙ౥౤౪౨౥ౢ Colony counts. Conventional plating techniques were used to determine

inoculum sizes, which were performed at the start of each incubation and when needed to corroborate extent of fluorescence with inoculum. This process involves plating one or more ten-fold dilutions of a bacterial suspension on an agar plate, incubating overnight, and counting colonies the following day. Using the spot inoculation technique, shown in Figure 3.2, several ten-fold dilutions of a bacterial suspension can be applied to one agar plate, using small volumes (10 µl). For

52 some samples, where the inoculum count was expected to be low (blue-purple colored wells), only about one to two 10-fold dilutions were needed for colony counting. Whereas, other samples (pink-colored wells) required at least six to seven 10-fold dilutions. CFU/ml was calculated with the following equation:

୒୭. ୭୤ େ୊୙ CFU/ml = ቀ ቁ ൈ dilution factor ୴୭୪୳୫ୣ ୮୧୮ୣ୲୲ୣୢ ሺ୫୪ሻ

Figure 3.2 Spot inoculation technique

Rationale for Selection of Antibiotic Testing Methods. An advantage of the

broth microdilution technique, compared to other susceptibility testing methods

(i.e. disk diffusion, agar dilution), is that once MICs are obtained, they can be used to derive bactericidal concentrations with an additional step (see MBC determinations). The addition of a growth indicator, such as alamar blue, provides even more of an advantage. Resazurin, the active ingredient in alamar blue, is a cell permeable non-toxic blue dye that undergoes an oxidation-reduction reaction after entering the cell, changing from non-fluorescent blue (oxidized form) to highly

53 fluorescent, pink-colored resorufin (reduced form).49 The extent of this color conversion represents cell viability and can be assessed qualitatively, by visual inspection of color change, and quantitatively, with fluorometric readings.50,51 Thus, the addition of alamar blue dye allows for an assessment beyond a simple dichotomous variable of growth versus no growth.

Data analysis

MICs and MBCs were recorded for each non-antibiotic drug and bacterial isolate and were interpreted as follows: MBC/MIC ratio ≥ 4 indicated bacteriostatic

activity and MBC/MIC ratio ≤ 2 indicated bactericidal activity. Assays were performed in duplicate, and repeated at least once. GraphPad Prism 7 software (San Diego, CA) was used to generate dose-response curves and calculation of

the half-maximal inhibitory concentration (IC50). Dose-response curves were generated using nonlinear regression and the data were fitted using ‘log (inhibitor) vs. normalized response–variable slope’.

Assay optimization

Quality control and selection of microplates. Since we were following CLSI

standard procedure, with the exception of adding alamar blue, we wanted to confirm that the addition of this step would not change susceptibility results. Additionally, CLSI specifically recommends round-bottom microplates for broth microdilution technique,52 however only flat-bottom plates can be used for measuring fluorescence, so we wanted to demonstrate that the change from a round-bottom to a clear flat-bottom 96-well microplate (Evergreen Scientific,

54 Vernon, CA) would not change the MIC results. We performed broth microdilution procedure with S. aureus quality control (QC) strain 29213, testing tetracycline MICs (QC range 0.12 - 1 µg/ml). This was performed in a round bottom plate, in which the MICs were compared to the same broth microdilution procedure with one additional step - the addition of alamar blue. The same procedure was performed again using a clear, flat-bottom microplate in place of the round-bottom microplate. Signal-to-background ratio. Generally, black-colored microplates are

considered the most ideal for obtaining the strongest fluorescence signal while minimizing background interference. However, it is more difficult to visualize the blue/pink colored wells when the microplate is black. This is especially true during the procedural step of adding alamar blue to the wells, as this is performed with minimal exposure to light. A lack of visualization of colored wells would defeat at least one of the purposes for adding alamar blue in the first place. To determine strength of fluorescence signal and background interference, a clear, flat-bottom microplate containing both positive and negative controls, after overnight incubation, was read using the Synergy HT Microplate Reader (excitation, 530 nm; emission 590 nm). Positive controls consisted of media, plus a growth control (S. aureus 29213), plus alamar blue, while negative control wells contained media with alamar blue.

