Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2013-08-31.

MISSING IMAGE

Material Information

Title:
Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2013-08-31.
Physical Description:
Book
Language:
english
Creator:
Naik,Runa S
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Pharmaceutical Sciences, Pharmaceutics
Committee Chair:
Derendorf, Hartmut C
Committee Members:
Hochhaus, Guenther
Palmieri, Anthony
Grant, Maria A

Subjects

Subjects / Keywords:
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

Statement of Responsibility:
by Runa S Naik.
Thesis:
Thesis (Ph.D.)--University of Florida, 2011.
Local:
Adviser: Derendorf, Hartmut C.
Electronic Access:
INACCESSIBLE UNTIL 2013-08-31

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2011
System ID:
UFE0043349:00001


This item is only available as the following downloads:


Full Text

PAGE 1

1 PHARMACOKINETIC/PHARMACODYNAMIC CHARACTERIZATION OF JNJ Q2, A NOVEL FLUOROQUINOLONE By RUNA S. NAIK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

PAGE 2

2 2011 Runa S. Naik

PAGE 3

3 To Swami

PAGE 4

4 ACKNOWLEDGMENTS First and foremost, I would like to extend my heartfelt gratitude to Dr. Hartmut Derendorf, my mentor for his constant guidance, support and encouragement. I sincerely thank him for having the confidence in me to accomplish a project of this nature. My special thanks go to the members of my supervisory committee, Dr. Guenther Hochhaus, Dr. Anthony Palmieri and Dr. Maria Gr ant for their support, encouragement and guidance. They have always accommodated me whenever I have approached them and guided me whenever I needed the help. My sincere thanks are due to Dr. Christoph Suebert and Dr. Maria Grant for their tireless su pport during the clinical study in spite of their busy schedules. They have not just been participating doctors in the study but also mentors and have learnt much from them. I am very grateful to Stephan Schmidt for his friendship as well as mentoring. He has been very helpful whenever I have reached out for any help. I would like to express my appreciation to Martina Sahre and Nina Kranz for their help while conducting the clinical study and the post doctoral fellows Sabarinath Sreedharan and Mike Bern wtiz for their immense help and support during the clinical study. I would like to especially thank Rajendra Pratap Singh for his constant support during the entire length of my projec t. I am thankful to all the personnel of General Clinical Research Cente r and Shands Angelo.

PAGE 5

5 Many thanks to my dear friends Fancy, Divya, Manuela, Daniela, Haripriya and Geeta for their love and friendship during my years here at Unive rsity of Florida and before. I would also like to acknowledge the unquestioning support of the administrative staff of the Department of Pharmaceutics, especially Pat Khan and Sarah Scheckner I would like to thank my beloved parents and brother for letti ng me achieve my dreams and supporting me in all ways to accomplish it. It is because of their faith in me and support I was able to come to this country and pursue my PhD degree. I would like to thank Ashish, my dearest friend and fianc for his patience, love, encouragement and support throughout these years and standing by me during the good and rough times I could not have completed this without his support and belief in me. Finally, I would like to thank God for giving me the strength in every step of the way and his constant grace in everything I did. I dedicate my work and education to God

PAGE 6

6 TABLE OF CONTENTS p age ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 12 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 FLUOROQUINOLONES AND THE APPLICATION OF PHARMACOKINETIC/PHARMACODYNAMIC APPRAOCHES IN THEIR DEVELOPMENT ................................ ................................ ................................ ..... 16 Fluoroquinolones ................................ ................................ ................................ .... 16 Introduction ................................ ................................ ................................ ....... 16 Mechanism of Action ................................ ................................ ........................ 16 Classes of Fluoroquinolones ................................ ................................ ............ 17 Phar macokinetics ................................ ................................ ............................. 17 Absorption ................................ ................................ ................................ .. 18 Distribution and protein binding ................................ ................................ .. 18 Metabolism ................................ ................................ ................................ 19 Elimination ................................ ................................ ................................ 19 Pharmacodynamics ................................ ................................ .......................... 20 JNJ Q2 ................................ ................................ ................................ .................... 22 Physicochemical Properties ................................ ................................ ............. 22 Antimi crobial Activity ................................ ................................ ......................... 22 Applications of PK/PD in Fluoroquinolone Development ................................ ........ 25 MIC based PK/PD Indices ................................ ................................ ................ 26 PK/PD Evaluation Based on Kill Curves ................................ ........................... 27 Summary ................................ ................................ ................................ ................ 28 Hypothesis ................................ ................................ ................................ .............. 28 Specific Aims ................................ ................................ ................................ .......... 29 2 APPLICATIONS OF MICRODIALYSIS IN PHARMACEUTICAL SCIENCES: CLINICAL MICRODIALYSIS IN SKIN AND SOFT TISSUES ................................ 32 Misconceptions about Tissue Drug Distribution ................................ ...................... 32 Microdialysis in the Skin: Tissue Bioavailability ................................ ...................... 34 Microdialysis in Soft Tissue: Antimicrobial Agents ................................ .................. 37 PK/PD Indices ................................ ................................ ................................ ......... 45 Conclusion ................................ ................................ ................................ .............. 46

PAGE 7

7 3 DETERMINATION OF JNJ Q2 IN SALINE BY HIGH PERFORMANC E LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (LC MS/MS) ............. 47 Objective ................................ ................................ ................................ ................. 47 Validation Procedure ................................ ................................ ............................... 47 Chemicals an d Equipment ................................ ................................ ...................... 48 Test Article ................................ ................................ ................................ ....... 48 Internal Standard (IS) ................................ ................................ ....................... 48 Reagent Preparation ................................ ................................ ............................... 48 JNJ Q2 Stock Standard Solution ................................ ................................ ...... 48 JNJ Q2 Working Standard So lutions ................................ ................................ 48 JNJ Q2 QC Samples ................................ ................................ ........................ 49 Internal Standard Stock Solution ................................ ................................ ...... 49 HPLC Mobile Phase ................................ ................................ ......................... 50 Mobile phase A: methanol with 0.05 % v/v formic acid .............................. 50 Mobile phase B: ammonium acetate buffer (10 mM, pH4.0) ...................... 50 HPLC wash solution ................................ ................................ ................... 50 Sample Prepara tion ................................ ................................ ................................ 50 Assay Procedure ................................ ................................ ................................ .... 5 1 Mass Spectroscopy Set Up ................................ ................................ .............. 51 HPLC Pump Conditions ................................ ................................ .................... 51 Autosampler Conditions ................................ ................................ ................... 51 Analysis procedure ................................ ................................ ........................... 51 Results ................................ ................................ ................................ .................... 52 Reproducibility of Calibration Curve Parameters ................................ .............. 52 Lower Limit of Quantification ................................ ................................ ............ 52 Intra batch Variability for Quality Control Samples ................................ ........... 52 Inter batch Variability for Quality Control Samples ................................ ........... 53 Freeze thaw Stability ................................ ................................ ........................ 53 Stability at Room Temperature ................................ ................................ ......... 54 Refrigeration Stability of QCs ................................ ................................ ........... 54 Freezer (Long term) Stability ................................ ................................ ............ 54 Auto injector Stability of QCs ................................ ................................ ............ 54 Ro bustness ................................ ................................ ................................ ...... 55 Summary ................................ ................................ ................................ ................ 55 4 IN VITRO MICRODIALYSIS OF JNJ Q2 ................................ ................................ 62 Objective ................................ ................................ ................................ ................. 62 Microdialysis ................................ ................................ ................................ ........... 62 Extraction Efficiency Method (EE) ................................ ................................ .... 63 Retrodialysis (RD) ................................ ................................ ............................ 64 Retrodialysis under Light Dark Conditions ................................ ....................... 65 Chemicals and Equipment ................................ ................................ ...................... 65 Test Article ................................ ................................ ................................ ....... 65 Internal Standard ................................ ................................ .............................. 65 Reagent Prepar ation ................................ ................................ ............................... 65

PAGE 8

8 JNJ Q2 Standards and Quality Control Solutions ................................ ............. 65 Mass Spectroscopy Mobile Phase ................................ ................................ ... 66 Mobile phase A: methanol with 0.05 % v/v Formic Acid ............................. 66 Mobile phase B: ammonium acetate buffer (10 mM, pH4.0) ...................... 66 Sample Preparation ................................ ................................ ................................ 66 Calibration Solution for Microdialysis ................................ ................................ 66 Dialysate Samples ................................ ................................ ............................ 67 Apparatus Setu p ................................ ................................ ................................ ..... 67 Sample Analysis ................................ ................................ ................................ ..... 68 Mass Spectroscopy Set Up ................................ ................................ .............. 68 HPLC Pump Conditions ................................ ................................ .................... 68 Autosampler Conditions ................................ ................................ ................... 68 Analysis P rocedure ................................ ................................ .......................... 68 Results ................................ ................................ ................................ .................... 68 Method Validation Summary ................................ ................................ ............ 68 In Vit ro Results ................................ ................................ ................................ 69 Conclusions ................................ ................................ ................................ ............ 70 5 CLINICAL TISSUE DISTRIBUTION STUDY OF A NOVEL FLUOROQUINOLONE, JNJ Q2 USING MICRODIALYSIS IN HEALTHY SUBJECTS ................................ ................................ ................................ ............. 74 Objectives ................................ ................................ ................................ ............... 75 Methods ................................ ................................ ................................ .................. 76 Demographics ................................ ................................ ................................ .. 76 Study Drug ................................ ................................ ................................ ....... 76 Sampling Technique ................................ ................................ ......................... 76 Study Design ................................ ................................ ................................ .... 77 Pilot ................................ ................................ ................................ ............ 77 Main ................................ ................................ ................................ ........... 78 Analysis ................................ ................................ ................................ ............ 79 Sample analysis ................................ ................................ ......................... 79 Sample preparati on: ................................ ................................ ................... 80 Data analysis ................................ ................................ ............................. 80 Results ................................ ................................ ................................ .................... 81 Discussion ................................ ................................ ................................ .............. 82 6 PHARMACOKINETIC/PHARMACODYNAMIC MODELING ................................ ... 89 Pharmacokinetic Modeling ................................ ................................ ...................... 89 Pharmacodynamic Modeling ................................ ................................ ................... 91 Materials and Methods ................................ ................................ ............................ 92 Population Pharmacokinetic Model Development ................................ ............ 92 PK/PD Analysis ................................ ................................ ................................ 93 AUC/ MIC ratio ................................ ................................ ........................... 93 Time kill curve approach ................................ ................................ ............ 93 Results ................................ ................................ ................................ .................... 93

PAGE 9

9 Population Pharmacokinetic Modeling ................................ .............................. 93 AUC/MIC Index ................................ ................................ ................................ 95 Modeling of Time Kill Curves ................................ ................................ ............ 96 7 CO NCLUSION ................................ ................................ ................................ ...... 118 APPENDIX A REAGENTS AND EQUIPMENT FOR LC MS/MS STUDY ................................ ... 122 B REAGENTS AND EQUIPMENTS FOR IN VITRO MICRODIALYSIS STUDY ...... 123 C STUDY CRITERIA ................................ ................................ ................................ 124 LIST OF REFERENCES ................................ ................................ ............................. 127 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 137

PAGE 10

10 LIST OF TABLES Table page 1 1 Classification of Fluoroquinolones [4] ................................ ................................ 30 3 1 Summaries of linearity data ................................ ................................ ................ 58 3 2 Intra batch variability of quality control samples (n=5 per day and per concentration) ................................ ................................ ................................ ..... 58 3 3 Inter batch variability of quality control samples (n=20 per concentration) ......... 58 3 4 Freeze/thaw stability of JNJ Q2 (n=3) ................................ ................................ 59 3 5 Room temperature Stability of JNJ Q2 (n=3). Time= 6hr ................................ .... 59 3 6 Refrigeration stability (4C) of JNJ Q2 (n=3). Time= 8hr ................................ .... 59 3 7 Freezer stability ( 70C) of JNJ Q2 (n=3).Time=62 days ................................ .... 59 3 8 Auto injector stability of JNJ Q2 (n=3). Time=62 days ................................ ....... 60 3 9 Intra batch variability of JNJ Q2 using a different column to check robustness (n=5) ................................ ................................ ................................ ................... 60 3 10 Calibration standard preparation of JNJ Q2 ................................ ....................... 61 3 11 Quality control sam ple preparation of JNJ Q2 ................................ .................... 61 3 12 Internal standard spiking solution ................................ ................................ ....... 61 4 1 Dilution schemes for calibration standard. ................................ .......................... 72 4 2 Dilution scheme for quality controls ................................ ................................ .... 72 4 3 Internal standard spiking solution ................................ ................................ ....... 72 4 4 Inter batch variability of quality control samples (n=20 per concentration) ......... 73 4 5 Recovery retrodialysis method ................................ ................................ ......... 73 4 6 Recovery extraction efficiency method ................................ ............................. 73 4 7 Recovery light & dark experiments by retrodialysis method ............................. 73 5 1 Patient demographics: main study ................................ ................................ ...... 86 5 2 Study procedure chart for pilot study ................................ ................................ .. 86

PAGE 11

11 5 3 Study procedure chart for main study ................................ ................................ 87 5 4 Mean (%CV) perfusate concentrations, cumulative dialysate concentrations, and the percent recovery of JNJ Q2 in interstitial fluids of adipose and skeletal muscle during the pilot study ................................ ................................ 87 5 5 Mean (%CV) perfusate concentrations, cumulative dialysate concentrations, and the percent recovery of JNJ Q2 in adipose and skeletal muscle in the main study ................................ ................................ ................................ .......... 87 5 6 Mean (SD) pharmacokinetic parameters of JNJ Q2 in plasma, adipose, and muscle following a single oral dose of JNJ Q2 (400 mg) in healthy subjects ...... 88 5 7 Mean (%CV) ratio of tissue to free and total p lasma JNJ Q2 concentrations ..... 88 6 1 Estimated PK model parameters with bootstrap 95% confidence intervals ...... 116 6 2 f AUC 24 /MIC ratio for JNJ Q2 ................................ ................................ ............ 117 6 3 Parameter estimates for 3 strains of MRSA ................................ ...................... 117

