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Pharmacokinetic/Pharmacodynamic Characterization of TR-700 (Torezolid)

Permanent Link: http://ufdc.ufl.edu/UFE0042485/00001

Material Information

Title: Pharmacokinetic/Pharmacodynamic Characterization of TR-700 (Torezolid)
Physical Description: 1 online resource (106 p.)
Language: english
Creator: Sahre, Martina
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: antibiotics, distribution, microdialysis, pharmacodynamics, pharmacokinetics, tissue, torezolid
Pharmaceutics -- 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: Infections with methicillin-resistant Staphylococcus aureus (MRSA) have become endemic in healthcare settings around the world and recently patients without risk-factors have become infected in the community-setting by MRSA that shows different characteristics compared to hospital strains. Most MRSA infections that are community associated present as skin and soft-tissue infections (SSTIs) and in lesser numbers as pneumonia or bacteremia. Antibiotics like vancomycin and linezolid are effective in treating infections, but prolonged use can cause resistances. Development of novel antibiotics is necessary in order to have an effective armamentarium against ever adapting bacteria. Pharmacokinetic/pharmacodynamic parameters like area under the curve over minimum inhibitory concentration (AUC/MIC), maximum concentration over MIC (Cmax/MIC) and time the concentration remains above the MIC (T > MIC) are used to correlate effect and drug levels, yet often the pharmacokinetic parameters are calculated from plasma concentrations. Most drugs, however, do not have their site of action in the blood, but in the tissue, either intracellularly or in the interstitial space fluid. TR-700 is a novel oxazolidinone found to be effective against MRSA, particularly strains that are also linezolid resistant. To characterize the time-course of drug concentrations not only in plasma, but also in skeletal muscle and subcutaneous adipose tissue, the site of action for skin and soft tissue infections, a microdialysis study has been conducted. In addition to the determination of pharmacokinetic parameters, the time-course of the effect of TR-700 has been elucidated in vitro, using time-kill experiments. The estimated pharmacodynamic parameters were then correlated to the kinetic parameters to model and simulate outcomes of various dosing regimens.
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 Martina Sahre.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Derendorf, Hartmut C.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042485:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042485/00001

Material Information

Title: Pharmacokinetic/Pharmacodynamic Characterization of TR-700 (Torezolid)
Physical Description: 1 online resource (106 p.)
Language: english
Creator: Sahre, Martina
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: antibiotics, distribution, microdialysis, pharmacodynamics, pharmacokinetics, tissue, torezolid
Pharmaceutics -- 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: Infections with methicillin-resistant Staphylococcus aureus (MRSA) have become endemic in healthcare settings around the world and recently patients without risk-factors have become infected in the community-setting by MRSA that shows different characteristics compared to hospital strains. Most MRSA infections that are community associated present as skin and soft-tissue infections (SSTIs) and in lesser numbers as pneumonia or bacteremia. Antibiotics like vancomycin and linezolid are effective in treating infections, but prolonged use can cause resistances. Development of novel antibiotics is necessary in order to have an effective armamentarium against ever adapting bacteria. Pharmacokinetic/pharmacodynamic parameters like area under the curve over minimum inhibitory concentration (AUC/MIC), maximum concentration over MIC (Cmax/MIC) and time the concentration remains above the MIC (T > MIC) are used to correlate effect and drug levels, yet often the pharmacokinetic parameters are calculated from plasma concentrations. Most drugs, however, do not have their site of action in the blood, but in the tissue, either intracellularly or in the interstitial space fluid. TR-700 is a novel oxazolidinone found to be effective against MRSA, particularly strains that are also linezolid resistant. To characterize the time-course of drug concentrations not only in plasma, but also in skeletal muscle and subcutaneous adipose tissue, the site of action for skin and soft tissue infections, a microdialysis study has been conducted. In addition to the determination of pharmacokinetic parameters, the time-course of the effect of TR-700 has been elucidated in vitro, using time-kill experiments. The estimated pharmacodynamic parameters were then correlated to the kinetic parameters to model and simulate outcomes of various dosing regimens.
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 Martina Sahre.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Derendorf, Hartmut C.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042485:00001


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1 PHARMACOKINETIC / PHARMACODYNAMIC CHARACTERIZATION OF TR 700 (TOREZOLID) By MARTINA D AGMAR SAHRE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR T HE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Martina D. Sahre

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3 To my mother

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4 ACKNOWLEDGMENTS I would like to thank Dr. Hartmut Derendorf for giving me t he chance to pursue this degree and my committee members, Drs. Gn ther Hochhaus, Kenneth Rand and Cary Mobley for their kind support. I would also like to acknowledge the personnel in the General Clinical Research Center for their hard work, support and care during the clinical trial and Drs. Sabarinath Sreedharan, April Barbour and Stephan Schmidt for their help with kill curves and the clinical trial. Finally, I would like to thank my mother, whose love and encouragement has given me the strength to pursue this degree.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 ANTIBIOTIC USE AND SPREAD OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS ................................ ................................ ............. 14 2 TR 700 AND ITS ROLE AMONG THE OXAZOLIDINONES ................................ ... 16 Chemistry and Physicochemical Properties ................................ ............................ 16 Mechanism of Action of Oxazolidinones and Resistance Development ................. 16 Comparative Pharmacokinetics, Pharmacod ynamics and Safety of TR 700 and Linezolid ................................ ................................ ................................ .............. 18 Absorption ................................ ................................ ................................ ........ 18 Distribution ................................ ................................ ................................ ....... 18 Metabolism ................................ ................................ ................................ ....... 19 Elimination ................................ ................................ ................................ ........ 19 Safety ................................ ................................ ................................ ............... 20 Pharmacodynamics ................................ ................................ .......................... 21 3 TISSUE DISTRIBUTION OF ANTIBIOTICS AN D MICRODIALYSIS ...................... 27 Dosing Rationale for Antibiotics ................................ ................................ .............. 27 Methods for Measurement of Tissue Distribution ................................ .................... 28 Microdialysis ................................ ................................ ................................ ........... 28 4 DETERMINATION OF TR HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH ULTRAVIOLET LIGHT DETECTION (HPLC/UV) ................................ ................................ ............. 31 Objective ................................ ................................ ................................ ................. 31 Experimental Procedures ................................ ................................ ........................ 31 Chemicals and Equipment ................................ ................................ ................ 31 Test article ................................ ................................ ................................ 31 Reagents ................................ ................................ ................................ .... 31 Equipment and disposables ................................ ................................ ....... 32 Rea gent Preparation ................................ ................................ ........................ 32

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6 TR 700 standard solution ................................ ................................ ........... 32 TR 700 calibration standards ................................ ................................ ..... 32 TR 700 quality control standards ................................ ............................... 33 HPLC mobile phase ................................ ................................ ................... 34 Sample Preparation ................................ ................................ .......................... 34 Sample Analysis ................................ ................................ ............................... 34 HPLC/UV s et up ................................ ................................ ......................... 34 HPLC pump conditions ................................ ................................ .............. 34 Auto sampler conditions ................................ ................................ ............. 34 Detector conditions ................................ ................................ .................... 35 Analysis Procedure ................................ ................................ .......................... 35 System Suitability ................................ ................................ ............................. 35 Data Analysis ................................ ................................ ................................ ... 35 Results ................................ ................................ ................................ .................... 35 5 TIME KILL EXPERIMENTS ................................ ................................ .................... 42 Objectives ................................ ................................ ................................ ............... 42 Time Line ................................ ................................ ................................ ................ 42 Methods ................................ ................................ ................................ .................. 43 Chemicals and Test Articles ................................ ................................ ............. 43 Bacteria ................................ ................................ ................................ ...... 43 Other supplies ................................ ................................ ............................ 43 Equipment ................................ ................................ ................................ .. 43 Preparation of TR 700 Solutions and Broth ................................ ...................... 44 TR 700 stoc k solution ................................ ................................ ................ 44 TR 700 solutions used for MIC and time kill experiments .......................... 4 4 Muller Hinton Broth ................................ ................................ .................... 44 Saline solution ................................ ................................ ............................ 44 MIC ................................ ................................ ................................ ................... 45 Time Kill Experiments ................................ ................................ ...................... 46 Calcula tions and Transformations ................................ ................................ .... 47 Results ................................ ................................ ................................ .................... 47 MIC ................................ ................................ ................................ ................... 47 Time Kill Experiments ................................ ................................ ...................... 48 6 IN VITRO MICRODIALYSIS ................................ ................................ ................... 52 Objective ................................ ................................ ................................ ................. 52 Microdialysis and Recovery ................................ ................................ .................... 52 Extraction Efficiency Method (EE) ................................ ................................ .... 53 Retrodialysis Method (RD) ................................ ................................ ............... 54 Experimental Procedures ................................ ................................ ........................ 55 Chemicals and Equipment ................................ ................................ ................ 55 Reagent Preparation ................................ ................................ ........................ 56 Sample preparation ................................ ................................ .......................... 57

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7 Microdialysis Setup ................................ ................................ .......................... 57 Sample Analysis ................................ ................................ ............................... 58 Results ................................ ................................ ................................ .................... 59 Method Validation Acceptance Criteria and Res ults ................................ ......... 59 In vitro Microdialysis Results ................................ ................................ ............ 60 Conclusions ................................ ................................ ................................ ............ 60 7 AN OPEN LABEL, SINGLE DOSE, MICRODIALYSIS AND PHARMACOKINETIC STUDY OF TR 701 IN NORMAL, HEALTHY ADULTS ....... 64 Objectives ................................ ................................ ................................ ............... 64 Study Design ................................ ................................ ................................ .......... 64 Pilot Phase ................................ ................................ ................................ ....... 64 Main Phase ................................ ................................ ................................ ...... 65 Subject Selection Criteria ................................ ................................ ................. 66 Inclusion criteria ................................ ................................ ......................... 66 Exclusion criteria ................................ ................................ ........................ 67 Flow Chart of Study Procedures ................................ ................................ ....... 69 Pharmacokinetic Measurements and Pharmacodynamic Assessments ........... 71 Results ................................ ................................ ................................ .................... 72 Pilot Phase ................................ ................................ ................................ ....... 72 Main Phase ................................ ................................ ................................ ...... 72 8 PHARMACOKINETIC/PHARMACODYNAMIC MODELLING ................................ 79 Modelling o f Pharmacokinetic Data ................................ ................................ ........ 79 Modelling of Kill Curve Data ................................ ................................ ................... 80 9 DISCUSSION AND CONCLUSION ................................ ................................ ........ 94 LIST OF REFERENCES ................................ ................................ ............................... 96 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 106

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8 LIST OF TABLES Table page 2 1 Oral Single Ascending Dose Pharmacokinetics of Linezolid and TR 70 0 (Mean SD) ................................ ................................ ................................ ....... 25 2 2 Oral Multiple Ascending Dose Pharmacokinetics of Linezolid and TR 700 (Mean SD) ................................ ................................ ................................ ....... 25 2 3 Observed MICs of T R 700, Linezolid and Vancomycin against Linezolid resistant bacteria ................................ ................................ ................................ 26 2 4 MICs in strains harboring varying numbers of mutated 23S rRNA genes ........... 26 2 5 f AUC 24 /MIC indices for TR 700 and linezolid ................................ ...................... 26 4 1 Intra batch variability of quality control samples* (Days 1 to 4) .......................... 38 4 2 Inter batch variability of quality control samples ................................ ................. 39 4 3 Freeze/thaw stability of TR 700 ................................ ................................ .......... 39 4 4 Freezer ( 70 C) long term stability of TR 700 ................................ ..................... 40 4 5 Bench top / Autosampler Stability of TR 700 ................................ ...................... 40 4 6 Short Term Stability of TR 700 ................................ ................................ ........... 41 5 1 Concentrations used for time kill experiments [g/mL] ................................ ....... 49 5 2 Results of MIC Determination ................................ ................................ ............. 49 5 3 Dates of Time Kill Experiments ................................ ................................ .......... 49 5 4 Maximum Reduction in Bacterial Numbers after 24 h ................................ ......... 49 6 1 Performance of Quality Controls for Extraction Efficiency Experiment ............... 61 6 2 Performance of Quality Controls for Retrodialysis Experiment ........................... 62 6 3 Results of Extraction Efficiency and Quality Control Experiments ...................... 63 7 1 Pilot Phase Timeline ................................ ................................ ........................... 69 7 2 Main Phase Timeline ................................ ................................ .......................... 70 7 3 Study Demographics ................................ ................................ .......................... 74

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9 7 4 Summary of Mean (SD) TR 700 Plasma, Adipose Tissue, and Muscle Tissue Pharmacokinetic Parameter Data (Main Study) ................................ .................. 78 7 5 Summary of Mean (SD) Unbound Plasma, Adipose Tissue, and Muscle Tissue Pharmacodynamic Parameter Data Corrected for Protein Binding (Main Study) ................................ ................................ ................................ ....... 78 8 1 Estimated model parameters ................................ ................................ .............. 88 8 2 Bootstrap results (N=500) ................................ ................................ ................... 88 8 3 P arameter estimates for all three S. aureus strains ................................ ............ 93