55 RESULTS

Assay optimization

QC tetracycline MICs were within range at 0.5 µg/ml for all broth microdilution procedures, including those with which alamar blue as added, and in both round-bottom plates (Figure 3.3) and flat-bottom microplates with alamar blue. The ‘test’ microplate performed well, demonstrating a signal to background ratio of > 25 (data not shown). The QC testing and fluorescence signal to background ratio confirmed that the selected microplates and other assay conditions were sufficient for testing antibiotic susceptibilities and fluorescence.

Figure 3.3 Tetracycline MICs (µg/ml) for QC strain S. aureus 29213 2 1 0.5 0.25 0.12 0.06 Mueller Hinton broth

Alamar blue

Non-antibiotic susceptibilities

Amlodipine, azelastine, and sertraline MICs were 64 µg/ml (112 µM), 200 µg/ml (480 µM), and 20 µg/ml (60 µM), respectively (Table 3.3), while ebselen ranged from 0.25 - 1 µg/ml (1 - 4µM). MBCs for amlodipine, azelastine, sertraline, were within one dilution of their MICs, indicating bactericidal activity for all test isolates. Ebselen MICs were 1 - 2 dilutions higher in isolates carrying two or more resistant determinants (A32, B72, B60).

56 Table 3.3 Non-antibiotic susceptibilities (µg/ml) Amlodipine Azelastine Sertraline Ebselen

Isolate MICs MBCs MICs MBCs MICs MBCs MICs MBCs

SA -29213 64 128 200 200 20 20 0.25 0.5

A1019 64 64 200 200 20 20 0.25 -0.5 0.5

A32 64 64 200 200 20 40 0.5 – 1 1

B72 64 64 200 200 20 40 1 1

B60 64 128 200 200 20 40 0.5 - 1 1

J1019 64 64 200 200 20 40 0.25 0.5

Fluorometric analysis

Though the microplate reader has the capability of continuous monitoring of fluorometric data, this functionality was limited in this case due to significant evaporation, likely from absence of the microplate lid during incubation. Evaporation was immediately evident upon visual inspection, as the volume loss created a more pale or colorless appearance, as pictured in the example microplate in Figure 3.4. Not surprisingly, volume loss from evaporation also affected antibiotic concentrations, which gave the appearance of increased

potency. We observed this with S. aureus 29213 after measuring fluorescence activity over 16 hours against non-antibiotics (Figure 3.5). Azelastine 100 µg/ml and sertraline 10 µg/ml appeared to inhibit growth by more than 60% compared to the control (Figure 3.5B, 3.5C). However, azelastine and sertraline MICs, determined prior to this experiment, were shown to be one dilution higher at 200 µg/ml and 20 µg/ml, respectively. Therefore, it less plausible that azelastine and sertraline inhibited growth to this extent at sub-MIC concentrations. The overall 57 level of fluorescence intensity, when tested as part of continuous monitoring, was also at least 30% lower than expected compared to initial testing for signal to background ratio. These inconsistencies can easily be explained by evaporation. Dose-response curves for non-antibiotics were plotted by drug

concentration (log [µg/ml]) and % inhibition in Figure 3.6. Calculated IC50 ranges were 37.9 - 47.2 µg/ml (66.3 - 82.6 µM) for amlodipine, 116 - 119 µg/ml (278 - 286 µM) for azelastine, 11.7 - 18.3 µg/ml (35.1 - 54.9 µM) for sertraline, and 0.17 - 0.62 µg/ml (0.67 to 2.5 µM) for ebselen (Table 3.4).