PAGE 12

12 LIST OF FIGURES Figure page 1 1 Chemical structure of JNJ Q2 ................................ ................................ ............. 31 3 1 Representative chromatograms for (A) blank JNJ Q2 (B) spiked JNJ Q2 (3.09 ng/mL) (C) blank int ernal standard (D) spiked internal standard (5 ng/mL) ................................ ................................ ................................ ................ 57 4 1 Clinical structure of JNJ Q2 ................................ ................................ ................ 71 5 1 Mean JNJ Q2 concentration time profiles in plasma (unbound concentration) and the interstitial fluids of subcutaneous adipose and skeletal muscle following single oral dose of JNJ Q2 (400 mg) in healthy subjects. .................... 85 6 1 Observations versus population and individual predictions for all co mpartments. Predicted concentrations ( g/mL): x axis and observed concentrations ( g/mL): y axis ................................ ................................ ........... 98 6 2 Observation versus population and individual predictions for plasma concentrations. Predicted concentrations ( g/mL): x axis and observed concentrations ( g/mL): y axis ................................ ................................ ........... 99 6 3 Individual fits for plasma. Time (hr): x axis and observed/ predicted concentrations ( g/mL) : y axis ................................ ................................ ......... 100 6 4 Observations versus population and individual predictions for adipose tissue. Predicted concentrations ( g/mL): x axis and observed concentrations ( g/mL): y axis ................................ ................................ ................................ .. 101 6 5 Individual fits for adipose tissue. Time (hr): x axis and observed/ predicted concentrations ( g/mL) : y axis ................................ ................................ ......... 102 6 6 Observations versus population and individual predictions for muscle. Predicted concentrations ( g/mL): x axis and observed concentrations ( g/mL): y axis. ................................ ................................ ................................ 103 6 7 Individual fits for muscle. Time (hr): x axis and observed/ predicted concentrations ( g/mL) : y axis. ................................ ................................ ........ 104 6 8 Weighted residuals plot versus time for all compartments ................................ 105 6 9 Weighted residuals versus population predictions for all compartments .......... 106 6 10 Curve fit for MRSA OC 8525 ................................ ................................ ............ 1 07 6 11 Curve fit for MRSA OC 11696 ................................ ................................ .......... 108

PAGE 13

13 6 12 Curve fit for MRSA OC 2838 ................................ ................................ ............ 109 6 13 Simulated curves for bacterial kill over a range of MIC strengths for MRSA OC 8525 & once a day oral administration of 400mg JNJ Q2 .......................... 110 6 14 Simulated curves for bacterial kill over a range of MIC strengths for MRSA OC 11696 & once a day oral administration of 400mg JNJ Q2 ........................ 111 6 15 Simulated curves for bacterial kill over a range of MIC strengths for MRSA OC 2838 & once a day oral administration of 400mg JNJ Q2 .......................... 112 6 16 Simulated PK/PD of JNJ Q2 against MRSA OC 8525 after once daily oral administration of 200mg, 300mg, 400mg and 500mg JNJ Q2 .......................... 113 6 17 Simulated PK/PD of JNJ Q2 against MRSA OC 11696 after once daily oral administration of 200mg, 300mg, 400mg and 500mg JNJ Q2 .......................... 114 6 18 Simulated PK/PD of JNJ Q2 against MRSA OC 2838 after once daily oral administration of 200mg, 300mg, 400mg and 500mg JNJ Q2 .......................... 115

PAGE 14

14 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 JNJ Q2, A NOVEL FLUOROQUINOLONE By Runa S. Naik August 2011 Chair: Hartmut Derendorf Major: Pharmaceutical Sciences For antimicrobial agents, selection of optimized dosing scheme for specific pathogens not only increases chances of cure but also reduces probability of resistance development. A tool helpful in dose selection is pharmacokinetic/pharmacodynamic (PK/PD) mod el. JNJ Q2, a novel fluorinated quinolone, with activity against key pathogens associated with complicated skin & skin structure infections including methicillin resistant S.aureus (MRSA). Most MRSA infections that are community acquired present as skin an d soft tissue infections and the standard care used in treating these infections such as linezolid is effective, but its prolonged use can cause resistance. To characterize the time course of JNJ Q2 at 400mg single dose, concentrations not only in plasma, but also in skeletal muscle and subcutaneous adipose tissue, where the site of action for infections is, a microdialysis study was conducted in healthy volunteers. For best correlation to clinical outcome, PK/PD index ratio of 24h area under the curve and MIC (AUC 24 /MIC) was evaluated. The time course effect of JNJ Q2 was defined using a modified Emax model using in vitro time kill data. Population PKPD model was developed to elucidate the dynamic relation between clinical PK and in vitro PD studies for dru g development and dosage recommendation.

PAGE 15

15 The project explored the usefulness of PK/PD relationships to support drug development and appropriateness of use of 400mg once a day oral dos ing regimen for JNJ Q2. Development of novel antibiotics such as JNJ Q2 is necessary in order to have an effective armamentarium against adapting bacteria.

PAGE 16

16 CHAPTER 1 FLUOROQUINOLONES AND THE APPLICATION OF PHARMACOKINETIC / PHARMACODYNAMIC APPRAOCHES IN THEIR DEVELOPMENT Fluoroquinolones Introduction The emergence of a new class of antibacterial agents began with the synthesis of nalidixic acid which was introduced for clinical use in 1962 [ 1 ] Modification of the nalidixic acid structure led to the synthesis of the first fluoroquinolone; norfloxacin, which exhibited enhanced activity for Gram negative organisms, including Pseudomonas aeruginosa [ 1 ] Further substitutions of the fluoroquinolone molecule resulted in th e development of ciprofloxacin with high activity against Gram negative bacilli but with questionable utility against important Gram positive pathogens Over the years we have seen n umerous manipulations of the fluoroquinolone structure in order to synthes ize a group of new fluoroquinolones with important advantages over their predecessors which exhibit enhanced potency against Gram positive organisms and/or anaerobes, while maintaining Gram negative activity These agents demonstrate significantly improved pharmacokinetic profiles, with oral and intravenous formulations an d the ability to concentrate in tissues and fluids at concentrations that exceed serum drug concentrations [ 2 ] Mechanism of Action Fluoroquinolones exert their bactericidal effect by inhibiting the type II topoisomerases DNA gyrase and topoisomera se IV DNA gyrase introduces negative superhelical twists into bacterial DNA, and thus is essential for replication and transcription. Topoisomerase IV appears to separate interlinked daughter DNA circles

PAGE 17

17 following a round of DNA replication, allowing segr egation of the replicated chromosomes. Resistance to the fluoroquinolones is mediated by mutations in the quinolone resistance determining regions of the genes coding for the target enzymes It has also been suggested that resistance occurs by active efflu x of fluoroquinolones [ 1 3 ] Classes of F luoroquinolones The quinolones can be classified into four generations based on antimicrobial activity [ Table 1 1] [ 4 5 ] First generation agents have moderate gram negative a ctivity and minimal systemic distribution. Second generation quinolones have expanded G ram negative activity and atypical pathogen coverage, but limited gram positive activity. These agents are most active against aerobic G ram negative bacilli. Ciprofloxac in remains the quinolone most active against Pseudomonas aeruginosa [ 2 5 6 ] Third generation quinolones retain expanded G ram negative and atypical intracellular activity but have improved G ram positive coverage. Finally, fo urth generation agents improve G ram positive coverage, maintain gram negative coverage, and gain anaerobic coverage [ 5 ] Pharmacokinetics Q uinolones exhibit concentration dependent bacterial killing. Bactericidal activity becomes more pronounced as the serum drug concentration increases to approximately 30 times the minimum inhibitory concentrat ion (MIC) [ 5 7 ] Higher drug concentrations paradoxically inhibit RNA and protein synthesis, thereby reducing bactericidal activity [ 5 ] Quinolones have a post antibiotic effect of about one to two hours [ 5 ]

PAGE 18

18 Absorption F luoroquinolones are well absorbed after oral administration, with bioavai lability ranging from approximately 70% for ciprof loxacin to 99% for levofloxacin [ 1 7 ] The majority of new fluoroquinolones are absorbed quickly, attaining their maximum concentration in plasma (Cmax) within approximately 1 to 2 hours after oral administration. A number of investigations into the effect of food on the pharmacokinetics (PK) of the new fluoroquinolones have demonstrated that although food intake can slow absorption, it does not cause clinically significant alterations in the extent of absorption [ i.e. area under the plasma concentration time curve (AUC)] or overall bioavailability of gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin, moxifloxacin, sitafloxacin, sparfloxacin, and trovafloxacin. Therefore, all of thes e new fluoroquinolones can be given orally without regard to food intake [ 3 8 12 ] However, quinolones chelate with cations such as aluminum, magnesium, calcium, iron, and zinc. This interaction significantly reduces absorption and bioavailability, resulting in lower serum drug concentrations and less target tissue penetration [ 5 7 ] Serum drug levels achieved after oral administration are comparable to those with intravenous dosing, which allows an early transition from intravenou s to oral therapy and a potential reduction of treatment costs [ 13 ] Distribution and p rotein b inding One of the most attractive PK characteristics of fluoroquinolones is their large volume of distribution (Vd) Distribution of fluoroquinolones to tissues is very good, owing to their physicochemical properties. Tissue penetration is higher than the concentration achieved in plasma, stool, bile, prostatic tissue, and lung tissue. Quinolones also penetrate well in urine and kidneys when renal clearance is the route of

PAGE 19

19 drug elimination. Penetration into prostatic fluid, saliva, bone, and cerebrospinal fluid is not known to exceed serum drug levels. The new fluoroquinolones have Vd ranging from 1.1 to 7.7 L/kg for gatifloxacin, levofloxacin, moxifloxacin, gemifloxacin, trovalfoxacin including ciprofloxacin [ 3 ] In rega rd to inflammatory fluid penetration, the new fluoroquinolones all demonstrate fluid to serum ra tios in the range of 0.9 to 1.3. This is not surprising as they have large Vd [ 14 23 ] Plasma protein binding of the quinolones varies, with the newer quinolones less bound to plasma proteins than nalidixic acid, ranging from 5 to 73%. Clinafloxacin and gatifloxacin have 5 and 20% protein binding respectively while l evofloxacin and moxifloxacin shown 31 and 47% and trevofloxacin of 73% [ 3 12 ] Metabolism The degree of the metabolism of fluoroquinolones varies widely [ 24 ] Biotransformation reactions involve predominantly the piperazinyl ring and its substituents. Most of the fluoroquinolone primary metabolites are active against bacteria however, these metabolites have a shorter elimination half life than their parent compound [ 24 ] Elimination Newer f luoroquinolones exhibit longer half lives than that of ciprofloxacin, ranging from 4.7 hours for sitafloxacin to 18.7 hours for sparfloxacin. Hence most are administered every 12 to 24 hours. The excretion of fluoroquinolones is primarily via the kidney and secondarily via the liver such as in case of sparfloxacin moxifloxacin, and trovaf loxacin To avoid toxicity, dosages often need to be adjusted in patients w ith renal or hepatic impairment [ 5 7 ] Clinafloxacin, gatifloxacin, levofloxacin, and sitafloxacin are excreted in the urine

PAGE 20

20 as the parent compound (>50%), indicating that these agents primarily undergo renal elimination. Conversel y, gemifloxacin, grepafloxacin, moxifloxacin, sparfloxacin, and trovafloxacin are all eliminated predominantly by non renal pathwa ys [ 3 18 21 ] A number of PK studies have been conducted in special populations. In the elderly, it has been demonstrated that the PK and bioavailability of levofloxacin, gatifloxacin, moxifloxacin, and sparfloxacin are not appreciably affected; however, grepafloxacin displays delaye d absorption, a 31% increase in the peak plasma concentration, and a 48% increase in AUC in the elderly [ 8 11 25 ] Excretion is decreased in individuals suffering from the renal failure and fluoroquinolones should be used in such patients with caut ion [ 24 ] Pharmacodynamics U nderstanding of clinical pharmacodynamics (PD) of anti infective agents has dramatically increased during the past decade [ 26 ] P D characteristics that best describe fluoroquinolones are concentration depend ent bactericidal activity and a significant post antibiotic effect for both G ram positive and G ram negative bacteria [ 7 27 ] The parameter most commonly used to quantify the antimicrobial activity of antibiotics against a certain bacterium is usually the minimum inhibitory concentration. It is defined as the lowest concentration of drug that prevents visible growth of the organism as detected by t he unaided eye Although the MIC is a well established PD parameter, this parameter has several disadvantages. For instance, the MIC does not provide information on the rate of bacterial kill. MIC determination depends on the number of bacteria at a single time bacterial activity and kill in vivo over time A two fold variability in the results is

PAGE 21

21 acceptable and it is not uncommon to see the MIC reported as a range of values instead of a single number. He nce, any PK/PD approach based on this PD indicator will carry with it the same amount of variability and uncertainty. An alternative PD approach, bacterial time kill curves, has been proposed to offer detailed information about the antibacterial efficacy as a function of both time and antibiotic concentration [3]. Time kill curves of many antibacterial agents have been studied in both in vitro kinetic models and animal infection models. The major limitation associated to the kill curve approach is that it is not a very practical method. Obtaining a complete set of kill curves that would allow a good evaluation of the concentration effect and time effect relationships between drug and bacteria is very labor intensive and time consuming. The presently availab le fluoroquinolones with in vitro activity against Streptococcus pneumoniae (including current penicillin resistant strains) are levofloxacin, sparf loxacin, gatifloxacin moxifloxacin, and trovafloxacin. Levofloxacin and sparfloxacin exhibit inferior in vitro streptococcal activity compared with gatifloxacin, moxifloxacin, and trovafloxacin. Gatifloxacin is two to four times more active than levofloxacin against S. pneumoniae in vitro and moxifloxacin is four to eight times more active [ 5 ] Compared with ciprofloxacin and levofloxacin, the fluoroquinolones gatifloxacin, moxifloxacin, and trovafloxacin have greater in vitro activity against S. aureus and some Enterococcus strains [ 5 ] Although gatifloxacin and moxifloxacin have in vitro anaerobic activity, only trovafloxacin is labeled for the treatment of anaerobic infections. C iprofloxacin, ofloxacin and the newer fluoroquinolones hav e exceptional intracellular concentrations.

