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Pharmacokinetics and Pharmacodynamics of the Lantibiotic Mu1140

Permanent Link: http://ufdc.ufl.edu/UFE0022470/00001

Material Information

Title: Pharmacokinetics and Pharmacodynamics of the Lantibiotic Mu1140
Physical Description: 1 online resource (139 p.)
Language: english
Creator: Ghobrial, Oliver
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: lantibiotic, mu1140, mutacin, pharmacodynamics, pharmacokinetics
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: One of the fastest growing medical concerns is the issue of antimicrobial resistance. Two possible approaches to reduce the emergence of resistance and control bacterial infections are the continuous production of novel antibiotics and selection of appropriate doses and dosing regimens that ensure maintenance of antimicrobial levels at inhibitory concentrations. MU1140 is an antibiotic peptide with a novel mechanism of action that is produced by the bacterium Streptococcus mutans JH1140. In this thesis, an initial evaluation of MU1140 is presented that includes improved production, analysis, and pharmacokinetic and pharmacodynamic assessment. The production of MU1140 has been improved by optimizing MU1140 fermentation conditions and its purification steps. At this point, the optimized yield is 1mg/Liter of fermentation broth. A bioanalytical method for the quantification of MU1140 in rat plasma was developed and validated, with a lower limit of quantitation of 0.39?g/ml. The pharmacokinetic behavior of MU1140 was investigated in Sprague Dawley rats following intravenous administration. The plasma concentration-time profile of MU1140 declined biexponentially with a mean elimination half-life of 1.7 ? 0.1 hours. In vitro susceptibility to MU1140, determined by MIC screening, showed activity against Gram positive organisms, even in case of drug resistant microorganisms. The in vitro pharmacodynamics of MU1140 were further investigated using time-kill studies in a constant concentration in vitro model using Enterococcus faecalis, Staphylococcus aureus, and Streptococcus pneumoniae. MU1140 was shown to act in a bacteriostatic fashion against E. faecalis and bactericidal fashion against S. aureus, and S. pneumoniae. A linked PK/PD model was developed to predict the in vivo counts of viable bacterial cells when MU1140 is administered in different dosing regimens. Collectively these findings illustrate the potential of MU1140 to serve as a therapeutic agent for the management of otherwise difficult to treat infections caused by Gram positive bacteria.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Oliver Ghobrial.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Derendorf, Hartmut C.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022470:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022470/00001

Material Information

Title: Pharmacokinetics and Pharmacodynamics of the Lantibiotic Mu1140
Physical Description: 1 online resource (139 p.)
Language: english
Creator: Ghobrial, Oliver
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: lantibiotic, mu1140, mutacin, pharmacodynamics, pharmacokinetics
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: One of the fastest growing medical concerns is the issue of antimicrobial resistance. Two possible approaches to reduce the emergence of resistance and control bacterial infections are the continuous production of novel antibiotics and selection of appropriate doses and dosing regimens that ensure maintenance of antimicrobial levels at inhibitory concentrations. MU1140 is an antibiotic peptide with a novel mechanism of action that is produced by the bacterium Streptococcus mutans JH1140. In this thesis, an initial evaluation of MU1140 is presented that includes improved production, analysis, and pharmacokinetic and pharmacodynamic assessment. The production of MU1140 has been improved by optimizing MU1140 fermentation conditions and its purification steps. At this point, the optimized yield is 1mg/Liter of fermentation broth. A bioanalytical method for the quantification of MU1140 in rat plasma was developed and validated, with a lower limit of quantitation of 0.39?g/ml. The pharmacokinetic behavior of MU1140 was investigated in Sprague Dawley rats following intravenous administration. The plasma concentration-time profile of MU1140 declined biexponentially with a mean elimination half-life of 1.7 ? 0.1 hours. In vitro susceptibility to MU1140, determined by MIC screening, showed activity against Gram positive organisms, even in case of drug resistant microorganisms. The in vitro pharmacodynamics of MU1140 were further investigated using time-kill studies in a constant concentration in vitro model using Enterococcus faecalis, Staphylococcus aureus, and Streptococcus pneumoniae. MU1140 was shown to act in a bacteriostatic fashion against E. faecalis and bactericidal fashion against S. aureus, and S. pneumoniae. A linked PK/PD model was developed to predict the in vivo counts of viable bacterial cells when MU1140 is administered in different dosing regimens. Collectively these findings illustrate the potential of MU1140 to serve as a therapeutic agent for the management of otherwise difficult to treat infections caused by Gram positive bacteria.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Oliver Ghobrial.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Derendorf, Hartmut C.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022470:00001


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PHARMACOKINETICS AN D PHARMACODYNAMICS OF THE LANTIBIOTIC MU1140 By OLIVER GEORGE GHOBRIAL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Oliver Ghobrial 2

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To God, my wife, and our family 3

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ACKNOWLEDGMENTS Many thanks are due to my advisors, Dr Derendorf for his insight, advice, and encouragement and to Dr. Hillman for his generosity, support, sense of humor, and the time he took to teach me to write in E nglish, I could not have finished this work without you both on my side. Sincere thanks also go to the member s of my committee, Dr. Genther Hochhaus, Dr. Veronica Butterweck, and Dr. Kenneth Rand, as well as Dr. Jeffery Hughes and Dr. Anthony Palmieri. I also would like to extend special thanks to the post-doc fellows in Dr. Derendorf lab, Sabarinath S and Vipul Kumar for the time they took to answer any of the many little questions that pop along the way. Special thanks are also due to my friends in the department of pharmaceutics, Immo Zdrojewski, Stephan Schmid t, Oliver Grundman, and Matt, and for my friends at OniBiopharma, Inc., Emily McDonnell and Terri Cram for all their help throughout the years. Without your help and support, this would have been much more painful. Last, but not least, I would lik e to extend sincere gratitude to my pearl Godien and my son Avanobe, for their encouragement, support, a nd sacrifices to get me to this point. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF TABLES................................................................................................................. ..........8LIST OF FIGURES.........................................................................................................................9ABSTRACT...................................................................................................................................12 CHAPTER 1 INTRODUCTION................................................................................................................. .14Antibiotic Resistance and Lantibiotics...................................................................................14Types of Bacterial Resistance.........................................................................................14Bacterial Resistance a nd Future Prospects......................................................................16The Potential of Antibacterial Peptides as Therapeutic Agents for Human Use....................17Antimicrobial Peptides Modes of Action........................................................................17Attractive Attributes of Antimicrobial Peptides..............................................................18Lantibiotics.............................................................................................................................18Lantibiotic Classifications...............................................................................................19Lantibiotics Biosynthesis.................................................................................................19MU1140...........................................................................................................................20MU1140 Mechanism of Action.......................................................................................20MU1140s Three Dimensional Structure........................................................................21Pharmacokinetic and other Essential Consider ations for Successful Antibiotic Therapy......22Antibiotics Mode of Action....................................................................................................2 2PK/PD Modeling MIC..................................................................................................24PK/PD Modeling Time Kill Studies.............................................................................25Conclusion..............................................................................................................................26Hypothesis and Objectives.....................................................................................................2 62 IMPROVEMENT OF Streptococcus mutans STRAIN JH1140 PRODUCTION OF MU1140 BY FERMENTATION AND PURIFICATION.....................................................35Introduction................................................................................................................... ..........35Material and Methods.............................................................................................................37Media and Reagents........................................................................................................37Bacteria and Starter Culture............................................................................................37Study Design................................................................................................................... 37Medium Composition......................................................................................................38Oxygen Tension...............................................................................................................38pH and Inoculum Size.....................................................................................................38Delayed Antagonism Assay............................................................................................38 5

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HPLC Column, Purification Conditions..........................................................................39Solubility and Stability Assessment................................................................................40Results and Discussion......................................................................................................... ..40Growth Medium..............................................................................................................40Oxygen Tension...............................................................................................................41Salts.......................................................................................................................... .......41pH and Inoculum Size.....................................................................................................42Purification......................................................................................................................42Solubility and Stability Assessment................................................................................43Conclusion and Discussion.....................................................................................................4 33 DEVELOPMENT AND VALIDATION OF AN EXTRACTION AND LC/MS QUANTIFICATION METHOD FOR THE LANTIBIOTIC MU1140 IN RAT PLASMA................................................................................................................................52Introduction................................................................................................................... ..........53Experimental................................................................................................................... ........54Materials and Stock Solutions.........................................................................................54Equipment and Analysis Conditions...............................................................................55Standards and Quality Control Samples..........................................................................55Sample Preparation..........................................................................................................56Method Validation...........................................................................................................56Preliminary Pharmacokinetic Study................................................................................58Results and Discussion......................................................................................................... ..58LC/MS Detection and Method Selectivity......................................................................58Linearity and Sensitivity..................................................................................................59Accuracy, Precision, and Recovery.................................................................................60Stability...................................................................................................................... ......60Preliminary Pharmacokinetic (PK) St udy of MU1140 in Sprague Dawley Rat.............62Conclusions.............................................................................................................................624 PHARMACOKINETIC/PHARMACODYNAMIC EVALUTATION OF THE LANTIBIOTIC MU1140 IN SPRAGUE DAWLEY RATS..................................................71Introduction................................................................................................................... ..........72Materials and Methods...........................................................................................................74Drug and Dose Administration........................................................................................74Animals............................................................................................................................74Experimental Design.......................................................................................................74PK Data Analysis............................................................................................................75Noncompartmental Analysis (NCA)........................................................................75PK Model.................................................................................................................76Statistical Analysis..........................................................................................................7 6Time-Kill Studies............................................................................................................77PD Model.........................................................................................................................77PK/PD Model and Simulation.........................................................................................78 6

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Results.....................................................................................................................................79Noncompartmental Data Analysis...................................................................................79Compartmental Data Analysis of MU1140 PK Data......................................................79Time-Kill Data and PD Model........................................................................................80PK/PD Model and Simulation.........................................................................................80Discussion...............................................................................................................................80Conclusions.............................................................................................................................825 PHARMACODYNAMIC ACTIVITY OF THE LANTIBIOTIC MU1140..........................88Introduction................................................................................................................... ..........89Materials and Methods...........................................................................................................91Bacteria and Media..........................................................................................................91Antimicrobial Agents......................................................................................................92Susceptibility Studies......................................................................................................92TimeKill Studies............................................................................................................92Development of Resistance.............................................................................................93Results.....................................................................................................................................93Susceptibility Studies......................................................................................................93Time-Kill Studies............................................................................................................94Resistance Development Study.......................................................................................95Discussion...............................................................................................................................956 In Vitro SERUM PROTEINS BINDING AND ITS EFFECT ON THE PHARMACODYNAMICS OF THE LANTIBIOTIC MU1140..........................................106Introduction................................................................................................................... ........106Materials and Methods.........................................................................................................109Determination of MU1140s Degree of Binding to Human Serum Proteins................109Broth Preparation...........................................................................................................109Bacterial Cu ltivation......................................................................................................110Time-Kill Studies..........................................................................................................110Bacterial Quantification.................................................................................................112Results...................................................................................................................................112Determination of MU1140 Unbound Fraction in Human Serum..................................112Effect of Protein Binding on MU1140s In vitro Activity: MIC Studies......................112Effect of Protein Binding On MU1140s In vitro Activity: Time-Kill Studies............113Discussion.............................................................................................................................114Conclusions...........................................................................................................................115Tables....................................................................................................................................1287 CONCLUSIONS.................................................................................................................. 129LIST OF REFERENCES.............................................................................................................132BIOGRAPHICAL SKETCH.......................................................................................................139 7

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LIST OF TABLES Table page 1-1 Current classification of Bacteriocins ...............................................................................342-1 Comparison of MU1140 concentrations obt ained with different commercial media........513-1 Intra-run and inter-day accuracy and precision of the bioanalytical method at the LLOQ and three concentrations of MU1140.....................................................................683-2 Recovery of MU1140 from plasma samples.....................................................................693-3 MU1140 stock solution (25 g/ml) stability at C for up to 30 days..........................693-4 Bench top stability of MU1140 in Sprague Dawley plasma at room temperature............693-5 Freeze/thaw stability assessment of MU1140 in plasma...................................................693-6 Post-preparative stability asse ssment of MU1140 after 4 days at 4oC in autosampler......703-7 MU1140 pharmacokinetic parameters determ ined using NCA analysis of plasma concentration-time data......................................................................................................704-1 Noncompartmental analysis of MU 1140 (12.5, and 25mg/kg) concentration-time data. PK parameters estimated were AUC, Cl, t, Vc and Cmax.........................................875-1 Tier 1 susceptibility StudyMU1140 MIC for various Gram positive and negative microorganisms, and y e 5-2 Tier 2 susceptibility StudyMU1140 MIC for va rious Gram positive and anaerobic microorganism 6-1 MU 1140 MICs against Streptococcus pneumoniae (ATCC 49619) in the presence of 0, 25, and 50% inactivated human serum........................................................................1286-2 MU 1140 MICs Multi Drug resistant Staphylococcus aureus in the presence of 0, 25, and 50% inactivated human serum..................................................................................128 8

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LIST OF FIGURES Figure page 1-1 Lateral or horizontal gene transfer (HGT).........................................................................281-2 Different mechanisms of bacterial antibiotic-resistance....................................................291-3 Lantibiotic maturation process...........................................................................................301-4 The mature MU1140.......................................................................................................... 311-5 Role of lipid II in bact erial cell wall biosynthesis.............................................................311-6 MU1140 three dimension struct ure as determined by NMR.............................................321-7 Representative antibiotics and their mode of action..........................................................331-8 MIC-Based Pharmacokinetic and Pharmacodynamic Indices...........................................331-9 PK/PD modeling as a combination of the two classical pha rmacology disciplines pharmacokinetics (PK) and pharmacodynamics (PD).......................................................342-1 The Sixfors Fermentor...................................................................................................... .442-2 Maximum MU1140 production is observed when yeast extract is at concentration equivalent to 7.5% (w/v) of the fermentation medium......................................................442-3 Maltose is a better induc er of MU1140 production when compared to glucose...............452-4 Comparison between MU1140 production cultu res biomass as a function of Oxygen saturation of fermentation medium....................................................................................452-5 The effect of fermentation medium pH on MU1140 production.......................................462-6 MU1140 concentration in the fermentation broth in relation to the inoculum size...........462-7 Bioassay of fermentation cultu re liquor containing MU1140...........................................472-8 Chromatogram of HPLC run of IPA extracted MU1140 from ammonium sulfate precipitation of culture liquor precipitate..........................................................................472-9 Bioassay of eluent fractions of C18 column......................................................................482-10 Chromatogram of HPLC methanol run..............................................................................482-11 Bioassay of eluent fractions of C18 column......................................................................492-12 Purification by lyophilization............................................................................................ 49 9

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2-13 Stability of MU1140 in saline............................................................................................5 03-1 Analyte and ISTD........................................................................................................... ...643-2 Representative LC/MS chromatogram of extracted drug-free rat plasma.........................653-3 Representative LC/MS chromatogram of plasma sample fortified with MU1140 (at LLOQ), m/z 1133 and gallidermin, m/z 1083....................................................................663-4 MU1140 plasma concentration-time profiles after IV bolus administration of a single dose of 12.5 mg/kg or 25 mg/kg to two different rats.......................................................674-1 MU1140..................................................................................................................... ........844-2 MU1140 PK profile after administration of 25 mg of MU1140 per kg rat body weight.................................................................................................................................844-3 Scheme of PK/PD model for antibacterial effect of MU1140...........................................854-4 Observed vs. predicted S. aureus concentration (cfu/ml)..................................................854-5 The result of the simulation of S. aureus viable cell count when MU1140 is administered at two dose levels (5 and 10mg/kg TID)......................................................865-1 MU1140..................................................................................................................... ........995-2 Bactericidal activ ity of MU1140 against S. pneumonia strain ATCC 49619. Symbols: Control, 0.5MIC, 1MIC, 2MIC, 4MIC, 8MIC...............995-3 Bactericidal activity of MU1140 against multidrug resistant S. aureus Symbols: Control, 0.5MIC, 1MIC, 2MIC, 4MIC, 8MIC, 16MIC........1005-4 Bacteriostatic activity of MU 1140 against vancomycin resistant E. faecalis Symbols: Control, x 0.25MIC, 0.5MIC, 1MIC, 2MIC, 4MIC, 8MIC, 16MIC....................................................................................................1015-5 MU1140 MIC values after 21 subculturing events for multidrug resistant S. aureus ( ) and S. pneumoniae ( )...............................................................................................1026-1 Lanthionine (Lan) and Methyl lanthionine (MeLan) structure.........................................1166-2 Time kill studies of MU1140 against S. pneumoniae in the absence and presence of 50% human serum............................................................................................................1176-2 Time kill studies of MU1140 against S. aureus in the absence and presence of 50% human serum.................................................................................................................... 1186-3 Side-by-side plot of S. pneumoniae viable cell counts in the presence of MU1140 and the presence and absence of human serum...............................................................122 10

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6-4 Side-by-side plot of S. aureus viable cell counts in the presence of MU1140 and the presence and absence of human serum............................................................................1266-5 Time kill studies of MU1140 at 0.5 time MIC against S. aureus in the presence of various human or rat serum concentrations.....................................................................127 11

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PHARMACOKINETICS AND PHARMACODYNAMICS OF THE LANTIBIOTIC MU1140 By Oliver Ghobrial August 2008 Chair: Hartmut Derendorf Major Department: Pharmaceutical Sciences One of the fastest growing medi cal concerns is the issue of antimicrobial resistance. Two possible approaches to reduce the emergence of re sistance and control bacter ial infections are the continuous production of novel antibiotics and selection of appropria te doses and dosing regimens that ensure maintenance of antimicrobi al levels at inhibitory concentrations. MU1140 is an antibiotic peptid e with a novel mechanism of action that is produced by the bacterium Streptococcus mutans JH1140. In this thesis, an initia l evaluation of MU1140 is pres ented that includes improved production, analysis, and pharmac okinetic and pharmacodynamic assessment. The production of MU1140 has been improved by optimizing MU1140 fe rmentation conditions and its purification steps. At this point, the optimized yield is 1mg/Liter of fermentation broth. A bioanalytical method for the quantification of MU1140 in rat plasma was developed and validated, with a lower limit of quantitation of 0.39g/ml. Th e pharmacokinetic behavior of MU1140 was investigated in Sprague Dawley rats followi ng intravenous administration. The plasma concentration-time profile of MU1140 declined biexponentially with a mean elimination half-life of 1.7 0.1 hours. 12

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13 In vitro susceptibility to MU1140, determined by MIC screening, showed activity against Gram positive organisms, even in case of drug resistant microorganisms. The in vitro pharmacodynamics of MU1140 were further investigated using time-kill studies in a constant concentration in vitro model using Enterococcus faecalis, Staphylococcus aureus, and Streptococcus pneumoniae MU1140 was shown to act in a bacteriostatic fashion against E. faecalis and bactericidal fashion against S. aureus, and S. pneumoniae. A linked PK/PD model was developed to predict the in vivo counts of viable bacterial cells when MU1140 is administered in different dosing regimens. Collectively these findings illustrate the poten tial of MU1140 to serve as a therapeutic agent for the management of otherwise difficult to treat infections caused by Gram positive bacteria.