Figure 3.4 Effect of evaporation on alamar blue assays

A Prior to incubation

B C

A). An example microplate containing media, drug concentrations, alamar blue dye, and bacterial isolate. B). Evaporation occurring post-incubation C). No evaporation post-incubation. 58 Figure 3.5 Fluorometric analysis to quantify the in vitro activity of non-antibiotic drugs against S. aureus 29213

A. Amlodipine B. Azelastine

C. Sertraline

Data points represent the mean ± standard error of the mean (SEM).

59

Figure 3.6 Dose-response curves for non-antibiotic drugs

A. Azelastine B. Sertraline

C. Amlodipine D. Ebselen

Data points represent the mean ± SEM. Note the difference in the x-axis scale for Figure 3.6D

60

Table 3.4 Calculated IC50 values

29213 A32 B60 J1019 A1019 38.4 41.4 47.2 38.1 37.9 Amlodipine (67.2) (72.5) (82.6) (66.6) (66.4)

116.2 118.3 118.5 118.8 117.5 Azelastine (278.9) (283.9) (284.4) (285.1) (282) 13.6 18.3 12.6 12.7 11.7 Sertraline (40.7) (54.9) (37.7) (38.2) (35.1) 0.22 0.62 0.36 0.26 0.17 Ebselen (0.88) (2.5) (1.5) (1.1) (0.7)

IC50 values are in µg/ml (µM)

DISCUSSION AND CONCLUSION

We quantified the antibiotic effects of four non-antibiotic drugs, amlodipine, azelastine, ebselen, and sertraline against S. aureus clinical strains using microplate alamar blue assays. Microplate alamar blue assays are, in fact, broth microdilutions, but with one difference: the addition of a colorimetric growth indicator, alamar blue. One simple, additional step yields several advantages. First, the appearance of bacterial growth versus no growth, with alamar blue added, is an irrefutably distinct difference in color (Figure 3.3) from royal blue to a bright pink. Without a growth indicator, the MIC is read simply as the lowest concentration for which there is no visible growth. Bacterial growth is otherwise indicated by turbidity or ≥ 2mm button collected in the well. Although the visual distinction of growth versus no growth may seem easily discernable based on the appearance of Figure 3.3 (top row), these results, unfortunately, are more often less clear. Even with a media control and growth control included for visual 61 comparison, there is still some difficulty distinguishing clear versus “cloudy” as the polypropylene material of the microplate itself has a slightly cloudy appearance. Furthermore, cellular debris due to dead organisms can, on occasion, collect in the

wells, creating an “appearance” of growth, and therefore falsely elevating MICs.53 When this occurs, even the use of a spectrophotometer to determine absorbance may not reliably discriminate viable from nonviable cells.54 Therefore, adding a colorimetric growth indicator, such as alamar blue, has the potential to eliminate errors or uncertainties when interpreting MIC endpoints. Another advantage of using alamar blue over standard broth microdilution is that the extent of antibiotic activity can be determined by measuring fluorescence or absorbance, which correlate with cell growth. Fluorescence provides more sensitive readings compared to absorbance, although either metric is appropriate for cell viability measurement. Determining inoculum size is also possible for either assay (with or without alamar blue) using a more traditional method of colony counting. However, this technique is considerably more time-consuming and labor- intensive. When multiple samples are taken at multiple time-points, as in a time- kill assay, this method also uses up resources quickly such as media, petri plates, pipette tips, and tubes. Antibiotic susceptibility testing with alamar blue and fluorometric readings can provide a simpler, more efficient approach to determining antibacterial activity and generate more information-rich results compared to standard broth microdilution technique. All four non-antibiotics demonstrated in vitro activity to varying degrees against S. aureus clinical isolates. Ebselen was the most potent of the four non- antibiotics tested, with MICs between 0.25 to 1 µg/ml, and MBC/MIC ratios of 1-2. This range is consistent with what prior investigations have observed against S. 62 aureus.9,30 In fact, ebselen has demonstrated bactericidal activity various multidrug resistant strains of S. aureus, including vancomycin-intermediate and vancomycin- resistant strains.21 Additionally, ebselen has been shown to inhibit toxin production as well as biofilm formation.21,33,35 Concentrations of non-antibiotics used in this study are higher than average plasma concentrations reported in humans (Table 3.2). This does not, by default, rule out their clinical utility, since plasma concentration is not the sole metric for assessing adequate drug exposure, even if it is the most practical. Sertraline plasma concentrations, for example, range from 0.1 to 0.5 µg/ml, depending on the dose.45 Sertraline MICs for Cryptococcus neoformans, a pathogen known to cause meningitis in AIDS patients range between 1 and 10 µg/ml.55,56 Although concentrations in this range are not achieved in blood with approved doses, sertraline may be 20 to 40-fold higher in brain tissue, which, in the case of cryptococcal meningitis, is the sight of infection. Furthermore, the standard of care for treating infections from Cryptococcus spp. and other difficult to treat infections is to use a combination of antimicrobials.57-59 (The topic of non-antibiotic/antibiotic combinations is explored further in Chapter Four). Amlodipine MICs were also considerably higher than mean plasma