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10 LIST OF FIGURES Figure page 2 1 Chemical Structure of Linezolid ................................ ................................ .......... 24 2 2 Chemical Structures and Conversion of TR 701 to TR 700 ............................... 24 3 1 Schematic of transfer process at the microdialysis membrane level .................. 30 4 1 .................... 37 4 2 Representative Chromatogram of TR e LLOQ (50 ng/mL) ................................ ................................ ................................ 37 4 3 Representative Chromatogram of TR ng/mL (Sensitivity Check) ................................ ................................ ................... 37 5 1 Time line for time kill curve ................................ ................................ ................. 42 5 2 TR 700 against S. aureus ATCC33591 (Mean, standard deviation (SD)) .......... 50 5 3 TR 700 again st S. aureus CM/05 (Mean, SD) ................................ .................... 50 5 4 TR 700 against S. aureus NRS271 (Mean, SD) ................................ ................. 51 7 1 Mean (SD) total plasma concentrations of TR 700 after a 600 mg oral dose ..... 75 7 2 Mean (SD) free adipose ( ) and muscle ( ) concentrations of TR 700 after a 600 mg oral dose ................................ ................................ ................................ 76 7 3 Individual total plasma concentrations of Torezolid after a single 600 mg oral dose ................................ ................................ ................................ .................... 77 8 1 Individual fits for free plasma concentrations ................................ ...................... 82 8 2 Observations, individual and population predictions for free plasma concentrations. ................................ ................................ ................................ ... 83 8 3 Observations versus population and individual predictions for free plasma concentrations. ................................ ................................ ................................ ... 83 8 4 Individual fits for muscle concentration s ................................ ............................. 84 8 5 Observations versus population and individual predictions for muscle concentrations. ................................ ................................ ................................ ... 85 8 6 Observations, individual an d population predictions for muscle concentrations. ................................ ................................ ................................ ... 85

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11 8 7 Individual fits for adipose concentrations ................................ ............................ 86 8 8 Observations versus population and individual predictions for adipose concentrations. ................................ ................................ ................................ ... 87 8 9 Observations, individual and population predictions for adipose concentrations. ................................ ................................ ................................ ... 87 8 10 Conditional weighted residuals versus time (independent variable) for all compartments. ................................ ................................ ................................ .... 89 8 11 Visual predictive check results for all tissues ................................ ...................... 90 8 12 Curve fits for S. aureus ATCC33591 ................................ ................................ .. 91 8 13 Curve fits for S. aureus CM/05 ................................ ................................ ........... 92 8 14 Curve fits for S. aureus NRS271 ................................ ................................ ........ 93

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PHARMACOKINETIC / PHARMACODYNAMIC CHARACTERIZATION OF TR 700 (TOREZOLID) By Martina Sahre December 2010 Chair: Hartmut Derendorf Major: Pharmaceutical Sciences Infections with methicillin resistant S taphylococcus aureus (MRSA) have become e ndemic in healthcare settings around the world and recently patients without risk factors have become infected in the community setting by MRSA that shows different characteristics compared to hospital strains. Most MRSA infections that are community assoc iated present as skin and soft tissue infections (SSTIs) and in lesser numbers as pneumonia or bacteremia. Antibiotics like vancomycin and linezolid are effective in treating infections, but prolonged use can cause resistances. Development of novel antibio tics is necessary in order to have an effective armamentarium against ever adapting bacteria. Pharmacokinetic/pharmacodynamic parameters like area under the curve over minimum inhibitory concentration ( AUC/MIC ) maximum concentration over MIC ( C max /MIC ) an d time the concentration remains above the MIC ( T >MIC ) are used to correlate effect and drug levels, yet often the pharmacokinetic parameters are calculated from plasma concentrations Most drugs, however, do not have their site of action in the blood, but in the tissue, either intracellularly or in the interstitial space fluid. TR 700 is a novel oxazolidinone found to be effective against MRSA, particularly strains that are also linezolid resistant. To characterize the time course of drug concentrations

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13 no t only in plasma, but also in skeletal muscle and subcutaneous adipose tissue, the site of action for skin and soft tissue infections, a microdialysis study has been conducted. In addition to the determination of pharmacokinetic parameters, the time course of the effect of TR 700 has been elucidated in vitro using time kill experiments. The estimated pharmacodynamic parameters were th en correlated to the kinetic parameters to model and simulate outcomes of various dosing regimens.

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14 CHAPTER 1 ANTIBIOTIC U SE AND SPREAD OF MET HICILLIN RESISTANT STAPHYLOCOCCUS AUREUS S. aureus is the cause of various disease syndromes, such as skin and soft tissue infections (abscesses, impetigo, furunculitis, cellulitis, etc.), endocarditis, infections of prosthetic joints, sepsis, etc. ( 18 ) In recent years the number of MRSA infections has increased steadily and the National Nosocomial Surveillance Report issued in 2004 shows that about 60% of S. aureus in hosp ital settings is methicillin resistant ( 63 ) Additionally, the occurrence of MRSA is no longer limited to healthcare settings, strains have emerged in the community setting that cause infection in otherwise healthy people ( 4 30 ) There are some distinct characteristics between hospital and community associated (HA CA ) MRSA, such as pulsed field gel electrophoresis (PFGE) types, staphylococcal chromosomal cassette ( SCC ) mec typ e and susceptibility patterns ( 38 42 ) In a study analyzing 1984 MRSA isolates collected from usually sterile sites, for the Active Bacterial Core (ABC) surveillance system 22 versus 19% of infections were HA and CA, respectively. 57% of infections were health care associated with community onset (HACO). PFGE types USA100 and USA300 were found most often associa ted with HA and CA MRSA infections, respectively. Of 627 genotyped USA300 strains 605 were carrying the genes for Panton Valentine leukocidin (PVL) but only 2 (of 1063) USA100 strains were PVL positive. ( 42 ) According to the ABC Surveillance Report from 2005 and 2006 USA100 strains are >95% and resistant to clindamy cin and levofloxacin, whereas 8 and 55% of USA300 were resistant to clindamycin and levofloxacin, respectively ( 21 22 ) S aureus is a predominant cause of SSTIs seen in emergency departments and ambulatory care and in one study 26.7% of these infections were due to CA MRSA. Most of the CA MRSA cases presented as skin infections (80%),

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15 respiratory tract infections (13%) and blood stream infections (6%) ( 25 30 55 82 ) In a study conducted in 11 university affiliated emergency departments in 2004, 76% of skin and soft tissue infections (SSTIs) were caused by S. aureus Of these about 78% were methicillin resistant. The majorit y of MRSA isolates were sent to the Centers for Disease Control and Prevention (CDC) for further characterization. 99% of strains geno and phenotyped were USA300, the rest was type USA400 and USA1000; 98% carried the SCCmec type IV chromosomal cassette an d 98% carried Panton Valentine Leukocidin ( pvl ) genes ( 59 ) Current guidelines for the treatment of HA MRSA include vancomycin as first line agent and the newer d rugs dalbavancin, linezolid and daptomycin for treatment of HA MRSA infections. Treatment of CA MRSA infections is recommended to employ trimethoprim sulfamethoxazole, tetracycline or linezolid, depending on the severity of disease for outpatient therapy a nd vancomycin, teicoplanin, linezolid or daptomycin for ventilator associated pneumonia, bacteremia or endocarditis ( 73 ) Surgical incision and drainag e of abscesses and furuncles is a major part of treatment and often cures patients without or with ineffective antibiotic treatment ( 30 )

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16 CHAPTER 2 TR 700 AND ITS ROLE AMONG THE O XAZOLIDINONES Chemistry and P hysicochemical P roperties effect on the growth of S. aureus and other gram positive cocci. ( 24 ) The baseline structure of the drugs of this class is the oxazolidinone ring, which together with an acyl group on its e nd fits into the peptidyl transferase center ( PTC ) binding pocket. The phenyl ring usually contains a strongly electronegative atom, fluorine in this case, which appears to facilitate the sterical conformation that inhibits tRNA binding. TR 700 (torezolid) is a member of the oxazolidinone class of antibiotics. It is structurally similar to linezolid which was the first oxazolidinone to be introduced to the market in the United States in April of 2000. See Figures 2 1 and 2 2 for the chemical structures. Lin ezolid and TR 700 are structurally different in their substituents at the phenyl ring. Whereas linezolid has a morpholino group, TR 700 has a pyridine tetrazole substituent Linezolid is easily and readily soluble in water with a solubility of about 3 mg/m L ( 99 100 ) whereas TR 700 has a low aqueous solubility of about 4 g/mL. As a result, TR 700 is adminis tered as a phosphate ester prodrug with a solubility of about 100mg / m L ( 34 ) In the intestines and the blood phosphatases are available to rapi dly convert the prodrug into the active moiety. Mechanism of Action of Oxazolidinones and Resistance Development Oxazolidinones have been found to interact with the 23S subunit of the rRNA in which the peptidyltransferase center is located. ( 39 ) The drug fits sterically in to the binding pocket that is formed to anneal a new amino acid to the forming protein chain.

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17 The result is that the tRNA cannot bind into the corr ect position of the ribosome and thus the drug inhibit s protein synthesis. ( 98 ) First reports of linezolid resistance included the development of target site mut ations at domain V in the 23S rRNA subunit that seemed to have occurred de novo during therapy ( 57 88 ) Recen tly, another resistance mechanism has appeared in the form of a mutant which was first isolated in Colombia and carried the chloramphenicol, florfenicol resistance ( cfr ) gene. ( 58 ) This marks the first linezolid resistance due to an acquired mechanism which utilizes a methyltransferase to posttranslationally methylate the 23S rRNA and thus makes binding of the drug harder Originally, oxazolidinones were thought to be less susceptible to plasmids conferring multi drug resistance, based on the fact that they are fully synthetic ( 58 85 ) Certainly, the development of resistance to many antibiotics warrants the research and consequently development of novel antimicrobials. TR 700, an oxazolidinone with a tetrazole substituent was shown to be effective against linezolid resistan t MRSA ( 34 ) The minimum inhibitory concentrations at which 90% of the isolates were covered ( MIC 90 ) for bacteria carrying the cfr gene were 1 and 32 mg/L for TR 700 and linezolid, respectively. MIC 90 s for Enterococci and Staphylococci harboring G2576U mutations were 8 and >16 mg/L for TR 700 and linezolid, respectively ( 35 51 ) Additionally, TR 700 shows effect against penicillin resistant Streptococcus pneumoniae (MIC 0.125 mg/L) and linezolid and vancomycin resistant Enterococcus faecalis (MIC 2 4 mg/L) ( 77 ) Another observation is that as the number of G2576U mutations increase, as there are always multiple genes coding for the rRNA, so does the MIC. This is seen for both, linezolid and TR 700, but to a lesser

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18 degree for the latter. ( 35 51 ) Mutation of S. aureus after serial passage was also observed to be of greater magnitude for linezolid as compared to tore zolid, so that serially torezolid passaged strains would show lower oxazolidinone resistance that serially linezolid passaged ones. ( 52 ) Comparative Pharmacokineti cs, Pharmacodynamics and Safety of TR 700 and Linezolid Absorption Linezolid is available in an intravenous (i.v.) and peroral (p.o.) application, as is planned for torezolid. Linezolid shows almost complete bioavailability at around 100% ( 97 ) and 91.7% of TR 700 are found after an oral dose of TR 701 compared to an i.v. dose. ( 16 ) Linezolid shows the maximum concentration after around 1.5 hours depending on the dose and TR 700 shows its T max (time at which maximum concentrations occurs) at around 2 hours. ( 15 66 81 ) Distribution The volume of distribution (V d ) of TR 700 is higher than 100 L, whereas linezolid shows a V d of less than 50 L at most doses in adults. Linezolid also had a much lower protein binding of around 35% compared to TR 700 were the bound fraction was 87%. ( 1 54 78 ) One study measuring linezolid concentration in tissues after either a single i.v. or mu ltiple oral doses found that there was no hindrance for the drug to enter the tissues as indicated by ratios of tissue to plasma AUCs of about one. After intravenous dose, the tissue to plasma ratios were 1.3 and 1.4 for muscle and subcutaneous adipose tis sue, respectively, which would indicate either a transporter mechanism, or possibly irreversible uptake of the drug into cells. ( 27 ) In a study done in critica lly ill patients, the tissue to plasma ratios were very variable, ranging from about