PAGE 22

22 Overall, these pharmacodynamic properties allow infrequent dosing of fluoroquinolones because prolonged activity, even when serum and tissue concentrations fall below the MIC, prevents bacterial re growth. In ad dition, optimal pharmacodynamic values are associated with preventing the development of bacterial resistance. JNJ Q2 Physicochemical Properties JNJ Q2 [ Figure 1 1 ] is one of the most potent anti staphylococcal agents in a series of aminoethylidenylpiperidine fluoroquino lones [ 28 ] The hydrochloride salt of JNJ Q2 has a low molecular weight of 4 55 8 and acceptable solubility profile and lipophilicity [ 29 ] T he experimentally determined value of pKa (6.13), pKb (8.59), and the logarithm of the distribution coefficient ( D ) at pH 7.4 is 0.37 for JNJ Q2 which is suggestive of absorption and permeability characteristics equivalent to those of curr ently approved fluoroquinolones. T he aqueous solubility of 4.52 mg/mL for the hydrochloride salt of JNJ Q2 is consistent with the development of oral and parenteral dosage forms [ 28 ] Antimicrobial Activity JNJ Q2 is indicated for its use in complicated skin and skin structure infections (cSSSIs), including diabetic foot infection, community acquired pneumonia (CAP) and other infections. JNJ Q2 is a broad spectrum, bactericidal fluoroquinolone antibiotic with potent activity against G ram positive pathoge ns, including methicillin resistant S. aureus (MRSA), G ram negative and anaerobic pathogens [ 29 ] JNJ Q2 has demonstrated potent in vitro microbiologic activity against S. pneumoniae including penicillin resistant (Pen R), erythromycin resistant (Ery R), and

PAGE 23

23 levofloxacin resistant (Lvx R) isolates, with MIC values against these organisms of 0.12 g/mL, a value that is 4 fold to 8 fold lower than gemifloxacin. JNJ Q2 also demonstrated potent activity against clinical ciprofloxacin non susceptible S. pneumoniae with characterized mutations in at least 2 DNA gyrase or topoisomerase IV genes. The MIC value for JNJ Q2 against these mutant S. pneumoniae isolates was 0.25 g/mL, 32 fold lower than moxifloxacin, and 8 fold lower than gemifloxacin. In addition to the potent anti pneumococcal activity, JNJ Q2 displayed more potent activity than marketed fluoroquinolones a gainst other Gram positive pathogens, including methicillin resistant S. aureus (MRSA). The newer fluoroquinolones (moxi and gemi ) are currently not indicated for the treatment of MRSA related infections due to their insufficient activity against the tar get pathogens. The activity of JNJ Q2 was 16 fold better than mo xifloxacin against MRSA, with a MIC value of 0.5 g/mL. Among the tested fluoroquinolones, JNJ Q2 was the most potent compound against methicillin resistant S. epidermidis (MRSE), Enterococcus spp, and S. pyogenes ( MIC values of 0.25, 4, and 0.015 g/mL, respectively). Against G ram negative pathogens, JNJ Q2 generally exhibited comparable activity to moxifloxacin and gemifloxacin, including potent activity against the respiratory p athogens H. influenzae and M. catarrhalis with MIC values of 0.015 and 0.015 g/mL, respectively. JNJ Q2 displayed potent bactericidal activity against S. pneumoniae MRSA, and E. coli in time kill studies exhibiting a 3 log 10 kill at 2 times the MIC. The in vivo efficacy of JNJ Q2 was superior to ciprofloxacin and comparable to moxifloxacin in lethal systemic infection models with methicillin susceptible S. aureus (MSSA). Against a community acquired MRSA isolate (CA MRSA), JNJ Q2 was superior to linezoli d and vancomycin when dosed

PAGE 24

24 subcutaneously. In a murine S. pneumoniae lower respiratory tract infection model, JNJ Q2 was more potent than moxifloxacin [ 29 ] CAP is a leading cause of death in the world, and the sixth most common cause of death in the United States. S. pneumoniae is the commonest pathogen responsible for 30% to 50% of CAP cases [ 30 ] Although CAP caused by S. aureus is uncommon, it is important because of emergence of the community acquired MRSA (CA MRSA). Current strategies for management of CAP are directed toward treating patients on an outpatient basis with accurate empirical therapy (e.g. monotherapy with either a lactam, a macrolide or a fluoroquinolone) whenever possible, by reducing the duration of therapy, or switching early on from i ntravenous to oral therapy However, t he incidence of infections caused by S. pneumoniae resistant to penicillin, macrolides and other anti microbial agents has increased at an alarming rate during the past two decades Fluoroquinolones such as levofloxa cin, moxifloxacin, gatifloxacin, and gemifloxacin have become some of the most widely used and most effective antibacterial agents in the world in treatment of respiratory tract infections (RTIs) due to their improved activity against major respiratory pathogens including multi drug resistant S. pneumoniae N ovel agents have been recommended by several recent guidelines as first line therapy for the management of most CAP patient categories [ 31 ] The newer fluoroquinolones play an important role in achieving these goals as they are highly bioavailable in both plasma and at the sites of infection and potent against major respirat ory pathogens and generally safer. However, increased use of fluoroquinolones in RTIs has also led to increasing resistance, especially to those most frequently prescribed fluoroquinolones, e.g., ciprofloxacin moxifloxacin and levofloxacin.

PAGE 25

25 Resistance of S. pneumoniae to fluoroquinolones has been reported in localized outbreaks and is associated with clinical failures [ 32 33 ] Therefore, concern is rising worldwide about the increased use of these agents that will result in increasing emergence of cross resistance to the whole class. Therefore, development of newer agents like JNJ Q2 with more potent activity is valuable. J NJ Q2 is anticipated to provide acceptable safety and tolerability with broa d spectrum activity against G ram positive and G ram negative pathogens. Applications of PK/PD in F luoroquinolone D evelopment The selection of the correct dose and dosing regimen is a fundamental step for therapeutic success with any pharmacological agent. For antimicrobial agents the selection of the best drug and dosing scheme for a specific pathogen not only increases the chances of cure while preventing toxic side effects, but als o decreases the probability of the infecting agent becoming resistant to the drug [ 26 34 39 ] However, deciding on the best drug and most effective dosing scheme is not an easy task and is often done based on trial and error rather than on rational design. Also f or older drugs dose was used as the surrogate for antibiotic exposure, being adjusted solely on a milligram per kilogram basis leading to a high rat e of clinical failure since it d id not take into consideration the inter subject variability in the PK parameters of the patient population [ 40 ] Dose alone is an incomplete surrogate for drug exposure [ 41 ] In addition, while the understanding of the time course of the drug in the body is a necessary step for dose optimization it alone cannot predict the time course or magnitude of the pharmacological effect [ 42 ] Today, rational drug design is strongly based on principles of PK/PD modeling. PK/PD modeling has also become an extremely import ant tool in dose optimization [ 43 ] By integrating the time concentration

PAGE 26

26 relationship of the antibiotic in the body and its concentration effect relationship at the infection site, PK/PD modeling allows clinicians to establish an educated selection of best drug and optimum dosing range in an attempt to maximize efficacy, minimize toxicity, limit development of resistance and reduce costly trial and error approaches [ 39 ] Much progress has been made in recent years towards the understanding the PK/PD relationship of anti infective agents. Several different strategies have been developed such as MIC based PK/PD indices, time kill analysis, and advanced modeling and simulation techniques, such as population pharmacokinetics and Monte Carlo simulations. Following is an overview of the most commonly used PK/PD approaches for fluoroquinolones and their potential for the evaluation and optimization of drug therapy MIC based PK/PD I ndices The two most important PK/PD indices associated with successful clinical and microbiologic outcomes for fluoroquinolones are the ratio of maximum serum concentration to the minimum inhibitory concentration ( Cmax/ MIC ) and ratio of 24h area under the curve and MIC ( AUC 24 / MIC ) [ 44 46 ] They are highly correlated with eac h other, particularly when a fixed dosing interval is used in clinical trials If the dosage is increased, both Cmax and AUC are increased. Preston et al [ 47 ] suggested that Cmax/ MIC is most likely correlated to clinical outcome as long as it is greater than 10 However, when the ratio is less than 10, AUC 24 / MIC is most important. The AUC 24 /MIC has been used to evaluate the efficacy of fluoroquinolones The AUC 24 /MIC target for fluoroquinolone therapy has been subject of debate over the years. Forrest el al [ 48 ] initially proposed a target of AUC 24 /MIC > 125 for the treatment of G ram negative infections with ciprofloxacin in elderly hospitalized patients

PAGE 27

27 has since then become a standard clinical break point for the treatment of G ram negative agents with fluoroquinolones although values between 100 125 are suggested to be sufficient [ 49 ] The target for treatin g infections caused by G ram positive agents with fluoroquinolones is believed to be lower. For S. pneumoniae an AUC 24 /MIC > 30 has been associated with high probability of success in patients with community acquired infections [ 50 ] Schentag et al [ 51 ] suggested that there should be no different iation between the targets for G ram negative and G ram positive bacteria and that area under the inhibitory curve ( AUIC ) values ( calculated as the AUC that remains above the MIC during a 24 h r interval ) should be above 250 to assure rapid killing of the infecting agent [ 52 ] However, there has been a lot of discussion on this approach and in general not been very well accepted. PK/PD Evaluation Based on Kill Curves Kill cu rves provide a more dynamic description of the effect of antimicrobials hence allowing for more precise predictions [ 35 ] An E max model has been successfully applied to describe the relationship between concentration and effect for fluoroquinolone s [ 35 36 53 ] There have been some attempts to evaluate antimicrobial activity with an adapted E max [ 54 ] P arameters such as the maximum number of bacteria ( N max) at the end of the growth phase, adaptation rate terms ( x y z ) and Hill factor ( h ) have been included in a PK/PD model to optimize the models [7]. Once these models have been established and validated, it is then possible to simulate the expected kill curves for different doses a nd dosing regimens of the antibiotic against the bacteria of interest by substituting the concentration term in the mathematical model by the fitted concentration versus time profile of the drug.

PAGE 28

28 Summary The new fluoroquinol ones are a valuable addition to the antibiotic group Their broader spectrum antimicrobial activity to include both G ram positive and G ram negative pathogens and favorable PK make these agents attractive alterna tives to traditional therapy. Optimization of fluoroquinolone pharmacodynamic s leads to maximizing pathogen eradication and clinical cure and minimizing the development of resistance. PK/PD indices are useful predictors of antibacterial therapy and the higher these parameters are the greater is the efficacy [ 45 ] PK/PD based concepts can be used as decision making tools in different stages of dug development leading to reduction in development cost and time [ 39 ] PK/PD characterizatio n of JNJ Q2 will allow streamlining drug development and selection of an appropriate dosage regimen Hypothesis JNJ Q2 has great po tential for treating infections due to its broad spectrum activity against pathogens However, in order to establish safe an d effective dosing regimens for JNJ Q2, PK and PD properties need to be explored and validated U sing an integrated PK/PD approach which combines both the free target site concentrations of the drug and the in vitro antibacterial activity will provide good insights towards understanding the potential of JNJ Q2 for the treatment of cSSSIs including diabetic foot infection CAP and other infections. The study hypothesizes, the recommended d osing regimen of 4 00mg administered as a single oral dose is efficacious at the site of action based on the pharmacodynamic endpoints of AUC 24 /MIC ratio and simulated kill curves

PAGE 29

29 Specific Aims The first aim is to p erform in vitro microdialysis experiments to validate the feasibility of using microdialysis as a sampling technique for JNJ Q2 The second aim is validate an LC MS/MS method for the analysis of JNJ Q2 in saline. The third aim is to p erform in vivo microdialysis study to evaluate the PK profiles of JNJ Q2 in interstitial space fluid of soft tissues (skeletal muscle and subcutaneous adipose tissue) and plasma of healthy subjects after single oral dose of 400mg of JNJ Q2 Next aim is to d evelop population pharmacokinetic (PO PPK) model to characterize JNJ Q2 PK in healthy subjects after 400 mg oral dose The fifth aim is d evelop ing a PD model to describe time kill profiles of JNJ Q2 against different bacterial strains Finally the last aim is d evelop ing a PK/PD model to optimi ze an appropriate dosage regimen for JNJ Q2

PAGE 30

30 T able 1 1. Classification of Fluoroquinolone s [ 4 ] Quinolon e Generations Antibiotic First generation Nalidixic acid Cinoxacin Second generation Lomefloxacin Norfloxacin Enoxacin Ofloxacin Ciprofloxacin Third generation Levofloxacin Sparfloxacin Gatifloxacin Moxifloxacin Grepafloxacin Fourth generation Trovafloxacin

PAGE 31

31 Figure 1 1 Chemical structure of JNJ Q2

PAGE 32

32 CHAPTER 2 APPLICATIONS OF MICRODIALYSIS IN PHARMACEUTICAL SC IENCES: CLINICAL MICRODIALYSIS IN SKI N AND SOFT TISSUES Microdialysis (MD) allows the measurement of actual free drug concentrations in different tissues and organs and subsequently to relate these PK findings with PD observations to predict clinical efficacy. The ability to measure the free concentrations at the site of drug act ion over time makes microdialysis a very valuable tool for assessment of bioavailability and bioequivalence and has been recognized by industry and regu latory authorities such as the United States Food and Drug Administration ( FDA ) [ 55 ] Earlier, PK research was restricted to measurement of drug concentrations from relatively easy to obtain biological matrixes such as tissue biopsies, urine, saliva or skin blister fluids, but the emerging knowledge of mic rodialysis and its benefits have slowly shifted the focus off of these methods and with good reason [ 56 ] In the following chapter we shall discuss various examples that help us understand why microdialysis is not just an important technique in the assessment of drug distribution in skin and soft tissues but also in general very crucial in the clinical drug development process. Misconceptions about Tissue Drug Distribution Until recent years research on PK was limited for practical reasons to concentration measurements derived from matrices which were easy to obtain, such as bloo d and total tissue specimens. These approaches, however, caused considerable confusion, as their interpretation was flawed by a few misconceptions : Drug based or plasma based models refer to the process of penetration into does not take into account the complexity of separate organ systems and disease processes. Therefore, although plasma based modeling may provide useful information