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CHAPTER 1 INTRODUCTION Antibiotic Resistance and Lantibiotics Antibiotics, also known as antimicrobial agents, are used to fight and control infectious diseases. After their initial discovery in the 1st half of the 20th century they transformed medical care and dramatically reduced illness and death from infectious diseases. However, during the second half of the 20th century, bacteria that antibiotics were able to kill and control began to develop resistance to these antibiotics. After almost fifty years of extensive antibiotic use, dis ease-causing bacteria are developing resistance to many antimicrobial drugs such that dise ases caused by these bacterial are once again causing serious morbidity and mortality. Now, bacterial antibiotic resistance has been called one of the world's most pressing public health problems (1). Types of Bacterial Resistance Bacterial resistance to antibiotics can be inherent. For example, Enterococci species are inherently resi stant to aminoglycosides, Pseudomonas species to tetracycline, and Gram negative bacteria ar e inherently resistant to gl ycopeptides (50). Inherent resistance could be due to stru ctural characteristics of the ce ll. A bacterial species could be missing the enzyme targeted by the antibiotic, or have an outer membrane that denies the antibiotic access to its intracellular site of action or on the cell membrane. On the other hand, previously sensitive bacteria may also acquire resistance to certain antibiotics. The emergence of bacteria that have acquired resistance to the antibiotics they are exposed to is a natu ral, evolutionary phenomenon resulting from a selective pressure exerted by the antibiotic (50). Acquired resist ance could develop by alterations of the microorganisms genetic ma keup and acquisition of genes that code for 14

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proteins that confer resistance to the antib iotic (e.g., acquisition of plasmids coding for lactamases which degrade -lactam antibiotics). Tr ansfer of DNA can occur by horizontal gene transfer (HGT). In HGT, genetic material is transferred from one bacterium to another and can occur by one of three mechanisms, transduction, transformation or conjug ation (Figure 1-1). Transduction is the process during wh ich DNA sequences are transferred from one bacterium to another via infections by bacteriophage. During bacteriophage DNA packaging, small pieces of bacterial DNA are inco rporated into the viral capsid which is injected into the next sus ceptible bacterium. The DNA co nferring resistance can either by incorporated into the bacterium genom e by recombination or into a plasmid. Phenotypically, the recipient bacterium is now resistant to the antibiotic. In transformation, free DNA is up-taken by the bacteria from the environment. This free DNA is released into the external e nvironment after the death of its carrier and that DNA is collected by other bacteria, wh ich integrates it into their genome by recombination, or it might exist as plasmid (20). The last mechanism is conjugation, where direct cell-cell contact between two bacteria lead to the transfer of plasmi d or chromosomal DNA from the donor to the recipient bacterium. This mechanism accounts for most of the resistance emergence among bacterial populations (20). Due to the relatively low inter-species specificity and ease of these DNA acquisition methods, transfer of genes encodi ng resistance from one microbe to another or from one species to another caused an explosive emergence and spread of antibiotic resistance bacteria (20). Such acquired resistance may be manifested through one of 15

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many mechanisms (Figure 1-2). Spontaneous mutations in the gene encoding the protein targeted by the antibiotic leading to the al teration of the antibiotic binding site and inability of the antibiotic to interact with its site of target molecule and exert its antimicrobial effect (3). Genes may encode transporter proteins that function to pump the antibiotic out of the cell. The development of alternative metabolic pathways to those inhibited by the drug is also a common physio logical mechanism of resistance resulting from alteration in the cells genome either by mu tations or gene transfer by plasmid (50). But, most common are plasmids encoding en zymes that degrade and inactivate the antibiotic molecule once it is inside the bacterial cell (e.g. as mention above, the lactamases which degrade all -lactam antibiotics). Bacterial Resistance and Future Prospects Medically, the most worrisome drug resist ant bacteria are the multidrug resistant Gram-negative (e.g. Pseudomonas, Enterobacter, and Salmonella species) and Gram positive organisms (Staphylococcus Enterococcus, and Streptococcus species) (3). Unfortunately antibiotic resistance is an una voidable consequence to any new antibiotic that bacteria get exposed to. This in turn leads to the rapid rise in bacterial populations that are resistant to developed antibiotics, and the subsequent stress to society in terms of morbidity, mortality, and increased health care expenditures. Fo r all the mentioned reasons, our policies regardi ng production, handling, and usage to antibiotics has to evolve. Less use of antibiotics, appropriate choice of antibiotic and dosing regimen, and the discovery and production of new antimicrobials are crucial fact ors to contain the problem in hand (1). Given the decreasing util ity of available antimic robials, attention to development of new antimicrobials is b ecoming increasingly important (4). 16

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The Potential of Antibacterial Peptides as Therapeutic Agents for Human Use Antimicrobial peptides have emerged as potential therapeutic agents for the treatment of various types of bacterial infecti ons due to their ability to kill Gram positive and Gram negative pathogenic microorgani sms and fungi as well as to activate components of the host innate immune system (6, 9, 34, 90). Some of these peptides were also shown to inhibit enveloped viruses re plication (57). So fa r, all discovered antimicrobial peptides share unique stru ctural characteristics required for their bioactivity, which include: 1An overall positive charge produced by the presence of multiple arginine and lysine amino acids, 2Approximately 50% of the peptides overall primary structure is composed of hydrophobic residues. This high hydrophobicity forces these peptides to assume an amphiphilic conformation when binding to bacterial membranes, thus allowing intercalation and subsequent perforation or penetration of the cell membrane into the cytoplasm (69). Antimicrobial Peptides Modes of Action Antimicrobial peptides can exert their e ffect either by interaction and perforation of the plasma membrane or by penetrati on through the membrane and binding to intracellular targets which can result in inhibition of pr otein or DNA synthesis, cell division, or induction of autolysi s. The cationic nature attrac ts the antimicrobial peptides to negatively charged bacterial membrane s rich in anionic phospholipids such as phosphatidylserine, anionic teichoic acids of Gram positive bacteria, and negatively charged lipopolysaccharides (LPS) of Gram ne gative bacteria. Upon interaction with the bacterial membranes, these cationic peptid es displace divalent cations from the membrane leading to membrane destabilizati on, which enables the peptide to translocate through the membrane, a mechanism known as self-promoted uptake (69). Other 17

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antibiotic peptides are know n to function as pore formers (13) which perforate the bacterial plasma membrane, or inhibitors of bacterial cell wall synthesis via a mechanism known as lipid II abduction (35) where the antibiotic removes lipid II from its site of activity thus disrupting ce ll wall synthesis. Attractive Attributes of Antimicrobial Peptides One of the prominent features of these peptides is that they can kill multidrug resistant bacteria at concentrations comparable to conventional antibiotics, but they kill at a much rapid rate (8). Anot her attractive attribute is the fact that there is no cross resistance between the currently used antibio tics and these peptides. Hence methicillinresistant S. aureus, multidrug resistant P. aeruginosa, as well as other multidrug resistant bacteria are susceptible to these antimicrobi al peptides at safe concentrations (91), making these peptides an attractive solution to the current problem of bacterial antibiotic resistance. The major hurdle preventing large scale ex ploitation of these peptides for human use is the high cost of manufacturing of antim icrobial peptides and inability to purify these molecules to homogeneity (15). Lantibiotics A unique class of antimicrobial peptides th at has gained increased attention in the last decade is the lantibiotics. Lantibioti cs were discovered the year before Fleming discovered penicillin. In 1928, L. A. Rogers mentioned a potent antimicrobial substance produced by certain lactic acid bacteria, but the nature of the inhibitory compound was not known at the time (71). La ntibiotics are peptides that function as bacteriocins. Lantibiotics are charac terized by the presence of thioet her bridged amino acids known as lanthionine (Lan) and/or met hyllanthionine (MeLan), as well as other modified amino 18

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acids such as didehydroalani ne (Dha) and didehydrobutarine (Dhb) (Figure 1-3 A). Lantibiotics are ribosomally synthesized a nd posttranslationally modified to their biologically active forms which are active ma inly against Gram positive bacteria. The word lantibiotic was first coined in 1988 as an abbreviation for lanthionine-containing antibiotic peptide. The defining posttranslational modification of lantibiotics is that they contain the lanthionine (Ala-S-Ala) or methyllanthionine (A bu-S-Ala) (13). Lantibiotic Classifications Lantibiotics are classified as type A or B based on th eir ring structure as well as their biological ac tivity. Type A lantibiotics are cationic peptides of 20-34 amino acids in length and assume an elongated and amphi pathic state in phys iological solution. Further, within the Type A lantibiotics, t hose which are posttranslationally modified by the action of a dehydratase enzyme (LanB) and a cyclase enzyme (LanC) are classified as Type AI, while those that ar e dehydrated and cyclized by a single enzyme (LanM) are classified as type AII. Type B lantibioti cs on the other hand are globular and compact in configuration with no net charge or anionic at pH 7 (13). Lantibiotics Biosynthesis Lantibiotics primary structure is encode d in a gene that is part of an operon containing other genes that encode for enzymes required for the posttranslational modification, processing, and translocation of the mature lantibiotic (Figure 1-3 B). The first gene, LanA, encodes the peptide precurs or preceded with an N-terminal leader sequence followed by the propeptide from whic h the mature lantibiotic is produced. Encoded in the same operon are LanB and LanC. LanB codes for a dehydratase which dehydrate serine and threonine amino acids of the propeptide to form didehydroalanine (dha) and didehydrobutarine (dhb) residues re spectively. LanC codes for a cyclase 19

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enzyme that catalyze the inter action of the dehydrated residue s with sulfhydryl groups of Cysteine residues to form the lanthionine amino acids. The lantibiotic operon also contains a LanT gene which codes for a transporter of the pr ecursor and inactive lantibiotic to the extracellular environment. At his point, the lantibio tic is inactive due to the presence of the N-terminal leader peptide. LanP encodes a protea se that is anchored to the extracellular leaflet of the plasma memb rane of the producer cell. LanP protease will cleave the leader sequence liberating the ma ture and fully functional lantibiotic (13). MU1140 A member of the Type A1 lantibioti cs is MU1140 (Figure 1-4). MU1140 is naturally produced by a strain of the common oral bacterium, Streptococcus mutans The prototype of type A lantibiotics, nisin, has been developed as a f ood preservative which has been given the generally recogn ized as safe status by the FDA. The primary mode of bactericid al activity of type A lantibiotics is believed to be disruption of the cytoplasmic membrane, causi ng the efflux of ions and metabolites and desynergization of the target cell (13). Howeve r, recent data suggest that this may not be the mode of bactericidal activity of MU1140 (35). MU1140 Mechanism of Action MU1140 acts to inhibit cell wall synt hesis by a novel mechanism known as Lipid II Hijacking. Lipid II hijacking (Fi gure 1-5) involves rem oval of the bacterial cell wall subunits carrier, lipid II, from its site of action on the bacterial cell membrane at the cell division septa (35) and aggrega tion to a different nonfunctional site. Lipid II (Figure 1-5 B) is a key molecu le in bacterial pep tidoglycan synthesis (Figure 1-5 A). Lipid II functions to transp ort cell wall subunits from the cytoplasm, their site of biosynthesis, across the bacter ial cytoplasmic membrane to the periplasmic 20

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space, where they are assembled into the growing peptidoglycan polymer. Lipid II is synthesized in the cytoplasm and is co mposed of N-acetyl glucosamine-N-acetyl muramic acid-pentapeptide units linked to an undecaprenyl lipid tail by a pyrophosphate. The assembled Lipid II transposes itself acr oss the membrane where the sugar subunits are transferred to the growing peptidoglycan ch ains and the isoprenoid carrier is recycled back to the cytoplasm where it picks up more peptidoglycan subunits and so on. MU1140s Three Dimensional Structure Elucidation of the native three dimensiona l structure of a molecule can provide us with an insight into the physiolo gical role the molecule assumes in its habitat. In the case of MU1140, a knowledge of the three dimens ional structure could allow for a better understanding of its antimicrobial activity, as well as the knowledge to manipulate and improve the molecules properties. Like nisin and gallidermin, the thioether ring structures of MU1140 were found to be rigid and well defined, and like nisin the regions not spanned by thioether linkages were quite flexible (83). This motif of two domains fixed by lanthionine rings joined by a flexible hinge seems to be a common feature of Type A lantibiotics. The molecule has an overall horseshoe-like shape kinked at the hinge region between rings B and C (Figure 1-6). The thioether bridges that form the ring structures of the molecule are exposed to the surface. Thioether bridges are resistant to enzymatic or non-enzymatic oxidation/reduc tion activities that w ould otherwise easily open disulfide bonds. The hinge region contains a potentially suscep tible arginine at residue 13 that appears to be sterically protected. The hinge region, quite flexible, may be important for bactericidal activity by allowing the lantibiotic to orient properly in the membrane and for membrane insertion properties of the molecule (53, 54). 21

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Pharmacokinetic and other Essential Cons iderations for Successful Antibiotic Therapy Antimicrobial therapy is a complex proce ss and its success is dependent on the appropriate selection of the right antibiotic and the righ t dosing regimen. Many factors are involved in the process of antimicrobi al chemotherapy. There are infective agentassociated factors like the bacterias suscepti bility to the available antibiotics as well as the infection site. There is also host-relate d factors which include the immune status and the patients overall physiologi cal state which could dramatically affect the antibiotics ADME (absorption, distribution, metabolism, and excretion) processes (24). The ADME process, also referred to as the drugs pharmacokinetics or drugs disposition in the body, is generally depicted as what the body does to the drug (21). The most important pharmacokinetic parameters that are used to predict the probability of therapys success are the peak serum level (Cmax), the trough (Cmin), and the area under the serum-concentration time curve (AUC) (47). Serum levels of an antibiotic need to be above a certain level during the dosing interval to eradicate the target microorganism and prevent the emergence of resistant strains (44). Pharmacokinetic (PK) studies will measure the time course of the antibiotic in the body. Antibiotics Mode of Action Antibiotics can exert their antimicrobial effect via one or more of multiple mechanisms. Shown in Figure 1-7 examples of certain antibiotics and their mode of action. The aminoglycosides and chloramphe nicol bind and inactive bacterial ribosomes thus inhibiting protein synthesi s resulting in bacterial cell death. Beta-lactam antibiotics (which include the penicillins, cephalosporins, carbapenems and monobactams) and glycopeptides, include vancomycin, act by inhi bition of cell wall synthesis via prevention 22

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of cross-linking of N-acetylglucosamine and N-acetylmuramic acid cell wall subunits, which renders the bacterial cell susceptible to osmotic lysis. The quinolones and rifampin inhibit DNA and RNA synthesis, respectively. Trimethoprim and sulfonamides function by blocking cell metabolism via inhi bition of folic acid biosynthesis. Pharmacodynamics is the study of th e relationship between the antibiotics concentration and its effect. When studyi ng antibiotic pharmacodynamics, two patterns of killing behavior emerge: Concentration-De pendent and Time-Depe ndent killing (33). More specifically concen tration-dependent antibio tics (e.g. aminoglycosides, daptomycin, and the fluoroquinolones) have activity that is proportional to the antibiotic concentration in the medium, and thus the goal of therapy when using a concentrationdependent antibiotic is to achieve a large peak serum concentration (Cmax) since the greater the concentration the faster and more complete bacterial eradication is achieved. Examples of Time-Dependent Antibiotics are the carbapenems, cephalosporins, and penicillins. For these antib iotics, the extent of killing is dependent on how long they persist in the medium. Thus, the extent and duration of e xposure needs to be maximized during therapy to ensure th e success of therapy. Using this classification, the clinician would design a dosing regimen that is optimized according to the type of bacteria and antibiotic pair to be used. Using an unoptimized dosing regimen may lead to failure of antibiotic therapy or, even worse, the emergence of bacterial subpopulations that have developed resistance to the antibiotic in use. Thus, the selection of the appropriate dos ing regimen is critical to successful therapy (73). Historically, the process of choosing an antibiotic dosing regimen has been based on trial and error or by following a dosing desi gn of another drug that worked. This 23

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approach is subject to high chance of fa ilure since it does not take into account differences between drugs and their mechanisms of action, as well as genetic differences between individuals, effect of difference di sease states on patients physiology and drug metabolism capabilities (49). The new and logical approach to dosing of an antibiotic and regimen design is based on pharmacokinetic and pharmacodynamic properties of the drug in a specific host (38). Due to the increas e of microorganism resi stance to currently used antibiotics, PK/PD modeling for antibiotic dosing regimen design is becoming an increasingly critical aspect of the antibioti cs development process. By incorporating pharmacokinetic and pharmacodynamic data obtaine d from in vitro as well as animal data into a mathematical PK/PD model it is su itable to develop a ra tional recommendations that involve the right drug and the right dos ing regimen to ensure clinical success of therapy (21). Several PK/PD modeling a pproaches have been devised for the optimization of antimicrobial therapy. Th ese approaches are based on either the minimum inhibitory concentration (MIC) of th e antibiotic or on the time-kill behavior as measured by time-kill studies. PK/PD Modeling MIC The minimum inhibitory concentration (MIC) is defined as the minimum amount of antibiotic needed to inhibit growth of an initial bacterial inoculum of 1x105colony forming units (cfu)/ml as is measured by lack of visible growth after 18-24 hours. The MIC of an antibiotic is currently the most wi dely used measure of antimicrobial activity. There are three MIC-based PK/PD indices (Figure 1-8) commonly used to predict and simulate the activity of an antibiotic. They are (i) the ratio of peak plasma concentration (Cmax) to MIC (Cmax/MIC), (ii) the time antibiotic plasma concentration is 24

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above the MIC (T>MIC), and (iii) the ratio of the area under the plasma concentrationtime curve to MIC (AUC/MIC). Cmax/MIC is the quotient of the maximum attained plasma concentration by the MIC of the antibiotic for that specific bacteria l species. To ensure success of therapy and prevent emergence of re sistant populations, a Cmax/MIC ratio of 10 or higher is the target when designing the dosing regimen (63). T>MIC is the duration when the plasma concen tration of the antibiotic is above the MIC of that antibiotic against that specifi c bacterial species. When designing a dosing regimen, the target is to achieve antibiotic concentration that exceeds the MIC more than 50% of the dosing interval (63). AUC24/MIC is the quotient of the attained area under the curv e (AUC) of the antibiotic plasma concentra tion over a 24 hour period divided by the MIC. This index provides information on the total exposure of the body to the antib iotic. For Gram negative bacteria, a ratio of >125 is a good indication of efficacy and therapy success, and a ratio of >50 is used for Gram positive bacteria. PK/PD Modeling Time Kill Studies Time-kill studies are frequently conducted by inoculating several vented cap flasks containing the appropriate media with the pathogen at midlog phase for a final bacterial concentr ation of 1-105 cfu/ml. Different concentr ations of the antibiotic are added to the flasks. Samples of the culture are removed at different time points, spotted on suitable agar media plates and incubated ove r night at optimal conditions of growth. Viable cells/colonies are counted to determine th e number of viable cel ls at different time points. Data is plotted as log cfu/ml vs. time and all antibiotic concentration are plotted on the same chart. Data from the time-kill curv es at different concentrations is translated 25