concentrations (32 µg/ml vs. 14 ng/ml). However, prior studies have suggested that doses required to effectively treat an infection in vivo may be significantly lower than doses used in vitro to inhibit bacterial growth.39 Amlodipine also appears to have an anti-inflammatory effect, which is something that cannot be reflected in the susceptibility test results. Dutta et al. observed a downregulation of expression of inflammatory cytokines interferon-γ, interleukin-1β, and tumor necrosis factor-α in mice treated with amlodipine compared to non-treated mice.60 63 With ebselen not yet FDA approved, there has been some uncertainty as to its pharmacokinetics and dosing. Ebselen doses of 50 mg/kg given orally and 1 mg/kg/hour by IV infusion produced peak plasma concentrations of up to 15 µg/ml

and 12 µg/ml, respectively in rat models.31,61 Ebselen MICs would then fall within a clinically achievable range, as concluded by previous investigators.9,21,62 However, a phase I study of ebselen at doses up to 1600mg orally per day performed in healthy volunteers found that peak plasma concentrations ranged between 30 ng/ml and 83 ng/ml.48 A follow-up phase II study determining safety and efficacy of ebselen, given at 200mg, 400mg, or 600 mg twice daily for prevention of noise-induced hearing loss was published last year.32 Additional trials are currently underway using similar oral doses (Appendix E), but it is unknown at this time whether this indication or dosage form will get FDA approval. Sound Pharmaceuticals, Inc. has been developing ebselen for several years for the treatment and prevention of noise-induced hearing loss. Given the many biological targets of ebselen, however, eventual pursuit of an unrelated indication and altered dosing regimen would not be surprising. While non-antibiotic MICs in this study may be too high to achieve in blood, concentrations may still be relevant at other tissue sites, even if minimal to no

systemic exposure. Furthermore, non-systemic medication applications such as antibiotic lock therapy, aerosolized antibiotics, antibiotic-impregnated catheters actually require high concentrations in order to be effective.43,44,63,64 Azelastine, an antihistamine available as a nasal spray, shows only negligible presence (picograms/ml) in blood, but can deliver a total daily dose of up to 274 micrograms/ml in each nostril.42 We observed inhibitory and bactericidal activity at concentrations of 200 µg/ml against S. aureus. It is therefore plausible that the 64 amount of azelastine exposure from the nasal spray could be enough to impact S. aureus nasal colonization or potentially other important bacterial flora. This also raises an important question as to whether or not frequent use of such a medication could impact not only colonization, but antibiotic resistance as well.