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19 20% to more than 100%, which indicates that tissue penetration does not seem to be easily predictable in these high risk patients. ( 19 ) Both TR 700 and linezolid are taken up into the cell, with TR 700 achieving this uptake quicker and to a higher degree (10 to 15 times the concentration measured extracellularly) at 37 C and pH 7.4. When temperature and pH were lowered however, TR 700 lost much of its ability to penetrate into the cell. ( 41 ) Cellular accumulation measured in Escherichia coli Citrobacter and Enterobacter showed that uptake can be inhibited by efflux pumps. ( 74 ) One study comparing plasma to blister concentrations of linezolid, fou nd that the drug equilibrated easily with blister fluid to an extent of approximately 100%. The T max was delayed two hours compared to the plasma T max of 0.7 hours. ( 3 2 ) Metabolism TR 701 is rapidly converted to TR 700 in heparinized blood and plasma. ( 2 ) See Figure 2 2. It is metabolized in various ways, including sulfatation, oxidation of the hydroxyl substituent on the o xazolidinone ring and reduction of a nitrogen in the tetrazole ring. When given with probes for major cytochrome P450 enzymes, no inhibition or increased activity was observed. Linezolid also shows a number of metabolites with an enzymatic and a non enzym atic pathway available ( 54 79 ) The major metabolites are either an enzymatically formed lactam or a lacton e that is formed non enzymatically. Both reactions occur at the oxazolidinone ring. Elimination Linezolid is mostly eliminated as parent drug, with 42.4% of it being eliminated in urine after a single dose, versus 27.6% and 8.2% of the lactone and lactams metabolite, respectively. After multiple doses, 30% of unchanged linezolid are recovered from urine, versus 39.2% of the metabolite from the non enzymatic pathway and 9.3% from the

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20 lactams (enzymatic). ( 79 ) For single and multiple ascending doses a nonlinearity in clearance has been observed in that the percentage of non renal clearance increases as total doses increase. ( 81 ) There is however no dose adjustment needed, as overall clearance remains linear. ( 3 ) Safety One adverse effect of linezolid is its activity as a monoamine oxidase (MAO) inhibitor. Pressure response rates after admi nistration of linezolid or moclobemid e together with foods rich in tyramine (air dried or fermented food like cheese, wine, sauerkra ut) were similar in that for both treatments, the dose needed to produce an increase in blood pressure was lower with tyrami ne rich food ( 7 ) When TR 700 was compared to linezolid pressure response in rats, it was found that a dose of up to 150 mg/kg did not show an increase in diastolic pressure of >30 mm Hg. Even though, TR 700 was weakly similar as an inhibitor of MAO a and b, the authors of the study argue that, since TR 701 is the only moiety absorbed (and shows no MAO inhibitory activity), it should not play a relevant role in obser ved side effects. ( 8 ) Hematological side effects are also seen with linezolid, particularly when the drug is used longer than 28 days (the approved treatment duration ), due to recurring infections. ( 29 33 ) A 600 mg twice daily (bid) regimen of linezolid was comparable in de creases of platelet, r eticulocyte and neutrophil counts to a 400 mg dose of TR 700 once daily (qd). Th e reductions in platelet, reticulocyte and neutrophil counts were 50, 91 and 66% for TR 700 and 54, 95 and 66% for linezolid, respectively. The 200 mg qd dose of TR 700 was similar to placebo dosing. ( 65 )

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21 Lactic acidosis as well as peripheral and optical neuropathies have also been reported after prolonged use of linezolid. ( 9 13 17 37 40 64 ) One explanation that is currently being studied is the possible interaction of oxazolidinones with mitochondrial 16S rRNA. The ribosomal RNA seems to be highly conserved between species. L inez olid shows significant inhib i t o ry ability on the mitochondrial protein synthesis. ( 56 ) This study also found that the ability to inhibit protein synthesis in the mitochondria was proportional to the efficacy of the drug against bacteria (in this case measured as MIC) or in other words, the stronger the drug effect, the higher the possibility to have an effect on mitochondrial protein synthesis. Another study t hat was done in patients undergoing linezolid treatment who experienced hyperlactatemia showed that protein synthesis for respiratory chain complexes was decreased significantly during the elevated lactate stage and normalized after linezolid was discontin ued. In some patients it appears to have taken several days to return to normal lactate levels. ( 31 ) Pharmacodynamics Linezolid is approved for the treatment of various gram positive microbial infections, such as vancomycin resistant Enterococcus faecium (VREF) infections hospital acquired pneumonia due to MRSA, methicillin susceptible S. aureus (MSSA) and resistant and susceptible Streptococcus pneumoniae Anot her indication is the treatment of CSSI, such as diabetic foot infections due to MRSA and MSSA, or Streptococcus spp. In the case of CA pneumonia, it is approved for use in multidrug resistant Streptococci and methicillin susceptible S. aureus only. ( 3 ) A study assess ing the pharmacodynamics of linezolid in neutropenic mice found that AUC/MIC was a good pharmacokinetic/pharmacodynamic index for linezolid. To a lesser degree, the percent of the dosing interval that the drug concentration remained above the MIC also show ed

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22 to be correlated to reduction in bacterial numbers. The maximum concentration to MIC ration showed a much smaller correlation. ( 6 ) Table 2 5 shows the AUC/MIC ind ices after a single 600 mg p.o. dose of TR 700, as determined in the study described in Chapter 7, and twice daily dosing of 600 mg linezolid p.o. Torezolid has been found to be effective against gram positive bacteria as well. ( 51 76 77 ) In studies comparing torezolid efficacy against linezolid resistant strains to rezolid showed an MIC 90 of 2 g/mL against S. aureus versus 8 g/mL for linezolid. In linezolid resistant S. epidermidis strains, the MIC 90 s were 8 and >128 g/mL, respectively and for Enterococci the MIC 90 was 4 g/mL for torezolid compared to 32 to 6 4 g/mL for linezolid. A recent study in Spain, assessing effectiveness of Staphylococci from blood isolates, found that torezolid had good efficacy against linezolid susceptible and resistant MSSA with an MIC of 0.5 g/mL The MIC against linezolid susc eptible coagulase negative Staphylococci (CoNS) was 0.25 and 0.5 g/mL for methicillin susceptible and resistant strains, respectively. Only for five strains of linezolid resistant CoNS was the MIC 4 g/mL. Linezolid showed MICs of 4 to 16 g/mL. Another observation this study made, is that the cfr gene is apparently readily spread among staphylococcal species. ( 14 ) Torezolid also shown an inter esting effect, when application in neutropenic versus immune competent mice is compared. ( 53 ) Based on the notion that TR 700 has better cellular uptake characteristics than linezolid ( 41 ) i t was found that torezolid shows two distinct kill mechanisms, one through direct action against bacterial organism s and one

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23 through granulocytes. The overall decrease in bacterial number was much higher for mice who were non neutropenic as compared to their neutropenic counterparts.

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24 Figure 2 1. Chemical Structure of Linezolid Figure 2 2 Chemical Structures and Conversion of TR 701 to TR 700

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25 Table 2 1. Oral Single Ascending Dose Pharmacokinetics of Linezolid and TR 700 (Mean SD) Drug Dose Level [mg] AUC 0 [mg*h/L] C max [mg/L] T max [h] T 1/2 [h] CL [L/h] V d [L] Linezolid 325 65.5 25 8.2 2 1.70.9 5 1 6.5 2.5 4414 500 74.328 10.4 2.5 1.40.9 4.6 1.8 7.5 2.6 4511 625 10230 12.7 3.4 1.30.6 4.9 1.4 6.7 2.7 4514 TR 700 400 56.1 13.2 3.8 1 3.5 (2 4) 10.8 0.8 7.4 1.6 116 24 600 79.3 31.3 5.2 0.7 2.5 (2 4) 11.4 2.6 8.8 4.0 134 31 800 91.8 12.9 5.5 1.2 4 (2 8) 10.6 1.3 8.9 1.3 135 39 *Median (Range) Sources: ( 15 54 81 ) Area under the curve from time 0 to infinity ( AUC 0 ), Maximum concentration (C max ), half life (T 1/2 ), clearance (CL) Table 2 2. Oral Multiple Ascending Dose Pharmacokinetics of Linezolid and TR 700 (Mean SD) Drug Dose Level [mg] AUC 0 [mg*h/L] C max [mg/L] C min [mg/L] T max [h] T 1/2 [h] CL [L/h] V d [L] Linezolid 375 83 23 13 3 3.9 1.8 10.3 5.41 4.8 1.4 388 (BID) 500 9937 15 4 5 2.4 1.31 5.72 5.5 1.5 4315 625 14758 19 6 8 3.6 2.11 5.40.9 4.7 1.4 3610 TR 700 300 31 6 2.5 0.4 0.5 0.2 1.8 (1.5 4) 9.4 1.2 10.1 1.8 136 23 (QD) 400 53 8 4.5 1 0.8 0.2 3 (2 8) 8.4 1 7.7 1 92 18 *Median (Range) Sources: ( 66 81 ) Minimum concentration (C min )

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26 Table 2 3. Observed MICs of TR 700, Linezolid and Vancomycin against Linezolid resistant bacteria Bacterium Drug MIC 90 /G2576T (Range) [mg/L] MIC 50 / cfr (Rang e) [mg/L] Staphylococcus spp. TR 700 8 (0.5 16) 1 (0.5 8) Linezolid >32 (8 >32) 32 (8 >32) Vancomycin 2 (0.5 2) 1 (1 2) E nterococcus faecalis TR 700 2 (0.5 8) E nterococcus faecium Linezolid 16 (4 32) Vancomycin >16 (0.5 >16) Table 2 4. MICs in s trains harboring varying numbers of mutated 23S rRNA genes MICs [mg/L] Species # Mutated Genes Linezolid TR 700 S. aureus 0 2 0.5 2 16 2 3 16 32 2 4 E. faecalis 0 2 0.5 2 16 2 3 32 4 E. faecium 0 2 0.5 2/3 8 16 1 2 4 32 4 5/6 32 64 4 8 Table 2 5. f AUC 24 /MIC indices for TR 700 and linezolid MIC f Plasma Adipose Tissue Muscle Tissue 0.25 g/mL TR 700 35.6 (12.2) 66.5 (63.2) 58.3 (42.0) 0.5 g/mL TR 700 17.8 (6.12) 33.3 (31.6) 29.2 (21.0) 2 g/mL Linezolid 108.5 (35.8) 93.3 (31.8) 117.7 (66.1) 4 g/mL TR 700 2.22 (0.76) 4.16 (3.95) 3.65 (2.62) Linezolid 54.3 (17.9) 46.6 (15.9) 58.9 (33.0)

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27 CHAPTER 3 TISSUE DISTRIBUTION OF ANTIBIOTICS AND M ICROD IALYSIS Dosing Rational e for Antibiotics Most antimicrobials are currently dosed based on in vitro measured minimum inhibitory concentrations (MICs) of a specific agent to a microbe and a pharmacokinetic parameter, such as C max and AUC. The resulting PK/PD parameters used are AUC 24 /MIC, C max /MIC and T >MIC ( 5 60 ) The MIC is a static parameter measured at two fold c oncentration intervals and gives no information about the time course of the drug action. Time kill curves have been used more recently to determine the time course of drug action. The fact that multiple concentrations are tested and at higher concentratio ns also the maximum effect, allows for estimation of efficacy parameters such as the concentration at which 50% of the effect is reached ( EC 50 ) and the maximum effect ( E max ) ( 50 ) Often, the pharmacokinetic parameters are measured in plasma and do not take unbound or tissue concentrations into account ( 50 ) In the case of microbial infections, this is disadvantageous, because most infections occur either intracellu larly or in the interstitial space fluid (ISF) of tissues. Distribution of drugs into tissues is contingent on many factors, such as protein binding in both plasma and tissues, pKa values of antibiotics, drug transport by passive diffusion or influence of active transport mechanisms and disease state ( 61 62 ) Thus, to assess tissue distribution of novel drugs, parti cularly antimicrobial agents, it is advantageous to measure unbound levels of the active pharmaceutical ingredient in the tissue of interest. The combination of biophase concentration and the time course of bacterial action can be used to predict the effec t of different dosing regimens in silico ( 84 ) Both the

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28 United States Food and Drug Administration ( FDA ) in its Critical Path whitepaper and the European Medical Agency (EMA ) acknowledge the need for information on biophase concentrations to facilitate more accurate pharmacokinetic/pharmacodynamic ( PK/PD ) Modeling. ( 28 95 ) Methods for Measurement of Tissue Distribution Traditionally there is a variety of approaches to measure tissue levels, including biopsy, skin blister formation and imaging techniques. Biopsy samples have the inhe rent disadvantage of providing whole tissue drug concentration only, due to the fact that they are homogenized before analysis, but tissues are not one homogenous compartment ( 62 ) Suction or chemical ly induced skin blisters were a convenient way to necessarily indicative of the real tissues of interest ( 62 ) T he most convenient and relatively minimally invasive technique is microdialysis ( 23 ) which is based on the principles of passive diffusion along concentrations gradients. The foremost advantage of microdialysis is the abi lity to sample from the tissue of choice directly and to sample free drug concentrations directly, without major sample cleanup. Microdialysis Microdialysis has been developed over the past decade and originated in neurological research, where it was used to measure concentrations of molecules in the brain. ( 12 44 49 69 70 80 93 94 ) Quickly, microdialysis was also used to observe concentrations of compound of interest in other tissues. ( 19 26 71 72 ) There are a number of studies that have been done to assess the tissue distribution of antibiotics in h ealthy volunte ers and patients. ( 10 11 19 83 86 ) For linezolid, there was also a study assessing the distribution into subcutaneous adipose and muscle tissue. ( 27 )