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to a mathematical formula that will predict the viable bacterial cell count when different doses and dosing regimens of the antibiotic are used. The incorporation of the drugs PK and PD data into a single model allows the use of simulation techniques to pr edict the effect time profile re sulting from a certain dosing regimen (Figure 1-9). Conclusion The current antibiotic resistance crisis di ctates the need for new antibiotics with novel mechanisms of action. Antimicrobial peptides, particularly lantibiotics, are attractive candidates to satisfy the current unmet medical need due to their broad spectrum of activity, an tibacterial action, and the lack of cross-resistance to currently prescribed antibiotics. MU1140, a bacteriocins produced by Streptococcus mutans is a lantibiotic with promising prospects for the co ntrol of Gram positive infection. The small size, good stability, low immunogenicity, a nd broad spectrum of activity of MU1140 make this molecule an extremely attractive candidate for use as a therapeutic agent for human use. Further studies are required to char acterize the PK/PD relationships of MU1140 in animal models. This data is required for MU1140 optimal dosing to ensure success of therapy and reduction of th e probability of resistant strains emergence. Hypothesis and Objectives The goal of this work was to investigate the applicability of MU1140 as an antimicrobial agent for human use. Ba sed on early discovery data, MU1140 shows activity against Gram positive microorganisms and hence is a promising antibiotic for treatment of a variety of Gr am positive infections. In preparation for an IND submission, a reproducible production and purification techniques that yield high quality MU1140 26

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had to be established, investigation of the pharmacokinetics of MU1140 in the rat model, as well as thorough understandi ng of the pharmacodynamic of this new chemical entity must be achieved. The specific aims of this work are: (1) Improvement of Streptococcus mu tans strain JH1140 Production of MU1140 by Fermentation and Purification (2) Development and Validation of an Extraction and LC-MS Quantification Method for the Lantibiotic MU1140 in Rat Plasma (3) Pharmacokinetic and Pharmacokinetic/Pharmacodynamic Evaluation of the Lantibiotic MU1140 in Sprague Dawley Rats (4) In Vitro Pharmacodynamic Activity Assessment of th e Lantibiotic MU1140 (5) Assess the degree of In Vitro Serum Proteins Binding and its Effect on the Pharmacodynamics of the Peptide Antibiotic MU1140 This dissertation will be presented as a compilation of manuscr ipts addressing the specific aims stated above. 27

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Figure 1-1. Lateral or horizont al gene transfer (HGT) (20). 28

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Cytoplasm Periplasm Cell Wall Outer Membrane 4 3 3 2 1 Modified Targets Drug Target Porin 5 Efflux Pump Drugs Figure 1-2. Different mechanisms of bacterial antibiotic-resistance. A Sensitive bacteria uptakes the antibiotic. Antibiotic binds to target and exerts it antimicrobial effect. B. Possible resistance mechan ism: (1) Alteration of target, thus antibiotic looses effect. (2 ) Acquisition of a modified target that the antibiotic will not bind to. (3) Enzymatic inactivation of the antibiotic, this is the most common antibiotic resistance mechanism, where an existing enzyme processes the antibiotic and modifies it so that it no longer affects the microorganism. Reduction of the cytoplas mic concentration of the antibiotic, either by (4) mutations of transporters that bring the antibiotic inside the cell, or (5) Actively pumping the antibiotic outside (20). 29

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Figure 1-3. Lantibiotic maturation process. A. Dehydration of Ser and Thr residues to produce Didehydroalanine (Dha) and didehydrobutyrine (Dhb) respectively and the formation of the thio ether bridge to yield a mature lanthionine. B. The process of Lantibiotic Biosynthesis. Lantibiotic posttranslational maturation process exemplified by the lantibiotic Nisin. The NisA prepeptide is composed of N-terminal leader sequence attached to the active moiety. NisB catalyzes the dehydration of Ser and Thr residues, wh ile NisC catalyzes the cyclization reaction by addition of sulf hydryl groups of Cys residues to Dha and Dhb residues to generate the cyclical thioether bridges. After completion of the dehydration/cyclization reactio n the protease NisP proteolytically removes the leader sequence to produce the mature Nisin. Reprinted, with permission, from the Annual Review of Microbiology, Volume 61 (c) 2007 by Annual Reviews www.annualreviews.org (13, 48). 30

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S Figure 1-4. The mature MU1140. Abbrev iations: dha, 2,3-didehydroalanine; dhb, 2,3didehydrobutyrine; abu, 2-am inobutyric acid (37). Figure 1-5. Role of lipid II in bacterial ce ll wall biosynthesis (7). Reprinted with permission from Nature Publishing Group. Phe Lys Ala Trp Dha Leu Ala S Abu Pro Gly Ala S Ala Arg Dhb Gly Ala Phe Asn Ala Tyr Ala S NH CH CH 31

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Figure 1-6. MU1140 three dimension structur e as determined by NMR. The overall backbone structure of MU1140 assumes a horseshoe shape. Rings A and B are on one side while the inte rtwined rings C and D are positioned on the other side. Ring A (red), Ring B (blue), Hinge (purple), Ring C and D (green) and thioether linkages (yellow) are visible. The first two N-terminal amino acids, Phe1 and Lys2, are shown in black (77). 32

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Figure 1-7. Representative antibioti cs and their mode of action (59). Figure 1-8. MIC-Based Pharmacokinetic and Pharmacodynamic Indices (55) 33

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34 Figure 1-9. PK/PD modeling as a combin ation of the two classical pharmacology disciplines pharmacokinetics (PK) and pharmacodynamics (PD) (51). Table 1-1. Current classificat ion of Bacteriocins (13).

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CHAPTER 2 IMPROVEMENT OF STREPTOCOCCUS MUTANS STRAIN JH1140 PRODUCTION OF MU1140 BY FERMENTATION AND PURIFICATION In this study, the effects of various para meters on the fermentative production of MU1140 by Streptococcus mutans strain JH1140 was tested and optimized using the Sixfors fermentor. The parameters we evaluated were the fermentation medium composition, oxygen tension, pH, and inoculum size. The highest MU1140 production was achieved using a fermentation medium th at consisted of 5% yeast extract, 0.5% CaCl2 and 4% glucose under microaerophilic cond itions. pH is to be maintained at a constant 5.1 0.1 by the addition of 5N NaOH. An inoculum size of 10% was used and fermentation lasted 24 hours. The purificat ion method consisted of a precipitation step using ammonium sulfate to precipitate the bioactivity followed by selective uptake in 80% IPA and two separation steps using C 18 reverse phase chromatography where the activity is eluted first using an acetonitrile gradient followed by a second run that used a methanol gradient. The activity-containing fraction was lyophilized and > 92% pire MU1140 was recovered as fluffy white strands. Introduction Antimicrobial therapy was revolutionized by the introduction of antibiotics in the 1930s and millions of lives have been saved since. Soon after th eir introduction, some broad spectrum and regularly prescribed antib iotics lost their ability to control certain bacterial infections (10), and currently more 70% of nosocomial infections are caused by drug resistant bacteria (1, 9). Annually, infections caused by Staphylococcus aureus alone, which represent 16% of infections na tionwide, results in 12,000 inpatient deaths, 2.7 million days in excess length of stay, and 9.5 billion dollars in extra hospital charges (11). 35

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Two factors contribute to th e continuous exacerbation of this health crisis. The first is the diminishing antibiotics pipeline ( 15), especially those with novel mechanisms of action. According to Rice et al since 1998, only ten antibioti cs have been approved; out of which only two possess a novel mechanis m of action. The other factor is the continuous and rapid emergence and spread of new bacterial strains th at are resistant to currently used antibiotics due to unoptimized dosing regimens (14). Thus, there is a need for new antibiotics and appropriate do se design can not be over emphasized. A class of antibiotics that is lately gaining much attention is the lantibiotics (2). These antibacterial peptides are produced by Gram positive bacteria and released into the environment to destroy organisms that compet e with the producer stra in for its ecological niche. Lantibiotics are ribosomally synthesized and posttranlationally modified to incorporate unusual amino acids such as lant hionine (Lan, ala-S-ala), methyllanthionine (MeLan, abu-S-ala), didehydroalanine (Dha) and didehydrobuterine (Dhb). MU1140 is a 22 amino acid lantibiotic that is produced by Streptococcus mutans strain JH1140 (7) and its novel mechanism of action, dubbed Lipid II Hijacking, involve s disruption of cell wall synthesis (6, 16). MU1140 is active against Gram positive bacteria including species of medical importance such as methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecalis (VRE) (4). Unfortunately, for the last 50 years lantibiotics have been utilized to a limited extent as food preservatives (3), to treat peptic ulcers caused by Clostridium difficile and Helicobacter pylori (5), and to treat acne caused by Propionibacterium acnes and bovine mastitis cause by S. aureus (12). The major obstacle to the clinical development of lantibiotics is their very low production titer and/or inability to be purif ied to homogeneity (17). In this study, 36

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MU1140 production and purificatio n procedures developed at Oragenics, Inc. are described. Material and Methods Media and Reagents Yeast extract (YEX), Todd Hewitt Broth (T HB), Tryptone Soy Broth (TSB), LuriaBertani (LB) broth, Brain Heart Infusion (B HI) Broth, Tryptic S oy (TS) Broth, were purchased from Difco Laboratorie s (Detroit, MI, USA). Acetonitrile, isopropyl alcohol, triflouroacetic acid (TFA) were purchased from Sigma Aldrich (St. Louis, MO). Bacteria and Starter Culture Stock cultures of S. mutans strain JH1140 and Microc occus luteus (ATCC 272) were kept at -80oC in 30% glycerol TSYEX (3% tr yptic soy broth and 0.3% yeast extract). Six hours prior to the fermentation reaction, an inoculum was grown at 37 C in TSYEX. The bacteria are grown for 5 hrs to an OD of 1.1 read at 600nm. This starter culture was grown in 2 lite r Erlenmeyer flasks contai ning 500 ml of media at 37oC with shaking at 200 rpm. Study Design The Sixfors fermentor (Infors, Bottminge n, Switzerland; figure 2-1) is composed of six vessels that can be controlled independently. Different cultivation parameters, namely, fermentation medium, carbon and nitr ogen source, salt content, oxygen tension, pH, and inoculum size, were varied and the subsequent effect on MU1140 titer was quantified using a delayed antagonism bioassay using M. luteus (ATCC 272) as the susceptible strain. This process was repeat ed until conditions resu lting in the optimal MU1140 concentration were identified. Du ring the optimization process, biomass formation was determined by centrifugation of culture and determining the weight of the 37

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cell mass. For determination of MU1140 titer, the supernatant of the culture broth was analyzed by bioassay. Medium Composition Different commercially available media were used in pilot fermentation processes to identify a medium with MU1140 inducing capability. On ce identified, the components of the medium were varied to determine the optimal mixture for MU1140 production. Oxygen Tension The production of MU1140 as a function of dissolved oxygen in the culture medium was optimized by vary ing the culture stirring speed and sparging with nitrogen in the fermentation vessel. Oxygen tension was monitored using an O2 probe and the amount of dissolved oxygen was varied betw een 0.1% to 100% satu ration throughout the fermentation period. Samples of the culture liquor were sampled for their MU1140 titer and biomass content. pH and Inoculum Size Fermentation medium pH was controlled by the automatic addition of 2M NaOH and was varied between pH 4 7.5. The MU1140 titer was measured. We also investigated the effect of inoculum size on the final MU1140 titer. Inoculum size was varied between 0 100% of the total fermentation volume. Delayed Antagonism Assay Stock cultures of M. luteus were maintained at -80oC in 30% glycerol TSYEX. The assay involved growing a culture of M. luteus to optical density (600 nm) of 0.2 in TSYEX. The culture was diluted 1:5 with mo lten top agar (3% tryp tic soy broth with 0.75% agar) maintained at 45C, and spread over the surface of a Trypticase Soy agar plate. Samples containing MU1140 activity to be tested were seri ally diluted in 50% 38

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acetonitrile and 5 l samples were spotted and allowed to air dry. After incubation overnight, the inverse of the hi ghest dilution producing a visi ble zone of clearing and was the MU1140 titer. HPLC Column, Purification Conditions The MU1140 purification scheme used a number of steps. The culture was centrifuged for 15 minutes at 8000 g at room temperature to remove the S. mutans cells. Antimicrobial activity was precipitated from the culture supernatan t by slow addition of ammonium sulfate to a final concentrati on of 30% (w/v), followed by overnight incubation at 4oC. The precipitated material was recovered by centrifugation (8,000xg for 20 minutes at 4oC). MU1140 was extracted from th e recovered prec ipitate using 3 treatment of 80% isopropyl al cohol for 24 hours each. The pH adjusted to 3.0 with 12N HCl, and the sample was stored at 4oC until used. The isopropanol was removed and two volumes of 40% acetonitril e were added. The material was passed through a 0.22 filter prior to reverse phase separation and elution using a 5 to 90% ACN gradient containing 0.1% trifluoroacetic acid. A reverse phase Dynamax (Palo Alto, CA) C18 column (250 41mm) was used and eluted prot eins were detected using an ultraviolet (UV) detector set to wavelength 280 nm. The MU1140-containi ng fraction, determined by bioassay, was collected, diluted with buffer B (acidified wa ter) and again loaded onto the C18 column. Elution was performed with a 0 to 100% methanol gradient containing 0.1% trifluoroacetic acid. The MU1140-contai ning fraction (52-58 minutes) was again collected and lyophilized to dryness. Lyophiliz ation resulted in separation of MU1140 as white fluffy strands over a dark, crusty pellet. The MU1140 fraction recovered and stored at -20oC until use. 39

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Solubility and Stability Assessment As MU1140 is intended to be administered as an IV solution, its solubility limit and stability in normal saline were tested. So lubility was tested by addition of various amounts of MU1140 to a fixed amount of nor mal saline at room temperature. The samples were vortexed and centrifuged at 16,000 g for 30 minutes at room temperature. Vials that showed a pellet were regarded to contain MU1140 in a concentration that surpassed MU1140s solubility limits. The stability of MU1140 in saline was tested by incubating the lantibiotic in salin e at room temperature and at 4oC for 24 hrs. Aliquots were removed at 1, 2.5, 5, 8, and 24 hrs, a nd bioassayed for their MU1140 content. Results and Discussion To determine cultivation parameters that produce optimal MU1140 production, various cultivation parameters were varied. The amount of MU1140 produced was determined by a deferred antagonism bioassay using M. luteus. Growth Medium Six different commonly used media (Table 1) were tested to identify a medium best suited for MU1140 production. It was found th at 1.25% maltose as the carbon source, 3% yeast extract (YEX) which served as th e cultures nitrogen source, and 1.25% CaCl2 proved optimal for MU1140 production. Further medium optimization was achieved by using TSYEX as a base model media, varyi ng the above mentioned components, and the subsequent MU1140 titer was noted. The percen tage of yeast extract in the fermentation medium was varied between 2.5 10% and maltose was varied between 2 4%. The increase in MU1140 titer was proportional to the mediums yeast ex tract concentration up to 5%, after which MU1140 titer plateau (figure 2-2). Th ere was no difference between 2% or 4% maltose in the fermentation medium but maltose was superior to glucose when 40

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compared in a side-by-side fermentati on. Figure 2-3 summarizes MU1140 formation when different carbon sources were used. Oxygen Tension To determine whether there is a co rrelation between the dissolbed oxygen concentration and the observ ed MU1140 titer, the oxygen c ontent of the fermentation broth was varied from slightly aerobic (2% oxygen saturation) to completely aerobic (100% oxygen saturation). The amount of dissolved oxygen was controlled and maintained at a constant level by varying the stirrer speed and thus medium aeration state. An oxygen-probe was used to measur e the medium oxygen content. Figure 2-4 summarizes the MU1140 titer and cells biomass observed as a function of fermentation medium oxygen tension. The highest MU1140 titer was observed when oxygen was maintained at 2% of maximum saturation. As oxygen tension in creases, and although the cultures biomass increases proportiona lly, the MU1140 titer decreased sharply with the lowest titer observed was at me dias oxygen at saturation (100%). Salts Production media of other la ntibiotics (8) were know n to contain the salt CaCl2, thus we investigated the e ffect of this salt on the produc tion of MU1140. To identify the CaCl2 concentration that is optimal for MU1140 production, different CaCl2 media content was varied from 0 2%. CaCl2 did not have any effect on the MU1140 titer. However, divalent cations have been implicat ed in lantibiotic resi stance of the producer strain (13). This could be due to neutraliz ation of the bacterial outer cellular anionic charge by the divalent cations and thus reduced interaction of the cationic lantibiotic to the bacterial cell. At this point the role of calcium is not clear. 41

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pH and Inoculum Size MU1140 fermentation is carried out using a constant pH that varied from pH 5 to 6 at increments of 0.2 pH units. It appear s that the amount of MU1140 produced is highly affected by the pH applied during fermentation. pH 5 5.6 resulted in almost the same titer with pH 5.2 inducing the highest MU1140 titer (figure 2-5). MU1140 titer significantly dropped at pH above 5.6 although S. mutans biomass did not change but rather plateau. MU1140 production increased as the percentage inoculum volume increased to peak at 10% which corresponds to a titer of 1600, after which the titer drops drastically to < 25 at 50% as well as 100% inoculum size (figure 2-6). Purification Before start of the purification proc ess, the presence of MU1140 activity is confirmed in the culture liquor by biasssys (fig ure 2-7). A typical chromatogram of a run of the MU1140 containing culture liquor afte r IPA extraction is shown in figure 2-8. Antimicrobial activity was found to be concen trated in a fraction between 25-30 minutes as shown using bioassay (figure 2-9). The ma terial eluted from the column is further diluted and separated on the same C18 column, activity is eluted using an acidified (0.1% TFA) methanol gradient (fi gure 2-10) which elutes as a single peak in fraction 55-60 minutes. Results are confirmed by bioassay of the mentioned fraction (figure 2-11). Post lyophilization of the methanol eluted material, MU1140 appear s as an off beige/ white matter (figure 2-12) with mo lecular weight 2266 Da. Analytical HPLC the MU1140 shows a single peak and MS anal ysis shows one major ion with m/z of 1133 which corresponds to the doubly charged MU1140 speci es. The above analysis suggests that according to these methods it appears that MU1140 produced in our laboratory is homogenous. 42

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Solubility and Stability Assessment Solubility has been estimated to be > 100 g/l. The stability study of MU1140 in saline shows a 20% drop in MU1140 concentratio n at both temperatur es which occur in the first few minutes followed by a stable and unchanging concentration of MU1140 for 24 hrs. Conclusion and Discussion In this work, growth medium components and environmental factors that trigger the production of the la ntibiotic MU1140 have b een described. It was observed that 5% yeast extract, 0.5% calcium chloride, and 4% glucose yielded the highest amount of MU1140. The purification method consisted of a precipitation step using ammonium sulfate to crash down the bioactivity followe d by selective uptake in 80% IPA and two separation steps using reversed phase chroma tography where the activity is eluted after the first separation using aceton itrile and after the second sepa ration using methanol. The activity containing fraction is lyophilized and MU1140 is left behind as a fluffy white matter. The yield of MU1140 according to this protocol is less than 1mg/liter which does not enable large scale commercialization of this antibiotic. This low yield could be explained by the susceptibility of the producer strain to the antibiotic. To prevent selfkill, the producer strain c ould utilize negative feedback loops to down-regulate the MU1140 production and maintain the antibioti c concentration below toxic levels. MU1140 was shown to be very soluble in saline and stable for over 24 hours. Collectively, the data demonstrates that the optimization process has led to an increase in MU1140 formation. Although the yield of MU1140 using our method does not support commercialization of this antibiotic, this work can be used to furthe r our understanding of the complex circuitry that triggers and controls the production of these potent cell killers. 43