PAGE 33

33 in many cases, it must be kept in mind t hat it assumes rapid, unrestricted, and homogeneous diffusion processes in hypothetical spaces, assumptions that do not always hold true. A second misconception was the reasoning that tissue is a uniform matrix. Measuring total antibiotic concentration mea surements from biopsy specimens may be misleading for several reasons. Most importantly, it must be considered that the actual target space for anti infective agents, with few exceptions, is the interstitial space fluid (ISF) [ 57 ] If only overall tissue drug concentrations are measured, then the effective site concentrations of drugs that equilibrates exclusively with the extracellu lar space, such as lactams, may be underestimated [ 57 ] This situation, in turn, will lead to an overestimation of the effective site concentrations of intracellularly accumulating drugs, such as quinolones or macrolides [ 57 ] Thus, homogenization of various tissue fluids and cells will lead to a hybrid tissue drug concentration which is difficult to interpret [ 57 ] Another misconcept ion was the notion that the entire drug fraction present in various tissue spaces is responsible for pharmacologic activity. In fact, it has been shown that only the unbound drug concentrations at the infection site has the ability to exert anti infective efficacy, both in vitro and in vivo [ 57 ] Besides the fact that only the free fraction exerts acti vity, it is also only the free d rug which has the ability to be distributed to the target site. This information was experimentally shown by various investigators, who found that differences in penetration were directly related to the free drug concentrations in serum. Although this conc ept is best described for antibacterial agents, one may safely assume that similar concepts hold true for many other classes of drugs

PAGE 34

34 It follows from these three considerations that an appropriate definition of tissue drug concentrations should imply in most cases the meaning of unbound interstitial drug measurement of tissue drug concentrations should allow for the direct measurement of unbound antibiotic concentration s in the ISF of a given organ. Microdialysis in the Skin: Tissue Bioavailability M D is an excellent method to assess local drug exposure at any site where a probe can be placed. Obviously, this includes the skin, where many microdialysis studies have been performed. The influence of changes in skin barrier properties on drug penetration was tested using the MD method [ 58 59 ] Tissue levels of salicylic acid after application of 5% solution in ethanol to intact skin or skin that was treated with 1% or 2% sodium laureth sulfate (SLS ), repeated tape stripping and drying by acetone wipe were measured [ 58 ] Skin irritation was highest in the 2% SLS and tape stripped regions, moderate in the 1% SLS treated skin and mild after treatment with acetone. While AUC of salicylic acid in tissue was only mildly increased after aceto ne treatment compared to intact skin, it increased 46 fold in moderately and > 140 fold in highly irritated skin The authors also assessed, whether application site had an influence on drug penetration and found slight differences which were not significa nt. The authors conclude that MD is a valuable tool for the assessment of concentrations of drugs in skin of various states of irritation and inflammation and for the evaluation of bioavailability of different study drug formulations. Acyclovir and pencicl ovir penetration through skin that was either intact, tape stripped and/or vasoconstricted with noradrenalin was tested [ 59 ] The results showed that after application of drug creams to intact, not vasoconstricted skin, there

PAGE 35

35 were no concentrations measurable above the lower limit of detection. When intact skin was vasoconstricted, concentrations became measurable and mean ( S.E.M.) AUC 0 5 was 13.3 2.9 and 27.6 10.6 ng*mL 1 *h for penciclovir and acyclovir, respectively. After tape stripping the skin to the extent o f removal of the stratum corneum (45 tape strippings) the mean ( S.E.M.) AUC 0 5 became 17.5 3.8 and 12.2 5 g*mL 1 *h. The authors concluded that for the two drugs under investigation, which are small molecules, water soluble and having logP values of 1.8 (acyclovir) and 2.12 (penciclovir) the stratum corneum thus presents the biggest barrier to drug penetration. Transepidermal water loss (TEWL) was used as a measure of the disruption of the stratum corneum barrier function. When TEWL was measured afte r every 5 tape strippings, water loss increased with increasing numbers of strippings until the stratum corneum had been removed completely after 40 45 strippings. Comparing the logarithm of the concentration of penciclovir per hour with the logarithm of t he TEWL showed a strong positive correlation (r 2 > 0.9). Since no in vivo recovery for both drugs was measured, values given here are relative concentrations only, which serve to compare and show relative changes. To assess the feasibility of using microdi alysis for drugs that are either highly protein bound or highly lipophilic, tissue levels of fusidic acid (protein binding 97%) and betamethasone 17 valerate were evaluated [ 60 ] In a human clinical trial with twice daily application for two days, no measurable levels for either drug was found. Microdialysis was used to assess skin penetration of acyclovir and salicylic acid into intact and tape stripped skin (acyclovir only) [ 61 ] For acyclovir, determinable dialysate levels were only found in tape stripped skin, whereas salicylic acid levels were

PAGE 36

36 found in intact skin. At the concentrations used, salicylic acid has keratolytic effect, so a certain amount of skin penetration is to be expected, the authors, conclude. The concentration time course of penciclovir in skin blister fluid and dialysate aft er an oral dose of its prodrug famciclovir was followed in a study by Borg et al. [ 62 ] Comparing plasma, blister and dialysate levels shows that plasma Cmax levels are similar to recovery adjusted dialysate Cmax levels. Cmax values found in blister fluid were slightly lower. The time to reach maximum concentrations in tissue (blister and dialysate) is about 60 min longer than in plasma, and tissue half life is also about 60 min longer compared to plasma. The authors point out that microdialysis measures free tissue concentrations only. For highly protein bound drugs, the concentration in tissue could then be much smaller than tota l concentrations in plasma. In the case of penciclovir, it is pointed out, which has a protein binding of about 20%, plasma and tissue levels can be comparable. The study also tested for the influence of vasoconstriction on penciclovir concentrations in ti ssues. When adrenalin was added to the perfusate or the area above the probe site was cooled using a cooling pack, AUC values for penciclovir in skin tissue was reduced to about one third of the value achieved without interference. The tissue concentration s of 8 methoxypsoralen after oral or topical application was also explored using microdialysis [ 63 ] The compound is used for psoralen plus ultraviolet A (PUVA) therapy for a number of dermatoses. T he study aimed to compare oral treatment and two topical treatments (bath and cream) to assess how to optimize treatment in regards to side effects such as the development of skin cancer after long term PUVA therapy. The three treatments were compared in e ight healthy volunteers in

PAGE 37

37 an open, randomized, three way crossover study. For three volunteers lower doses were given, the rest received higher doses to guarantee measurable concentrations, due to the assumption that 8 methoxypsoralen recovery may be low, because of relatively high protein binding and lipophilicity. Maximum skin tissue concentrations after application of lower doses of 8 methoxypsoralen (i.e. 0.6 mg/kg oral dose, 0.001% cream or 1 mg/L bath) were highest in volunteers treated by bath compa red to cream and oral application, what is more, total plasma concentrations were up to a thousand fold higher after oral application compared to cream or bath application. Compared to tmax after oral application, the maximum concentrations in tissues afte r topical applications were reached faster (one hour compared to one 4 hours) and with less variability. Maximum plasma concentrations were reached at 1 4 hours after oral application and after 1 3 hours after cream and 1.5 hours after bath application. Ti ssue concentrations of 8 methoxypsoralen were measurable after only 20 minutes and peaked within one hour. The authors conclude that topical treatment achieves high efficacy while having low systemic exposure, which could make topical treatment more desira ble than oral application. Microdialysis in Soft T issue : Antimicrobial Agents Infections of skin and soft tissue can be caused by a variety of G ram positive and G ram negative pathogens and are routinely treated with antibiotics. Whereas penicillins and cep halosporins are drugs of first choice, agents of different classes (e.g. oxazolidinones, glycopeptides, macrolides, tetracyclines, etc) have to be used in case of lactam resistance. In order to increase the chances of clini cal success and to decrease the likelihood of toxic side effects as well as resistance development, selection of an appropriate antibiotic dosing regimen becomes

PAGE 38

38 extremely important [ 55 ] The most rati onal approach is to link active drug concentrations to the respective pharmacodynamic outcome. However, efficacy predictions based on total plasma concentrations might be misleading, as most infections are not located in the bloodstream but rather in the I SF of tissues, which is the usual target site for bacterial infections [ 64 ] In fact, it is the free, unbound drug in the ISF that is responsible for antimicrobial efficacy. The chapter sha ll discuss in detail different examples of how this can be achieved by use of microdialysis as a pha rmacokinetic sampling technique and how it has the potential to streamline the decision process on proper drug dosing in drug development. Moxifloxacin is clinically used in the treatment of uncomplicated skin and skin structure infecti ons. A study by Burk hardt et al [ 65 ] used microdialysis to compare the free protein unbound moxifloxacin concentrations in normal as well as infected subcutaneous tissue. A single oral dose of 400mg moxifloxacin was administered to patients with spinal cord injury a nd decubitus ulcers and drug concentrations were determined from serum, saliva and subcutaneous tissue. The study found that moxifloxacin reaches adequate concentrations in normal s.c. tissue and decubitus ulcer tissue in patients with spinal injury. The c oncentrations measured in the ulcer tissue (C max : 2.4 1.6 mg/l) and healthy tissue (C max : 2.7 2.3 mg/l) were similar. The mean C max in serum was found to be 4.4 2.7 mg/l and in saliva 1.4 0.4 mg/l. AUC for normal (9.2 8.6 mg*h/l) and infected sub cutaneous tissue (9.6 6.8 mg*h/l) were approximately same as the free AUC in serum. [ 65 ] Also the tissue levels measured in the study correspond to free protein bound fraction of Moxifloxacin in seru m which is

PAGE 39

39 about 50 60%, by which they concluded that poor blood flow does not affect tissue levels of moxifloxacin in patients with the injury. Traditionally, plasma samples were taken to determine pharmacokinetic properties of a compound and make predict ions on the efficacy. However, these drug concentrations were sometimes presented as total drug concentrations where as in reality only the free drug is pharmacologically active and using total concentrations would overestimate the target site concentratio ns and hence clinical efficacy. Microdialysis has proven useful in the measurement of free drug concentrations in muscle and s.c. adipose tissue [ 66 69 ] A study by Barbour et al [ 70 ] looked at the tissue penetration of ceftobiprole from plasma into skeletal muscle and adipose tissue after a single i.v. dose of 500mg using microdialysis in healthy volunteers. The study measurements allowed showing that ceftobiprole distributes into the ISF of skeletal muscle ( f AUC muscle / f AUC plasma of 0.69 0.13 ) and s.c. adipose tissue ( f AUC s.c./ f AUC plasma of 0.49 0.28) The f AUC of plasma (76.0 8.81 h.mg/l) and f AUC of both tissues were significantly different and a difference between the muscle (50.6 10.9 h.mg/ L ) and s.c. tissue (34.3 19.0 h.mg/ L ) was also noted These findings confirmed that it is important to measure free active drug in each tissue and not make the ass umption that free drug levels in plasma equal free drug levels in tissue, even in well perfused tissues. The reason for difference in penetration ratios of drug can be attributed to several factors such as perfusion of particular tissue, local capillary de nsity, the degree of tissue binding, the possibility of active transporters, loss of drug from peripheral compartments and lipophilicity of the com pound [ 70 ] The MIC for ceftobiprole against methicillin resistant S. aureus [ 71 ] and penicillin resistant S.

PAGE 40

40 pneumoniae [ 72 ] has been reported as 2 mg/l. Measurements from the study showed that concentrations in both skeletal muscle and adipose tissue met the efficacy breakpoint i.e. remained above 2 mg/l for about 50% of the dosing interval. Hence, ceftobiprole qualifies as a potential agent with penetration capabilities to treat complicated skin and skin structure infections, as it is the key determinant in clinical efficacy. A comparison of the kinetic profiles of two cephalosporins (cefpodoxime and cefixime) in soft tissue g iven the same oral dose was conducted in six healthy volunteers [ 69 ] In this study, AUC in plasma of cefpodoxime was 22.4 mg/L*h 8.7 and for cefixime, 25.6 mg/L*h 8.5, which was similar. Interestingly, their target site exposure was markedly different, i.e. AUC muscle was 15.4 mg/L*h 5.1 an d 7.3 mg/L*h 2.2 for cefpodoxime and cefixime, respectively. The C max in plasma for cefpodoxime and cefixime was found to be 3.9 mg/l 1.2 & 3.4 mg /l 1.1 and the in muscle C max was 2.4 mg/l 0.9 & 0.9 mg/l 0.3, respectively The reason for the dif ference in target site exposure of the two drugs in spite of similar profile in plasma is the difference in the degree of protein binding. The study provided insight into the free interstitial levels of cefpodoxime and cefixime which is more meaningful and showed that it is not sufficient to only consider plasma concentration profiles when evaluating pharmacokinetic properties of anti infective agents. Using total plasma concentrations may overestimate the therapeutic outcome because only the unbound fract ion in plasma is able to cross the capillary membrane and reach the interstitial space where the infection is. This may be the reason why some antibiotic treatments fail despite good activity in vitro as well as resistance development [ 73 74 ] The use of total pla sma concentrations in a PK/PD