LIMITATIONS

Broth microdilution technique is regarded as the “gold standard” of antibiotic susceptibility testing, but MICs can be somewhat limited when translating to the “bug-drug” interactions in human hosts. Antibiotic concentrations in broth microdilutions remain fixed throughout the incubation period and therefore do not represent the dynamic concentrations that occur in vivo. The concept of bacteriostatic versus bactericidal, and whether or not they

are clinically applicable terms, remains controversial.65,66 Currently no standardized definition exists. The MBC/MIC ratios have been arbitrarily assigned based on concept. Furthermore, MBC determinations have been shown to be less reproducible compared to MIC determinations by broth microdilution, which maintains a precision of ± one dilution.67

65 REFERENCES

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Repurposing FDA-approved drugs to combat drug-resistant Acinetobacter baumannii. J Antimicrob Chemother. 2010;65(12):2598-2601. 9. Younis W, Thangamani S, Seleem MN. Repurposing Non-Antimicrobial Drugs and Clinical Molecules to Treat Bacterial Infections. Curr Pharm Des. 2015;21(28):4106-4111. 10. Yssel AEJ, Vanderleyden J, Steenackers HP. Repurposing of nucleoside- and nucleobase-derivative drugs as antibiotics and biofilm inhibitors. J Antimicrob Chemother. 2017;72(8):2156-2170. 11. Leamer NK, Clemmons NS, Jordan NN, Pacha LA. Update: Community- acquired methicillin-resistant Staphylococcus aureus skin and soft tissue infection surveillance among active duty military personnel at Fort Benning GA, 2008-2010. Mil Med. 2013;178(8):914-920. 12. Lee GC, Long SW, Musser JM, et al. Comparative whole genome sequencing of community-associated methicillin-resistant Staphylococcus aureus sequence type 8 from primary care clinics in a Texas community. Pharmacotherapy. 2015;35(2):220-228. 13. Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler VG, Jr. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev. 2015;28(3):603-661. 14. Torres NS, Abercrombie JJ, Srinivasan A, Lopez-Ribot JL, Ramasubramanian AK, Leung KP. Screening a Commercial Library of Pharmacologically Active Small Molecules Against Staphylococcus aureus Biofilms. Antimicrob Agents Chemother. 2016. 66 15. Aguinagalde L, Diez-Martinez R, Yuste J, et al. Auranofin efficacy against MDR Streptococcus pneumoniae and Staphylococcus aureus infections. J Antimicrob Chemother. 2015;70(9):2608-2617. 16. Machado D, Pires D, Perdigao J, et al. Ion Channel Blockers as Antimicrobial Agents, Efflux Inhibitors, and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis. PLoS One. 2016;11(2):e0149326. 17. Perlmutter JI, Forbes LT, Krysan DJ, et al. Repurposing the antihistamine terfenadine for antimicrobial activity against Staphylococcus aureus. J Med Chem. 2014;57(20):8540-8562. 18. El-Nakeeb MA, Abou-Shleib HM, Khalil AM, Omar HG, El-Halfawy OM. In vitro antibacterial activity of some antihistaminics belonging to different groups against multi-drug resistant clinical isolates. Braz J Microbiol. 2011;42(3):980-991. 19. Bhardwaj AK, Mohanty P. Bacterial efflux pumps involved in multidrug resistance and their inhibitors: rejuvinating the antimicrobial chemotherapy. Recent Pat Antiinfect Drug Discov. 2012;7(1):73-89. 20. Karine de Sousa A, Rocha JE, Goncalves de Souza T, Sampaio de Freitas T, Ribeiro-Filho J, Melo Coutinho HD. New roles of fluoxetine in pharmacology: Antibacterial effect and modulation of antibiotic activity. Microb Pathog. 2018;123:368-371. 21. Thangamani S, Younis W, Seleem MN. Repurposing ebselen for treatment of multidrug-resistant staphylococcal infections. Sci Rep. 2015;5:11596. 22. Sun LH. 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Wall G, Chaturvedi AK, Wormley FL, Jr., et al. Screening a repurposing library for inhibitors of multi-drug resistant Candida auris identifies ebselen as a repositionable candidate for antifungal drug development. Antimicrob Agents Chemother. 2018;62(10). 36. Williamson DA, Carter GP, Howden BP. Current and Emerging Topical Antibacterials and Antiseptics: Agents, Action, and Resistance Patterns. Clin Microbiol Rev. 2017;30(3):827-860. 37. Ayaz M, Subhan F, Ahmed J, et al. Sertraline enhances the activity of antimicrobial agents against pathogens of clinical relevance. J Biol Res (Thessalon). 2015;22(1):4. 38. El-Nakeeb MA, Abou-Shleib HM, Khalil AM, Omar HG, El-Halfawy OM. Membrane permeability alteration of some bacterial clinical isolates by selected antihistaminics. Braz J Microbiol. 2011;42(3):992-1000.