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Figure 2-1. The Sixfors Fermentor. A. Illust ration of a single vessel. B. The complete sixfors apparatus with six fermenta tion vessels shown at the bottom. Effect of % Yeast Extract in Fermentation Medium on MU1140 Titer0 200 400 600 800 1000 051 0 % Yeast ExtractMU1140 Titer1 5 MU1140 Titer Figure 2-2. Maximum MU1140 production is observed when yeast extract is at concentration equivalent to 7.5% (w/v) of the fermentation medium 44

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Effect of Carbon Source on MU1140 Titer0 1000 2000 3000 4000 5000 6000 7000 GlucoseGlucoseGlucoseMaltoseMaltoseMaltose Carbon Source MU1140 Titer Figure 2-3. Maltose is a better inducer of MU1140 production when compared to glucose. In all Sixfors vessels containing maltose as the sole carbon source in the medium, MU1140 titer was double that observed in vessels containing glucose as the carbon source. Effect of Fermentation Medium Oxygen Content on MU1140 Titer 0 100 200 300 400 500 600 700 05 01 0 0 Oxygen % of SaturationMU1140 Tite r 0 5 10 15 20 25 30 MU1140 Titer Biomass Figure 2-4. Comparison between MU1140 producti on cultures biomass as a function of Oxygen saturation of fermentation medium. 45

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The Effect of Fermentation Medium pH on MU1140 titer and S. mutans Biomass 0 100 200 300 400 500 600 700 4.555.566.5 pHMU1140 Titer0 2 4 6 8 10 12 MU1140 Titer Biomass Figure 2-5. The effect of fermen tation medium pH on MU1140 production. Effect of Inoculum Size on MU1140 Titer0 500 1000 1500 2000 05 01 0 01 5 0 Inoculum Size (% of total culture volume)MU1140 Titer MU1149 Titer Figure 2-6. MU1140 concentration in the fermen tation broth in relation to the inoculum size. 46

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Figure 2-7. Bioassay of fermentation culture liquor containing MU1140. Spots labeled (A) are 1:100 dilutions from the culture liquor followed by 2-fold dilutions. Spots labeled (B) are standard MU1140 solu tion. This technique is used to monitor and quantify the amount to MU1140 produced per batch. Figure 2-8. Chromatogram of HPLC run of IPA extracted MU1140 from ammonium sulfate precipitation of cultu re liquor precipitate. 47

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Figure 2-9. Bioassay of eluent fractions of C18 column. The lane labeled Mutacin peak shows the most bioactivity which corres ponds to the peak collected in the 2628 minutes fraction. Figure 2-10. Chromatogram of HPLC methanol run. Material eluted off the C18 column using ACN diluted and rechromatographed again on the C18. MU1140 eluted using an acidified methanol gradient. 48

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Figure 2-11. Bioassay of eluent fractions of C18 column. Th e lane labeled Mutacin peak shows the most bioactivity which corres ponds to the peak collected in the 5258 minutes fraction. Figure 2-12. Purification by lyophilization. Th e material eluted from the C18 column using acidified methanol lyophilized. A) Fluffy white MU1140. B) Crusty pellet containing contaminants. 49

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Figure 2-13. Stability of MU1140 in saline. An initial drop in concentration is observed followed by no change in concentration. 50

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51 Tables Table 2-1. Comparison of MU 1140 concentrations obtained with different commercial media. Media* MU1140 Titer Media 1: 1% YEX, 0.5% CaCl2, 4% Maltose 1000 Media 2: THB, 0.5% CaCl2, 4% Maltose <25 Media 3: TSB, 0.5% CaCl2, 4% Maltose <25 Media 4: LB, 0.5% CaCl2, 4% Maltose <25 Media 5: BHI, 0.5% CaCl2, 4% Maltose <25 Media 6: THB, 0.3%/YEX, 4% Maltose <25 YEX (yeast extract), THB (Todd Hewitt Broth) TSB (Tryptic Soy Broth), LB (Lauriat Broth), BHI (Brain Heart Infusion).

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CHAPTER 3 DEVELOPMENT AND VALIDATION OF AN EXTRACTION AND LC/MS QUANTIFICATION METHOD FOR THE LA NTIBIOTIC MU1140 IN RAT PLASMA This study reports the first ever developm ent and validation of an extraction and quantification method for a lan tibiotic in plasma. This method was developed for the quantification of total MU1140 in Sprague Dawl ey rat plasma. The procedure involved acidification of plasma samples with form ic acid followed by precipitation of plasma proteins using isopropanol. The samples were analyzed by RPLC/MS. Gallidermin was used as an internal standard (ISTD). The an alyte and ISTD were el uted using a gradient of isopropanol and water, both acidified with 0.3% formic acid (v/v), at a flow rate of 250l/min. Positive electrospray ionization was utilized at the ion source and the analyte and ISTD were both detected by selected-i on monitoring (SIM). Total run time was 15 minutes. This method was validated for sele ctivity, sensitivity, linearity, recovery, accuracy, and precision. The method was s hown to be selective, with a quantitative linear range of 0.39 100 g/ml using 25 l sa mples. The mean extraction recovery for MU1140 was 96% 0.4%. The bias, intraand inter-day percent relative standard deviation at all concentrations tested was lo wer than 15%. The analyte was shown to be stable to freeze/thaw and for short and long term storage. Extracted MU1140 was stable at 4oC for over 5 days. This method was successfully applied to a preliminary pharmacokinetic study of intravenously administered MU1140 in Sprague Dawley rats. Overall, this method is shown to be applicable for quantification of MU1140 in plasma samples for the purpose of further MU1140 ADME or bioequivalence studies. 52

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Introduction The excessive and improper use of antibiotics has lead to the selection and spread of bacterial strains resistant to many of the currently used antibiotics. The United States FDA noted that antibiotic resistance problems must be det ected as they emerge, and actions taken to contain them, or else the world could be faced with previously treatable diseases that have again become untreatable as in the days before antibiotics were developed (40). This sharp increase in bact erial antibiotic resistance can be contained by the development and commercialization of new classes of antimicrobials (75). MU1140 (Figure 3-1A) is a bacterio cin produced by the microorganism Streptococcus mutans strain JH1140 (37). It belongs to the family of antimicrobial peptides known as lantibiotics, so named for their content of lanthionine residues (79). Lanthionines are amino acids that are com posed of two alanine residues linked by a thioether bridge through their -carbons. MU1140 has been shown to exert its antimicrobial effect on Gram positive bacter ia by a novel mechanism involving lipid II abduction, in which aggregates of MU1140 bind to molecules of lipid II and translocate them from sites of active cell wall biosynthesis (35). The result is i nhibition of cell wall synthesis. Although the first lantibiotic was discove red in 1928 (71), and approximately 50 more have been identified s ubsequently, their development as pharmaceutical agents for treatment of infectious diseas es has been hindered by the lack of cost effective production and/or purification (15). In the case of MU1140, produc tion of sufficient amounts of essentially pure product has been achieved in order to perform a number of pre-clinical tests (29). These indicate the potential useful ness of MU1140 in the treatment of certain 53

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Gram positive infections, including those cause d by wild-type and drug resistant variants of Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis. A reliable bioanalytical method for the quan tification of drugs is crucial for their development (61). Determination of the drugs concentration is needed for many reasons, among which is the assessment of th e pharmacokinetic properties of the drug and the subsequent dose design, evaluation of st ability, assessment of patient compliance, therapeutic drug monitoring, and determinati on of the bioequivalence of generics and follow on biologics. To date, no analy tical method has been reported for the quantification of free or total lantibiotics in a biological matrix. This study describes the development and validation of an LC/MS me thod for the quantific ation of total MU1140 in rat plasma. The method was validate d with regard to its accuracy, precision, selectivity, sensitivity, repr oducibility, and stability. Experimental Materials and Stock Solutions MU1140 was produced by Oragenics, Inc. (Alachua, FL) and gallidermin (Figure 3-1B), which was used as an internal st andard (ISTD), was purchased from Alexis Biochemicals (San Diego, CA). MU1140 and the gallidermin ISTD stock solutions were prepared in 1:1 (v/v) mixture of isopropyl alcohol (IPA):wat er at a concentration of 25 g/ml and stored at -80oC until used. Mass spectrometry grade IPA, water, and formic acid were purchased from Sigma (St Louis, Mo). Microcon Centrifugal Devices (10 KDa cutoff) were purchased from Millipore (Bedford, MA). Drug-free, male Sprague Dawley rat plasma with EDTA was purch ased from Rockland Immunochemicals, Inc. (Gilbertsville, PA) and aliquoted and stored at 20oC until used. 54

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Equipment and Analysis Conditions The LC/MS analysis system used consiste d of a Surveyor plus autosampler and pump (ThermoFisher Scientific, San Jose, CA) coupled to an API SCIEX 150EX single quadrupole mass spectrometer (Concord, ON, Canada) equipped with electrospray ionization. A Clipeus C-18 an alytical column (100x2.1 mm; 5 m particle size; Higgins, MA, USA) with a pre-column in-line filter (0.5m, MacMod, PA) was used for separation at room temperature. Sample s (25 l) of standards and unknowns were injected onto the column. Proteins were elut ed with an acidified (0.3% v/v formic acid) IPA:water gradient at a flow rate of 250 l/min. The gradient went from 5% to 95% IPA:water (v/v). Electrospray ionization wa s used for ions generation, with positive ion detection. Optimal sensitivity was achieved when ion source temperature was maintained at 475C and a voltage of 5.5kV was applied to the sprayer needle. Nitrogen was used as the nebulizer and curtain gas. Single ion monitoring (SIM) was used for detection of analyte and ISTD. SCIEX Analyst softwa re 1.4 was used for data collection and integration of the chromatographic peaks. Th e peak area ratios of MU1140 to ISTD were plotted as a function of MU1140 concentration in standard solutions A linear curve fit with no weighing was used to generate the regression line. The regression equation of the calibration curve was used to calculate the concentrations of the quality control samples and all unknowns. Standards and Quality Control Samples Working solutions of MU1140 (1 g/l) and gallidermin (0.1 g/l) were prepared in 10% IPA. These solutions were used to prepare calibration curve standards and QC samples. Calibration standards of MU1140 in rat plasma were prepared by addition of MU1140 working solution to an in itial concentration of 100 g/ml and nine serial 2-fold 55

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dilutions were prepared. Quality cont rol (QC) samples were prepared at 3 concentrations, including low (1 g/ml, LQ C) medium (10 g/ml, MQC), and high (50 g/ml, HQC). Both the calibration standard s and quality control samples were spiked with the working solution of the ISTD to a final concentration of 6 g/ml. Sample Preparation Plasma samples were spiked with the IS TD working solution to give a final concentration of 6 g/ml. Samples from th e pharmacokinetic study were allowed to thaw unassisted and fortified with th e ISTD for a final concentration of 6 g/ml. The samples were mixed for 30 seconds at medium speed using a vortex (VWR, Chicago, IL, USA). MU1140 and ISTD were extracted by acidifyi ng the samples by addition of 100% formic acid to a final concentration of 2% (v/v) a nd vortexing for 30 seconds. Each sample was diluted with an equal volume of 100% IP A and vortexed for 10 seconds to ensure complete mixing, after which the sa mples were centrifuged at 16,000 x g for 30 minutes in a table top centrifuge (Eppendorf, Hamburg, Germany) at room temperature. The supernatant was transferred to Microcon ul trafiltration device and centrifuged to dryness at 10,000 g at room temperature. The ultrafiltrate was analyzed by LC/MS as described above. Method Validation The method was validated for selectivity (specificity), sens itivity, linearity, accuracy, precision, recovery, and stability. Th e selectivity of this method was verified by treating blank rat plasma samples from six different lots and analyzing the samples for interfering peaks with the same m/z ratio at the analyte and ISTD retention times. Sensitivity was assessed by determining the lowest quantifiable concentration (LLOQ) of MU1140. The LLOQ was establis hed as the lowest concentration of 56

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MU1140 used in the calibration cu rve with accuracy and precision of 100% %. Bias and relative standard deviat ion were used as measures of accuracy and precision respectively, and we re computed using 100 ion Concentrat lTheoretica ion Concentrat Observed Mean ion Concentrat lTheoretica Bias and % Relative Standard Deviation = 100 Mean Deviation Standard Linearity was assessed by plotting MU1140:ISTD peak area ratios versus concentrations of calibration curve standards. Accuracy and precision were assessed by in jecting QC samples in pentuplicate and quantifying the MU1140 concentration using the regression line equation of the calibration curve. Bias and relative standa rd deviation were used as measures of accuracy and precision, respectively, and cal culated as mentioned above. A run was rejected if more than a third of the QC sample concentrations showed a deviation from the theoretical concentration equal to or greater than 20%. The developed methods ability to recover MU1140 was estimated by quantifying the MU1140 content of QC samples extrac ted by the developed method using a calibration curve constructed from unextracted standards. Unextracted standards were prepared by fortifying extracted, drug-fr ee plasma filtrate with MU1140 and ISTD. These samples represent 100% recovery and normalize for matrix effect, if any. Stability of MU1140 under different condi tions was assessed as part of the methods validation procedure. MU1140 stoc k solution stability was assessed at -80oC for up to 30 days. Every 10 days, 3 aliquots were thawed, spiked with ISTD, extracted, and analyzed. Bench top (short term) stability was determined at three concentrations (LQC, MQC, and HQC). Plasma aliquots were fortified with MU1140 and incubated at 57

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room temperature for 1.5, 3, and 6 hours. After incubation, the ISTD was added to the samples and the samples were extracted and analyzed for their MU1140 content. Freeze and thaw stability was evaluated by subject ing rat plasma samples spiked with MU1140 at three different concentrations (2.5, 10, and 40 g/ml) to three freeze-thaw cycles. Samples were frozen for 24 hours at -80oC then allowed to thaw unassisted at room temperature. This process was repeated two more times, and after the third cycle, samples were spiked with ISTD, extracted and analyzed. To determine the postpreparative stability of MU1140, plasma samp les were spiked with the MU1140, samples were extracted as per the developed method and incubated at 4oC for up to 4 days. ISTD was added to the samples prior to analysis. Preliminary Pharmacokinetic Study MU1140 doses equivalent to 12.5 mg/ kg or 25 mg/kg rat body weight were administered via the indwelli ng jugular cannula to two rats as a rapid iv infusion (< 1 minute) and plasma samples were drawn via the cannula at 5, 10, 20, 30 minutes, and 1, 2, 4, and 6 hours post dosing in anticoagulan t containing tubes. Blood samples were centrifuged at 500 g for 10 minutes to separate the plasma. Plasma samples were immediately collected and stored at -80C until analyzed. The validated method was used to quantify the rat plasma samples MU1140 content. Results and Discussion LC/MS Detection and Method Selectivity Analysis conditions for LC/MS were optimized using MU1140 and gallidermin (ISTD) in 50% IPA. The run time of the chromatographic method was 15 minutes with retention times of the anal yte and ISTD being approxima tely 5.2 and 5.3 minutes respectively. These methods were used in the following studies. Chromatograms of rat 58

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plasma spiked with MU1140 and ISTD and then extracted revealed that each molecular species was dominant in its doubly protonated molecular ion form (M+2H)2+, detected at m/z of 1133 and 1083, respectively. These va lues accord with their known molecular formulas. Six different lots of drug-free ra t plasma were treated as per the developed method and analyzed by LC/MS. No e ndogenous matrix ions were observed at m/z 1133 or 1083 at the retention times of MU1140 and ISTD. Data ar e presented in Figures 3-2 and 3-3. This ensured the selectivity of the method and its applicab ility to quantify these lantibiotics in rat plasma. The relatively short run time allowed in creased sample throughput, thus making this method specifically suitable for quantitation needs of studies of large sample size such as pharmacokinetic or bioe quivalence studies. To increase sensitivity, selective ion monitoring (SIM) was used for quantification. Gallidermin was found to be a suitable internal standard due to its structural similarity to MU1140. Linearity and Sensitivity The calibration curve was linear over the range of 0.39 100 g/ml when 25l of sample was injected onto the column. The relatively small sample volume allowed multiple injections from the same sample, thereby improving precision of quantitation. The correlation coefficient (r2) was > 0.995 for all validation batches. The limit of quantification for MU1140 wa s far below the established MIC of MU1140 for susceptible orga nisms (Ghobrial et al, submitted). This finding suggests that accurate quantification of MU1140 in the concentration range of interest should be readily achieved, and lead to accurate determination of the pharmacokinetic and pharmacodynamic parameters, and robust PK/PD modeling. 59

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Accuracy, Precision, and Recovery The inter-day accuracy and precision of the method were determined at the LLOQ as well as at three different QC concentrations in two different days. The accuracy of the method was described by the bias of theoreti cal versus measured concentrations, while the percentage of the relativ e standard deviation (% RSD) served as a measure of precision. Table 1 summarizes the intra-run, as well as the inter-day accuracy and precision of this bioanalytical method, meas ured on two different days. The intra-run bias was < 11% for all concentrations with intra-run %RSD of < 16 at the LLOQ level, and < 11% for all other concentrations. The mean inter-day deviation from the nominal concentration was < 8% and the inter-day RSD was < 2% for all tested concentrations. These data are in compliance with FDA guidance on bioanalytical method validation (82). The developed methods percentage recove ry of MU1140 from the plasma samples was estimated by comparing the ratio of the an alyte peak areas from extracted samples to that from the unextracted samples. The mean recovery of MU1140 for all samples was 96.05% with RSD of < 11%. Data are summarized in Table 2. The data presented above confirms that the developed method is capable of accurately and precisely qua ntifying MU1140 in Sprague Dawley plasma. This high recovery ratio improves the ability to detect and quantify MU1140. Stability Stability of the MU1140 stock solution cons tituted at 25g/ml in 50% IPA /water (v/v) was assessed after freezing for 10, 20, and 30 days at -80C. The original solution was aliquoted into twelve tubes. Three tube s were analyzed immediately and the others were frozen at -80oC. Three tubes were thawed and an alyzed at the indicated times. 60

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Each vial was sampled in triplicate. In al l cases, MU1140 was detectable at levels equal to or greater than 96% (RSD < 8 %) compared to MU1140 freshly prepared at the same concentration. Data are presented in Table 3. This suggests that th e standard solution of MU1140 was stable for at least 30 days when stored at -80C. Bench top stability at room temperature of MU1140 in plasma was investigated at the three QC concentration levels, LQC, MQC, and HQC. Just before termination of the incubation period the ISTD was added. Incuba tion at room temperature was stopped at 0, 1.5, 3, and 6 hrs by addition of formic acid and isopropanol as per the extraction procedure and samples were analyzed for MU1140 content. There was no measurable loss of MU1140 in plasma at room temperat ure for more than 6 hrs. Data are summarized in Table 4. Plasma samples spiked with MU1140 were subjected to three freeze and thaw cycles, after which ISTD was added, and th e samples were processed by extraction and quantification. A mean per centage change of < 0.5% with a % RSD of < 10% was observed. Data are summarized in Table 5. This result confirms that multiple freezing and thawing of MU1140-containi ng plasma did not affect th e stability of MU1140. Post-preparative stability of MU1140 was al so determined at three concentrations at 4C for up to 4 days. Less than 5% (< 12 % RSD) change in the intensity of the MU1140 signal was evident. Data are summarized in Table 6. This result indicates that MU1140 extracted from plasma was stable for at least 4 days at 4C. Optimal storage and handling condition of MU1140-containing plasma samples were tested by the long-term and short-te rm stability studies, multiple freeze/thaw studies, and post-preparativ e stability studies which all showed no appreciable 61