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29 The method of microdialysis relies on passive diffusion of molecules across a semipermeable membrane (see Figure 3 1). ( 91 92 ) The pore diameter of the membrane defined the cut off and the membranes that were used in the experiments in this work were impermeable to compounds larger than 20 Kilodalton ( kD ) therefore eliminating major proteins found in tissues (e.g. albumin) from passing the membrane. Due to the fact that microdialysis is passive diffusion driven, it is reliant on a number of factors, such as the flow rate through the microdialysis memb rane, temperature, area of diffusible membrane, tissue properties (e.g. tortuosity), among others. To assess the true concentrations in tissue there is another factor that needs to be considered. The microdialysis membrane is constantly perfused by a physi ological between the phases in the membrane and outside of the membrane cannot be achieved. The magnitude of the difference between the two phases is assessed by measuring the relative recovery, which is explained in Chapter 6. Briefly, the probes are calibrated to assess the recovery factor, i.e. how much of drug is found in the dialysate compared to what was present in tissue or the perfusate. Since every probe is unique, they have to be calibrated individually. There are several methods available for probe calibration, the most convenient method for human microdialysis experiments being retrodialysis. Retrodialysis assumes that diffusion through the membrane is equal for f lux into and out of the probe. To determine whether this can be assumed, the loss and gain through the probe is determined by in vitro experiments. For this the dialysis probe is either placed into a centrifuge tube containing the drug of interest at a kno wn concentration (mimicking the tissue during in vivo microdialysis) and is perfused by a

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30 blank physiological solution (gain is measured), or the probe is perfused with a known concentration of drug solute, while resting in a blank physiological solution ( loss is measured). In clinical studies using microdialysis ( 36 ) probes are implanted into the tissues of interest using an introducer cannula, which is then removed. The part remaining in the tissue consists of flexible plastic material and should cause minimal discomfort only. The probe is then connected to a syringe that contains a blank physiological solution ched to a pump that delivers a constant flow rate. The perfusate then flows through the inlet tubing into the probe, at the tip of which is the dialysis membrane, where diffusion occurs. From there the, now, dialysate flows through the outlet tubing into a collection vial. *Courtesy of CMA, Sweden Figure 3 1. Schematic of transfer process at the microdialysis membrane level

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31 CHAPTER 4 DETERMINATION OF TR 700 IN LACTATED RING HIGH PERFORMANCE LIQUID C HROMATOGRAPHY WITH U LTRAVIOLET LIGHT DETECTION (HPLC/UV) Objective To validate an HPLC UV procedure for the determination of TR 700 in lactated was used for the determination of TR 700 levels in samples generated by the clinical microdialysis sampling method for study TR701 102. The basis for the validation was formed by an FDA guideline and an update from a workshop on method validation. ( 75 90 96 ) Experimental Procedures Chemicals and Equipment Test a rticle TR 700 (Anal ysis # 68189) used for the HPLC/UV method were obtained fro m Trius Therapeutics, Inc. The compounds were stored at 2 8 C and protected from light. Reagents All reagents were of HPLC grade unless otherwise stated. They were obtained from indicated sources or equivalent suppliers. Double distilled water (ddH 2 0), in house, Department of Pharmaceutics Acetonitrile (ACN), Fisher, #A998 4 35% Acetonitrile in double distilled Water: In a graduated cylinder measure 650 mL ddH 2 0 and 350 mL ACN independently. Pour into a 1 L bottle and add magnetic stir bar. Stir for 10 mi nutes. Cover and degas under Helium atmosphere for ten minutes. Dimethyl Sulfoxide (DMSO) Fisher, #BP231 1

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32 Equipment and d isposables The f ollowing pieces of equipment or equivalent su bstitutes were used. Micropipettes, Eppendorf, BCL Pipette, repeating Eppendorf, BCL Vortex, Kraft Apparatus Volumetric Flasks, 10 mL, Pyrex, #5640 Corning 15 mL centrifuge tubes, #430052 Corning 50 mL centrifuge tubes, #430828 Microcentrifuge tubes, Fishe rbrand, 1.5 mL, #02 681 272 HPLC vials, Fisherbrand, 8*40 mm, HPLC vial inserts, Polypropylene, 200 L, conical, Sun Sri #600013 Discovery C18 Column, Supelco, #504955, Column ID: 9157 7 03 HPLC Guard column, in house C18 packing HPLC Pump, Waters 515 Au tomatic Injector, Waters 717 Plus LC Autosampler Balance AB104, Mettler Toledo,Hightstown,NJ,USA Waters Empower software No glass and plastic ware wa s re used. Reagent Preparation TR 700 s tandard s olution TR 700 was dissolved in 100% DMSO to give a nominal concentration of 1mg/ml. This solution was used to spike the calibration and quality control plasma samples and was prepared freshly for the preparation of calibration standards and quality controls. The solution wa s stored at approximately 80C or lower for not more than 6 months for determination of the HPLC retention times. T R 700 c alibration s tandards TR 700 calibration standards was to the following pipetting sc heme: Standard 1 (3200 ng/mL): Add 0.032 ml of TR 700 standard solution to 9.968 ml of

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33 Standard 2 (1600 ng/mL): Standard 1 (0.5 solution Standard 3 (800 ng/mL): Add Standard 2 (0.5 mL) to 0.5 solution solution solution Standard 6 (100 ng/mL): Add Standard 5 (0.5 mL) to 0.5 solution solution T R 700 quality control standards Quality control (QC) samples were prepared from the Stock solution. The final concentrations were 0.05 ( lower limit of quantitation ( LLOQ) ) 0.1 ( low ( L) ) 1.6 ( mid ( M) ) and 2.4 ( high ( H) ) g/ml. QC solution (1 mg/mL) Dilution 1 (3200 ng/mL): Add 0.032 L stock solution to 9.968 mL lactated solution QC Dilution 2 (800 ng/mL): Add 0.5 mL QC QC QC LLOQ (50 ng/mL): Add 0.5 mL QC

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34 H PLC mobile phase 650 m L of ddH 2 O and 350 mL ACN were independently measured in a graduated cylinder and added into a glass bottle. A magnetic stir bar was added and the solution stirred for 10 minutes. The mobile phase wa s degassed in the same bottle under helium atmosphere for approximately 10 min. The mobile phase was not used longer than 7 days after preparation. Mobile phase w as also used for needle washing purposes. Sample P reparation All microdialysis samples were stored on ice and frozen within two hours of collection. Af ter thawing the samples on the day of analysis, they were vortexed and analyzed. Samples from microdialysis, that contain ed solution were injected directly, however, to have enough volume for injection, samples may ha d to be diluted. Sample Analysis HPLC/UV set up The HPLC system consist ed of a Waters 515 LC pump, Waters 717 Plus LC autoinjector, an adequate precolumn filled in house with reversed phase material, a HPLC Column Supelco Discovery C18, 150mm 4.6mm, follow ed by a Waters 2487 software. HPLC pump conditions Mobile Phase: 35% Acetonitrile, 65% double distilled Water Flow rate: 0.75 mL/min Auto sampler conditions Injection Volume: 10l Cycle time: 6 min 1 injectio n per vial 1 vial per sample

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35 Detector conditions Wavelength: 300nm Detector Output: mV Analysis P rocedure Each Batch of test Sample was run with Calibration C urve points run in duplicates. 6 sets of Quality Control Standards at 4 levels (LLOQ, low, medium and high) w ere included in random positions in the run. The number of quality controls sets used depend ed upon the number of samples analyzed. The approximate retention time for TR 700 is 4.5 0.5 min. System Suitability Three injections of the 2400 g/m L quality control sample we re made. The (coefficient of variation in percent) CV% for these samples was less than 5%. The symmetry was The pressure during sample analysis did not exceed 2500 psi. Data Analysis Data acquisition and processing was performed with the Waters Empower Software. The calibration curves were plotted as the peak response on the y axis vs. analyte concentration on the x axis. The coefficient of determination was calculated for the regressed calibration curve. Results The results from the validation can be seen in Tables 4 1 to 4 6. In short, the could be frozen and thought at least three times before stability became a problem. The compo und was stable in the freezer at 70 C The quality controls were all well within

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36 the limit of 20% the nominal value at the lower limit of quantitation and 15% that at all other concentrations. Representative chromatograms are shown in Figures 4 1 to 4 3.

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37 Figure 4 Figure 4 2 Representative Chromatogram of TR 700 in lactated the LLOQ (50 ng/mL) Figure 4 3 Representative Chromatogram of TR 700 in lactated R 400 ng/mL (Sensitivity Check)

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38 Table 4 1 Intra batch variability of quality control samples (Days 1 to 4) TR 700 ng/mL 50 100 400 1600 2400 Day 1 (14 May 08) Mean 46.56 92.23 391.48 1668.88 2355 .06 SD 1.83 2.26 28.73 34.30 31.97 CV% 3.93 2.45 7.34 2.05 1.36 Accuracy % 93.12 92.23 97.87 104.30 98.13 Day 2 (15 May 08) Mean 58.28 104.82 414.16 1773.08 2529.52 SD 2.44 4.54 20.89 58.69 42.19 CV% 4.19 4.33 5.04 3.31 1.67 Accuracy % 116.5 5 104.82 103.54 110.82 105.40 Day 3 (16 May 08) Mean 46.63 92.36 393.28 1738.81 2501.38 SD 2.14 5.30 14.30 48.13 33.71 CV% 4.59 5.74 3.64 2.77 1.35 Accuracy % 93.26 92.36 98.32 108.68 104.22 Day 4 (17 May 08) Mean 47.24 85.19 340.44 1527 .31 2206.08 SD 2.29 2.00 6.36 34.12 70.26 CV% 4.84 2.35 1.87 2.23 3.18 Accuracy % 94.48 85.19 85.11 95.46 91.92 Day 5 (19 May 08) Mean 52.03 94.19 386.35 1640.69 2350.62 SD 2.36 3.17 9.94 27.04 36.75 CV% 4.53 3.37 2.57 1.65 1.56 Accuracy % 10 4.06 94.19 96.59 102.54 97.94 Day 6 (22 May 08) Mean 50.02 94.24 392.68 1686.83 2412.34 SD 2.31 3.44 13.34 29.58 25.34 CV% 4.63 3.65 3.40 1.75 1.05 Accuracy % 100.04 94.24 98.17 105.43 100.51 Day 7 (23 May 08) Mean 53.58 99.40 396.53 164 3.87 2422.08 SD 2.00 2.73 22.52 35.71 73.64 CV% 3.73 2.75 5.68 2.17 3.04 Accuracy % 107.16 99.40 99.13 102.74 100.92 n=6 per day and per concentration SD (standard deviation)

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39 Table 4 2 Inter batch variability of quality control samples TR 700 [ng/mL] 50 100 400 1600 2400 Day 1 to 7 (14 to 23 May 2008) Mean [ng/mL] 50.62 94.63 387.85 1668.49 2396.73 SD 4.35 6.15 22.67 79.09 107.76 CV [%] 8.59 6.50 5.85 4.74 4.50 Accuracy [%] 101.24 94.63 96.96 104.28 99.86 n=5 6 per concentration Table 4 3 Freeze/thaw stability of TR 700 T R 700 Concentrations [ng/mL] ng/mL 50 100 400 1600 2400 Cycle 1 Mean 56.09 103.56 400.05 1783.64 2589.50 SD 1.01 0.45 7.09 48.81 110.22 CV (%) 1.80 0.44 1.7 7 2.74 4.26 Accuracy (%) 112.19 103.56 100.01 111.48 107.90 Cycle 2 Mean 46.56 93.85 417.17 1783.60 2546.32 SD 2.00 2.42 32.76 36.06 57.81 CV (%) 4.39 2.58 7.85 2.02 2.27 Accuracy (%) 91.11 93.85 104.29 111.47 106.10 Cycle 3 Mean 43.80 85.83 362. 39 1673.74 2244.18 SD 3.12 3.54 12.11 89.88 64.01 CV (%) 7.12 4.13 3.34 5.37 2.85 Accuracy (%) 87.59 85.83 90.60 104.61 93.51 *n=3 per concentration

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40 Table 4 4 Freezer ( 70 C) long term stability of TR 700 Standard 33 Day stability Concentration Mean S D Accuracy ng/ml ng/ml % 50 42.26 3.92 84.5 1600 1754.43 75.84 109.7 2400 2574.48 101.93 107.3 Quality controls of 0.05, 1.6 and 2.4 g/ml standards were stored over 33 days, calibration curve was prepared freshly. Dat a were generated on Mar 19 2008. Table 4 5 Bench top / Autosampler Stability of TR 700 Nominal Time Conc. Difference Mean SD CV Accuracy ng/ml h ng/ml % % 50 5 48.14 1.65 3.43 96.29 100 5 93.67 2.54 2.71 93.67 400 5 402.57 13.10 3.25 100.64 1600 5 1723.12 42.16 2.45 107.70 2400 5 2466.05 65.10 2.64 102.75 50 18 47.46 4.72 9.94 94.93 100 18 94.20 5.59 5.93 94.20 400 18 425.37 27.79 6.53 106.34 1600 18 1580.61 144.47 9.14 98.79 2400 18 2610.04 59.35 2.27 108.7 5 5h stability samples were generated and analyzed 22 May 08, 18h stability samples were generated and analyzed 15 Jul 08.