68 39. Mazumdar K, Asok Kumar K, Dutta NK. Potential role of the cardiovascular non-antibiotic (helper compound) amlodipine in the treatment of microbial infections: scope and hope for the future. Int J Antimicrob Agents. 2010;36(4):295-302. 40. Lewis RJ, Angier MK, Williamson KS, Johnson RD. Analysis of sertraline in postmortem fluids and tissues in 11 aviation accident victims. J Anal Toxicol. 2013;37(4):208-216. 41. Tremaine LM, Welch WM, Ronfeld RA. Metabolism and disposition of the 5-hydroxytryptamine uptake blocker sertraline in the rat and dog. Drug Metab Dispos. 1989;17(5):542-550. 42. Bernstein JA. Azelastine hydrochloride: a review of pharmacology, pharmacokinetics, clinical efficacy and tolerability. Curr Med Res Opin. 2007;23(10):2441-2452. 43. Justo JA, Bookstaver PB. Antibiotic lock therapy: review of technique and logistical challenges. Infect Drug Resist. 2014;7:343-363. 44. Onder AM, Billings AA, Chandar J, et al. Antibiotic lock solutions allow less systemic antibiotic exposure and less catheter malfunction without adversely affecting antimicrobial resistance patterns. Hemodial Int. 2013;17(1):75-85. 45. Rhein J, Morawski BM, Hullsiek KH, et al. Efficacy of adjunctive sertraline for the treatment of HIV-associated cryptococcal meningitis: an open-label dose-ranging study. Lancet Infect Dis. 2016;16(7):809-818. 46. Abernethy DR. The pharmacokinetic profile of amlodipine. Am Heart J. 1989;118(5 Pt 2):1100-1103. 47. Elliott HL, Meredith PA. The clinical consequences of the absorption, distribution, metabolism and excretion of amlodipine. Postgrad Med J. 1991;67 Suppl 3:S20-23. 48. Lynch ED, Kil J. Development of ebselen, a glutathione peroxidase mimic, for the prevention and treatment of noise-induced hearing loss. Semin Hear. 2009;30:47-55. 49. Baker CN, Tenover FC. Evaluation of Alamar colorimetric broth microdilution susceptibility testing method for staphylococci and enterococci. J Clin Microbiol. 1996;34(11):2654-2659. 50. Pettit RK, Weber CA, Kean MJ, et al. Microplate Alamar blue assay for Staphylococcus epidermidis biofilm susceptibility testing. Antimicrob Agents Chemother. 2005;49(7):2612-2617. 51. Saxena S, Gomber C. Comparative in vitro antimicrobial procedural efficacy for susceptibility of Staphylococcus aureus, Escherichia coil and Pseudomonas species to chloramphenicol, ciprofloxacin and cefaclor. Br J Biomed Sci. 2008;65(4):178-183. 52. Clinical and Laboratory Standards Institute (CLSI). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard. CLSI document M07-A10. 10th ed. Wayne, PA2015. 69 53. Pfaller MA, Messer SA, Boyken L, et al. Further standardization of broth microdilution methodology for in vitro susceptibility testing of caspofungin against Candida species by use of an international collection of more than 3,000 clinical isolates. J Clin Microbiol. 2004;42(7):3117-3119. 54. Riss TL, Moravec RA, Niles AL, et al. Cell Viability Assays. In: Sittampalam GS, Coussens NP, Brimacombe K, et al., eds. Assay Guidance Manual. Bethesda (MD)2004. 55. Butts A, DiDone L, Koselny K, et al. A repurposing approach identifies off- patent drugs with fungicidal cryptococcal activity, a common structural chemotype, and pharmacological properties relevant to the treatment of cryptococcosis. Eukaryot Cell. 2013;12(2):278-287. 56. Zhai B, Wu C, Wang L, Sachs MS, Lin X. The antidepressant sertraline provides a promising therapeutic option for neurotropic cryptococcal infections. Antimicrob Agents Chemother. 2012;56(7):3758-3766. 57. Whitney LC, Bicanic T. Treatment principles for Candida and Cryptococcus. Cold Spring Harb Perspect Med. 2014;5(6). 58. Bassetti M, Righi E. Multidrug-resistant bacteria: what is the threat? Hematology Am Soc Hematol Educ Program. 2013;2013:428-432. 59. Le T, Bayer AS. Combination antibiotic therapy for infective endocarditis. 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70 67. Pankey GA, Sabath LD. Clinical relevance of bacteriostatic versus bactericidal mechanisms of action in the treatment of Gram-positive bacterial infections. Clin Infect Dis. 2004;38(6):864-870.