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degradation and loss of the lantib iotic. These findings indicate that plasma samples from dosed animals can be conveniently stored at -80C and thawed, processed on the bench top at room temperature, and placed in a re frigerated autosampler for extended periods without significan t loss of MU1140. Preliminary Pharmacokinetic (PK) St udy of MU1140 in Sprague Dawley Rat The purpose of this PK study was to evaluate the validated method for the quantification of MU1140 content of in vivo samples. The method was successfully applied to a preliminary pharmacokinetic study of MU1140 in two rats which received either 12.5 or 25 mg/kg dose. All samples we re analyzed within 1 day and precision and accuracy for QC samples were within acceptabl e limits. Plasma concentration-time data were subjected to noncompartmental analysis (NCA) and dose linearity of the calculated pharmacokinetic parameters was established. Cmax and AUC0were dose dependent and measured to be 8.86, 15.9 g/ml and 12.39, 24.69 hr. g/ml for the 12.5, 25mg/kg doses, respectively. The half life and clea rance were dose independent indicating the linearity of the pharmacokinetics of MU1140 in that dose range. The pharmacokinetic profiles are presented in Figur e 3-4 and the pharmacokinetic data are presented in table 36. Overall, it appears that the develo ped method is reliable for the in vivo of quantification of MU1140. Conclusions This paper describes the first bioanaly tical method for the quantification of a lantibiotic in a biological matrix. The method was developed specifically for the quantification of MU1140 in ra t plasma samples, and uses a simple and inexpensive liquidliquid extraction followed by a rapid, sensitive LC/MS separation and detection procedure. This method was validated to be selective, accurate, prec ise, and sensitive, 62

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and the stability of MU1140 was not co mpromised during sample handling and processing. The method was tested for the quantification of MU1140 in rats following IV administration. Clear dose-dependent respon se was observed, ensuring the validity of this quantification method. Collectively, this method should be applicable to all quantitative studies of MU1140 development and should serve as a starting point for optimization of bioanalytical me thods for other lantibiotics. 63

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A Ile Ala Ala L y s Phe Leu Ala S Abu Pro Gl y Ala S Ala L y s Dhb Gl y Ala Phe Asn Ala T yr Ala NH CH CH S Al a Trp Dh Le Al a Ab Pro Gl Al a Al a Ar Dh Gl Al a Ph As Al a Tyr Al a NH CH CH S S S S Ly Ph S B Figure 3-1. Analyte and ISTD. A) MU1140 (37) B) Gallidermin (42). Amino acids different from MU1140 are highlighted. 64

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Figure 3-2. Representative LC/MS chromatogr am of extracted drug-free rat plasma A. Total ion current (TIC) of the two ions B. Extracted ion chromatographs (XIC) for m/z 1082-1084 (gallidermin) C. XIC for m/z of 1132-1134 (MU1140). 65

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Figure 3-3. Representative LC/MS chromat ogram of plasma sample fortified with MU1140 (at LLOQ), m/z 1133 and gallidermin, m/z 1083. A. TIC of the two ions B. XIC for m/z 1082-1084. C. XIC for m/z of 1132-1134. 66

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0 2 4 6 8 10 12 14 16 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time (hr) 12.5 mg/kg Dose 25 mg/kg Dose Figure 3-4. MU1140 plasma con centration-time profiles after IV bolus administration of a single dose of 12.5 mg/kg or 25 mg /kg to two different rats. 67

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Table 3-1. Intra-run and inter-day accuracy and precision of the bioanalytical method at the LLOQ and three con centrations of MU1140. Day 1 Theoretical Concentrations LLOQ (0.39g/ml) 1g/ml 10g/ml 50g/ml Measured Concentrations (g/ml) Within Run Mean (n=5) 0.36 1.07 9.64 49.39 % Bias -6.67 6.86 -3.58 -1.23 % RSD 10.7 11.87 8.47 5.1 Day 2 Theoretical Concentrations LLOQ (0.39g/ml) 1g/ml 10g/ml 50g/ml Measured Concentrations (g/ml) Within Run Mean (n=5) 0.35 1.10 9.85 48.9 % Bias -10.26 10.0 -1.50 -2.20 % RSD 15.7 7.92 6.69 4.68 Inter-day Comparisons Theoretical Concentrations LLOQ (0.39g/ml) 1g/ml 10g/ml 50g/ml Inter-day Mean (n=2) Concentration 0.36 1.09 9.70 49.2 % Bias -8.97 8.50 -2.55 -2.14 % RSD 1.99 1.95 1.52 0.70 68

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Table 3-2. Recovery of MU1140 from plasma samples. Theoretical Concentration (g/ml) 2.5 10 40 Mean (n=6) Observed Concentration (g/ml) % RSD 2.39 10.7 9.64 9.1 38.46 6.4 Recovery (%) 95.6 96.4 96.15 Table 3-3. MU1140 stock solution (25 g/ml) stability at C for up to 30 days. Day Mean (n=3) Concentration (g/ml) % Bias % RSD 0 24.94 0.24 6.38 10 24.09 3.64 7.65 20 25.73 -2.92 6.4 30 25.13 -0.52 7.89 Table 3-4. Bench top stability of MU 1140 in Sprague Dawley plasma at room temperature. % RSD is shown in parenthesis. Theoretical Concentration (g/ml) Time (hr) 1 10 50 Calculated Concentration (g/ml) 0 0.98 (9.5) 10.2 (7.9) 51.1 (4.7) 1.5 1.10 (10.6) 9.95 (8.2) 49.9 (4.5) 3 1.20 (11.3) 10.3 (8.6) 51.6 (3.3) 6 0.95 (8.8) 9.50 (10.5) 50.3 (5.5) Table 3-5. Freeze/thaw stabilit y assessment of MU1140 in plasma. Theoretical concentration (g/ml) Mean (n=6) Concentration (g/ml) % Bias % RSD 2.5 2.29 0.06 9.1 10 10.0 0.36 8.4 40 42.5 0.49 6.3 69

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Table 3-6. Post-prepara tive stability assessment of MU1140 after 4 days at 4oC in autosampler. Theoretical Concentration (g/ml) Day 2.5 10 40 Mean (n=3) Concentration (g/ml) 0 2.30 (11.3) 8.64 (8.1) 38.9 (5.3) 1 2.40 (9.6) 9.30 (10.2) 37.2 (6.2) 2 2.30 (9.2) 10.2 (9.7) 37.7 (5.8) 3 2.55 (9.8) 10.0 (8.7) 40.5 (7.4) 4 2.45 (10.3) 10.1 (9.1) 36.5 (6.1) % Bias -3.00 -3.37 -4.60 Table 3-7. MU1140 pharmacokinetic paramete rs determined using NCA analysis of plasma concentration-time data. Pharmacokinetic Parameters Dose Cmax (g/ml) AUC0(hr. g/ml) t. (hours) Clearance (l/hr/kg) 12.5 mg/kg 8.86 12.39 1.33 1.00 25 mg/kg 15.9 24.69 1.56 1.01 70

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CHAPTER 4 PHARMACOKINETIC/PHARMACODYNAMIC EVALUTATION OF THE LANTIBIOTIC MU1140 IN SPRAGUE DAWLEY RATS This is the first study to report the pharmacokinetics (PK) of a lantibiotic in an animal model and a linked in vivo pharmacokinetic/ in vitro pharmacodynamic (PK/PD) model to predict its in vivo activity. This work was done using the lantib iotic MU1140, an antibiotic in preclinical development that is indicated for the management of Gram positive infections. Following intravenous administration of MU1140 at 25 mg/kg rat body weight, the plasma concentration-time profile of MU1140 in rats declined biexponentially with a mean elimination half-life of 1.7 0.1 hours and a mean maximum concentration of 18.7 5.5 g/ml. The mean volume of distribution and systemic clearance, calculated from the noncompartmental analysis of MU1140 plasma concentration-time profiles, were 3.48 1.14 L/kg and 1.44 0.42 L/hr/kg, respectively, and the mean total area under the plasma concentration-time curve was 18.7 5.5 g.hr/ml. Plasma concentrations of MU1140 were measurable up to 6 hours postadministration. The best fit of plasma concentration-time data was achieved using an open two-compartment model with elimination from the central compartment. It was observed that rapid injection of this lantibiotic is associated with a histamine release/hypersensitivity reaction similar to vancomycins red man syndrome. Premedication with diphenhydramine blocked th is response. Using time-kill data of MU1140 vs. Staphylococcus aureus (S. aureus), a pharmacodynamic (PD) model was developed to explain the observed time-kill profile. The PD model was based on the two subpopulation (susceptible and resistant) st rategy. The utility of the developed in vitro PD model was further extende d by incorporation of the in vivo PK model of MU1140 in rats as an input function to yield the pers pective linked PK/PD model. This model was 71

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used to simulate in vivo time-kill behavior of MU1140 against S. aureus in rat. The developed PD model was predictive of the in vitro time-kill data and valuable MU1140 PD parameters were estimated, namely the concentration of MU1140 that produced half the maximum kill for susceptible and resistant bacteria (EC50S and EC50R) were calculated to be 0.0001 and 2 g/ml respectively. Co llectively, these findings suggested that by using simulation the developed PK/PD model can be used to study the effect of variation of dose, bioavailability, and dosing regimen on the outcome of therapy and to optimize the dosing regimen for MU1140 and other la ntibiotics with similar properties. Introduction Soon after they are approved and are in widespread human use, some broad spectrum and regularly prescribed antibiotics lost their ability to control certain bacterial infections(46), and currently more than 70% of nosocomial infections are caused by drug resistant bacteria(12, 41). Annually, infections caused by S. aureus alone, which represent 16% of infections nationwide, re sults in 12,000 inpatient deaths, 2.7 million days in excess length of stay, and 9.5 billi on dollars in extra hospital charges(60). Two factors contribute and further exacerbated this an tibiotic resistance healthcare crisis. The first is the diminishing antibio tics pipeline(74) especially th ose with novel mechanisms of action. According to Rice et al, since 1998, only ten antibiotic s have been approved, out of which only two out possess a novel mechanis m of action. The other factor is the continuous and rapid emergence and spread of new bacterial strains th at are resistant to currently used antibiotics(70). Thus, there is a need for new antibiotics and appropriate dose and dosage regimen design are crucial to prevent the emerge nce and spread of bacterial strains resistant to ne wly developed an tibiotics (73). 72

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MU1140 (Figure 4-1) is a lantibiotic produced by the indigenous oral microorganism Streptococcus mutans strain JH1140 (37). Lantibiotics (lanthioninecontaining antibiotics) are ribosomally synthe sized peptides containing various modified amino acids such as lanthionine (Lan, ala-Sala), methyllanthionine (MeLan, abu-S-ala), didehydroalanine (Dha) and dide hydrobuterine (Dhb) (13). Th ese antimicrobial peptides are thought to be excreted in order to e liminate microorganisms competing with the producer microorganism for its habitat. MU1140 has a novel mechanism of action known as lipid II abduction, which involves inhibition of pe ptidoglycan cell wall synthesis by binding to and sequest ering lipid II(35, 78) away fr om its site of action, thus, destabilizing the bacter ial cell wall resulting in cell lysis. Currently, MU1140 is in the preclinical phase of development as an an tibiotic for the management of Gram positive infections. The purpose of this study was to characterize the plasma concentration-time profile and systemic exposure of a single dose of intravenously administered MU1140 in Sprague Dawley rats. The plasma concentration-time profile will be used to generate a PK model that is predictive of MU1140 concentrations produced by different dosing regimen. Previous time-kill studies show that MU1140 was bactericidal against S. aureus when tested in vitro (29). By assembling a pharmacodynamic model using time-kill data, very important pathogen and antibiotic speci fic parameters can be elucidated. Such parameters as the pathogens growth rate constant (g), the antibiotics maximum kill effect (Kmax) and the concentrati on that produce 50% of Kmax (EC50)(55). To further improve the utility of the PD model, integrati on of the PK data of the antibiotic with the PD model will create a powerful tool (the linked PK/PD model) that is capable of 73

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predicting the results of therapy in an anim al model of infectio n and can be further modified to design a rational and efficacious c linical dosing regimen that reduces toxicity and the chance of resistance development to MU1140. Materials and Methods Drug and Dose Administration MU1140 was produced by Oragenics, Inc. and was administered over a one-minute period via an indwelling jugular catheter as a single intr avenous dose with a volume of administration of 5ml/kg. Animals Jugular vein cannulated Spra gue Dawley male rats (200-220 g) were purchased from Charles River Laboratories (Raleigh, NC). Animals were housed and allowed to acclimatize for 5 days prior to experiment st art day. Experiments were conducted at the University of Florida rodent facility (Alachua Fl) where they were given Harlan rat chow (7912) and water ad libitum. Rats rooms were temperatur e and humidity controlled. Experimental Design All rats were weighed immediately before initiation of the study and the weights were recorded and used for dose calculati ons. One hour prior to dosing with MU1140, all animals were injected with diphenhydram ine (DPA, 20mg/kg) subcutaneously. The jugular cannulae were used to draw the blood samples at times 5, 10, 20, 30 min, and 1, 2, 4, and 6 hrs post-dosing. Blood was mi xed 10:1 with a 10 anticoagulant stock solution (15 mg/ml sodium EDTA plus 17 mg /ml sodium chloride) and centrifuged at 500 gs for 20 min at room temperature to separate the plasma. Plasma samples were immediately collected and stored at 80 C. Normal saline (1 ml) was used to cleanse 74

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the cannulae after MU1140 administration and after blood sampling. Hepranized saline (1000 IU/ml) was used as the cannula lock solution. Quantification of MU1140 in rat blood samples was carried out using the validated LC/MS method developed by Ghobrial et al (28). Briefly, prior to analysis, plasma samples were allowed to thaw at room temperature. Gallidermin, the internal standard, was added prior to sample preparation at a concentration of 6 g/ml. The sample preparation procedure involved addition of formic acid to a final concentration of 2% (v/v) and vortexing for 30 seconds. Plas ma protein precipitation was achieved by addition of an equal volume of isopropa nol, followed by centrifugation at 16,000xg for 30 minutes at room temperature. The supe rnatant was filtered through a 10 KDa MWCO Microcon filter (Millipore, Bedford, MA). The ultrafiltrate was analyzed for its MU1140 content by LC-MS. MU1140 and ISTD were detected by an API 100 single quadrupole mass spectrometer (Applied Biosystems, Concor d, ON, Canada) operated in the positive mode with electrospray ionization. Singl e ion monitoring was used to improve the assays sensitivity. Quantitation was achieved by monitoring ions at m/z 1133 (MU1140) and m/z 1083 (gallidermin). The stan dard curve was linear (r2 > 0.998) and ranged from 0.039 to 100 g/mL, representing the lower a nd upper limits of quantitation (LLQ and ULQ), respectively. The relative standard de viation (RSD) for with in-run precision was < 15%. Bias was < 7% of the nominal values. PK Data Analysis Noncompartmental Analysis (NCA) WinNonlin (Pharsight Corporation, M ountain View, California) was used to perform the NCA to calculate the area under the concentration-time curve (AUC), total clearance (CL) half-life (t), volumes of distribution (Vss and Varea), and the maximum 75

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concentration (Cmax). The trapezoidal rule was used to estimate AUC0-tlast. The AUCtlast(the AUC from the last measured time point to infinity) was determined by dividing the last measured concentration by the elimination rate constant of the terminal phase. The total AUC was the sum of AUC0-tlast and AUCtlast. The clearance was calculated as CL = Dose/AUC. The slope of the terminal phase of the plasma concentration-time profile, ke, was estimated from the terminal slope of the log-linear plot of individual plasma concentrations versus time. The terminal half-life (t) was calculated using t =ln2/ke. The mean residence time (MRT) was calcula ted as the ratio of area under the first moment curve (AUMC) divided by AUC. Vss was calculated as CLMRT. The volume of distribution (Varea) was calculated using Varea = clearance/ke. PK Model PK model parameters were estimated using the nonlinear least squares regression software program WinNonlin version 5.2 (Pharsight Corporation, Mountain View, California) using the Gauss Newton algorith m and uniform weighting. The PK model was selected based on goodness of fit using th e Akaikes Information Criterion (AIC) and Schwarz Criterion (SC)(86) resi dual analysis, and overall correlation coefficient. Plasma concentration-time data for each rat were fitted using a two-compartment open body model described by where C is the total plasma concentration, t is the time in hours, A is the y axis intercept for the dist ribution phase, B is th e y axis intercept for the linear elimination phase. t tBeAeC Statistical Analysis Statistical analysis of PK parameters was performed using unpaired Students t-test where a p value 0.05 was considered significant. 76

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Time-Kill Studies Previous time-kill data of MU1140 vs. S. aureus (29) were used. The time-kill studies were performed as desc ribed earlier. Briefly, bact erial inocula were prepared from test organisms grown for 4 h in the ap propriate broth media and diluted in saline to 0.5 McFarland to obtain 100 ml of a starting culture containing 106 colony forming units (cfu)/ml, which was verified by col ony counts of replicate samples. Aliquots (10 ml) of the culture were transf erred to sterile plastic 25 cm2 culture flasks (Corning Inc, Corning, NY) and MU1140 was added from a sterile stock solution to give final concentrations equal to 0.5, 1, 2, 4, 8, and 16 times the MIC for S. aureus strain ONI33. The assay included a growth c ontrol tube with no antibiotic. The cultures were incubated at 37C and sample s were obtained at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 hours following addition of MU 1140. The samples were serially diluted 10-fold in ice cold normal saline and 10 l samples spotted onto duplicate BAPs. Following incubation at 37C for 24 h, colonies that arose on plates with 30 colonies were counted. PD Model Observed from previous studies of the time-kill profile of MU1140 is the re-growth of S. aureus after the sharp decline of the inoc ulum starting numb er(29). A common approach to model such data is the use of the two subpopulation model which assumes that the initial bacterial population was composed of two subpopulations with different susceptibility to MU1140 (designated by two different EC50). The rise in bacterial numbers at later time points was explaine d by the continued growth of the less susceptible subpopulation. The model (10) we used assumes equal growth rate constant 77