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41 Table 4 6 Short Term Stability of TR 700 TR 700 Concentration [ng/mL] 50 100 400 1600 2400 Mean 55.81 100.28 413.52 1692.98 2416.91 SD 1.59 0.93 12.32 76.23 45.01 CV [%] 2.86 0.93 2.98 4.50 1.86 Accuracy [%] 111.62 100.28 103.38 105.81 100.70 *n=3 per concentration

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42 CHAPTER 5 TIME KILL EXPERIMENTS Objectives The objective of this study was to e lucidate the effect of TR 700 on three strains of methicillin resistant Staphylococcus aureus (MRSA) in a time kill experiment The strains were S. aureus ATCC 33591, NRS271 and CM/05. S. aureus NRS 271 harbors a G2576U mutation in the domain V of the 23S rRNA subunit of the 50S ribosomal subunit and S. aureus CM/05 harbors the mobile cfr gene, both determinants render these strains resistant against linezolid. To assess the concentrations to be used for later time kill studies, the minimum inhibitory conce ntrations (MIC) for each strain were determined on three individual days. The Clinical and Laboratory Standards Institute (CLSI) guideline M07 was followed for the evaluation. Time Line Figure 5 1. Time line for time kill curve

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43 Methods Chemicals and Te st Articles Bacteria Three strains of methicillin resistant Staphylococcus aureus (MRSA) were used for this study. S. aureus ATCC 33591 and two linezolid resistant strains S. aureus NRS271 and CM/05. Bacteria were supplied by Trius Therapeutics, Inc. The d ates of shipment and receipt were 27 November, 2007 and 29 November, 2007, respectively, for S. aureus ATCC 33591 and 23 January, 2008 and 24 January, 2008, respectively, for both S. aureus NRS271 and CM/05. The strains were subcultured daily onto fresh sh eep blood agar plates and incubated at 37 C. Once the experiment was finished the strains were stored in a 25% glycerol solution and sterile saline at 80 C. Other supplies Muller Hinton broth (Beckton Dickinson, #211443) Muller Hinton broth II (Beckton Dickinson, #212322) Dimethyl sulfoxide (DMSO, #BP231 1) Sterile saline (in house production) Double distilled water (ddWater, in house distillation) Equipment Scale (Mettler Toledo, Highstown, NJ, USA) CO 2 Culture Incubator (#460, Lab Line Instruments, Mel rose Park, IL) SteriGard Hood (Baker SG400) BactiCinerator IV (McCormick Scientific, # 004002 ) Stirring Hot Plate (#310T, Fisher Scientific) Vortexer (Thermolyne MaxiMix Plus TM # M63215) Angled neck flasks 75 mL, 25 cm 2 (Nunc, #156367) 96 well plates, fla t bottom (Corning,, #3595) Sterile pipettes 10 mL (Corning, #4488) Automatic pipette (Pipet Aid, Drummond Scientific Co., Broomall, PA) Autoclave ( Con solidated Stills & Sterilizers, #SSR 3A MO) Sterile hood (Baker SG 400) Turbidimeter (A JUST TM Turbidity M eter, Abbott Laboratories, USA) McFarland standards (0.5, #R20410 and 1, #R20411)

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44 Inoculation loop (Remel, R503154) Sheep blood agar plates (SBA, Remel, #01202) Pipette tips (Fisherbrand SureOne, #02707451) Pipettes 10 100 L (Eppendorf, #s 22470302, 02247 2003) Pipette 2 20 L (Eppendorf, #022472054) Pipette 100 1000 L (Eppendorf, #022472101) Mul ti Channel Pipettes (Eppendorf 10 100 L and 30 300 L) Parafilm (PM 999, Menasha, WI) Preparation of TR 700 Solutions and Broth TR 700 stock solution A TR 700 sto ck solution was prepared by weighing 10 mg and dissolving in 5 mL DMSO in a volumetric flask ( stock 1 : 2 mg/mL). The solution was then vortexed for up to five minutes to facilitate dissolution. TR 700 s olutions used for MIC and t ime k ill e xperiments In ord er to obtain a final concentration of 20g/mL the following stocks were prepared on the day of use: stock 1: 10 mg TR stock 2: stock 3: Muller Hinton Broth Muller Hinton broth (MHB) and then autocl instructions. Saline solution To prepare a 0.9% (m/V) solution of sodium chloride (NaCl) in water, 9 g NaCl were weighed and dissolved to 1 L with double distilled water in a volumetric flask. T he solution was then autoclaved at 121 C for 30 min.

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45 MIC MIC experiments were done according to the Clinical and Laboratory Standards CLSI ) M07 microbroth dilution method ( Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically ). Experiments were done in triplicate, each on a different, consecutive day. A bacterial starting suspension was made using an overnight culture of each of the three tested strains. A bacterial suspension in sterile saline was adjust ed to a 0.5 McFarland standard (10 8 colony forming units ( CFU ) /mL) and 2 mL of this original suspension were diluted in 38 mL of sterile saline (10 7 CFU/mL). 96 well plates were prepared with blank cation adjusted MHB Well A1 contained the positive contro l (broth and inoculum, no study drug) ; well A2 contained a negative control (only broth). All wells were filled with 100 L MHB II except for well B1, which contained 200 L MHB II and well A2 which contained 110 L of broth only. The highest concentration of TR 700 was then added to well B1 (to attain a concentration of 4 g/mL). 100 L from well B1 were then diluted in well C1 containing 100 L blank MHB II and so forth, until the lowest concentration of 0.25 g/mL was reached. 100 L from this well were then taken and discarded to yield an even 100 L in all wells. Immediately after this the bacterial suspension was prepared and 10 L of the dilution containing approximately 10 7 CFU/mL were added to the wells. This yielded 110 L in each well (10 6 CFU/mL) evaporation and then placed into an incubator at 37 C, in ambient air, for 20 h. Following the incubation time, the plates were removed from the incubator and the parafilm was r emoved. Plates were read using light from below the plates. The positive

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46 and negative controls were checked for growth. Only when the negative control showed no growth and the positive control showed growth were the samples read. The first concentration (b eginning with the lowest) that showed no visible growth was taken as the MIC. Results are shown in Paragraph 0 Time Kill Experiments This model wa s used to investigate the effect of constant concentrations of T R 700 against bacteria as a function of time. The in vitro infection model consist ed of a 50 ml canted neck, vented cap, tissue culture flask containing 20 ml of Muller Hinton broth. The bacteria we re exposed to a series of concentrations (0.25, 0.5, 1, 2, 4, 8 and 16xMIC) of TR 700 without dilution in order to obtain time kill curves for 24 hours. Bacteria from an overnight culture were suspended in sterile saline as described in Paragraph 0 and the final conce ntration of bacteria was adjusted to 10 6 CFU/mL. Canted neck flasks were filled with Muller Hinton broth and inoculated with the bacterial suspension. Thereafter the inocula were incubated at 37 C in ambient air, under constant rocking for 2 hours. After that, the flasks were removed from the incubator shortly, a 20 L sample was taken from each flask and TR 700 added at the appropriate concentrations. The concentrations used for each individual strain are shown in Table 5 1 Samples (20 L ) wer e collecte d from the in vitro model at times zero and every two hours up to 24 hours. Bacterial counts we re determined by plating serial dilutions of the samples on 5% sheep blood agar (SBA) plates. The dilutions are made in sterile normal saline. Aliquots ( 50 L ) o f each dilution were plated in duplicate. The plates were incubated at 37 C for 2 0 24 hours before reading.

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47 The procedure wa s repeated 3 times per bacterial strain and dose. Positive con trols (with bacteria, no drug) we re run simultaneously in order to as sess the method. Following incubation, colonies we re counted in all readable plates that showed up to approximately 200 colonies. The number of colonies in e ach dilution tube at each time wa s determined by averaging the counts obtained. The data wa s entere d into Microsoft Excel spreadsheets. The mean values we re plotted against time. Bacterial counts obtained from the kill curves were fitted simultaneously to a mathematical model in Equation 5 2 ( 87 ) using Scientist 3.0 software (Micromath, Saint Louis, MO) Calculations and Transformations The following calculations were used to transform the counted colony forming units on an SBA plate into CFU/mL: 5 1 5 2 w here ks is the bacterial growth rate constant, N and Nmax are the bacterial and maximum achievable bacterial number, respectively and dk as a factor of describing delay in onset of kill. Results MIC Results from MIC determination on three consecutive days are shown in Table 5 2 The results show that TR 700 shows efficacy against all three strains Also, the domain V mutation in strain NRS271 confers a signific antly decreased susceptibility, while carriage of the cfr gene does not diminish the effect of TR 700.

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48 Time Kill Experiments Dates on which experiments were performed are shown in Table 5 3 Results for S. aureus ATCC33591, CM/05 and NRS271 are shown in Table 5 2 The graphical outcome for the kill curve s can be seen in Figures 5 2 to 5 4 For all three kill curves, the bacteria grew well in the medium until they reach a stagnant phase after about twelve hours. This growth stagnation occurs when the broth is depleted of nutrients and the bacterial populat ion has outgrown its space. Also, for the effective concentrations, there is a delayed kill observable. This was included in the model as a delay kill factor (dk). This could be due to the mechanism of action for this drug. Finally, for all three strains, a maximum effect, i.e. an effect after which an increase in concentration did not produce an increase in kill, was reached. TR 700 showed relatively slow, but sustained kill at the higher concentrations, over the period of 24 h. The total reduction in CFU/ mL is shown in figure 5 4. This effect continues after the observation period. However, as was shown in experiments comparing granulocytopenic and non granulocytopenic mice, the direct effect on the bacterium is not the only mechanism at play, as it seems. ( 53 )

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49 Table 5 1 Concentrations used for time kill experiments [ g/mL ] S. aureus strain (MIC) ATCC 33591 (0.25 g/mL ) CM/05 (0.5 g/mL) NRS271 (4 g/mL) Times MIC Concentration [g/mL] 0 (Growth Control) 0 0 0 0.25 0.0625 0.125 1 0.5 0.125 0.25 2 1 0.25 0.5 4 2 0.5 1 8 4 1 2 16 8 2 4 32 16 4 8 64 Table 5 2 Results of MIC Determinatio n Determined MIC [ g/mL ] Day of Analysis ATCC 33501 CM/05 NRS271 29 Feb, 2008 0.25 0.5 2 01 Mar, 2008 0.25 0.5 4 02 Mar, 2008 0.25 0.5 4 Reported MIC 0.25 0.5 4 Table 5 3 Dates of Time Kill Experiments S. aureus Strain Dat es of Experiment ATCC33591 04, 08*, 12 Mar, 2008 CM/05 05 Apr and 11 Apr, 2008 NRS271 12 Mar and 01 Apr, 2008 *One kill curve from 08 Mar, 2009 was discarded due to contamination Table 5 4. Maximum Reduction in Bacterial Numbers after 24 h S. aureus Strain Maximum Average Reduction [log10 CFU/mL] ATCC33591 1.63 CM/05 0.63 NRS271 0.47

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50 Figure 5 2 TR 700 against S. aureus ATCC33591 (Mean, standard deviation ( SD) ) Figure 5 3. TR 700 against S. aureus CM/05 (Mean, SD)

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51 Figure 5 4. TR 700 against S. aureus NRS271 (Mean, SD)

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52 CHAPTER 6 IN VITRO MICRODIALYSIS Objective The aim of this study wa s to determine the ability of TR 700 to cro ss the microdialysis (MD) membrane using two dose ranging, in vitro microdialysis experiments. The two experiments were done using the extraction efficiency (EE) and retrodialysis (RD) methods. In both of these methods the recovery percent (R%) was determi ned. The R% had to be more than 10% if microdialysis wa s to be used for sampling TR 700 from soft tissues. Microdialysis and Recovery Microdialysis (MD) is a very useful tool for sampling from soft tissues, the most common site of infection, because only t he free, pharmacologically active drug is able to pass through the probe membrane. In this technique, the microdialysis probe is placed into the tissue of interest and continuously perfused with a physiological solution (perfusate). Based on diffusion the drug passes from the tissue into the probe and is collected (dialysate). Ideally, an absolute equilibrium between the tissue and perfusate will be established. In reality, due to the fact that the MD probe is perfused at a constant flow rate of 1.5L/min, an absolute equilibrium will not be reached. The ability of the drug to pass through the membrane and establish equilibrium at this flow rate is established before or after the experiment It is termed the recovery (R) and this value has to be known to bac k calculate the actual concentration at the sampling site, C tissue from the concentration in the dialysate, C dialysate This is done by using the following equation: 6 1