71 CHAPTER 3

Translational application

Translational science involves the movement of clinically relevant scientific discoveries from the laboratory to the bedside or clinic. Translation and application of newly acquired knowledge is essential for bridging the gap between scientific breakthroughs and unmet clinical needs, thereby improving overall human health.1,2 Although there is some debate as to how translational research should be defined, the general consensus is that it is a continuous spectrum consisting of a five-phase bidirectional process (Figure 6.1).3 These phases include T0, the application of basic sciences to define mechanisms of disease and identify targets; T1, the initial proof of concept studies in humans; T2, controlled studies in patients with specific disease or condition of interest; T3, dissemination and implementation of new findings into clinical practice; and T4, the impact on population health.4 The translational research spectrum is especially integral to the drug discovery process, as each T-phase bridges an important milestone towards development and launch of a new treatment (Figure 6.2). In fact, the drug discovery and development pathway for a novel compound follows a very similar process, though with a more defined timeline, ranging 10-14 years. Unfortunately, drug

development is also notoriously riddled with barriers and has a high failure rate.5,6 One of the major benefits of drug repurposing is that it can significantly reduce this lengthy timeline, thereby making treatments more readily available for clinical unmet needs.7-9 Drug repurposing is simply a more expedited form of traditional drug development.

72 Figure 6.1 Translational research spectrum

Forward translation, “bench to bedside”

Figure 6.2 Translational phases incorporated in drug discovery and development

73 The conventional understanding of translational science is that it is a “bench-to-bedside” forward movement that begins in a laboratory and ends with

the treatment of patients.3,4 Not only is drug repurposing a translational process, but it is also the embodiment of a concept known as “reverse translation.” This is defined loosely as the use of scientific research from an earlier phase to answer questions arising from clinical data.10 Reverse translation typically begins with patients, in which a question is identified during patient care experiences or clinical trials, and works backwards to test the new hypothesis. Reverse translation is therefore often referred to as a “bedside-to-bench” approach, as it occasionally requires revisiting basic sciences to answer a clinically relevant question.11 The same principle applies with drug repurposing, in which existing drugs are reevaluated for new indications. Drugs that tend to have the highest probability of success and shortest turnaround time to a new indication are those that are reevaluated within the clinical development phases (i.e., T1 - T3) that have at least cleared phase I clinical trials (i.e., safety and tolerability). Even drugs that failed to meet clinical efficacy endpoints for their desired indications can be excellent candidates for repurposing. Discovery of an unanticipated drug effect (e.g., a side effect or therapeutic effect unrelated to the primary indication) occurs most commonly during patient care. Investigation of this unexpected effect is a frequent driver for working backwards to reevaluate an old drug. Though it is more ideal to stay within the T1- T3 phases during reevaluation, many of these clinically relevant questions require investigations at the basic science level.11,12 Figure 6.3 provides a schematic representation of when retroactive scientific investigations are driven by questions arising from clinical patient observations. 74 The traditional process of drug discovery and development of a novel compound has a notoriously low probability of success. Drug repurposing, a reverse translational process, is a strategy that can dramatically increase this probability. Using pre-existing scientific knowledge, usually in the form of human clinical data, drug repurposing has the potential to turn even a failed drug into a success.