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and MU1140 maximum kill rate for susceptible (S) and resistant (R) subpopulations. The following differential equations describe th e changes of both populations over time. ) )( (50 max max max S gECC C K N NNSK S dt dS ) )( (50 max max max R gECC C K N NNRK R dt dR RSN Where S and R are the numbers of susceptible and resistant bacteria at time t, kg is the microbial net growth rate constant, Nmax is maximum achievable number of bacteria in the in vitro medium, Kmax is the antibiotic maximum kill rate, EC50 is the MU1140 concentration that produces 50% of maximal killing, and C is the concentration of MU1140 at time t. PK/PD Model and Simulation The PK/PD model was assembled using the developed PD model and substitution of the concentration term in the PD model with the concentration function from the pharmacokinetic model(30). The scheme of the model is presented in Figure 4-3. V1 and V2 are the MU1140 volumes of di stribution in the central an d peripheral compartments, k10 is the elimination rate constant, k12 and k21 are the rate constants for the transfer of MU1140 from the central to the peripheral and from the peripher al to the central compartments, respectively. The simulation function of WinNonlin Regre ssion Analysis software was used to simulate the concentration of S. aureus resulting from a certain dosing regimen and thus in designing a dosing regimen fo r MU1140 to be used in this S. aureus infection model. MU1140 concentrations resulting from two dosing regimens, 5mg/kg TID and 10mg/kg 78

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TID were simulated. Model parameters used in the simulation were as follows: The model parameters, V1, V2, k12, k21, k10, N, kg, EC50s, EC50R, Kmax were estimated using WinNonlin. Results The plasma concentration-time profile of MU1140 in Sprague Dawley rats after a single IV bolus dose at 25 mg /kg is presented in Figure 4-1. MU1140 was measurable in plasma for up to 6 hours post administration. MU1140 plasma concentration-time profile declined in a biexponential mode post administration. Noncompartmental Data Analysis MU1140 PK parameters estimated by noncom partmental analysis of the MU1140 plasma concentrations are summarized in Table 1. To test the linearity of the estimated PK parameters, the PK study was repeated using a MU1140 dose equivalent to 12.5 mg/kg rat body weight. For both dose groups, there was no statistical significance between CL, t, Vss, or Varea. On the other hand, Cmax and AUC0were dose linear. Data is summarized in table 1. Compartmental Data Analysis of MU1140 PK Data MU1140 plasma concentration-time data we re fitted using the two-compartment open model equation. The log concentrationtime profiles were characterized by a short (less than 30 minutes) but clear distribution phase follo wed by a slower elimination phase, as seen in Figure 4-1. The mean half-life of the distribution phase (t ) was 4.2 min, while the terminal elimination phase mean half life (t ) was 1.6 hours. Using compartmental analysis the volume of the central compartment (V1) was 0.37 0.23 L/kg and the volume of the peripheral compartments was estimated to be 1.4 0.56 L/kg. CL and CLD2 were estimated to be 1. 67 0.56 L/h/kg and 2.55 1.29 L/h/kg. 79

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Time-Kill Data and PD Model The kill profile of MU1140 against S. aureus is characterized by a rapid and significant decline (> 3 log drop) in bacterial counts within the first 2 hours independent of the antibiotic concentration. Regrowth is observed at all MU1140 concentrations. The experimentally observed time course of the bacterial concentrations (CFU/ml) and the bacterial concentrations predicted by PD m odel are presented in Figure 4-4. The model accurately predicted the bacter ial concentration for all te sted MU1140 concentrations. Estimates of the PD model parameters are S. aureus growth rate of 0.25 hr-1, EC50S and EC50R were estimated to be 0.0001 a nd 2 g/ml, respectively. PK/PD Model and Simulation The PK/PD model and the si mulation function of WinNon lin was used to predict the concentration of viable S. aureus cells when MU1140 is administered and its concentration is decaying as per the pharmacokinetic model developed in rats. Figure 4-5 shows the simulated S. aureus counts resulting from vari ous MU1140 dosing regimens. Discussion Vancomycin, the first peptide antibiotic to be used in the clinic and the current drug of last resort, is loosing ground with the emergence of vancomycin resistant S. aureus and E. faecalis. The spread of community and hos pital acquired infections due to drug resistant Gram positive pathogens stresses the need for new antibiotics with novel mechanisms of action. MU1140 is active against a wide range of Gram positive organisms, including MRSA, VRE, and VI SA. MU1140s unique mechanism of action involves binding and translocati on of lipid II away from cell division septa. Due to its unique MOA on cell wall biosynthesis, there is a chance that MU1140 will not be subject to commonly known mechanisms of antibiotic resi stance. In a previous study (29) S. 80

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aureus and S. pneumoniae were unable to develop resistance to MU1140 after continuous sub-culturing in sub-inhibitory concentra tions of MU1140 for 21 days, just a merely 3 fold increase in MIC values. The PK profile of MU1140 was investigated after administration of an intravenous dose equivalent to 12.5 and 25 mg/kg. Afte r intravenous administration of MU1140, its disposition was consistent w ith a two-compartment open model with elimination from the central compartment. The Cmax achieved after a 25mg/kg dose is approximately 20 g/ml and the concentration drops below 1 g/ml within four hours, with a relatively short halflife (1.7 hr). Noncompartmental analysis revealed th at the mean volume of distribution of MU1140 in rats was about 3500 ml/kg. Given that the extr acellular fluid volume of a Sprague Dawley rat is around 320 ml/kg, MU11 40s volume of distribution is more than 10 times the rats total body wa ter. This indicates partit ioning of MU1140 to blood cells, tissues and other extravascular sites. Rapid injection of MU1140 was not well tole rated. A hypersensitivity reaction, similar to stimulation of histamine rel ease, is observed with in 5 minutes postadministration of the first dose. This reacti on is characterized by redness of the ears and paws, swelling, and lethargy, and usually wi ll last for no more than 20 minutes, after which the condition subsides. Subcutane ous administration of diphenhydramine (DPA, 20mg/kg) 1 hour before dosi ng of MU1140 is enough to block most of these symptoms. A similar reaction, known as red man syndrome, is observed when vancomycin is introduced as a rapid infusion (76). In a cl inical setting, pre-medication with DPA or 81

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administration of MU1140 as a slow infusion might be needed to block or avoid this reaction. The development of a mathematical model to describe the killing behavior allows quantitative correlation of the antibiotic concen tration and the bacter ial concentration at any time point. Thus, a rational dosing regimen can be conceptualized A PD model that can explain the time-kill experiment data where MU1140 was maintained at a constant concentration was developed. Although the model fit the observed data well, such a simple model has limited utility in an in vivo scenario where the antibiotic concentration is changing. Thus, the PD model was extended for the simulation of S. aureus counts when MU1140 concentrations are fluctuati ng in a way that mirrors the plasma concentration-time profile of MU1140 observed in Sprague Dawl ey rats. The values of the PK/PD model parameters (Emax, kg, EC50S, and EC50R) were fixed to the values estimated from the PD model derived from the time-kill data. The model may be a useful guide to identify target PK/PD indices predic tive of therapys success in humans as well as determining initial dosing regimens in the clinic. Conclusions After IV administration of MU1140 in male Sprague Dawley rats, MU1140 showed two-compartmental model plasma kinetics. The developed PK model is predictive of MU1140 concentrations in Sp rague Dawley rats following intravenous administration. A PD model to descri be the activity prof ile of MU1140 against S. aureus using time-kill data was developed. The goodness of the model was tested by curve fitting to obtain the S. aureus and MU1140 specific parameter estimates. The applicability and usefulness of the PK model was enhanced by incorporating it into the 82

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developed PD model to create the PK/PD m odel of MU1140 activity. Using this model, alterations in dose, and dosing regimen on th e outcome of therapy can be evaluated by simulation. 83

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Phe L y s Ala Tr p Dha Leu Ala Abu Pro Gl y Ala Ala Ar g Dhb Gl y Ala Phe Asn Ala T yr Ala NH CH CH S S S S Figure 4-1. MU1140 (37) Figure 4-2. MU1140 PK profile after admi nistration of 25 mg of MU1140 per kg rat body weight (symbols). Shown also is the simulated plasma concentrationtime profile generated by the PK model (line). 84

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Figure 4-3. Scheme of PK/PD model fo r antibacterial effect of MU1140. Figure 4-4. Observed vs. predicted S. aureus concentration (cfu/ml). 85

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Figure 4-5. The result of the simulation of S. aureus viable cell count when MU1140 is administered at two dose levels (5 a nd 10mg/kg TID). The simulated rat PK profile was taken in to consideration. 86

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87 Table 4-1. Noncompartmental analysis of MU1140 (12.5, and 25mg/kg) concentrationtime data. PK parameters estimated were AUC, Cl, t, Vc and Cmax. AUC0(g.hr/ml) Clearance (ml/hr/kg) t (hr) Vss* (ml/kg) Varea (ml/kg) Cmax (g/ml) 12.5mg/kg 8.7 (.1) 1343.1 (.3) 1.46 (.2) 2207.1 (.5) 2768.6 (.6) 10.7 (.1) 25mg/kg 18.7 (5.5) 1441.4 (420.7) 1.7 (0.1) 2283.7 (1010.5) 3478.3 (1135.6) 31.1 (12.6) Vss is the estimated volume of distributi on at steady state calculated as MRT0Cl, based on the last observed concentration. Varea is the volume of distribu tion, calculated as Cl/ke.

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CHAPTER 5 PHARMACODYNAMIC ACTIVITY OF THE LANTIBIOTIC MU1140 This study evaluated the in vitro pharmacodynamics of the lantibiotic MU1140 as well as the ability of selected agents to develop resistan ce to this novel antibiotic. Susceptibility to MU1140 of over 30 Gram positive and 32 Gram negative bacteria representing 28 species was assessed. MU1140 demonstrated high activity against all tested Gram positive organisms including Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, and Staphylococcus aureus and moderate activity was observed against Enterococcus faecalis as well as Bacillus species. MU1140 showed antimicrobial activity against oxacillin and vancomycin resistant S. aureus, as well as vancomycin resistant E. faecalis and E. faecium. No antibacterial activity was observed at the concentrations tested against Gram negative bacteria and yeast. Time kill studies were used to assess the kill prof ile of MU1140 against clinical isolates of multidrug resistant S. aureus, vancomycin resistant E. faecalis, and an ATCC S. pneumoniae strain. According to CLSI susceptibility breakpoints, MU1140 was bactericidal against S. pneumoniae as well as multidrug resistant S. aureus, and bacteriostatic against vancomycin resistant E. faecalis. In vitro resistance development of S. aureus and S. pneumoniae to MU1140 was tested by sequential subculturing of the microorganisms in media containing subinhibi tory concentrations of MU1140. After 21 subculturing even ts, the MIC of S. aureus and S. pneumoniae increased by only three fold. Subsequent subculturing of the strains with elevated MIC values in antibiotic free media for 7 days did not result in a reducti on of their MU1140 MIC values. Collectively these findings illustrate the potential of MU1140 to serve as a therapeutic agent for the management of infections caused by Gram positive bacteria. 88

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Introduction The ability of microorganisms to develop resistance to antimicrobial agents has recently limited our ability to effectively control certain microbial infections and hindered effective antibiotic th erapy. Infections due to an tibiotic resistant pathogens have become a current, major health crisis in the world (1, 64). The situation has dramatically deteriorated during the last tw o decades since excessive and improper use of existing antibiotics has created multi-drug resi stant bacteria, also known as the superbugs. In addition, discovery and development of new antibiotics has dwindled during this period. As a consequence, infections caused by these multi-drug resistant pathogens have emerged as a major cause of morbidity and mortality in the United States and worldwide (1, 81). This problem is particul arly apparent in the cases of methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecalis (VRE). About 500,000 infections caused by MRSA are acquired in hospitals every year, costing about 8 billion dollars to treat (87). Treatment of these infections will be a major challenge in the near future and will re quire the discovery and development of novel antimicrobial agents (87). A novel class of antibiotics that has long attracted much attention is the antimicrobial peptides. Antimicrobial pept ides have emerged as potential therapeutic agents for the treatment of various types of bact erial infections due to their ability to kill Gram positive and Gram negative pathogenic mi croorganisms and fungi as well as to activate components of the host innate immune system (6, 9, 34, 90). Some of these peptides were also shown to inhibit envel oped viruses replication (57). So far, all discovered antimicrobial peptides share certai n similar structural characteristics required for their bioactivity, which include an overall positive charge inferred by the presence of 89

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multiple arginine and lysine amino acid residues as well as ~50% of the peptides overall primary structure is composed of hydrophobic residues (90). It is thought that these amphiphilic structural features promote bind ing to and intercalation into bacterial membranes, which then allows the peptide to carry out its antibacterial activity (69). A promising class of the antimicrobial pept ides are the lantibiotics. Lantibiotics (lanthionine-containing antibioti c) are peptides with antimicrobial properties that are secreted by certain Gram positive bacteria ( 13). Although to date, lantibiotics have not been utilized as pharmaceutical agents, several have been used in commercial applications. Nisin, for example, is a lantibiotic produced by the bacterium Lactococcus lactis, which has been used extensively as food preservative sin ce the 1920s. Lantibiotics are ribosomally synthesi zed and then undergo extensive posttranslational modification. Lantibiotics ar e characterized by unusual amino acids such as lanthionine (Lan, ala-S-ala) methyllanthionine (MeLan, abu-S-ala), di dehydroalanine (Dha) and didehydrobuterine (Dhb). MU1140 (M utacin 1140, Figure 5-1) is a 22 amino acid lantibiotic that is produced by Streptococcus mutans (37). It has been extensively characterized in regard to its physical and ch emical properties (37, 78) and its role in promoting colonization of the oral cavity by the producer strain. Its unique mechanism of action involves inhibition of the peptidoglycan synthesi s by binding to and abducting lipid II from its site of action at points of peptidoglycan synthesis (35). The aim of this study was to evaluate the potential efficacy of MU1140 against a broad spectrum of Gram positive organisms as well as to assess the potential of their resistance development to MU1140. Measure of the MIC was used to assess the 90

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organisms susceptibility to MU1140 and timekill studies provided a dynamic picture of MU1140 antimicrobial action. Materials and Methods Bacteria and Media Bacterial strains, shown in Tables 1, 2 and 3, used in the spectrum of activity studies were clinical isolates as well as ATCC strains. Kill curves were performed using a multidrug resistant strain of Staphylococcus aureus (ONI33) and a multidrug resistant strain of Enterococcus faecalis (ONI47), both obtained as fres h clinical isolates from Shands Hospital (Gainesville, FL ). These studies were also performed using a strain of Streptococcus pneumoniae (ATCC49619). Strains ONI33 and ATCC49619 were also used in the development of resistance study. S. aureus strain ONI33 was shown to be resistant to amoxicillin, am picillin, cefazolin, cefepim e, cefotaxime, ceftriaxone, cefuroxime, cephalothin, ciprofloxacin, clindamycin, erythromycin, imipenem, levofloxacin, meropenem, oxacillin, penicill in, sparfloxacin, ticarcillin, azithromycin, amikacin and chloramphenicol. E. faecalis strain ONI47 was shown to be resistant to ampicillin, ciprofloxacin, erythromycin, le vofloxacin, penicillin and vancomycin. Bacterial strains were stored as 50% glycerol stabs at -80C. Starter plates of bacterial strains were prepar ed by inoculation of samples from glycerol stabs onto blood agar plates (BAP) consisting of casein peptone (1.5%; Remel, Lenexa, KS), soy peptone (0.5%; Remel), sodium chloride (0.5%; Remel), sheeps blood (5%, Lampire, PA) and agar (1.5%, Fisher, NJ). S. aureus strain ONI33 and E. faecalis strain ONI47 were grown in cation-adjusted Muller-Hinton broth (CAM H; Becton Dickinson Biosciences, Franklin Lakes, NJ) at 37C in a CO2 (5%) incubator. S. pneumoniae strain ATCC49619 was 91

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grown in Todd-Hewitt broth (THB; Becton Dickinson Biosciences) under the same conditions. Antimicrobial Agents MU1140 was manufactured by Oragenics, Inc. Purity was estimated to be greater than 90% as determined by analytic al reverse phase HPLC (RP-HPLC). Susceptibility Studies The minimum inhibitory concentrations (MIC) of MU1140 against target microorganisms were determined by Focus Bio-Inova (Herndon, VA). Aerobes MU1140 MIC values were determined by broth micr odilution method according to CLSI-defined methodology (M7-A6), while MU1140 MIC values for anaerobes were determined by the agar dilution according to CLSI-defined methodology (M11-A5). TimeKill Studies The MICs for S. aureus strain ONI33, E. faecalis strain ONI47 and S. pneumoniae strain ATCC49619 used in the time-kill and development of resistance studies were determined using the microbroth dilution met hod. Inocula were prepared from test organisms grown for 4 h in the appropriate broth media and diluted in saline to 0.5 McFarland to obtain 100 ml of a starting culture containing 106 colony forming units (cfu)/ml, which was verified by colony counts of replicate samples. Aliquots (10 ml) of the culture were transferre d to sterile plastic 25 cm2 culture flasks (Corning Inc, Corning, NY) and MU1140 was added from a sterile stoc k solution to give final concentrations equal to 0.5, 1, 2, 4, 8, and 16 times the MIC for S. pneumoniae strain ATCC49619 and S. aureus strain ONI33, and 0.25, 0.5, 1, 2, 4, 8, and 16 times the MIC for E. fecalis strain ONI47. Each assay included a growth control tube with no antibiotic. 92

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The cultures were incubated at 37C and sample s were obtained at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 hours following addition of MU 1140. The samples were serially diluted 10-fold in ice cold normal saline and 10 l samples spotted onto duplicate BAPs. Following incubation at 37C for 24 h, colonies that arose on plates with 30 colonies were counted. Development of Resistance S. aureus strain ONI33 and S. pneumoniae strain ATCC 49619 were grown overnight on BAPs. Cells were scraped from the surface and dilute d with saline to 0.5 McFarland. Cells were then diluted 1: 100 in appropriate broth media to give approximately 106 cfu/ml, and 100 l samples were a dded to microtiter wells (Corning Inc, Corning, NY) containing 100 l of doubling concentrations of MU1140 in broth to achieve a final bacteria l concentration of 55 cfu/ml. The microtiter plates were incubated overnight at 37C in an atmosphere of 5% CO2. Wells containing the highest concentration of MU1140 that showed turbidity (equivalent to 0.5 MIC) were diluted to 0.5 McFarland and used as the inocula to repeat the above process. This process was repeated daily 21 times and the MIC after each subculture was recorded. After the 7th, 14th, and 21st repetition, a sample of cells from th e 0.5 MIC well was used to inoculate 1 ml of MU1140-free broth, which was grown overnight to saturation. These cells were subcultured in the ab sence of MU1140 an additional 6 times, after which MICs for MU1140 were determined using the broth microdilution method. Results Susceptibility Studies The results of the tiers 1 and 2 susceptibil ity studies are summarized in Tables 1 and 2. The tier 1 study demonstrated that MU1140 was biological ly active against all 93