PAGE 41

41 approach for predicting the clinical efficacy of antibiotics is common where MIC values are compared with plasma concentrations of the antibiotic, usually without considering protein binding. It improves the accuracy of predic ting clinical efficacy if free antibiotic tissue levels by use of microdialysis. Cefpodoxime with low protein binding of 25% had more than twice higher peak concentration in the muscle than cefixime (2.1 vs. 0.9 mg/l). The average tissue penetration (AUC ti ssue,free /AUC plasma,total ) of cefpodoxime (70%) was much higher than that of cefixime (29%) which is consistent with their protein binding values, suggesting that higher protein binding of the drug would lower the tissue penetration [ 73 ] Burkhardt et al [ 75 ] used microdialysis to measure and compare the free protein unbound ertapenem concentrations in the interstitial space fluid of two soft tissues, skeletal muscle and subcutaneous adip ose tissue, following the administration of 1 g infusion, and compared them with the respective plasma concentrations. Results from the study indicate that free, unbound ertapenem profiles in the ISF of both skeletal muscle and subcutaneous adipose tissue are lower than corresponding total plasma concentrations. While free ISF concentrations of the skeletal muscle correlated well with free, unbound concentrations in plasma (4% 16% of total plasma concentration), they were comparably higher than free ISF con centrations in subcutaneous adipose tissue. This was observed in other studies as well and differences in blood flow in these two tissues may be a possible explanation [ 76 ] Free ertapenem concentrations of 1.13 0.68 mg/L in the muscle, observed 12 h after single dose IV infusion of ertapenem, exceeded the MIC of meth icillin susceptible S aureus (0.25 mg/L), Streptococcus spp (0.5 mg/L), extended spectrum b lactamase (ESBL) producing Enterobacteriaceae

PAGE 42

42 (0.03 0.06 mg/L), Bacteroides fragilis least 50% of the entire dosing interval. In comparison, free levels of 0.31 0.16 mg/L in the subcutaneous adipose tissue (at 12 h) exceeded the MIC of the same SSSI pathogens for at least 30% of the dosi ng interval [ 55 ] The authors concluded that free, active ertapenem concentrations reached sufficient levels in non infected ISF of muscle and subcutaneous adipose tissue. When a drug is given orally, i t gets absorbed from the intestine following which it reaches systemic circulation. Depending on the oral bioavailability of the drug, oral doses need to be adjusted to compare it with the respective IV dose in order to achieve similar therapeutic drug lev els in the body. As a case in point, microdialysis was employed to compare free, active ciprofloxacin concentrations in the ISF of skeletal muscle and subcutaneous adipose tissue, after IV(400mg) or oral ciprofloxacin(500mg) administration, respectively [ 55 77 ] Ciprofloxacin is a broad spectrum anti infective agent of the fluoroquinolone class used in the treatment of upper respiratory infection, urinar y tract infections and skin infections. Free ciprofloxacin concentrations were measured in the ISF of skeletal muscle and subcutaneous adipose tissue, saliva, and cantharis induced skin blister, as well as capillary plasma, and compared to total venous pla sma concentrations. Mean f AUCs of both muscle and subcutaneous adipose tissue were significantly lower than the corresponding AUC for plasma after oral and iv administration. C skin blister /C plasma ratio >4 is an indicated that ciprofloxacin mostly accumula tes in the inflamed lesions, while saliva and capillary blood concentrations were similar to total plasma [ 55 77 ]

PAGE 43

43 In another study, Hollenstein et al [ 78 ] addressed the issue of tissue penetration of ciprofloxacin in obese subjects with a mean weight of 122kg using microdialysis. They found significantly lower AUC tissue /AUC plasma ratios in obese subjects (0.45 0.27 vs. 0.82 0.36)[14]. The results helped them conclude that the penetration of ISF is highly impaired in obese subjects, most prob ably due to a reduced capillary permeability surface area in fat tissue. An interesting application of microdialysis was extended to evaluate the effects of simulated microgravity (sG) on the pharmacokinetics of ciprofloxacin [ 79 ] Astronauts have been taking drugs during flights since the early days but little information is available regarding the efficacy of drugs administered during space flights. Astronauts run into the risk of infections due to long term confinement of the spacecraft and impairment of the immune system [ 80 81 ] Also, physiological changes indu ced by microgravity may affect the pharmacokinetics of antibiotics, resulting in altered concentrations at the infection site, thus affecting the way antibiotics are given in space. The study determined and compared the unbound soft tissue concentrations o f them to plasma concentrations. Also, the disposition of ciprofloxacin in humans after 3 interstitial ciprofloxacin concentrations measured by microdialysis in the medial vastus muscle (AUC free,tissue) g/mL*h) than during 1G (1861 109 1 ng/mL*h). The free plasma concentrations were not different in s 274 ng/mL*h) and 1G (2518 97 1 ng/mL*h), a slightly lower value of f was

PAGE 44

44 altered in microgravity [ 79 ] However, the differences were not statistically significant, probably due to the small number of su bjects that participated in our study. Linezolid, an oxazolidinone, is approved for the treatment of nosocomial pneumonia and complicated SSSIs [8] It shows good antimicrobial activity against various resista nt G ram positive bacteria, including methicilli n and glycopeptide resistant S. aureus. Although only free, unbound data is considered for antimicrobial efficacy and most relevant pathogens are located in the ISF, most of the available linezolid PK data is based on total plasma concentrations [ 55 66 ] Hence, a clinical microdialysis study was performed that evaluated the penetration of linezolid into soft tissu es of healthy volunteers after single and multiple dose administration. [ 66 ] After calibration and b aseline determination, 600 mg linezolid was infused intravenously for 30 min and dialysate as well as blood samples were taken for up to 8 h. After withdrawal of the MD probes, volunteers were started on oral linezolid (600 mg) BID for 5 consecutive days [ 55 ] The second set of MD experiments was started simultaneously with the last oral dose. Results show that after single IV administration of linezolid, 0 8 was 65.3 18.2 mg*h/L and 75.8 24.2 mg*h/L for skeletal muscle tissue and subcutaneous adipose tissue respectively which was significantly higher than the 0 8 of plasma (53.0 11.6 mg*h/L) [ 66 ] However, at steady state, no significant differences between concentrations in the ISF of skeletal muscle and subcutaneous adipose tissue could be detected. These findings furthe r indicated that steady state 24 muscle/MIC 58.9 33.0 mg*h/L) and adipose 24 s.c./MIC 46.6 15.9 mg*h/L) were sufficient to treat infections that are caused by pathogens with MICs of up to 4 mg /L.

PAGE 45

45 PK/PD Indices The minimum inhibitory concentration is a well established and routinely determined susceptibility breakpoint parameter for antibiotics. Combinations of this PD marker with free unbound PK parameters to MIC based PK/PD indices such as free >MIC ), max /MIC have led to a much better understanding of antibiotic dosing [ 55 ] The first PK/PD index was developed for penicillin s. It correlates in vivo efficacy with the amount of time free >MIC ) [ 55 ] A common threshold >MIC lactam antibiotics max /MIC index values of 10 to 12 seem to be a good predictor for aminoglycosides [ 55 ] 24 /MIC values o f 1 00 to 125 (G ram negatives), 25 to max /MIC index values of 10 have been identified for >MIC [ 55 ] Onc e these MIC based PK/PD indices are identified, they can support the identification of optimized dosing regimens and the prediction of treatment outcome. Knowledge of the free antibiotic concentration time course in the ISF is necessary in order to establi sh >MIC max 24 / MIC index values. While several techniques are available for determination of free, unbound concentrations, they are not all capable of characterizing dynamic changes in free ISF concentrations. Only microdia lysis allows the combination of these two properties which is essential in predicting clinical efficacy with accuracy. Therefore, it is a very valuable sampling tool and has become an inherent part of evaluation and establishment of PK/PD approach.

PAGE 46

46 Conclus ion The examples presented in this review show that m icrodialysis is appli cable for a variety of purposes Being that the method is comparatively young, there are several methodological issues still to be addressed Analytical methods for analyzing samples gathered via microdialysis frequently have to be very sensitive due to the limited amount of sample volume available [ 82 ] Pr obe calibration is another important part of any microdialysis experiment, especially when absolute interstitial space concentrations need to be reported. Recovery should be measured for each probe and individual experiment and is dependent on a variety of factors, e.g. the tissue surrounding the probe, the flow rate, length of the diffusible membrane, composition of the perfusion fluid, the molecule to be perfused, and many more [ 82 83 ] In summary, microdialysis is an exciting new sampling technique to evaluate pharmacokinetics of drugs in skin and soft tissues serving as an important contri butor to PK/PD approaches.

PAGE 47

47 CHAPTER 3 DETERMINATION OF JNJ Q2 IN SALINE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROM ETRY (LC MS/MS) Objective To validate a High Performance L iquid C hromatography Tandem Mass Spectrometry ( LC MS/MS ) meth od for the determination of JNJ Q2 in saline. The assay is designed to be used for a microdialysis study of JNJ Q2 in soft tissues. The validation parameters included inter/intra batch accuracy and precision measured over 4 days, robustness, stability stud ies including freeze thaw stability, stability at room temperature, in autosampler and long term storage at 80 C and stability studies. Validation Procedure Two standard curves (one at start and second at end of batch) and five of each quality control ( QC ) concentration s were assayed on each analytical day for four days to find the lower limit of quantification (LLOQ) and linear range using inter/intra batch accuracy and precision. The acceptance criteria are defined as accuracy between 80 120% at the (LL OQ) and 85 115% for all other standards and QCs. Precision is defined as relative standard deviation and must be <20% at the LLOQ and <15% at all other concentrations. The accuracy% is calculated as follows: (3 1) The acceptan ce criterion for accuracy of QC samples is 85% 115% except at LLOQ where it is 80% 120%. At least 67% (4 of 6) of the QC samples should be within the above limits; 33% of the QC samples (not all replicates at the same concentration) can be outside the lim its. If there are more than 2 QC samples at each level, then 50%

PAGE 48

48 of QC samples at each level should pass the above limits of deviation. A minimum of 75% standards (at least 6 nonzero points) should be within the limits (85% 115%) for the analytical run to qualify. Chemicals and Equipment Test Article JNJ Q2 was provided by Johnson and Johnson, Pharmaceutical Research and Development, L.L.C. The compound was stored in the original shipping vial at 2 8C in the GLP refrigerator in an amber colored pouch. Internal Standard (IS) The internal standard used for the LC MS/MS assay was JNJ 28312141 AAC (Molecular weight: 460), provided by Johnson and Johnson, Pharmaceutical Research and Development, L.L.C. The compound was stored in the original shipping vial at 2 8C in a GLP refrigerator in an amber colored pouch. Reagent Preparation JNJ Q2 Stock Standard S olution 1 mg/mL stock standard solution of JNJ Q2 is prepared by weighing 10 mg of JNJ Q2 and dissolving it in 10 mL methanol. This is done by adding the J NJ Q2 to a 10 mL volumetric flask and filling with the solvent. A secondary stock of 10 g/mL is prepared by adding 10 L of stock standard solution to 990 L of dilution solution (50:50 v/v methanol:saline). The amount of secondary stock can vary as long as the ratio of stock standard to saline remains the same. JNJ Q2 Working Standard S olutions After the 10 g/mL secondary stock solution is prepared, the working standards are prepared with serial dilutions u sing methanol:saline (1:1 v/v) [ Table 3 10 ] The

PAGE 49

49 starting volume and total volume can vary as long as the ratio remains the same so that the same final concentrations are obtained. The highest concentration in the standard calibration curve will be 792 ng/mL. JNJ Q2 QC S amples Five sets of qualit y controls (QC) are prepared, each set containing a lower limit of quantification, low, two mids, and high QC. The high QC should be between 75 90% of the highest standard. The mid QC should be 40 50% of the highest standard and the low QC should be no m ore that 3x the lower limit of quantitation (LLOQ). Additionally, there should be one QC at the lower limit of quantification. From the stock solution of 1 mg/mL a solution of 10 g/mL is prepared. From here dilutions are made to get the desi red concen trations for the QCs [ Table 3 11 ] The starting volume and total volume can vary as long as the ratio remains the same so that the same final concentrations are obtained. All quality control solutions should be prepared fresh daily. In this dilution sc heme [ Table 3 11 ] the concentrations of 3.12, 6.93, 69.3, 139 and 693 ng/mL would be used as the LLOQ, low, Medium1, Medium2, and High QCs respectively, if the standard curve concentration range is 3.1 792 ng/mL. Internal Standard Stock S olution 1 mg/mL st ock solution was prepared by weighing 10 mg of JNJ 28312141 and dissolving in was prepare dilution scheme for IS preparation is shown in Table 3 12.

PAGE 50

50 HPLC M obi le P hase Mobile p hase A: m ethanol with 0.05 % v/v formic a cid Formic acid was added to HPLC grade methanol in a 1 liter glass bot tle to get a final concentration of 0.05 % v/v. The mobile phase was degassed for 20 min in a ba th sonicato r for 20 min prior to use. Mobile p hase B: a mmonium acetate buffer (10 mM, pH4.0) Accurately weighed ammonium acetate (enough to make 10 mM in 1L) a nd dissolved in 1L triple distilled water and adjusted the pH to 4 using glacial acetic acid. Filtered through a 0.22 micron filter and degassed by sonication for 20 min for use. Mobile phase A and B were used in an isocratic 70:30 ratio for HPLC analysis at 0.5 mL/min flow rate. HPLC wash solution Mix 250 mL of triple distilled water with 2 mL Formic Acid and 50 mL Acetonitrile. To this add 700 mL methanol and mix well. The wash solution is degassed. Sample Preparation All samples that have saline as the m atrix can be directly injected onto the HPLC column. However, samples obtained from microdialysis, both in vivo and in vitro, need to be diluted with methanol to obtain a sufficient sample volume for LC MS/MS analysis. This is done by dividing the test s ample into two equal aliquots of one with 20 L and adding 20 L of 10 ng/mL internal standard in methanol The second aliquot is available as backup sample. The sample results found from the analysis need to be multiplied by two to get the true concentrat ion.