Figure 6.3. Reverse translation process

75 REFERENCES

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76 APPENDICES

APPENDIX A. TWO METHODS OF CULTURING MICROORGANISMS FROM SOIL1

Comparison of the traditional method of culturing, shown at the top, compared to the novel iChip method (bottom of picture)

77 Appendix B. Cost simulation for discovering and developing an antibiotic2

The financial return on the investment required to bring an antibacterial to market, even using optimistic estimates, is predicted to occur only in the final years of the patent life.

78 Appendix C. Four-tiered approach to consideration of non-clinical

79 Appendix D. Regulatory pathways for drug repurposing: marketed versus shelved/withdrawn drugs

NCE = new chemical entity, ROA = route of administration

80 BIBLIOGRAPHY

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CHAPTER 2

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CHAPTER 3

1. Blumberg RS, Dittel B, Hafler D, von Herrath M, Nestle FO. Unraveling the autoimmune translational research process layer by layer. Nat Med. 2012;18(1):35-41. 2. Wehling M. Principles of translational science in medicine : from bench to bedside. Second edition. ed. Amsterdam: Elsevier/AP, Academic Press is an imprint of Elsevier; 2015. 3. Fort DG, Herr TM, Shaw PL, Gutzman KE, Starren JB. Mapping the evolving definitions of translational research. J Clin Transl Sci. 2017;1(1):60-66. 96 4. Fernandez-Moure JS. Lost in Translation: The Gap in Scientific Advancements and Clinical Application. Front Bioeng Biotechnol. 2016;4:43. 5. DiMasi JA. Pharmaceutical R&D performance by firm size: approval success rates and economic returns. Am J Ther. 2014;21(1):26-34. 6. DiMasi JA, Grabowski HG, Hansen RW. Innovation in the pharmaceutical industry: New estimates of R&D costs. J Health Econ. 2016;47:20-33. 7. Austin BA, Gadhia AD. New therapeutic uses for existing drugs. In: Posada de la Paz M, Taruscio D, Croft S, eds. Rare diseases epidemiology: Update and overview. Vol 1031. Cham, Switzerland: Springer; 2017:233-247. 8. Barratt MJ, Frail D. Drug repositioning : bringing new life to shelved assets and existing drugs. Hoboken, N.J.: John Wiley & Sons; 2012. 9. Ashburn TT, Thor KB. Drug repositioning: identifying and developing new uses for existing drugs. Nat Rev Drug Discov. 2004;3(8):673-683. 10. Shakhnovich V. It's Time to Reverse our Thinking: The Reverse Translation Research Paradigm. Clin Transl Sci. 2018;11(2):98-99. 11. Becker ML, Funk RS. Reverse Translation in Advancing Pharmacotherapy in Pediatric Rheumatology: A Logical Approach in Rare Diseases with Limited Resources. Clin Transl Sci. 2018;11(2):106-108. 12. McWilliam SJ, Antoine DJ, Pirmohamed M. Repurposing Statins for Renal Protection: Is It a Class Effect? Clin Transl Sci. 2018;11(2):100-102.

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