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Gram positive bacterial strains tested, with MICs ranging from 0.5 32 g/ml. It was most potent against Streptococcus pyogenes (MIC is 0.5 g/ml) and least potent against E. fecalis and E. faecium (MICs range 16 32 g/ml). In the tier 2 studies (Table 2), MU1140 showed greater activity (MIC <8 g/ml) against S. pyogenes, L. monocytogenes, and C. difficile than against S. aureus, E. fecalis, and Bacillus sp. (MIC >8 g/ml). Results of the tier 3 studies (T able 3) revealed that MU1140 was as active as vancomycin against vancomycin intermediate S. aureus, but showed superiority to vancomycin against all tested vancomycin resistant S. aureus, and vancomycin resistant E. faecalis and E. faecium. Vancomycin had lower MIC values when compared to MU1140 when tested against all vancomycin sensitive strains. Time-Kill Studies One isolate each of S. pneumoniae (Figure 5-2), MDR S. aureus (Figure 5-3) and VRE (Figure 5-4) were selected as test organisms for the time-kill analysis. Very similar kill profiles were observed for S. pneumoniae and S. aureus, characterized by a rapid and significant decline (> 3 log drop) in bacterial counts within the first 2 hours independent of the antibiotic concentrati on. Regrowth is observed at lower MU1140 concentrations (0.5X, 1X MIC for S. pneumoniae, and 0.5X, 1X, 2X MIC for S. aureus) but not in concentration > 8X MIC for all strains. For S. pneumoniae, time to 99.9% killing after exposure to MU1140 at 1 and 2 times MIC (1X and 2X MIC) was 5 hrs while at 4 and 8 times MIC (4X and 8X MIC) was 2.5 hrs. For MDR S. aureus, time to 99.9% killing after exposure to MU1140 at 0.5 times MIC (0.5X MIC) was 1.5 hrs and at 4, 8, and 16 times MIC (4X, 8X, 16X MIC) was 0.5 hrs. CLSI defines a bactericidal agent as one that a given concentration reduces the or iginal inoculum by 99.9% (>3 log10 cfu/ml) for each time period and bacteriostatic if th e inoculum was reduced by 0 log10 cfu/ml. 94

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According to that definition, time-kill studies reveal that MU1140 is bactericidal against S. pneumoniae at concentrations at or above 1X MIC and bacter icidal against S. aureus at concentrations at 0.5X MIC or above. The time-kill studies also reveal that MU1140 is bacteriostatic against vancomycin resistant E. fecalis (Figure 5-4) at all concentrations tested and maintained bacter ial counts at approximately the initial inoculum size. Resistance Development Study MIC values resulting from daily subculturing of S. aureus strain ONI33 and S. pneumoniae strain ATCC 49619 are summarized in Fi gure 5-6. Sequen tial subculturing of the these strains resulted in emergence of variant stai ns with elevated MU1140 MIC values. The MICs for the parent S. aureus was 3.2 g/ml and doubl ed after the second and the twelfth subculturing to stabilize at 12.8 g/ml. The MIC of MU1140 against the S. pneumoniae parent started at 0.4 g/ml and doubled after the second, third, and fourth subculturing event, stabilizing at 3.2ug/ml. Subculture of the resistant variants in the absence of MU1140 did not affect their respective MICs, indicating that the observed resistance was genetically stable and not an adaptive response. Discussion The rise in bacterial resistance to curren tly used antibiotics is an alarming reality that has attracted considerable worldwide atte ntion (84). The emergence of antimicrobial resistance is not a new phenome non. It is an inevitable resu lt of the large number of bacterial species, their rapid replication, a nd the frequent and misu se of antibiotics. A mutation or acquisition of a ge ne that helps a microbe survive in the presence of an antimicrobial agent will quickly become predominant throughout the microbial population and may spread from person to pers on. Clearly, there is a need for more sensible use of existing antibiotics, but more importantly there is a pressing need for the 95

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development of new antibiotics. The class of antibiotics known as lantibiotics has been known for decades, and throughout this period many investigators, reviewed by cotter et al. (15), have predicted their potential for us e as therapeutic agents. The goal of this study was to evaluate MU1140 as a potential antimicrobial agent. Determination of the MICs of MU1140 fo r select microorganisms was used as a measure of their susceptibility to MU1140. Th e testing was performed in three stages in which Tier 1 results indicated that all 30 Gram positive species tested were sensitive according to CLSI susceptibility breakpoint de finitions (58), while none of the 28 Gram negative species or the yeast species tested showed sensitivity. These findings are in accord with previously reported studies (36, 37 ). The lack of Gram negative bacteria and yeast sensitivity to MU1140 is likely to be a function of its mode of action. MU1140 exerts its antimicrobial effect by a novel mechanism (88), which involves abduction of lipid II from the plasma membrane near areas of active peptidoglycan synthesis. The presence of an MU1140-absorbing outer memb rane in Gram negative bacteria and the absence of lipid II in yeast provide explanat ions for the observed sp ectrum of activity of MU1140. The results of the Tier 2 study c onfirmed the effectiveness of MU1140 against multiple strains of selected pathogenic Gram positive species, including ones resistant to various, currently used antibiotics. The results of the Tier 3 study added further evidence for the effectiveness of MU1140 against drug resistant Gram positive pathogens. In particular, this study demonstrated the sus ceptibility of vancomycin and oxacillin resistant S. aureus, E. faecalis and E. faecium strains to MU1140. The MICs of susceptible organisms showed a wide ra nge of interspecies variability, with S. pyogenes and C. difficile being highly susceptible to MU1140 and E. faecalis and E. feacium being 96

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less susceptible. At present, there is no definitive explanation for these observed differences. Although MIC determination is still the gold standard for characterizing the potency of an antimicrobial agent, it does not provide information about the time course of the antibiotics action. This limitation is overcome by the use of time-kill studies (55), which were performed using strains of medically important Gram positive species, S. aureus, S. pneumoniae, and E. faecalis. The results of time-kill investigations showed that MU1140 exhibit rapid ini tial killing against MDRSA and S. pneumoniae, whereas a bacteriostatic activity was observed agai nst a vancomycin resistant strain of E. faecalis. Vancomycin also exhibits this species-dep endent difference in activity (2, 25). MU1140 and vancomycin both target lipid II, but at different moieties on this complex molecule. Thus, it is likely that the involvement of lipid II is important in the observed speciesspecific differences of MU1140 activity, alt hough the actual basis for this phenomenon remains unknown. The ability of susceptible microorganisms to devel op resistance to MU1140 was tested using an in vitro model. After 21 daily, sequent ial passages in subinhibitory concentrations of MU1140, MDRSA and S. pneumoniae mutants with modest 3-fold elevated MU1140 MICs were selected. This phenotype was stable, indicating the selection of genetic variants. Resistance deve lopment to lantibiotic has been extensively studied using nisin, reviewed by Chatterjee (13), and involved such diverse mechanisms as decreased nisin binding due to changes in the net negative charge of the cell envelope, increased cell wall thickness that altered cell surface hydrophobicity, and the possible existence of inactivating enzymes. In the present study, there was no evidence to support 97

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any of these or other mechanisms for the obser ved modest increase in MICs. This will be the subject of future investigations. The present studies indicate that MU1140 has a spectrum of activity that includes a number of medically important bacteria. The observed time-kill profiles for certain of these species is consistent with vancomycin, one of the current drugs of last resort, which is currently losing its effectiveness due to the rise of drug resistant pathogens. Low level increase in MICs of select pathogenic specie s during repeated cultivation in the presence of sublethal concentrations of MU1140 indicates that development of significant resistance to this molecule will not be easily accomplished. In support of this last contention is the observation th at the producer strain of Streptococcus mutans, JH1140, has an MIC comparable to other streptococci indicating that it has not been able to develop effective immunity agai nst its own bacteriocin. Add itional work is in progress to determine the usefulness of MU1140 as a clinically useful therapeutic agent for the treatment of infectious diseases. 98

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Al a Tr p Dh Le Al a Ab Pro Gl Al a Al a Ar Dh Gl Al a Ph As Al a Tyr Al a NH CH CH S S S S Ly Ph Figure 5-1. MU1140 1.E-01 1.E+01 1.E+03 1.E+05 1.E+07 1.E+09 012345678Time (h)CFU/mL Figure 5-2. Bactericidal activity of MU1140 against S. pneumonia strain ATCC 49619. Symbols: Control, 0.5MIC, 1MIC, 2MIC, 4MIC, 8MIC. 99

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1.00E-01 1.00E+01 1.00E+03 1.00E+05 1.00E+07 1.00E+09 012345678Time (h)CFU/mL Figure 5-3. Bactericidal activity of MU1140 against multidrug resistant S. aureus. Symbols: Control, 0.5MIC, 1MIC, 2MIC, 4MIC, 8MIC, 16MIC 100

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0.1 10 1000 100000 1E+07 1E+09 1E+11 01234567Time [hr]CFU/mL Figure 5-4. Bacteriostatic activity of MU1140 against vancomycin resistant E. faecalis. Symbols: Control, x 0.25MIC, 0.5MIC, 1MIC, 2MIC, 4MIC, 8MIC, 16MIC 101

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0 2 4 6 8 10 12 14 16 18 20 22 24 0123456789101112131415161718192021SubculturesMIC [ug/ml] Figure 5-5. MU1140 MIC values after 21 s ubculturing events for multidrug resistant S. aureus ( ) and S. pneumoniae ( ). Decrease in suscep tibility after repeated subculturing in subinhibitory MU114 0 concentrations is evident by the increase in organisms MU1140 MIC values. 102

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Table 5-1. Tier 1 susceptibility Study. MU1140 MIC for various Gram positive and negative microorganisms, and yeast. Microorganism (number of isolates) MU1140 MIC (g/ml) Gram Positive Organisms Enterococcus faecalis (3) 16-32 ATCC 29212 Enterococcus faecalis (1) 32 Enterococcus faecium (4) 8-32 Staphylococcus aureus (4) 16 ATCC 29213 Staphylococcus aureus (1) 16 Staphylococcus epidermidis (4) 16 Staphylococcus saprophyticus (2) 4-16 Streptococcus agalactiae (2) 4 Streptococcus intermedius (1) 2 Streptococcus mitis (1) 4 Streptococcus pneumoniae (3) 1 ATCC 49619 Streptococcus pneumoniae (1) 4 Streptococcus pyogenes (2) 0.5 Clostridium difficile (2) 1 Gram Negative Organisms Acinetobacter baumannii (2) >32 Acinetobacter calcoaceticus (2) 32 Citrobacter freundii (2) >32 Citrobacter koseri (diversus) (2) >32 Enterobacter cloacae (2) >32 ATCC 25922 Escherichia coli (1) >32 Haemophilus influenzae (1) >32 ATCC 49247 Haemophilus influenzae >32 Klebsiella oxytoca (2) >32 Klebsiella pneumoniae (2) >32 Morganella morganii (2) >32 Proteus mirabilis (2) >32 Proteus vulgaris (2) >32 Providencia stuartii (2) >32 Pseudomonas aeruginosa (1) >32 ATCC 27853 Pseudomonas aeruginosa >32 Serratia marcescens (2) >32 Stenotrophomonas (Xanthomonas) maltophilia (2) >32 ATCC 25285 Bacteroides fragilis >32 Yeast Candida albicans (1) >32 ATCC 90028 Candida albicans (1) >32 103

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Table 5-2. Tier 2 susceptibility Study. MU1140 MIC for various Gram positive and anaerobic microorganism Microoganism (number of isol ates) Antibiotic MIC (g/ml) MU1140 Vancomycin Enterococcus faecalis Vancomycin S (9) 16-32 1-2 Vancomycin R (9) 16 >64 Streptococcus pyogenes Erythromycin (S) 0.5-2 0.5 Erythromycin (R) 0.5-1 0.5 Staphylococcus aureus Vancomycin S MRSA (9) 8-32 1 Inpatient Vancomycin S MRSA (10) 16-32 1 Community Acquired Vancomycin S MRSA (4) 16 1 Streptococcus pneumoniae Penicillin S (9) 0.5-8 0.25-0.5 Penicillin R(9) 0.25-8 0.25-0.5 Listeria monocytogenes (9) 4 1 (9) 16-32 0.5-2 Clostridium difficile (9) 0.5-2 NA Bacillus species (9) 16-32 0.5-2 104

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105 TABLE 5-3. Tier 3 susceptibility study. MICs of MU1140 in comparison to vancomycin against selected clinical isolates Microorganism (number of isolates) Antibiotic MIC (g/ml) MU1140 Vancomycin Staphylococcus aureus OXA-S (22) 2-8 0.5-1 OXA-R(33) 2-8 0.5>128 VAN S (51) 2-8 0.5-2 VAN I (1) 4-4 4-4 VAN R (3) 4-8 >128 Enterococcus faecalis VAN S (17) 4-8 0.5-4 VAN R (14) 4-8 32>128 Enterococcus faecium VAN S (12) 2-8 0.5-1 VAN R (13) 1-8 64>128

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CHAPTER 6 IN VITRO SERUM PROTEINS BINDING AND ITS EFFECT ON THE PHARMACODYNAMICS OF THE LANTIBIOTIC MU1140 The lantibiotic MU1140s degree of binding to serum proteins and the effect of serum components on the bactericidal activity of this novel antibiotic were investigated. The percentage of MU1140 bound to human serum proteins was determined by ultrafiltration to be 92.7% 2% when te sted in the range of 6.25-200 g/ml. The presence of inactivated serum in creased the average MU1140 MICs for Streptococcus pneumoniae (S. pneumoniae), but decreased the average MICs for Staphylococcus aureus (S. aureus). Time-kill studies of MU1140 against S. pneumoniae and S. aureus in serumcontaining medium showed that in serum containing media higher drug concentrations were needed to achieve the bactericidal effect against S. pneumoniae than were needed in broth, while lower amounts of the antibiotic were needed to achieve the bactericidal effect against S. aureus than were needed in broth. Pooled serum exerts a protective effect for S. pneumoniae against MU1140, but enhanced MU1140s antibacterial activity against S. aureus suggesting a possible synergistic eff ect between this lantibiotic and serum components. Introduction Bacterial resistance to currently available an tibiotics is a public health crisis that deserves worldwide attention (1). The clinical selection of bacterial isolates resistant to currently used antibiotics is an evolutiona ry force that derives the emergence of the superbugs (64). Currently, 50% of hospita l acquired infections are due to multidrug resistant bacteria which annually result in ov er 100,000 deaths and 100 billion dollars in associated healthcare cost (12, 41). Of sp ecial importance are the methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecalis (VRE), 106

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which are very difficult to treat and contribu te heavily to hospital associated deaths. Thus, the need for new antibio tics is great, especially thos e with novel mechanisms of action as this will ensure the lack of cross resistance to the newly developed antibiotics (16). MU1140 is a 22 amino acid bacteriocin w ith a novel mechanism of action. MU1140 is produced by Streptococcus mutans strain JH1140 (37) a nd it belongs to the lantibiotics which is a group of ribosomally synthesized and posttranslationally modified bacteriocins. Lantibiotics contain unusual am ino acids such as la nthionine, as well as methyllanthionine and didehydroalanine. Lant hionine amino acids are composed of two alanine residues cross-linked via a thioether linkage that connects their -carbons (S(alaninyl-3-yl)-cysteine) (Figure 6-1). Lantibiotics are produced by a large number of Gram-positive bacteria and have their lanthi onines imbedded within cyclic peptides (13). MU1140 has been characterized in regard to its physical and chemical properties (37, 78) and its role in promoting the pr oducer strain ability to colonize the oral cavity. Its novel mechanism of action involves inhibition of the peptidoglycan cell wall synthesis by binding to and abducting lipid II fr om its site of action at site s of peptidoglycan synthesis (35). Inappropriate dosing has been implicated as one of the major f actors contributing to the continuous emergence and sp read of antibiotic resistant bacterial strains (73). In order to optimize dosing of a new antibiotic, all factors that determine the pharmacologically active fraction has to be ta ken into account, one being the degree of antibiotic binding to serum proteins. Once drug molecules enter the systemic circulation, they exist in a state of dynamic equ ilibrium between two forms, bound to blood 107

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components and unbound or free. A common att achment site for dr ugs in the blood is serum proteins, usually albumin, formi ng a reversible complex by hydrogen bonding (45). Binding to serum proteins is an impor tant property of a dr ug since the degree of binding determines the pharmacologically act ive free fraction (17, 80). Only the unbound drug is able to exert the pharmacological effect because this is the fraction of drug molecules free to diffuse and reach the biophase (80). Overall, plasma protein binding influences the disposition profile as only free drug is avai lable for elimination and distribution into periphera l tissues. The effect of pr otein binding on antibiotic action has been well documented and reviewed (18, 19) Since most infections take place in various body tissues, for an antibiotic to be efficacious it has to pe netrate the tissue and there it will exert its antimicrobial effect. In that sense, an antibiotics degree of protein binding is a factor of great im portance since it determines the fraction of the dose that is free to illicit its antimicrobial effect and consequently determines the outcome of therapy (85). Generally, reduction in activity is found to correlate direc tly with decreasing free fraction, i.e. increased per centage binding (52) and the in vivo activity inversely correlated with the extent of binding, thus the highly bound molecules have a much higher ED50 values and a longer in vivo half life than the com pounds with lower binding coefficients. The flip side of the coin is also true, only the free drug molecules are available for clearance, either by kidney filtration, uptake and metabolism by liver cell, or any of the other clearance mechanism. Since binding to blood components can affect the drugs distribution, tissue pene tration, metabolism, and e limination from the body (17, 23, 68) it can have a profound effect on the drugs pharmacodynamic and pharmacokinetic behavior (5). 108

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The aim of this study was to investigate the degree of MU1140s binding to serum proteins and the effect of protein binding on MU1140s pharmacodynamic activity against multidrug resistant S. aureus clinical isolate and S. pneumoniae (ATCC 49619) as measured by MIC determination and time-kill studies. Although MIC can serve as an indicator of a drugs in vitro potency, it does not provide a ny data on the drugs killing kinetic profile. Time-kill curves on the othe r hand provide a dynamic view of the drugs antimicrobial activity that is of great clinical relevance (55). The bactericidal activity of MU1140 has been investigated (4), but the degree of protein bindi ng and bactericidal activity in serum has not been reported for any of the lantibiotics. Materials and Methods Determination of MU1140s Degree of Binding to Human Serum Proteins Human serum was spiked with MU1140 to yiel d final concentrati ons of 6.25, 12.5, 25, 50, 100, and 200g/ml. Following incubation at 37oC for 1 h, an aliquot was transferred to an ultrafiltration device with a molecular weight cut-off of 10 Dalton (Amicon Co., Danvers, MA, USA), which was centrifuged at 15000 g for 1 h at 37oC. Samples ultrafiltrate were analyzed by LC -MS. The concentrations of MU1140 in the filtrate were determined using standard samples prepared by spiking known amounts of MU1140 in plasma followed by ultrafiltration in the same fashion. The unbound fraction was estimated from the ratio of drug concentra tion in the filtrate to that in the original plasma samples. Parallel studies using prot ein-free plasma instead of plasma indicated that MU1140 was bound minimally to the ultrafiltration device. Broth Preparation Cation adjusted Mueller-Hinton broth (MHB; Becton Dickinson, Franklin Lakes, NJ, USA) was used to grow S. aureus, T odd Hewitt broth (THB; Difco, Detroit, USA) 109