PAGE 51

51 Assay Procedure Mass Spectroscopy Set Up An API 4000 LC MS/MS system consisting of PE200 series Perkin Elmer pumps and autosampler connected to the API 4000 triple quad mass spectrometer was used for the analysis. See Figure 3 1 for representative chromatograms of JNJ Q2 and internal standard. HPLC Pump Conditions Mobile Phase: (1) 70% Metha nol with 0.05% v/v Formic Acid ; (2) 30% Ammonium acetate buffer (10 mM, pH 4) Flow rate: 0.5 mL/min JNJ Q2 Retention Time: ~1.4 min Internal Standard Retention Time: ~1.9 min Autosampler Conditions Injection Volume: 10 L Run time: 4 min 1 injection per vial (blanks may be injected more than once) 1 vial per sample Analysis procedure Each batch of samples will be run with two calibration curves, one at the start and one at the end and quality control standards at 3 levels (Low, Medium and High) in duplicate placed throughout the run. With each run a blank injection of 50:50 v/v methanol:saline will be made at the start to ensure system equilibration. Blank 50:5 0 v/v methanol:saline injections can be made periodically throughout run.

PAGE 52

52 Results Reproducibility of Calibration Curve P arameters The calibration curves (4 days) generated during the determination of inter and intra day precision and accuracy were linear. Consistently good correlation coefficients (r 2 >0.99) were obtained in these experiments [ Table 3 1 ] A 1/X 2 weighting scheme was used. This option provided a similar correlation coefficient to the linear model and a y intercept closer to 0. Lower Limit o f Q uantification The limit of quantifica tion (LLOQ) was identified as 3. 12 ng/mL. It was defined as the lowest concentration of quality controls used in this validation for which the accuracy% was between 80 120%. Intra batch V ariability for Q uality C ontr ol S amples Intra batch evaluation of the analyte was performed on four different days. Intra batch variability on day 1 was 5.88%, 7.31%, 4.50%, 2.08% and 2.39% for the 3.12, 6.93, 69.3, 139 and 693 ng/mL quality control samples, respectively (n=5, Day 1). Accuracy for day 1 of all samples ranged from 94.3% to 116 (Day 1, Table 3 2 for averages). Intra batch variability on day 2 was 13.2%, 4.62%, 6.94%, 5.21% and 2.35% for the 3.1 2, 6.93, 69.3, 139 and 693 ng/mL quality control samples, respectively (n=5, Day 2). Accuracy for day 2 of all samples ranged from 105% to 113% (Day 2). Two LLOQ QC out of five fell out of the specified limit on validation day 2; therefore accuracy and precision for fourth day validati on was determined. Intra batch variability on day 3 was 5.64%, 4.76%, 2.54%, 3.78% and 2.88% for the 3.1 2, 6.93, 69.3, 139 and 693 ng/mL quality control samples, respectively (n=5, Day 3). Accuracy for day 3 of all samples ranged from 94.8% to 113% (Day 3 Table 3 2 for averages). Intra batch

PAGE 53

53 variability on day 4 was 1.98%, 7.09%, 5.29%, 3.09% and 4.12% for the 3.12, 6.93, 69.3, 139 and 693 ng/mL quality control samples, respectively (n=5, Day 4). Accuracy for day 4 of all samples ranged from 99.1% to 114 % (Day 4, Table 3 2 for averages). Inter batch Variability for Quality Control S amples Inter batch variability was 7.25%, 5.97%, 6.45%, 4.00% and 4.42% for the 3.12, 6.93, 69.3, 138.6 and 693 ng/mL quality control samples, respectively [ Table 3 3 ] Mean ac curacy was between 98.2% and 114% [Table 3 3] The accuracy range based on individual QCs is already stated above. Freeze thaw S tability Three cycles of freeze/thawing were performed on the LOQ, mid, and high quality controls (6.93, 138.6 and 693 ng/ mL n =3) between 05 Jan 09 and 10 Jan 09. The QCs were also injected with the standard curve before freezing to ensure correct preparation. The samples were stable for three f reeze/thaw cycles [ Table 3 4 ] One each of the 6.93 and 693 ng/ mL QC samples fell out of acceptance criteria after the first freeze/thaw cycle with an accuracy of 118 and 120%. One each MQC and HQC was out of acceptance criteria after second freeze/thaw cycle with accuracy 117 and 117%. No QC fell out of acceptance criteria after third fre eze/thaw cycle. The ranges for accuracy are 106% 115%, 109% 113%, and 93.3% 98.8% for the 1st, 2nd, and 3rd, freeze/thaw cycles respectively. Therefore, it is concluded that the QCs are stable for 3 freeze thaw cycles. To test freeze/thaw stability, st andard curves were prepared fresh and analyzed with quality control samples. The standard curves were prepared fresh from the powdered compound on 07 Jan 09 for 1 st and 2 nd freeze/thaw cycles and on 10 Jan 07 for 3 rd freeze/thaw cycle.

PAGE 54

54 Stability at Room T emperature The stability of JNJ Q2 QCs was tested at room temperature. To do so a set of QCs were prepared and left out on the bench top. Then after a defined length of time a fresh standard and second set of QCs were prepared from the stock standard, a nd injected onto the column. The concentrations of QCs left on bench top were determined using the standard curve injected. The stock standard QCs was shown to be stable f or 6 hours at room temperature [ Table 3 5 ] The accuracy of each individual QC samp le ranged from 101% 106%. Refrigeration S tability of QCs To test the stability of QC samples at 4C a set of QCs were prepared and stored at 4C. Then after a defined length of time a fresh standard and second set of QCs were prepared from the stock sta ndard, and injected onto the column. The concentrations of QCs stored at 4C were determined using the standard curve injected. The QCs were shown to be stable for 8 hours at 4C. The mean accuracy of QC sample after 48 h ours ranged from 98.9% to 101% [ Ta ble 3 6 ] Freezer (Long term) S tability Long term stability of the stock standard solution was demonstrated for 62 days by comparing QCs stored at 70C for 62 days with freshly prepared standards. An aliquot of each the samples prepared on 14 June 09 was frozen at 70C and analyzed on 15 Aug 09. The Mean accuracy at each QC level ranged from 107 111% [ Table 3 7 ] Auto injector S tability of QCs To test the stability of QC samples in auto injector a set of QCs were prepared and stored in auto injector. Then after a defined length of time a fresh standard curve was prepared from the stock standard, and injected onto the column. The concentrations o f

PAGE 55

55 QCs stored in auto injector were determined using the standard curve injected. The QCs were shown to be stable for 8 hours in the auto injector. The mean accuracy of QC sample after 8 hours ranged from 100% to 102% [ Table 3 8 ] Robustness To test the a bility of this method to endure changes a different column was used to instead of column no. 016835292102, which was used for most other validation experiments. The intra bat ch precision was 10.8%, 7.72%, 4.53%, 6.04% and 3.19% for the 3.12, 6.93, 69.3, 139 and 693 ng/ mL quality contr ol samples, respectively (n=5) [ Table 3 9 ] Accuracy ranged from 102% 113% for individual samples. Summary Range of Standard Curve Linear over the range of 3.09 792 ng/mL Lower Limit of Quantification 3.12 ng/mL Selectivity No interfering chromatographic peaks were observed in injections with blank saline at the retention time of JNJ Q2 and internal standard ( JNJ 28312141 ) Intra batch Precis ion of Quality Controls (QCs) 1.98% 13.2% for the range of 3.09 792 ng/mL Inter batch Precision of QCs 4.00% 7.25% for the range of 3.09 792 ng/mL Intra batch Accuracy of QCs 94.3% 116% for the range of 3.09 792 ng/mL Inter batch Accuracy of QCs 98.2% 114% the range of 3.09 792 ng/mL Freeze/thaw Stability of QCs Stable over 3 cycles for the range of 3.09 792 ng/mL Stability in saline at Room Temperature of QCs Stable over 6 hours for the range of 3.09 792 ng/mL Freezer Stability of QCs S table over 2 month for the range of 3.09 792 ng/mL

PAGE 56

56 Auto Inj ector Stability of QCs Stable over 8 hours for the range of 3.09 792 ng/mL Refrigeration Stability of QCs Stable over 8 hours for the range of 3.09 792 ng/mL Robustness method: (1) Intra batch Precision of QCs 3.19% 10.8% for the range of 3.09 792 ng/mL; (2) Intra batch Accuracy of QCs 102% 113% for the range of 3.09 792 ng/mL An LC MS/MS method for the analysis of JNJ Q2 in s aline was developed for the analysis of microdialysis samples. The calibration curve range, 3.09 792 ng/mL is appropriate ly based on the expected in vivo concentrations. The compound is stable over 6 hrs at room temperature, can be frozen and thawed at least 3 times before stability becomes a concern. In conclusion, JNJ Q2 was observed to be stable under the conditions which will be encountered during an in vivo clinical study and its analysis

PAGE 57

57 Figure 3 1. Representative chromatograms f or (A) blank JNJ Q2 (B) spiked JNJ Q2 (3.09 ng/mL) (C) blank internal standard (D) spiked internal standard (5 ng/mL) B C D A

PAGE 58

58 Table 3 1 Summaries of linearity data Slope Intercept r 2 Day 1 0.0105 0.0090 0.9920 Day 2 0.0082 0.0002 0.9974 Day 3 0.0115 0.0070 0.9952 Day 4 0.0105 0.0038 0.9916 Mean 0.0102 0.0049 0.9941 SD 0.0014 0.0040 0.0027 Table 3 2 Intra batch variability of quality control samples (n=5 per day and per concentration) QC LLOQ Low Med 1 Med 2 High Conc. (ng/mL ) 3.12 6.93 69.3 139 693 Day 1 12/23/08 Mean 3.63 6.97 65.4 139 720 SD 0.21 0.51 2.94 2.88 17.2 % CV 5.88 7.31 4.50 2.08 2.39 % Accuracy 116 101 94.3 99.7 104 Day 2 01/02/09 Mean 3.52 7.29 72.5 146 783 SD 0.47 0.34 5.03 7.62 18.4 % CV 13.2 4.62 6.94 5.21 2.35 % Accuracy 113 105 105 106 113 Day 3 01/03/09 Mean 3.52 6.95 65.7 141 723 SD 0.2 0.33 1.67 5.33 20.8 % CV 5.64 4.76 2.54 3.78 2.88 % Accuracy 113 100 94.8 102 104 Day 4 01/05/09 Mean 3.58 7.23 68.7 143 744 SD 0.07 0.51 3.63 4.41 30.6 % CV 1.98 7.09 5.29 3.09 4.12 % Accuracy 115 104 99.2 103 107 Table 3 3 Inter batch variability of quality control samples (n=20 per concentration) QC LLOQ Low Med 1 Med 2 High Conc. (ng/mL ) 3.12 6.93 69.3 139 693 Mean 3.56 7.11 68.1 142 742 SD 0.26 0.42 4.39 5.69 32.8 % CV 7.25 5.97 6.45 4.00 4.42 % Accuracy 114 103 98.2 103 107

PAGE 59

59 Table 3 4 Freeze/thaw stability of JNJ Q2 (n=3) QC Low Med 2 High Concentration (ng/mL ) 6.93 139 693 Cycle 1 Mean 7.35 153 795 SD 0.76 4.91 31.2 CV (%) 10.3 3.21 3.92 Accuracy (%) 106 110 115 Cycle 2 Mean 7.76 151 786 SD 0.17 9.77 23.1 CV (%) 2.14 6.49 2.94 Accuracy (%) 112 109 113 Cycle 3 Mean 6.71 129 684 SD 0.39 4.25 45.9 CV (%) 5.88 3.29 6.7 Accuracy (%) 96.8 93.3 98.8 Table 3 5 Room temperature Stability of JNJ Q2 (n=3) Time= 6hr QC Low Med 2 High Concentration (ng/mL ) 6.93 139 693 Mean 7.38 145 697 SD 0.66 7.54 25.3 CV (%) 8.88 5.22 3.63 Accuracy (%) 106 104 101 Table 3 6 Refrigeration stability (4C) of JNJ Q2 (n=3) Time= 8hr QC Low Med 2 High Concentration (ng/mL ) 6.93 139 693 Mean 7.02 139 685 SD 0.11 6.63 33.8 CV (%) 1.51 4.77 4.93 Accuracy (%) 101 100 98.9 Table 3 7 Freezer stability ( 70C) of JNJ Q2 (n=3) .Time=62 days QC Low Med 2 High Concentration (ng/mL ) 6.93 139 693 Mean 7.70 148 770 SD 0.17 1.59 13.3 CV (%) 2.20 1.07 1.73 Accuracy (%) 111 107 111

PAGE 60

60 Table 3 8 Auto injector stability of JNJ Q2 (n=3) Time=62 days QC Low Med 2 High Concentration (ng/mL ) 6.93 139 693 Mean 7.10 140 694 SD 0.61 9.85 44.6 CV (%) 8.63 7.04 6.43 Accuracy (%) 102 101 100 Table 3 9 Intra batch variability of JNJ Q2 using a different column to check robustness (n=5) QC LLOQ Low Med 1 Med 2 High Conc. (ng/mL ) 3.12 6.93 69.3 139 693 Mean 3.50 7.51 70.9 146 781 SD 0.38 0.58 3.21 8.82 24.9 % CV 10.8 7.72 4.53 6.04 3.19 % Accuracy 112 108 102 105 113

PAGE 61

61 Table 3 10 Calibration s tand ard p reparation of JNJ Q2 CS Code CS 9 CS 8 CS 7 CS 6 CS 5 CS 4 CS 3 CS 2 CS 1 Vol of DS (L) 1840 1000 1000 1000 1000 1000 1000 1000 1000 Vol of CS added (L) 160 of SS1 1000 of CS9 1000 of CS8 1000 of CS7 1000 of CS6 1000 of CS5 1000 of CS4 1000 of CS3 1000 of CS2 Final Conc (ng/mL ) 792 396 198 99.0 49.5 24.8 12.4 6.19 3.09 Note: Dilution Solution (DS): Methanol:Saline (50:50 v/v) JNJ Q2 Primary Stock: 1 mg/mL in Methanol JNJ Q2 Secondary Stock1(SS1): 10 g/mL Table 3 11. Quality co ntrol sample p reparation of JNJ Q2 QC High Medium 2 Medium1 Low LLOQ Vol of DS (L) 930 800 500 900 550 Vol of QC added (L) 70 of SS1 200 of QC H 500 of QC M2 100 of QC M1 450 of QC L Final Conc (ng/mL) 693 139 69.3 6.93 3.12 Table 3 12 Internal s ta ndard spiking s olution Solution ID Prepared from Stock Volume added (L) Total Volume (mL) Solution Conc IS Stock1 added (L) 1mg/mL IS stock solution 10 1 10 g/mL IS Stock 2 added (mL ) IS Stock1 25 1 250 ng/mL Note: 10 L of 250 ng/mL IS Stock2 solution was added to all calibration standards and QC samples to have a final concentration of 5 ng/mL of I S The starting volume and total volume can vary as long as the ratio remains the same so that the same final concentrations are obtained.