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53 In microdialysis, the sampling time is determined by the flow rate. A higher flow rate would lead to a shorter sampling time since there is a minimum volume requirement, but the recovery is decreased because there is not enough time for equilibration between the solution inside the probe and th e surrounding media to occur. Therefore, a compromise has to be made between sample volume and flow rate. Depending on the sensitivity of the assay, the sample volume should not be smaller are collected. The equilibration period is determined by the dead volume of the MD probe tubing which can be ca was performed using five different concentrations, all of the tubing has to be completely flushed before the sampling procedure can start. The equilibration time is the calculated as de ad volume divided by flow rate. Extraction E fficiency M ethod ( EE ) containing analyte solution, starting with the lowest concentration. Drug will diffuse from the calibration tube into the MD probe, and the dialysate is collected for analysis. Each sample is collected for 20min after the end of a 15min equilibration period. To ensure that the prepared solution is the concentration expected within the calibratio n tube, two samples are taken from this tube, one before the sampling period and one after. It is important to sample from the calibration tube since it is critical to know the actual concentration in the tube to perform the calculations. Additionally, the two samples can be compared to see if the concentration within the calibration tube was consistent

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54 throughout the sampling period. The same procedure is done for the remaining five samples. After the highest concentration is completed the probe is flushed for one hour percent recovery, R%, for the EE method is calculated as follows: 6 2 Retrodialysis M ethod ( RD ) In the RD method, the syringe cont ains the analyte solution that is pumped is place d into a calibration tube that i The analyte diffuses out of the probe into the calibration tube. The loss of analyte through the membrane is then determined from the concentration in the dialysate. Samples are taken for 20min after the end of the 15min equilibration period. To ensure that the prepared solution has the concentrat ion expected within the syringe, a sample is taken from the syringe after the sampling period. Additionally, two samples are taken from the calibration tube, one before and one after the sampling period, to see if the concentration within the tube was cons istent throughout sampling. Again the lowest concentration i s sampled first. In this method, the calibration tube, which contains a small amount of analyte after the sampling period, has to be exchanged with a new tube containing fresh blank lactated Ringe is done for the remaining five samples. After the highest concentration was sampled the probe is we re

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55 performed in triplicate. The percent recovery R%, for the RD method wa s calculated as follows: 6 3 Experimental Procedures Chemicals and Equipment TR 700 (Lot numbers GH 8S 16 2/DP 90 29 5, received 08 Feb 2008 and DUG AE 129 (3)/ DD 90 54 14, received 08 Apr 2008) used for the HPLC/UV method was obtained from Trius Therapeutics, Inc. The compound was stored at 2 8 C in its original shipping vial and protected from light. See structure below. All reagents were of HPLC grade unless otherwise stated. They were obtained from t he indicated sources or equivalent suppliers. Double distilled water, Filtered in house by Corning AG 3 Acetonitrile, Fisher Scientific A998 4 DMSO, Fisher Scientific BP231 1 The f ollowing pieces of equipment or e quivalent substitutes were used. Micropipettes, Eppendorf, Westbury, NY Repeater Pipette, Eppendorf, Westbury, NY Microcentrifuge tubes, Fisherbrand, #02 681 272 Centrifuge tubes, Corning, #430052 Pipette tips, Fisherbrand, #02 707 304 Pipette tips, US Sci entific, #1111 2021 Volumetric flask, Pyrex, #5642 C 18 column, Supelco Discovery C18 Bellefonte, PA, #504955, ID: 9157 7 03 HPLC Guard column, pellicular C18 packing, In house packing Polypropylene inserts conical (200l,) Sun Sri, Rockwood, TN, #600 013 Waters 515 HPLC Pump Waters 717 Plus LC Autoinjector

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56 Waters Empower software Weighing balance, Mettler Toledo, Hightstown, NJ, USA AB104 Double distilling apparatus, Corning, NY # AG 3 Balance, Mettler AE240 Vortex, Kraft Apparatus Inc. Model PV 5 Autosampler vials, S un Sri 200 046 Aluminum seals, Sun Sri 200 100 Agilent 1100 Series HPLC D iode A rray D etector G1315B Autosampler, G1329A HPLC Column Oven, G1316A Degasser, G1379A Quaternary Pump G1311A Work Station, hp Compaq p4 Agilent Technologies Agilent ChemStation for LC and LC/MS Systems Syringes, Becton Dickinson 309603 Syringe Pump, Harvard Apparatus Model 55 4150 Heated Stir Plate, Fisherbrand Isotemp Thermometer, Fisherbrand 76mm Immersion 14 997 Microdialysis Probes, CMA 60 P000002 No glass and plastic ware were reused. Reagent Preparation TR 700 standards and quality control solutions The preparation of TR 700 standards and quality controls was done as follows: TR 700 was provided as pure white to off white powder. 10 mg were weighed and dissolved in 10 mL of DMSO using a volumetric flask. Thereafter, solutions were made The standard curve ranged from 50 to 3200 ng/mL and q uality controls of 50, 100, 400 (only for retrodialysi s experiment), 1600 and 2400 ng/mL were used. For the mobile phase, 650 mL ddH2O and 350 mL ACN were measured in individual graduated cylinders and mixed. After stirring for five to ten minutes the mixture was degassed in a helium gas atmosphere for 10 m inutes minimum.

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57 Sample preparation Calibration and quality control samples. Seven calibration standards of TR 700 (3200, 1600, 800, 400, 200, 100 and 50 ng/mL) were prepared. This wa s done by preparing a 1mg/mL stock solution of TR 700 in DMSO. From here s erial dilutions we re Quality control samples were prepared in the same way, but from individual stock solutions. The concentrations selected were chosen because the expected maximum conc entration in soft tissues is approximately 1000 ng/mL. Dialysate samples During the sampling period approximately 45 L of sample were collected. For the extraction efficiency experiment samples were diluted with an ion, except for the 100 ng/mL perfusate sample from the third replicate, which was not diluted. For the retrodialysis method, no sample was diluted. Samples were also taken from the tube containing the calibration solution before and after dialysis. To get the true concentrations for the extraction efficiency method, observed values from dialysate samples were multiplied by two, except for the one non diluted sample. Microdialysis Setup Before the experiments we re started each MD probe has to be checked for functionality. To do so, the inlet of the MD probe wa s connected to a syringe containing wa s then, carefully, flushed manually. The probe was considered functional and ready to use when no liquid drops appear ed on the MD probe membrane. The solution should only exit the probe from the outlet tubing. To control temperature, a beaker filled with water was placed on a heated stir plate and the system was maintained at 37 o C. During setup a 5 mL syringe wa s filled ei ther

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58 the enclosed air wa s cleared from the syringe. The syringe wa s put in place on the syringe pump and fastened. The pump was then connected to the inlet of the probe a nd wa s (RD method) or analyte solution (EE method). The dialysate wa s collected into a 0.5 mL microcentrifuge tube covered with parafilm, which help ed to fix the outlet tubing from the microdialysis probe in place and prevent ed evaporation. Sample Analysis For extraction efficiency the HPLC system wa s the Agilent 1100 series which in cludes a quaternary pump (G1311A), a degasser (G1379A), a column oven (G1316A), an autosampler (G1329A), a guard column with pellicular C18 packing, done in house, a Supelco Discovery C18 column and a DAD detector (G1315B). The work station for data retent ion wa s a hp Compaq p4 using ChemStation software. Retrodialysis samples were analyzed using a Waters system consisting of a Waters 515 HPLC pump, a Waters 717 Plus LC autoinjector, a guard column filled in house with C18 packing, a Supelco Discovery C18 absorbance detector and Waters Empower software. HPLC Pump Conditions Mobile Phase: 65% Distilled Water, 35% Acetonitrile Flow rate: 1.0 mL/min Retention Time: 4 5 min Autosampler Conditions Injection Volume: 10l Run time: 6 min

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59 Injections per vial: 1 (Blanks may be injected more than once) Samples per vial: 1 Temperature: 4C (Agilent system), no temperature control on Waters system Detector Conditions Wavelength: 300nm Reference Wavelength: 360nm (only for Waters system) H PLC procedure The test samples were run with a calibration curve and 3 sets of quality control standards at 5 levels (LLOQ, low, mid 1, mid 2, high) placed throughout the run. Results Method Va lidation Acceptance Criteria and Results Analytical runs were accepted if the correlation coefficient for the calibration curve (R 2 ) was at least 0.98, the calculated concentration of the low standard was within 20% of the nominal concentration, the calcul ated concentrations for all other standards were within 15% of their nominal concentrations, the low, mid range and high quality controls (QC) were within 15% (20% at the LLOQ) of their nominal concentrations and at least three out of five of the QCs met t he acceptance criteria at each concentration. A calibration curve ranging from 50 to 3200 ng/mL was used. Quality controls of 50, 100, 1600 and 2400 ng/mL were interspaced with samples. For the retrodialysis sample analysis done using the Waters system an additional quality control at 400 ng/mL was added. The analysis was performed using weighted linear regression (1/X2) to achieve a better curve fit for smaller concentrations. For extraction efficiency, which was done using the Agilent system, the slope, i ntercept and R2 value of the curve were 0.035, 0.43 and 0.990, respectively. For retrodialysis, which was done using the

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60 Waters system, the slope and intercept for the calibration curve were 51.22 and 339.9, respectively. Also, the R2 value was 0.994. Th e results can be studied in Tables 6 1 and 6 2. In vitro Microdialysis Results In this experiment two in vitro microdialysis methods were used, the extraction efficiency (EE) and the retrodialysis (RD) methods. Both methods are acceptable to characterize how the compound interacts with the MD membrane and if the compound can freely pass through the membrane. These in vitro experiments were done as a preliminary study to an in vivo experiment. The results are summarized in Table 6 3. There are some fundamen tal differences in the setup of both experiments which can explain the difference in R%. The retrodialysis method is the same method which was used in the in vivo experiment for MD probe calibration. Conclusions This experiment confirmed that TR 700 has th e ability to freely cross the microdialysis membrane. The R% was well over 10% which was the minimum requirement, and therefore it was concluded that microdialysis can be used as a sampling technique to obtain a PK profile.

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61 Table 6 1 Performance of Quality Controls for Extraction Efficiency Experiment Quality Controls Concentration (g/mL) Observed Concentration [ng/mL] Mean Observed Concentration [ng/mL] SD CV% Accuracy% Mean Accuracy % LLOQ 50 51.06 49.74 2.19 4.41 102.11 99.48 LLOQ 50 52.05 1 04.11 LLOQ 50 48.17 96.34 LLOQ 50 50.94 101.88 LLOQ 50 50.08 100.17 LLOQ 50 46.15 92.29 L 100 99.18 97.10 2.14 2.20 99.18 97.10 L 100 95.35 95.35 L 100 98.98 98.98 L 100 98.95 98.95 L 100 94.81 94.81 L 100 95.30 95.30 M 1600 1685.58 1636.54 98.28 6.01 105.35 102.28 M 1600 1552.99 97.06 M 1600 1689.49 105.59 M 1600 1476.47 92.28 M 1600 1692.26 105.77 M 1600 1722.43 107.65 H 2400 2293.97 2294.70 156.54 6.82 95.58 95.61 H 2400 2302.39 95.93 H 2400 2531.37 105.47 H 2400 2048.76 85.37 H 2400 2241.23 93.38 H 2400 2350.46 97.94

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62 Table 6 2 Performance of Quality Controls for Retrodialysis Experiment Quality Controls Concentration (g/mL) Observed Concentration [ng /mL] Mean Observed Concentration [ng/mL] SD CV% Accuracy% Mean Accuracy% LLOQ 50 52.7 52.10 0.87 1.67 105.4 104.2 LLOQ 50 51.1 102.2 LLOQ 50 52.5 105.0 L 100 94.1 94.63 0.84 0.89 94.1 94.6 L 100 94.2 94.2 L 100 95.6 95.6 M1 400 377.2 396.60 17.50 4.41 94.3 99.2 M1 400 401.4 100.4 M1 400 411.2 102.8 M2 1600 1720.4 1765.20 66.98 3.79 107.5 110.3 M2 1600 1733 108.3 M2 1600 1842.2 115.1 H 2400 2521.9 2578.47 59.21 2.30 105.1 107.4 H 2400 2573.5 107.2 H 2400 264 0 110.0

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63 Table 6 3 Results of Extraction Efficiency and Quality Control Experiments Concentratio n (g/mL) EE Method R% (average of triplicate) SD CV% RD Method R% (average of triplicate) SD CV% 100 40.86 57.15 4.64 8.13 250 89.86 17.22 19.17 76. 16 3.69 4.84 500 78.35 7.68 9.80 81.43 0.74 0.91 1000 67.97 2.97 4.38 86.83 2.13 2.45 2000 67.02 2.49 3.72 85.30 5.82 6.83 Average 68.81 77.37 SD 18.16 12.03 CV% 26.39 15.55

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64 CHAPTER 7 AN OPEN LABEL, SINGLE DOSE, MICRODIALYSIS AND PH ARM ACOKINETIC STUDY OF TR 701 IN NORMAL, HEALT HY ADULTS Objectives The part of an antibiotic dose that is going to show efficacy is the fraction that will show up at the site of action. Both the FDA and the EMA have issued guidelines to that effect and the de finition of bioavailability ifs the rate and extent of drug becoming available at the site of action, or biophase. Nonetheless, tissues are not easily accessible. The objective of the study was to assess the tissue distribution of TR 701 into subcutaneous adipose and skeletal muscle tissue of healthy volunteers, after administration of a single oral dose by applying the microdialysis sampling technique. Study Design The study was planned as a single dose, open label study. No randomization was done. The ra tionale for a microdialysis study was to measure the amount of drug that becomes available at the site of action, in this case skin and soft tissue, after a single oral dose of 600 mg TR 701. Since TR 701 was reportedly, very quickly converted to TR 700, t he active ingredient, prodrug concentrations were not measured in vivo The FDA, in its critical path document ( 89 ) suggests the use of concentrations at the site of action as preferred for exposure response analysis. Yet, as mentioned before tissue concentrations were not always easy to generate. The method of microdialysis as a simple, relatively minimally invasive sampling tool is well suited to assess these concentrations. The study was done in two phases a pilot phase and a main phase. Pilot Phase During the p ilot phase of the study, the feasibility of using the microdialysis in vivo and the duration for the washout period was determined. To achieve this, healthy