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was used for S. pneumonia. Both were prep ared at 4 times (4X) the manufacturers suggested concentration and autoclaved prior to use at 121oC (15 min per 1L). Sprague Dawley rat serum was purchased from Innova tive Research (Southfield, MI) and human serum was purchased from Rockland Immunoc hemicals (Gilbertsville, PA). Serum containing media was prepared as follows; 0% serum medium contained one part of 4 broth and 3 parts of autoclaved 0.9% sodi um chloride, 25% serum medium contain one part of 4 broth, one part of heat inactiv ated (55C 30 min) serum, and 2 parts of autoclaved 0.9% sodium chloride, 50% serum medium contain one part 4 broth, 2 parts of heat inactivated serum, and one part au toclaved 0.9% sodium chloride, and the 75% serum medium contain one part 4 broth and three parts of h eat inactivated serum. Bacterial Cultivation The bacterial inocula were prepared from colonies grown overnight on blood agar plates (BAP; 5% sheep blood agar plates Remel Microbiology Products, Lenexa, KS, USA). Cells were scraped from the plate using an inoculation loop and suspended in sterile saline solution to 0.5 McFarland units, which is equiva lent to a concentration of 1x108 cfu/mL. Time-Kill Studies Two types of time-kill studies were conducted: 1. Studies in which the amount of serum in the growth medium was fixed at 50% and varied the concentration of MU1140 (0.1-16 times MIC). 2. In the other study fixed the amount of MU1140 was 0.5 MIC, and varied the amount of serum in the growth media (0, 25, 50, and 75%). For all time-kill studies an in vitro kinetic model was used. This system consisted of eight 50 ml vented cap tissue cu lture flasks with canted necks (nuncTM, Nunc A/S, Roskilde, Denmark), each containing 20 ml of the appropriate broth media. 110

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For the first study, growth media was pr epared as follows: 0% serum flasks contained one part of 4 broth and 3 parts of autoclaved 0.9% sodium chloride; 50% serum flasks contain one part 4 broth, 2 pa rts of heat inactivated serum, and one part autoclaved 0.9% sodium chloride. Untreate d (not inactivates) human serum was used for this study. For the second study the growth media was prepared as follows: 0% serum flask contained one part of 4X br oth and 3 parts of autoclaved 0.9% sodium chloride; 25% serum flask contained one part of 4X broth, one part of heat inactivated (55C for 30 min) serum, and 2 parts of autoclaved 0.9 sodium chloride; 50% serum flask contained one part 4X broth, 2 parts of heat inactiv ated serum, and one part autoclaved 0.9% sodium chloride; 75% serum flask contained on e part 4X broth and three parts of heat inactivated serum. A 100 l aliquot of the 0.5 McFarland inocul um was added to each flask to produce a final inoculum of approximately 5x105 CFU/mL. The bacteria were incubated standing for 2 hours at 37oC in an atmosphere of 5% CO2 to allow them to reach the exponential growth phase before adding different MU1140 concentrations. The selection of MU1140 concentrations tested for each bacterial stra in was based on their previously determined MIC values. At least six different concentra tions were investigated besides the MIC, which included subinhibitory (0.06, 0.12, 0.25 and 0.5 times MIC) and suprainhibitory concentrations (2, 4, 8 and 16 times MIC). A control with bacteria and no drug was run simultaneously. Fifty microliter samples were rem oved, diluted in saline, and plated at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, and 24 hours post-antibiotic add ition for quantification. 111

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Bacterial Quantification Bacterial counts were determined by plati ng 50 L of serial 10-fold dilutions in 0.9% saline on BAPs, where plat es were divided into four qu adrants. Using a pipette, five 10 L droplets of the chosen dilution were spotted equidistantly onto one of the quadrants. Replicate spots were plated onto the adjacent quadrant. The plates were then incubated at 37oC in 5% CO2 for 18-24 hours before reading. Positive controls with bacteria but no drug were run simultaneously Following incubation, colonies that arose were counted. Data were used from quadr ants that contained 15-150 colonies. The experiments were performed independently in triplicate. The means and standard deviations were calculated. Results Determination of MU1140 Unbou nd Fraction in Human Serum MU1140 was spiked in human serum to achieve concentrations of 6.25-200 g/ml. The samples were subjected to ultrafiltration using Ultrafree-MC Centrifugal Devices (50 KDa cut off). The average MU1 140 percentage binding is 92.7%. Effect of Protein Binding on MU1140s In vitro Activity: MIC Studies The effect of inactivated human serum upon the in vitro activity (MICs) of MU1140 against S. pneumoniae and S. aureus was studied. A microdilution method was employed using either Muller Hinton Broth al one or Muller Hinton broth supplemented with 25 or 50% (v/v) inactivated human serum for S. aureus. For S. pneumoniae, Todd Hewitt broth (THB) or THB supplemented w ith 25 or 50% (v/v) inactivated human serum was used. A bacteria l final inoculum of 5 105 cfu/mL was employed. Following incubation for 18 h at 35C in an atmos phere enriched with 4% carbon dioxide for S. pneumoniae and S. aureus. The MIC was defined as the lowest concentration at 112

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which there was no visible growth. The pres ence of 25 and 50% (v/v) human serum lead to a two fold and four fold increa se in MU1140 MIC respectively against S. pneumoniae. On the other hand, the presence of 25 and 50% inactivated human serum increased MU1140 activity against S. aureus causing a re duction in the MIC value from 4.8g/ml in 0% serum to 1.6g/ml in the 25 and 50% se rum wells. Data is summarized in tables 6-1 and 6-2. Effect of Protein Binding On MU1140s In vitro Activity: Time-Kill Studies In the presence of 50% human serum, MU 1140 antibacterial activity was decreased against S. pneumonia. Figure 2C illustrates the significan t increase in bacterial viable cell counts at all MU1140 concentration when co mpared to those in the absence of serum (Figure 6-2A). By plotting the viable cell counts in flasks contai ning equivalent MU1140 concentration in the presence and absence of serum on the same plot (Figure 6-3 panels A-H), the inhibition of antibacterial activity is apparent. It seems that the presence of serum inhibits MU1140 by four fold as shown in panel F, where the bacterial cell counts for the 1 MIC in broth and the 4 time s MIC in serum (Figure 3 panel F) are superimposable. Human serum seems to exert the opposite effect on MU1140 antibacterial activity against S. aureus. Unlike the picture observed with S. pneumoniae, S. aureus viable cell count decreased dramatically in the presence of serum when compared to the viable cell count in the absence of serum. Figures 6-2B and 6-2D illustrates the decrease in bacterial cell counts at all MU1140 concentration when comp ared to those in th e flasks with broth only. Again, by plotting the bacterial viable cell counts from flasks containing equivalent MU1140 concentration in the presence and absence of 50% serum on the same plot (Figure 6-4 panels A-H) the effect exerted by serum on MU1140 activity is more evident. 113

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To quantify human serums degree of augmen tation of MU1140s activity, the viable cell count plots were superimposed in the presence and absence of serum. Again, the profile of 1 times MIC in serum and 4 times MIC in broth seem to be superimposable indicating that the presence of serum augments MU1140 antibacterial effect against S. aureus by four fold which suggests a possible synerg istic effect between serum components and MU1140 against S. aureus. In a further attempt to understand the co rrelation between the human or rat serum concentration in the growth medium and MU1140 antibacterial effect on S. aureus a second set of time-kill studies were performed where the bacterial inoculum (5x105 CFU/ml) and MU1140 concentration (0.5 time MIC) were fixed, but varied the human or rat serum concentrations (0, 25, 50, and 75% ). As shown in Figure 6-6, the most significant kill is observed when human (Figure 6-6 panel A) or rat (Figure 6-6 panel B) serum is in small amounts (25%). There was no difference in the viable cell counts in the 50% and 75% serum flasks. The growth control of S. aureus in the various serum containing media are comparable, suggesting th at the observed kill behavior was due to MU1140 antibacterial activity. Similar beha vior is observed when testing the MU1140 kill behavior against S. aureus using Sprague Dawley rat serum. Discussion Only the free (non protein bound) fraction of an antibiotic exerts antib acterial activity and is able to diffuse rapidly from plasma to the extravascular compartments where most bacterial infections are located (11, 43, 62, 72). For highly bound drugs (percentage bound exceeds 70-80%), binding to serum proteins is a crucial factor since small changes in binding will produce large variations in th e percentage of free and pharmacologically active drug (18). 114

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The results in this study show that MU 1140 is 92.7% 2% protein bound and that the MIC values increase proportional to the concentration of serum in growth media when tested against S. pneumoniae. A different picture is a pparent when testing the bactericidal activity against S. aureus where a synergy/augmentation of bactericidal activity is noticed when serum proteins are present in the culture broth, a phenomenon that has not been reported before for any lant ibiotic. Overall, it was concluded that the pharmacodynamic activity of MU1140 in the presen ce of serum suggests clearly that it is the free fraction of MU1140 that correl ates with its an tibacterial effect. Conclusions The serum protein binding and its effect on the bactericidal activity of MU1140 against S. pneumoniae and a multi drug resistant S. aureus was investigated. Protein binding was determined in human serum by ultr afiltration to be 92.7% 2% when tested in the range of 6.25-200 g/ml. The presence of inactivated human serum (25 and 50%) increased the average MU1140 MICs for Streptococcus pneumoniae in a concentration dependent manner, but it decr eased the average MICs for S. aureus by a nonproportional factor of two. Time-kill studies of MU1140 against S. pneumoniae and S. aureus in serum-containing media (25, 50, and 75%) show ed that much higher drug concentrations were needed to achieve the bactericidal effect against S. pneumoniae than were needed in broth, while lower amounts of the antibiotic we re needed to achieve a bactericidal effect against S. aureus than were needed in broth. Th e high level of protein binding of MU1140 appears to influence its antibacterial activity against S. pneumoniae, but a synergistic effect between MU 1140 and low concentrations of serum components is observed against S. aureus. 115

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Figure 6-1. Lanthi onine (Lan) and Methyllanthi onine (MeLan) structure. 116

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1.00E+00 1.00E+02 1.00E+04 1.00E+06 1.00E+08 1.00E+10 1.00E+12 024681012141618202224Time (h)S. pneumoniae Lo g CFU/ml Control 0.25X MIC 0.5X MIC 1X MIC 2X MIC 4X MIC 8X MIC 16X MIC A 1.00E+00 1.00E+02 1.00E+04 1.00E+06 1.00E+08 1.00E+10 1.00E+12 024681012141618202224 Time (h)S. pneumoniae Log CFU/ml Control 0.25X MIC 0.5X MIC 1X MIC 2X MIC 4X MIC 8X MIC 16X MIC B Figure 6-2. Time kill studies of MU1140 against S. pneumoniae in the absence and presence of 50% human serum, panels A and B, respectively. 117

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1.00E-01 1.00E+01 1.00E+03 1.00E+05 1.00E+07 1.00E+09 024681012141618202224 Time (h)S. aureus Log CFU/mL control 0.06X MIC 0.125X MIC 0.25X MIC 0.5X MIC 1X MIC 2X MIC 4X MIC A 1.00E-01 1.00E+01 1.00E+03 1.00E+05 1.00E+07 1.00E+09 024681012141618202224 Time (h)S. aureus Log CFU/mL control 0.06X MIC 0.125X MIC 0.25X MIC 0.5X MIC 1X MIC 2X MIC 4X MIC B Figure 6-2. Time kill studies of MU1140 against S. aureus in the absence and presence of 50% human serum, panels A and B, respectively. 118

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MU1140 x S. pneumoniae ATCC 49619 Control1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 0 51015202530 Time [hr]Log CFU/ml Control 0% Serum Control 50% Serum A MU1140 x S. pneumoniae ATCC 49619 0.25X MIC1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 051015202530 Time [hr]Log CFU/ml 0.25X MIC 0% Serum 0.25X MIC 50% Serum B 119

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MU1140 x S. pneumoniae ATCC 49619 0.5X MIC1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 051015202530 Time [hr]Log CFU/ml 0.5X MIC 0% Serum 0.5X MIC 50% Serum C MU1140 x S. pneumoniae ATCC 49619 1X MIC1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 051015202530 Time [hr]Log CFU/ml 1X MIC 0% Serum 1X MIC 50% Serum D 120

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MU1140 x S. pneumoniae ATCC 49619 2X MIC1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 051015202530 Time [hr]Log CFU/ml 2X MIC _0% Serum 2X MIC 50% Serum E MU1140 x S. pneumoniae ATCC 49619 4X MIC1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 051015202530 Time [hr]Log CFU/ml 4X MIC_ 0% Serum 4X MIC_ 50% Serum F 121

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MU1140 x S. pneumoniae ATCC 49619 8X MIC1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 051015202530 Time [hr]Log CFU/ml 8X MIC_0% Serum 8X MIC_50% Serum G MU1140 x S. pneumoniae ATCC 49619 16X MIC1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 0 51015202530 Time [hr]Log CFU/ml 16X MIC_0% Serum 16X MIC_50% Serum H Figure 6-3. Sideby-side plot of S. pneumoniae viable cell counts in the presence of MU1140 and the presence and abse nce of human serum. MU1140 concentrations varied from 0.25-16 times MIC. 122

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MU1140 XS. aureus Controls1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 1.00E+10 051015202530 Time [hr]Log CFU/ml NoSerum 50% Serum A MU1140 X S. aureus 0.06X MIC1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 051015202530 Time [hr]Log CFU/ml 0% Serum 50% Serum B 123

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MU1140 X S. aureus 0.12X MIC1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 051015202530 Time [hr]Log CFU/ml No Serum 50% Serum C MU1140 X S. aureus 0.25X MIC1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 051015202530 Time [hr]Log CFU/ml No Serum 50% Serum D 124

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MU1140 X S. aureus 0.5X MIC1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 051015202530 Time [hr]Log CFU/ml No Serum 50% Serum E MU1140 X S. aureus 1X MIC1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 051015202530 Time [hr]Log CFU/ml No Serum 50% Serum F 125

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MU1140 X S. aureus 2X MIC1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 051015202530 Time [hr]Log CFU/ml No Serum 50% Serum G MU1140 X S. aureus 4X MIC1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 051015202530 Time [hr]Log CFU/ml No Serum 50% Serum H Figure 6-4. Sideby-side plot of S. aureus viable cell counts in the presence of MU1140 and the presence and absence of human serum. MU1140 concentrations varied from 0.064 times MIC. 126

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MU1140 vs S. aureus 0, 25, 50, 75% Human Serum 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 051015202530Time [hr]S. aureus CFU/ml GrowthControl_0%Serum GrowthControl_25%Serum GrowthControl_50%Serum GrowthControl_75%Serum MU1140_0%Serum MU1140_25%Serum MU1140_50%Serum MU1140 75%Serum A MU1140 vs. S.aureus 0, 25, 50, 75% Rat Serum1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 051015202530Time [hr]S. aureus CFU/ml GrowthControl_0% Serum GrowthControl_25% Serum GrowthControl_50%Serum GrowthControl_75%Serum MU1140_0%Serum MU1140_25%Serum MU1140_50%Serum MU1140 75%Serum A B B Figure 6-5. Time kill studies of MU1140 at 0.5 time MIC against S. aureus in the presence of various human or rat serum concentrations. 127

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128 Tables Table 6-1. MU 1140 MICs against Streptococcus pneumoniae (ATCC 49619) in the presence of 0, 25, and 50% inactivated human serum. MIC (g/ml) 0% Serum MIC (g/ml) 25% Serum MIC (g/ml) 50% Serum Mean (n=3) 0.8 1.6 3.2 SD 2.43 e-16 2.43 e-16 2.43 e-16 Table 6-2. MU 1140 MICs Multi Drug resistant Staphylococcus aureus in the presence of 0, 25, and 50% inactivated human serum MIC (g/ml) 0% Serum MIC (g/ml) 25% Serum MIC (g/ml) 50% Serum Mean (n=6) 4.8 1.6 1.6 SD 1.75 2.43 e-16 2.43 e-16

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CHAPTER 7 CONCLUSIONS As antibiotic resistant bacterial strains emer ge, the need for new antibiotics with novel mechanisms of action is great. Appropriate dose design and proper use are the tools available to ensure success of therapy and efficacy mainte nance of these newly developed antimicrobial agents. The work presented here is part of the preclinical development plan of the lantibiotic MU1140, an antibiotic indicated fo r the management and control of infectious diseases caused by Gram positive pathogens. Studies presented here are focused on the evaluation of the druglike properties of MU1140 and its applicability to se rve as a pharmaceutical agent for human use. Production of MU1140 by fermentation and its purification process has been improved. The fermentation media composed of 5% yeast ex tract, 0.5% calcium chloride, and 4% glucose is the media mixture that trig gered the highest pro duction of MU1140. The purification method consisted of a precipitation step using ammoni um sulfate followed by selective uptake of the activity in isopropanol a nd two separation steps using revers ed phase chromatography where the activity is eluted with acetonitrile and methanol after the first and s econd separation steps, respectively. The yield of MU1140 according to th is protocol is less than 1mg/liter which does not enable large scale commercialization of this antibiotic. To study the in vivo behavior of MU1140, a bioanalytical method for its quantification in rat plasma was developed and validated. The developed method involves a simple and inexpensive liquidliquid sample preparation followed by protein precipitation, filtration, LCMS separation and detection procedure. This method was validated to be selective, accurate, precise, and sensitive, and th e stability of MU1140 was not compromised during sample handling and processing. 129

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The pharmacokinetics of MU1140 was investigat ed at a dose equivalent to 25 mg/kg rat body weight. The plasma concentration-time prof ile of MU1140 in rats d eclined biexponentially and was best fitted using an open two-compartment model with elimination from the central compartment and uniform weighing. MU1140s mean elimination half life was 1.7 0.1 hrs. During the PK study, it was observed that rapid injection of MU1140 was not well tolerated. A hypersensitivity reaction, characterized by redne ss of the ears and paws and swelling, was observed within 5 minutes post-administration of the first dose. Subcutan eous administration of diphenhydramine 1 hour prior to MU1140 administrati on is sufficient to block most of these symptoms. In a clinical setting, pre-medica tion with DPA or administration of MU1140 as a slow infusion might be needed to block or avoid this reaction. MU1140 in vitro pharmacodynamic investigation sugge sts a broad spectr um of activity against medically important Gram positive pathogens, including MRSA, VISA, VRSA, and VRE, which are responsible for most of infecti ous disease related deaths in US hospitals and worldwide. Time-kill studies reveal that MU1140 is bactericidal against S. aureus and S. pneumoniae in a concentration-independent ma nner, but bacteriostatic against E. faecalis. A PD model capable of predicting S. aureus concentrations resulting fr om different MU1140 static concentrations was developed. A PK/PD m odel for MU1140s activities was assembled which enables quantitative correlations of the antibiotic concentration and the bacterial concentration at any time point and thus a rational dosing regimen can be conceptualized. MU1140 was found to highly bind (92.7% 2%) to serum proteins. In vitro, human or rat serum displayed a synergisti c effect with MU1140 against S. aureus, where the addition of human/rat serum strongly augmente d MU1140 bactericidal activity. In vitro efficacy warranted further investigation of the therapeutic pot ential of MU1140. Coll ectively these findings 130

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illustrate the potential of MU1140 to serve as a therapeutic agent for the management of infections caused by multidrug resistant Gram positive bacteria. 131

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BIOGRAPHICAL SKETCH Oliver Ghobrial developed a pa ssion for life science at an early age. He earned his bachelors degree in biological sciences and his masters degree in molecular and microbiology from the University of Central Florida. Oliver moved to Gainesville, Florida to join Encor Biotec hnology. He worked for Dr. Jerry Shaw where the focus of his project was recombinant DNA technology and antibodies production. Recently after, he joined the PhD program in the Department of Pharmaceutics at the University of Florida, working in the lab of Dr. Hartmut Derendorf. Oliver received his PhD in Pharmaceutics in summer 2008. Oliver plans to work hard to improve human life on this planet. 139