PAGE 62

62 CHAPTER 4 IN VITRO MICRODIALYSIS OF JNJ Q2 Objective The aim of this study was to determine the ability of JNJ Q2 to cross the MD membrane using two dose ranging, in vitro microdialysis experiments. The two experiments performed used the extraction efficiency (EE) and retrodialysis (RD) methods. In both of these methods, the percent recovery (R%) was determined. The R% had to be m ore than 10% if microdialysis was to be used for sampling JNJ Q2 from soft tissues. Microdialysis MD is a unique tool for antibiotic sampling because the samples are taken from soft tissues, the most common site of infection, and because only the free, pha rmacologically active drug is able to pass through the probe membrane. In this technique, the MD 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 equilibr ium will not be reached. The ability of the drug to pass through the membrane and establish equilibrium at this flow rate will be established. It is termed the recovery (R) and this value has to be known to back 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: (4 1)

PAGE 63

63 In MD 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 the surrounding media to occur. Therefore, a compromise has to be made between sample volume and flow rate. Depending on the sensitivity of L are collected. The equilibration period is de termined by the dead volume of the MD probe tubing which can be performed using five different concentrations, all of the tubing has to be completely flushed before the sampl ing procedure can start. The equilibration time is the product of dead volume multiplied by flow rate. Extraction Efficiency Method (E E ) In the EE method, blank saline is pumped through the MD probe at a flow rate of into the calibration tube 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 30min after the end of a 25min equilibration period. To ensure that the prepared solution is the concentration expected within the calibration tube, two samples are taken from this tube, one before the sampling period and one after. It is important to sample from the calibrati on tube since it is critical to know the actual concentration in the tube to perform the calculations. The same procedure is done for the remaining five samples. After the highest concentration is completed the probe is flushed for two hours

PAGE 64

64 with blank sa line. All experiments are performed in triplicate. The percent recovery, R%, for the EE method is calculated as follows: (4 2) Retrodialysis (RD) In the RD method, the syringe contains the analyte solution that is pumped th calibration tube that is filled with approximately 10mL of saline. The analyte diffuses out of the probe into the calibration tube. The loss of analyte through the membrane can t hen be determined from the C dialysate Samples are taken for 30min after the end of the 25min equilibration period. To ensure that the prepared solution is the concentration expected within the syringe, a sample is taken from the syringe before and after the sampling period. Again the lowes t concentration is 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 saline. The same procedure is done for the remaining fiv e samples. After the highest concentration is sampled the probe is flushed for two hours with blank saline. All experiments are performed in triplicate. The percent recovery, R%, for the RD method is calculated as follows: ( 4 3 )

PAGE 65

65 Retrodialysis under Light D ark Conditions To check if JNJ Q2 shows any instability due to its exposure to light during the in vitro MD study, experiments were performed in presence and in the absence of white laboratory light (set up was covered with amber films to avoid laboratory light) using RD technique. RD was chosen as it is represents the method to be used for in vivo experiments. This comparison was performed at a concentration level, which is the expected maximum tissue concentration after pr otein binding. All other conditions were kept identical. Chemicals and Equipment Test Article JNJ Q2 was provided by Johnson and Johnson, Pharmaceutical Research and Development, L.L.C. The compound was stored in the original shipping vial at 2 8C in the GLP refrigerator in an amber colored pouch. The c hemical structure of JNJ Q2 (Molecular weight: 455 .88) is shown in Figure 4 1. Internal Standard The internal standard used for the LC MS/MS assay was JNJ 28312141 AAC (Molecular weight: 460.3) provided by Johnson and Johnson, Pharmaceutical Research and Development, L.L.C. The compound was stored at 2 8 C in a GLP refrigerator in an amber colored pouch. Reagent Preparation JNJ Q2 Standards and Quality Control Solutions JNJ Q2 was provided as a pure powder. To obtain a 1 mg/mL primary stock solution, 10 mg of JNJ Q2 was accurately weighed out and dissolved in 10 mL of pure methanol. The 1 mg/mL primary stock solution was diluted to a secondary stock solution

PAGE 66

66 of 10 g/mL with dilution solution (50:50 v/v methanol:saline). Calibration standards were then prepared by se rial dilution with dilution solution. The standard curve ranged from 3.09 792.08 ng/mL. Quality controls (QC) standards of 3.12, 6.93, 69.31, 138.61 and 693.07 ng/mL were used for LLOQ, LOQ, Medium1, Medium2, and High, respectively. The dilution scheme for the calibration standards (CS) and QCs are shown in Table 4 1 and Table 4 2. Preparation scheme and addition of IS is described in Table 4 3. Mass Spectroscopy Mobile Phase Mobile phase A: m ethanol with 0.05 % v/v Formic Acid Formic acid was added to HPLC grade methanol in a 1 liter glass bottle to obtain a final concentration of 0.05 % v/v. The mobile phase was degassed for 20 min in a sonicator bath prior to use. Mobile phase B: a mmonium acetate buffer (10 mM, pH4.0) Accurately weighed ammonium acetate (enough to make 10 mM in 1L) and dissolved in 1L triple distilled water and adjusted the pH to 4 using glacial acetic acid. Filtered through a 0.22 micron filter and degassed by sonication for 20 min for use. Mobile phas e A and B were used in an isocratic 70:30 ratio for HPLC analysis at 0.5 ml/min flow rate. Sample Preparation Calibration Solution f or M icrodialysis Five calibration solutions of JNJ Q2 over concentration range of 37.4 513.7ng/mL were prepared. From the 1mg/mL stock solution of JNJ Q2 in pure methanol a 10

PAGE 67

67 making further dilutions. These concentrations were selected for vitro studies as the expected maximum concentrat ion of JNJ Q2 in soft tissues is ~300 ng/mL. Dialysate Samples During the sampling period approximately 45 L of sample were collected. Accurately 30 L of the sample was diluted ( 1:1 by volume) with 10 ng/m L internal standard in methanol. The samples ta ken from the calibration tube and/or the syringe were also diluted 1:1 with 10 ng/m L internal standard in methanol, before and after the dialysis procedure (dilution factor of 2 for the samples). Apparatus Setu p Prior to the start of experiments, each MD probe was checked for functionality. To do so, the inlet of the MD probe was connected to a syringe containing blank saline. The probe was then flushed manually. The probe was functional and ready to use when no liquid drops appeared on the MD probe membra ne. The solution should only exit the probe from the outlet tubing. If droplets appeared on the membrane, the probe could not be used. To control temperature the sampling device was assembled on a heated stir plate and was maintained at 37 o C. During setup a 5mL syringe was filled either with blank saline solution (EE method) or analyte solution (RD method) and the enclosed air was cleared from the syringe. The syringe was put in place on the syringe pump and fastened. T he pump was then co nnected to the inlet of the probe and run at a flow rate of 1.5 min to allow for equilibration. The probe itself was in a 15 mL centrifuge, or calibration, tube containing either blank saline (RD method) or analyte solution (EE method). Att ention was paid to ensure that t he membrane of the probe was completely covered with fluid and that it d id not touch the wall of the tube. The

PAGE 68

68 dialysate was collected in a microcentrifuge tube covered with parafilm, which helps to fix the outlet tubing fr om the microdialysis probe in place, and protected from laboratory light by aluminium wrap. Sample Analysis Mass Spectroscopy Set Up An API 4000 LC MS/MS system consisting of PE200 series Perkin Elmer pumps and autosample r connected to the API 4000 tri pl e quad mass spectrometer was used for the analysis. HPLC Pump Conditions Mobile Phase: 70% Methanol with 0.05% v/v Formic Acid, 30% Ammonium acetate buffer (10mM, pH 4) Flow rate: 0.5 mL/min JNJ Q2 Retention Time: ~1.4 min Internal Standard Retention Time: ~1.9 min Autosampler Conditions Injection Volume: 10 L Run time: 4 min 1 injection per vial (blanks may be injected more than once) 1 vial per sample A nalysis Procedure The test samples were run with a calibration curve and quality contr ol standards at 5 levels (LLOQ, A blank injection of methanol:saline::1:1 was included. Results Method Validation Summary Range of Standard Curve Linear over the average concentration range of 3.0 9 792.08 ng/mL

PAGE 69

69 Lower Limit of Quantification 3.09 ng/ml (% error < 20%) Selectivity N o interfering chromatographic peaks were observed in blank saline Procedure QCs (LLOQ, LOQ, Medium1, Medium2, and High) were used to test the accuracy and precisio n. The analysis was performed using a 1/X 2 weighted linear regression. Accuracy and Precision t he mean accuracies for the LLOQ, LOQ, Medium1, Medium2, and High were 114.2%, 102.6%, 98.2%, 102.5% and 107.1% respectively. The average precision (CV %) wa s less than 7%. 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 calculated concentrations for a ll other standards were within 15% of their nominal concentrations, the low, mid range and high quality controls were within 15% (20% at the LLOQ) of their nominal concentrations and at least three out of five of the QCs met the acceptance criteria at each concentration. In Vitro 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. Thes e in vitro experiments were done as a preliminary study to the in vivo experiment. The retrodialysis method is the same method which will be used in the in vivo experiment f or MD probe calibration See T able 4 5 and Table 4 6 for results. The results f rom the comparison of exposure to light and under dark conditi ons for concentration ~300 ng/mL by retrodialysis are shown in T able 4 7.

PAGE 70

70 Conclusions The in vitro experiments confirmed that JNJ Q2 has the ability to freely cross the microdialysis membrane. The R% was well over 10% (77.8% 105.6%) and therefore microdialysis may be used as a sampling technique to obtain a PK profile in soft tissues. Also it was noted that carrying out the in vitro microdialysis under exposure to laboratory light had no sig nificant impact on the drug stability. The recovery was found to be similar under light exposure and with no light.

PAGE 71

71 Figure 4 1. Clinical structure of JNJ Q2

PAGE 72

72 Table 4 1. Dilution schemes for calibration standard CS Code CS 9 CS 8 CS 7 CS 6 CS 5 CS 4 CS 3 CS 2 CS 1 Vol of DS (L) 920 500 500 500 500 500 500 500 500 Vol of CS added (L) 80 of SS1 500 of CS9 500 of CS8 500 of CS7 500 of CS6 500 of CS5 500 of CS4 500 of CS3 500 of CS2 Final Conc (ng/mL ) 792.08 396.04 198.02 99.01 49.50 24.75 12.38 6.19 3.09 *Dilution Solution (DS): Methanol:Saline (50:50 v/v) JNJ Q2 Primary Stock: 1 mg/m L in Methanol JNJ Q2 Secondary Stock1 (SS1): 10 g/mL (990L DS + 10L of Primary Stock). Table 4 2. Dilution scheme for quality controls QC High Medium 2 Medium1 LOQ LLOQ Vol of DS (L) 930 800 500 900 550 Vol of QC added (L) 70 of SS1 200 of QC H 500 of QC M2 100 of QC M1 450 of QC L Final Conc (ng/mL) 693.07 138.61 69.31 6.93 3.12 Table 4 3. Internal standard spiking solution Solution ID Prepared from Stock Volume added (L) Total Volume (mL) Solution Conc IS Stock1 added ( L ) 1mg/m L IS stock solution 10 1 10 g/mL IS Stock 2 added (mL ) IS Stock1 25 1 250 ng/mL 10 L of 250 ng/mL IS Stock2 was added to all calibration standards and quality control samples to have a final concent ration of 5 ng/mL of Internal standard.