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65 volunteers checked in at the General clinical research center on the morning of the microdialysis p rocedure and two microdialysis probes were implanted. Both probes were initially perfused with lactated solution to let the tissue rest after the trauma of probe insertion. Afterwards both probes were connected to syringes containing a solution wi th a known concentration of TR 700 of 2 g/mL. The probes were perfused for 30 minutes at a perfusion speed of 1.5 L/min. The resulting exposure to TR 700 was thus in the range of 0.36 g from both probes. The perfusion with a solution of known concentrat ion was intended to assess, whether the recovery rate that was observed in vitro was also achievable in vivo which was the case here. The washout duration was observed by reconnecting the syringes containing concentration of TR 700 in the tissue decline over a period of three hours. Immediately following that, participants underwent final safety laboratory tests and physical examination and were then free to leave. Main Phase The main part of the study was des igned similarly for the first hours. Subjects would check in at the General Clinical Research Center ( GCRC ) the evening before the study procedures were to begin so that they would be well rested and check in assessments like medication consumption and vi tal signs were made. On the morning of the procedure, two probes were inserted into the upper thigh and initially perfused give the tissue time to settle. Following that, the retrodialysis procedure was performed, whereby a 2 g/mL solution of TR 700 was perfused through the microdialysis probes for the purpose of assessing the recovery of TR 700 from the solution during sample analysis.

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66 Immediately after the retrodialysis perfusion was finished the probes were reconnected observed. At the end of the washout period a sample was taken to measure the concentration of TR 700 shortly before doing, then a 600 mg oral dose of TR 701 in form of three capsules 20 0 mg was given and observed. Immediately after the dose was swallowed the microdialysis was started at time point 0 and samples were collected every 20 minutes for 12 hours. Plasma samples were taken at 13 time points for 24 hours. Protein binding was also measured for each subject individually in two samples. Once the microdialysis sampling period was finished, probes were discontinued from the microdialysis pump and subjects were free to move around. Subject Selection Criteria Subjects who met all of the inclusion criteria and none of the exclusion criteria were enrolled into the study. The eligibility was determined by a medical doctor during the screening portion of the study and then again in the morning before any procedures were begun, to add an extra level of safety. Inclusio n criteria Males or females, between 18 and 50 years of age inclusive; Body mass index (BMI) of 20 to 29 kg/m 2 inclusive; In good health, determined by no clinically significant findings from medical history, 12 lead electrocardi ogram ( ECG ) clinical laboratory evaluations and vital signs; Corrected QT interval ( QTc ) females; Resting heart rate, by ECG, in the range of 45 bpm to 100 bpm; Agree not to consume any products containing tobacco, alcohol, quinine, grapefruit, caffeine, or high levels of tyramine within 24 hours be fore dosing of

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67 study drug until after completion of all PK assessments (approximately 24 hours after study drug dosing). Agree not to use any other medication (prescription or nonprescription) [including but not limited to vitamins, minerals, antacids (inc luding H2 blockers and proton pump inhibitors), analgesics, anti inflammatory drugs, dietary supplements and phytotherapeutic/herbal/plant derived preparations], within 1 week before TR 701 dosing until after the microdialysis and last blood collection. Ad ministration of hormonal contraceptives, hormonal replacement therapy and paracetamol (acetaminophen, for symptomatic treatment of pain, etc.) will be allowed as described in Section 6.9, Concomitant Medications. Subjects with normal renal function ( Creati nin clearance ( CrCl ) 80 mL/min as determined by the Cockcroft Gault equation presented below). Men: 7 1 Women: 0.85 x above value Female subjects Exclusion criteria History or clinical manifestations of metabolic, hepatic, renal, hematological, pulmonary, cardiovascul ar, gastrointestinal, urological, neurological, or psychiatric disorders that is significant in the opinion of the Investigator History of hypersensitivity or allergies to any drug compound, including Zyvox (linezolid) unless approved by the Investigator H istory of gastric or duodenal ulcer within 1 year before enrollment Has known or suspected hypersensitivity or intolerance to heparin, if an indwelling cannula (e.g., heparin lock) is used Relevant history or any current conditions that might interfere wit h TR 701 absorption, distribution, metabolism, or excretion

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68 Recent febrile illness (less than 72 hours before the first intake of study medication). Significant blood loss (300 mL) or donation of blood within the 60 days before the Screening visit History of alcoholism or drug abuse within 5 years prior to Check in; Women who are pregnant or breast feeding Employees of the Investigator or study center, with direct involvement in the proposed study or other studies under the direction of that Investigator or study center, as well as family members of the employees or the Investigator. Use of any foods or beverages with high levels of tyramine within 24 hours prior to the microdialysis procedure, unless deemed acceptable by the investigator. Use of any investi gational drug within 30 days prior to study entry and during the entire study.

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69 Flow Chart of Study Procedures Table 7 1. Pilot Phase Timeline Study Procedures Screening ( 21 to 1) Day 1 Study Completion Visit (Day 7 14) Confined to Clinic X Infor med Consent X Demographics X Medical History X X Physical Exam X X 12 Lead ECG X Vital Signs c X X Microdialysis probe insertion X TR 700 Administration X Microdialysis probe removal X Dialysate collection X AE Evaluations X X C linical Laboratory Evaluations X X Pregnancy Test X X

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70 Table 7 2. Main Phase Timeline Study Procedures Screening ( 21 to 2) Check in (Day 1) Day 1 Day 2 Study Completion Visit (Day 7 14) Confined to Clinic X X X Informed Consent X Demogr aphics X Medical History X X Physical Exam X X X 12 Lead ECG X X X Vital Signs X X X X AE Evaluations X X X Dose j X Microdialysis probe insertion X Microdialysis probe removal X Dialysate collection X PK Blood Sample s k X X Blood samples for Plasma Protein Binding Analysis X Clinical Laboratory Evaluations X X X Pregnancy Test X X X X

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71 Pharmacokinetic Measurements and Pharmacodynamic Assessments Based on the individual concentration time data, given actu al sampling times, the following PK parameters of TR 700 in plasma and dialysate were estimated: C max T max AUC last (AUC from time 0 to the last measured time point) AUC z (terminal phase elimination rate constant) t 1/2 CL/F (clearance adjusted divi ded by bioavailability) and V z /F (volume of distribution over bioavailability) As a measure of tissue penetration, the ratios of free tissue AUC to free plasma AUC ( f AUC tissue / f AUC plasma ) ratios were determined for subcutaneous ( s.c. ) adipose and skeleta l muscle tissues. The plasma concentration was corrected to account for plasma protein binding. If deemed necessary, additional PK data analyses would have been performed. For the Pilot Study, only exogenously administered TR 700 recovery data are presente d. TR 700 plasma concentration versus time profiles for each subject were plotted, mean plasma concentration time profiles were plotted, and plasma and tissue (s.c. tissue and skeletal muscle) concentration data at each timepoint were summarized by descrip tive statistics. Pharmacokinetic parameters, pharmacodynamic estimates, and protein binding measurements (% bound and unbound) were summarized with mean, median, geometric mean, minimum, maximum, standard deviation (SD), and coefficient of variation. For p lasma and microdialysis samples that were drawn at the same time, the mean and SD for the concentration ratio of TR 700 in tissue versus plasma was plotted versus time. For the Pilot Study, only exogenously administered TR 700 recovery data are presented. Minimum, maximum, mean, and median values were determined and standard deviations were calculated. No formal statistical analyses were planned. Non

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72 compartment a l pharmacokinetic calculations were performed, if appropriate, using the commercial software Win Nonlin (Pharsight Corporation, Version 5.2) Pharmacodynamic parameter AUC/MIC in plasma corrected for protein binding, in s.c. adipose tissue and skeletal muscle were summarized with mean, median, minimum, maximum standard deviation and coefficient of va riation. Results Pilot Phase The primary objectives of the Pilot Study were to investigate the feasibility of using the microdialysis technique to measure TR 700, a biologically active moiety of TR 701 prodrug, by determining its recovery by the microdialy sis probe and to determine the duration of the washout period necessary to ensure clearance of all calibration solution from the tissues and microdialysis probes before oral dosing. The results of the pilot study indicate that TR 700 recovery from the micr odialysis probe was approximately 90%. The originally estimated 3 hr washout period had to be extended to 4 hrs because levels of TR 700 found in the washout dialysate were still close to the lower level of quantitation after 3 hrs. Main Phase Data sets of all twelve Main Study subjects were included in the PK analysis. For subject 007 adipose data were missing due to a broken probe. For subject 004 the 140 min muscle dialysate sample was missing. For subject 005 the 140 min dialysate sample was missing. Fo r subject 008 time point 220 min adipose and muscle samples were missing. Table 7 3 shows the demographics for the participants in the main study Mean C max SD was 5373 1514, 664 164, and 742 155 ng/mL for total plasma, subcutaneous adipose and ske letal muscle tissue, respectively. Interstitial fluid

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73 concentrations of TR 700 are thus close to free, unbound plasma concentrations, based on the measured mean plasma protein binding of 87.2% and a free fraction of 0.13 (free C max = 5375; 5375 0.13 free fraction = 699 ng/mL). T max values were 2.42 1.12, 4.27 2.36, and 3.72 1.5 hrs for plasma, adipose and muscle tissue, respectively. The in plasma, adipose and muscle tissue was 8.88 2.4, 19.7 25.5, and 13.0 11.6 hrs, respectively. Ha lf life in tissues showed a great variability, with median values being close to those in plasma (9.22 (range 5.98 85.9) hours in adipose tissue and 9.59 (range 6.15 48.2) hours in muscle tissue). Mean CL/F and V z /F values were 9.46 2.91 L/hr and 113 19.3 L, respectively. Total TR 700 plasma concentrations and protein binding were consistent with results from a previous study. Mean f AUC tissue / f AUC plasma values were 1.1 0.2 and 1.2 0.2 for adipose and muscle tissues, respectively. The PD paramete r f AUC /MIC for subcutaneous adipose and skeletal muscle tissue was calculated for each subject individually and summarized using descriptive statistics, including mean, SD, minimum, median, maximum, geometric mean, and coefficient of variance. Datasets fo r plasma and skeletal muscle TR 700 concentrations were available from 12 healthy volunteers. For subcutaneous adipose tissue 11 datasets were available. The CV% for AUClast was 25.8, 24.3 and 18.7% for plasma, adipose and muscle tissue, respectively. For Cmax the CV% values were 28.2, 24.6 and 20.9%, respectively. For calculation of f AUC tissue / f AUC plasma the AUC0 12 in tissues and plasma was used, this allowed the use of only measured data.

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74 Table 7 3. Study Demographics Demographic Variable Pilot Study N = 3 Main Study N = 12 Mean age in years (range) 21 (19 to 22 years) 24 (19 to 29 years) Mean weight in kg (range) 65.9 (63.0 to 70.3 kg) 71.7 (52.5 to 95.9 kg) Mean height in cm (range) 170.5 (163.8 to 175.0 cm) 171.5 (160.0 to 191.9 cm) Body mass in dex ( BMI ) in kg/m2 (range) 22.7 (21.1 to 24.0 kg/m2) 24.1 (19.7 to 28.7 kg/m2) Gender (n[%]) Male --5 (41.7%) Female 3 (100.0%) 7 (58.3%) Ethnicity (n[%]) Hispanic or Latino --1 (8.3%) Not Hispanic or Latino 3 (100.0%) 11 (91.7%) Race (n [%]) White 3 (100.0%) 10 (83.3%) Black or African American --2 (16.7%)

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75 Figure 7 1. Mean (SD) total plasma concentrations of TR 700 after a 600 mg oral dose

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76 Figure 7 2 Mean (SD) free adipose ( ) and muscle ( ) concentrations of TR 700 aft er a 600 mg oral dose

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77 Figure 7 3. Individual total plasma concentrations of Torezolid after a single 600 mg oral dose

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78 Table 7 4 Summary of Mean (SD) TR 700 Plasma, Adipose Tissue, and Muscle Tissue Pharmacokinetic Parameter Data (Main Study) Para meter Plasma Adipose Tissue Muscle Tissue C max (ng/mL) 5373 (1514) 664 (164) 742 (155) T max (hr) 2.42 (1.12) 4.27 (2.36) 3.72 (1.50) AUC 0 12 (ng*h/mL) 38813 (7548) 5266 (1279) 5948 (1109) AUC last (ng*h/mL) 57105 (14740) 5266 (1279) 5948 (1109) AUC (ng*h/mL) 70021 (24784) 16634 (15802) 14582 (10494) f AUC 0 12 (ng*h/mL) 4959 (1093) NA NA f AUC last (ng*h/mL) 7276 (1906) NA NA f AUC (ng*h/mL) 8895 (3059) NA NA t (hr) 8.88 (2.40) 19.7 (25.5) 13.0 (11.6) z (hr 1 ) 0.08 (0.02) 0.07 (0.04) 0.07 (0.03) CL/F (L/hr) 9.46 (2.91) NA NA V z /F (L) 113 (19.3) NA NA f AUC tissue / f AUC plasma NA 1.08 (0.22) 1.22 (0.18) NA: not applicable. Table 7 5 Summary of Mean (SD) Unbound Plasma Adipose Tissue, and Muscle Tissue Pharmacodynamic Parameter Data Corrected for Protein Binding (Main Study) Parameter Plasma Adipose Tissue Muscle Tissue f AUC /MIC 0.25 g/mL 35.6 (12.2) 66.5 (63.2) 58.3 (42.0) 0.5 g/mL 17.8 (6.12) 33.3 (31.6) 29.2 (21.0) 4 g/mL 2.22 (0.76) 4.16 (3.95) 3.65 (2.62)

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79 CHAPTER 8 PHARMACOKINETIC/PHAR MACODYNAMIC MODELLIN G Modelling of Pharmacokinetic Data Population pharma cokinetic estimates of free, unbound plasma, subcutaneous adipose and muscle tissue concentrations were assessed using NONMEM VI software (ICON, Ellicott City, MD). Based on Figures 7 1 to 7 3, most subjects either showed a one or a two phasic concentrat ion time course in plasma and mostly a one phasic course in the tissues. Therefore, to model the concentrations in plasma and tissues simultaneously, a two compartment model was developed. See Equations 8 1 and 8 2 below 8 1 8 2 The model was fit in NONMEM VI using ADVAN6, which allows the user to write differential equations, instead of using the pre programmed prediction tools. NONMEM was run using the Perl based interface, Perl speaks NONMEM (PsN). ( 43 ) A covariate

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80 analysis was done by assessing the impact of covariates on inter individual errors and parameters. As expected and reported in literature ( 67 ) due to the fact that this population is comprised of twelve individuals, which were selected on the basis of a stringent protocol, elucidating any covariate impact was difficult. The specific compartments were labeled by using a FLAG indicator. In this case FLAG= 1, FLAG=2, FLAG=3 indicates the central, muscle and adipose compartment, respectively. The fits of the model for the free plasma concentrations are shown in Figure s 8 1 to 8 3 The fits for the tissue compartments are shown in Figures 8 4 to 8 9. The fits for plasma tissue are generally better than those for the free tissues. The fit of the model was assessed using visual methods and a visual predictive check (VPC) (see Figure 8 10). The VPC showed that, even though the variability is high, all predictions fall within the 90% confidence interval at the higher concentrations. Lower concentrations, particularly in the distribution phase into muscle and adipose tissues are not very well predicted by the model. The results of the model validation by bootstrappin g the model with 500 samples of the data are shown in Table 8 2. The bootstrap had a success rate of 93.2%, meaning that for most of the data sets that were newly samples, the estimates could be found and the objective function could be minimized. Modellin g of Kill Curve Data The resulting counts from the kill curve experiment were saved in an Excel spreadsheet and analyzed using Scientist software. To describe the bacterial growth or kill over time, the following modified Emax model was employed: 8 3

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81 The results are shown in Table 8 2 and in Figures 8 11 to 8 13. The results show that the growth rate constant for all three strains is very similar and that the EC50 correlates well with the determined MIC. The model predicted the k ill and growth curves well.

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82 Figure 8 1. Individual fits for free plasma concentrations *IPRED=individual predictions, PRED=population predictions, DV=observation

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83 Figure 8 2. Observations, individual and population predictions for free plasma con centrations. Figure 8 3. Observations versus population and individual predictions for free plasma concentrations

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84 Figure 8 4. Individual fits for muscle concentrations IPRED=individual predictions, PRED=population predictions, DV=observation

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85 Figu re 8 5. Observations versus population and individual predictions for muscle concentrations. Figure 8 6. Observations, individual and population predictions for muscle concentrations.

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86 Figure 8 7. Individual fits for adipose concentrations IPRED=ind ividual predictions, PRED=population predictions, DV=observation

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87 Figure 8 8. Observations versus population and individual predictions for adipose concentrations. Figure 8 9 Observations, individual and population predictions for adipose concentra tions.

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88 Table 8 1. Estimated model parameters Parameter Estimate SE CV% TVCL [L/h] 75.9 8.56 11.3 TVV2 [L] 592 55.2 9.3 TVK23 [1/h] 0.21 0.11 53.2 TVKA [1/h] 0.94 0.16 16.6 TVK32 [1/h] 0.73 0.06 7.8 TVMSC 1.39 0.05 3.7 TVADP 1.24 0.08 6.2 ETA(1) (o n V2) 0.11 0.04 40 ETA(2) (on K23) 0.01 0.02 190 ETA(3) (on K32) 2.23 1.26 57 ETA(4) 0.51 0.14 28 EPS(1) 0.15 0.03 17 EPS(2) 0.01 0.00 25 EPS(3) 0.02 0.01 45 Table 8 2. Bootstrap results (N=500) Parameter Estimate SE 95% CI TVCL [L/h] 76.6 8.92 ( 59.2, 94.1) TVV2 [L] 630 126 (382.3, 877.7) TVK23 [1/h] 0.21 0.17 ( 0.1, 0.5) TVKA [1/h] 0.99 0.20 (0.6, 1.4) TVK32 [1/h] 0.77 0.12 (0.5, 1) TVMSC 1.39 0.06 (1.3, 1.5) TVADP 1.24 0.07 (1.1, 1.4) ETA(1) (on V2) 0.10 0.04 (0, 0.2) ETA(2) (on K23) 0.0 2 0.04 (0, 0.1) ETA(3) (on K32) 2.34 1.42 ( 0.4, 5.1) ETA(4) 0.48 0.15 (0.2, 0.8) EPS(1) 0.14 0.03 (0.1, 0.2) EPS(2) 0.01 0.00 (0.01, 0.02) EPS(3) 0.02 0.01 (0.01, 0.04)

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89 Figure 8 10. Conditional w eighted residuals versus time (independent variabl e) for all compartments.

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90 Figure 8 11. Visual predictive check results for all tissues

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91 Figure 8 12. Curve fits for S. aureus ATCC33591

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92 Figure 8 13. Curve fits for S. aureus CM/05

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93 Figure 8 14. Curve fits for S. aureus NRS271 Table 8 3 Parameter estimates for all three S. aureus strains Strain/ Parameter S. aureus ATCC 33591 S. aureus NRS 271 S. aureus CM/05 EC50 [g/mL] 0.07 1.32 0.14 MIC [g/mL] 0.25 4 0.5 ks [h 1] 0.89 0.82 0.89 Nmax [CFU/mL] 3.85*10 9 1.04*10 10 3.85*10 9 Ema x [h 1] 1.02 0.9 1.02 Dk [h 1] 1.65 3.04 1.65 h 1 1.4 1

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94 CHAPTER 9 DISCUSSION AND CONCLUSION In recent years, the technique of microdialysis has become available to access otherwise hard to come by free drug concentrations. In this study microdialysi s was used to evaluate the extent and rate of distribution of TR 700, the active moiety of the prodrug TR 701, into the interstitial space fluid of subcutaneous adipose and skeletal muscle tissues. The objectives of the Main Study were 1) to measure the TR 700 distribution into subcutaneous adipose tissue and skeletal muscle interstitial fluids after a single oral dose of 600 mg TR 701, 2) to determine the concentration versus time profiles of free TR 700 in s.c. adipose tissue and skeletal muscle using the microdialysis technique and in plasma after administration of a single oral dose of 600 mg TR 701, and 3) to further assess the safety and tolerability of TR 701 after a single oral dose of 600 mg administered in fasting conditions. TR 700 appeared in bot h subcutaneous adipose and muscle tissue dialysate samples after approximately 60 min of oral TR 701 administration and was followed for 12 hrs after dosing. The measured plasma levels were consistent with previous data of a single ascending dose study. P rotein binding was assessed by determination of plasma ultrafiltrate TR 700 concentrations using HPLC MS/MS and was consistent with already existing data. Plasma concentrations adjusted for protein binding were similar to those found in s.c. adipose and mu scle tissues, indicating that free, unbound TR 700 is able to penetrate into the tissues. Mean ratios of f AUC tissue / f AUC plasma are 1.08 and 1.22 for s.c. adipose and muscle tissues, respectively.

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95 The results from this study show, that TR 700 concentrations in the two observed tissues are comparable to its unbound concentration in plasma. Thus, for future clinical studies, it is reasonable to use free, unbound plasma concentrations as surrogate for unbound concentrations in ISF of s.c. adipose and skeletal m uscle tissues. While the microdialysis technique is a means to sample from ISF of tissues of interest, it is not, by itself, a means to measure intracellular concentrations. Studies have shown, that, especially for drugs that penetrate inside the cell (e.g macrolides, tigecycline), unbound concentrations in plasma and interstitial space fluid of infected tissues are not necessarily indicative, as a result, the AUC/MIC targets are smaller than expected based on free, unbound plasma concentrations. One examp le for this is tigecycline, where plasma concentrations are significantly lower than intracellular concentrations. ( 20 68 ) Another limitation of this study is that tissue concentrations were not measured in infected tissue. AUC/MIC values achieved in healthy tissue may differ from the AUC/MIC targets in infected tissues based on potential differences in physiol ogic parameters in severely ill patients compared to healthy individuals. This study provides an assessment of the time course and extent of tissue exposure of TR 700, the active moiety of prodrug TR 701. More studies are needed to fully understand the int racellular concentrations and their significance in the determination of appropriate breakpoints and therapeutic outcome. In conclusion, the study showed that unbound concentrations of TR 700 in the interstitial space of subcutaneous adipose and skeletal m uscle tissue are comparable to unbound plasma concentrations.

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96 LIST OF REFERENCES 1. 2009. Clinical Study Report An open label, single dose, microdialysis and pharmacokinetic study of TR 701 in normal, healthy adults. 2. 2008. Invest igator's Brochure TR 701. 3. 2010. Zyvox Prescription Information. 4. Abrahamian, F. M., D. A. Talan, and G. J. Moran. 2008. Management of skin and soft tissue infections in the emergency department. Infect. Dis. Clin. North Am. 22: 89 116, vi. 5. Andes, D. J. Anon, M. R. Jacobs, and W. A. Craig. 2004. Application of pharmacokinetics and pharmacodynamics to antimicrobial therapy of respiratory tract infections. Clin. Lab. Med. 24: 477 502. 6. Andes, D., M. L. van Ogtrop, J. Peng, and W. A. Craig. 2002. In vi vo pharmacodynamics of a new oxazolidinone (linezolid). Antimicrob. Agents Chemother. 46: 3484 3489. 7. Antal, E. J., P. E. Hendershot, D. H. Batts, W. P. Sheu, N. K. Hopkins, and K. M. Donaldson. 2001. Linezolid, a novel oxazolidinone antibiotic: assessmen t of monoamine oxidase inhibition using pressor response to oral tyramine. J. Clin. Pharmacol. 41: 552 562. 8. Atterson, P. R., K. Takacs, and M. J. Schlosser. 2008. Absence of a pressure response to oral tyramine in conscious telemeterized rats treated wit h the novel oxazolidinone TR 701: comparison to linezolid. In t. I. C. o. A. A. a. Chemotherapy (ed.), Washington, D.C. 9. Azamfirei, L., S. M. Copotoiu, K. Branzaniuc, J. Szederjesi, R. Copotoiu, and C. Berteanu. 2007. Complete blindness after optic neuro pathy induced by short term linezolid treatment in a patient suffering from muscle dystrophy. Pharmacoepidemiol Drug Saf 16: 402 404. 10. Barbour, A., S. Schmidt, W. R. Rout, K. Ben David, O. Burkhardt, and H. Derendorf. 2009. Soft tissue penetration of cef uroxime determined by clinical microdialysis in morbidly obese patients undergoing abdominal surgery. Int. J. Antimicrob. Agents 34: 231 235. 11. Barbour, A., S. Schmidt, S. N. Sabarinath, M. Grant, C. Seubert, D. Skee, B. Murthy, and H. Derendorf. 2009. So ft tissue penetration of ceftobiprole in healthy volunteers determined by in vivo microdialysis. Antimicrob. Agents Chemother. 53: 2773 2776.

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106 BIOGRAPHICAL SKETCH Martina D. Sahre was born in Berlin, Germany. She studied pharmacy at the Freie Universit t Berlin from 2000 to 2005 and received her accreditation to work as a pharmacist (R.Ph) in Germany in 2005. After a brief experience working in a public pharmacy in Germany, she was accepted into the PhD program in the College of Pharmaceutics, University of Florida. Her supervisor was Hartmut Derendorf, PhD. She graduated from University of Florida in December 2010.