PAGE 73

73 Table 4 4. I nter batch variability of quality control samples (n=20 per concentration) QC LLOQ LOQ MQC1 MQC2 HQC Conc. (ng/mL) 3.12 6.93 69.31 138.61 693.07 Mean 3.45 7.04 68.21 141.17 729.78 SD 0.32 0.35 4.09 5.53 36.75 % CV 9.33 5.00 6.00 3.92 5.04 % Accuracy 114.26 102.58 98.21 102.53 107.13 Table 4 5. Recovery retrodialysis m ethod Average Observed Concentration (ng/mL) Mean R% (n=3) SD 37.4 77.8 4.8 53.1 89.7 2.0 325.3 95.3 2.2 513.7 95.7 0.6 Average 89.6 Table 4 6 Recovery extraction efficiency m ethod Average Observed Concentration (ng/mL) Mean R% (n=3) SD 66.9 105.6 7.79 111.6 100.7 9.12 168.3 93.1 10.97 318 96.2 9.36 485 100.6 5.37 Average 99.2 Table 4 7 Recovery light & dark e x periments by retrodialysis m ethod Average Observed Concentration (ng/mL) Dark Mean R% (n=3) Average Observed Concentration (ng/mL) Light Mean R% (n=3) 307.7 89.7 325.3 96.6 96.1 92.7 98.7 96.4 Average 94.8 Average 95.3 SD 4.6 SD 2.2

PAGE 74

74 CHAPTER 5 CLINICAL TISSUE DIST RIBUTION STUDY OF A NOVEL FLUOROQUINOLON E, JNJ Q2 USING MICRODIALYS IS IN HEALTHY SUBJEC TS JNJ Q2 is a novel fluorinated 4 quinolone oral antimicrobial agent. It is a broad spectrum fluoroquinolone with potent in vitro bactericidal act ivity against a diverse set of G ram positive bacteria including the methicillin resistant S. aureus strains (MRS A), G ram negative, atypical respiratory and anaerobic pathogens. It is indicated for its use in treating community acquired bacterial pneumonia and complicated skin and skin structure infections (cSSSIs), including diabetic foot infection. JNJ Q2 displays a mechanism of action that is consistent with other members of the fluoroquinolone class, in that the compound inhibits the target enzymes DNA gyrase and topoisomerase IV in vitro with IC50 values comparable to those of marketed fluoroquinolones [ 29 ] The in vitro MIC value against MRSA is 32 fold lower than moxifloxacin, and 8 fold lower than gemifloxacin. The in vivo efficacy of lethal infection efficacy models with methicillin susceptible S. aureus strains (MSSA) was comparable to moxif l oxacin and improved over ciprofloxacin. JNJ Q2 was more pote nt against community acquired MRSA (CA MRSA) i solate rather than linezolid or vancomycin when dosed subcutaneously. In a murine S. pneumoniae lower respiratory tract infection model JNJ Q2 was more potent on an exposure basis than moxifloxacin in a murine MRSA skin infection model,JNJ Q2 reduced the bacterial levels and skin lesion volumes to a greater extent than linezolid or vancomycin [ 29 ] The planned clinical lead indication for JNJ Q2 is cSSSIs including diabetic foot infection. Efficacy predictions based on total plasma concentrations might be misleading, as most infections are not located in the bloodstream but rather in the interstitial fluid (ISF) of tissues, which is the usual target site for bacterial infections [ 84 ]

PAGE 75

75 In fact, it is the free, unbound drug in the ISF that is responsible for antimicrobial efficacy. The tissue penet ration and distribution of an tibiotic is of great importance, since most of the infections occur in the tissue. At the infection site, the free, unbound fraction of the antibiotic is responsible for the anti infective effect. [ 85 ] It is therefore important to access the in vivo penetration of JNJ Q2 into the interstitial space fluid (ISF) of sof t tissues such as s.c. adipose tissue and skeleton muscles. A technique that has been proven useful for the measurement of unbound concentrations in target tissues in human is MD Microdialysis is a minimall y invasive sampling technique which allows the measurement of actual free drug concentrations in different tissues and organs and subsequently uses these PK findings to relate with PD observations to predict clinical efficacy. The ability to measure the free concentrations at the site of drug action over time makes microdialysis a very valuable tool for assessment of bioavailability and bioequivalence and has been recognized by industr y and regulatory authorities such as the FDA [ 86 87 ] Furthermore, guidance from both European Medicines Evaluation Agency and the Unites States Food and Drug Administration (F DA) support the exploration of PK/PD relationships in plasma and target tissues th a t support dose and dose regimen selections. As such, tissue distribution studies are recommended to evaluate the PK/PD relationships for anti infectives. Objectives The overall objective of this study is to evaluate penetration of the JNJ Q2 into the ISF of s.c. and skeletal muscle tissue s in healthy volunteer s at a projected human

PAGE 76

76 efficacious dose (400 mg) using the micro dialysis technique. Thi s study is divided into 2 parts Pilot and Main study. The hypothesis for testing is that JNJ Q2 concentrations are measurable in the target tissues indicating the tissue penetration of the study drug. Methods Demographics A total of 15 subjects participated in the study (3 in the pilot and 12 in the main). Of these subjects 10 were male and 5 were female. The ages of subjects ranged from 19 43 years old. This study enlisted 1 Asian, 2 Black, and 12 Caucasians [Table 5 1] Study Drug JNJ Q2 was supplied as tablets by the sponsor, Johnson & Johnson Pharmaceutical Research and Development. Subjects received a 400mg single oral dose of JNJ Q2 with 240mL of water. To ensure that an accurate dose is given, the study medication was administered direct of the investigator or designated study personnel. Sampling Technique Microd ialysis is a sampling technique based on simple diffusion of free analyte through the semi permeable membrane at t he tip of a microdi alysis probe placed in the tissue of interest. The feasibility of this technique has been previously demonstrated in soft tissues [ 73 85 88 90 ] A physiologic al solution, i.e. saline, is continuously perfused through the probe at a low flow rate, i.e. 1.5 l/min. Drug passes from the tissue (C tissue ) into the dialysate in the probe (C dialysate ) and is collected and analyzed. This principle is based on the fac t that diffusion is equal in both directions across the semi permeable membrane of the microdialysis probe. However, the equilibrium between the tissue and dialysate is incomplete, C tissue >C dialysate and the amount which is actually recovered has

PAGE 77

77 to be ca lculated typically by retrodialysis [ 84 ] This is done by performing an in vivo calibration as described below. (5 1) The average of concentrations of calibration solution at the start of calibration and at the end of calibration was used for the C perfusate value. The concentration of the sample collected from 30 to 60 minutes after the start of the calibration time was taken as C dialysate Once the recovery value has been determined the concentration in the tissue is calculated as [ 84 ] : (5 2 ) The C dialysate represented here is the dialysate collected after dosing has started. Once the probes are implanted they remain in place until all study related procedures are completed. Therefore, each recovery value is specific for each probe implanted for use in the study. Study Design Study subjects were admitted to University of Florida Clinical Research Center (CRC) Refer to Appendix C for study criteria. The study was a single center, open label, one arm, non ran domized study in healthy subjects. It consist ed of 2 parts: a pilot study and a main study. Pilot A pilot study with 3 subjects was conducted first to validate the microdialysis method with JNJ Q2 At least 10% recovery is needed in the dialysate in order to quantify JNJ Q2 in the target tissues recovery, it

PAGE 78

78 were followed by the main study [ 55 87 88 91 ] Pilot study subjects participated in a screening examination, a 1 day open label treatment phase with end of study procedures, and a follow up visit 1 2 weeks after the discharg e [Table 5 2]. Three eligible subjects were admitted to the CRC for the microdialysis procedure on Day 1. Two microdialysis probes were inserted into the thigh, one into the medius vastus muscle and the other into the s.c. adipose tissue. JNJ Q2 is locally administered via the MD probe at a concentration of approx. 2 g/mL in saline at a flow rate of 1.5 L /min for 60 minutes into the muscle and adipose tissue. Cumulative dialysate collected from 30 to 60 min utes after the start of the calibration and then individual dialysate samples every 30 minutes for 3 hours during the washout period. Pharmacokinetic plasma samples were collected before the start of the procedure, immediately following the equilibration period (1 hour) and upon completion of the washout period (4 hours). Main The ma in study involved 12 healthy male and female subjects. It consist ed of a screening examination, 2 day open label treatment phase, end of study procedures on Day 2, and a follow up visit 1 2 weeks later to assess ongoing or new adverse events T he expected duration of the main study was approximately 5 weeks [Table 5 3] Subjects were admitted on Day 1 to confirm eligibility to the study. On Day 1, microdialysis probes were inserted into the target tissues of subjects and the dialysis probe calibrated Probe calibration was performed for 60 minutes followed by a washout period of a length determined in the pilot study. Following the washout period, microdialysis probes were continue d to be infused with saline Subjects receive d a single 400 mg oral dose of JNJ Q2 on Day 1. Subjects were given a standardized lunch

PAGE 79

79 by the CRC 30 min before dosing and allowed 20 min to consume it. Plasma samples were taken within 30 min predose and at 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, 16, 24 hours postdose. Blood samples for plasma protein binding analysis were also collected at 4 and 12 hours postdose Dialysate samples were taken at 20 minute intervals for up to 12 hours Concentrat ions of JNJ Q2 in plasma (total drug concentration) and dialysate samples (free unbound concentration) were determined using the validated analytical methods. Safety was assessed by review of adverse events, physical examination, vital signs, 12 lead ECGs and clinical laboratory tests. Subjects were discharged from the unit on Day 2 fol lowing the 24 hour blood sampling and completion of safety assessments at the predefined time points. A pharmacogenomic blood sample w as collected from subjects who consen t ed separately to the pharmacogenomic component of the study. Subject participation in pharmacogenomic research is optional. While in the study unit the subjects were provided dinner on day 1; standardized breakfast, lunch, dinner and snack on day 1; and breakfast and lunch on day 2. Meals other than those standardized were provided from Shands Hospital Foodservice at University of Florida and were modified for the protocol restrictions. Analysis Sample analysis The JNJ Q2 concentration in dialysate was determined by a validated HPLC MS/MS assay method. An API 4000 LC MS/MS system consisting of PE200 series Perkin Elmer pumps and autosampler connected to the API 4000 triple quad mass spectrometer was used for the analysis. The mobile phase consisted of a mixture of

PAGE 80

80 70% m etha nol with 0.05% v/v Formic Acid, and 30% a mmonium acetate buffer (10mM, pH 4) The flow rate was set to 0.5 mL/min and injection volume to 10 L The lower limit of quanti fic ation (LLOQ) for this method was 3.12 ng/mL. The linearity for calibration curve was 3.09 792 ng/mL. T o ensure system functionality throughout each run, quality controls standards (QCs) were injected at three concentration levels. Sample prep aration : Dialysate s amples obtained from MD study were diluted with m ethanol to obtain a sufficient sample volume for LC MS/MS analysis. This is done by by a 1:1 dilution of dialysate with 10 ng/m L internal standard in methanol Following the analyses free adipose and muscle ISF tissue concentrations were calculated from measured dialysate concentrations by correcting for determined recovery% values using the formula C tissue = 100 x C dialysate x recovery% 1 where C tissue is the drug concentration in the ISF of respective tissue. Data analysis Plasma and microdialysis fluid concentrations of JNJ Q2 were subjected to pharmacokinetic analyses using non compartmental methods. Pharmacokinetic parameters were determined by noncompartmental analysis using the commercially available software program Wi nNonlin 5.2 (Pharsight Corp., Mountain View, CA). The points selected for analysis were chosen using the best fit as supplied by WinNonlin. The area under the concentration time curve from 0 to the last measured value (AUC 0 last ) was calculated using the linear trapezoidal rule and AUC 0 was calculated as AUC 0 last + C last / z where C last is the last concentration measured. Clearance (CL) was calculated as the dose divided by the AUC 0 and the volume of distribution ( V d ) was calculated as the CL z The plasma concentration s were corrected for the

PAGE 81

81 free fraction based on individual protein binding As a measure of tissue penetration, ratios of free muscle AUC to free plasma AUC ( f AUC muscle /AUC plasma ) and free adipose AUC to free plasma A UC ( f AUC adipoe /AUC plasma ) concentrations will be determined. Results Mean (%CV) in vivo recovery values of JNJ Q2 from subjects in the pilot study for the adipose and muscle tissues were 70.8% (6.7) and 73.7% (5.5), respectively [Table 5 4] In the pilot study, one probe malfunctioned after insertion and therefore the mean recovery value for the muscle was calculated from two subject s. These recoveries were considered high and sufficient (i .e. measurable and reproducible) to proceed to the main stud y Additionally, the average concentrations at the end of the 3 hr wash out were higher than the effective LOQ of 6 ng/mL and therefore wash out was increased to 4 hrs. Mean ( %CV) in vivo recovery in the main study w as 56.5% (29.3) and 68.1% (13.8) in adi pose and muscle, respectively and the measured concentrations were adjusted accordingly [Table 5 5] Mean pharmacokinetic parameters are summarized in Table 5 6 MD data for adipose was calculated from only 11 subjects owing to probe failure during the ex periment. Individual protein binding estimates were used to calculate unbound plasma concentrations. Mean C max ( SD ) was 1.95 (0.45), 0.67 (0.14), 0.83 (0.39) and 0.89 (0.32) ng/mL for total plasma, free plasma, subcutaneous ad ipose and skeletal musc le, respectively. t max values were 4.08 ( 1. 7 2 ) 4. 80 ( 1.11) and 4.81 (1.02) hrs for plasma, adipose and muscle was 13.6 (5.48). Mean CL/F and V d /F values were 12.5 ( 3.87 ) L/hr and 223.9 ( 42.8 ) L, respectively.

PAGE 82

82 The mean protein binding was 65% and mean ( SD) free fraction in the plasma was 0.35 ( 0.049) The mean concentration time profiles for plasma, free plasma, free skeletal muscle ISF, and free s.c. adipose tissue ISF are presented in Figure 5 1 The mean f A UC 0 12 ( %CV ) ratios of tissue ISF compared to plasma were 1.15 (3 0. 8) and 1.06 (44.7) for skeletal muscle and s.c. adipose tissue, respectively [Table 5 7]. Based on the AUC ratios, JNJ Q2 penetrates the skeletal muscle and s.c. adipose tissues very well. JNJ Q2 was well tolerated at 400mg single dose. No serious adverse events or discontinuations from the study due to adverse events were observed in the study. Discussion The ability to penetrate the soft tissue at the infection site for JNJ Q2 is importa nt f or the desired clinical outcome [ 92 ] Thus, this MD study was designed to measure free, unbound JNJ Q2 concentrations in the ISF of muscle and s.c. adipose tissue of 12 healthy volunteers following a single oral dose of 400 mg. The result s of these studies revealed that free unbound concentrations in the ISF of muscle and adipose tissues are comparable to the free concentrations in the plasma which is typically expected. [ 75 93 95 ] Th e study shows that JNJ Q2 penetrates well into muscle and s.c. adipose tissue based on the f AUC muscle / f AUC plasma and f AUC adipose / f AUC plasma values calculated as 1.15 and 1.02, respectively. The degree of tissue penetration for JNJ Q2 correlates well with other fluoroquinolones which are known to have good distribution in soft tissues The respective f AUC tissue / f AUC plasma ratios in muscle and adipose tissues for the following fluoroquinolones are: levofloxacin 0.85 and1.1 [ 96 ] moxifloxacin 0.86 and 0.81 [ 21 ] gemifloxacin 1.7 and 2.4 [ 95 ] ciprofloxacin 0.57 and 0.57 [ 77 ] There is no significant

PAGE 83

83 difference between the f AUC of plasma and f AUC of both tissues and between the f AUC of muscle and the f AUC of adipose tissue. T here is evidence that tissue penetration by antibiotics depends on several key characteristics and chief among those are chemical and physical properties of the drug, including lipophilicity or hydrophilicity, the molecular weight, and p lasma protein binding. Molecular weights are comparable between fluoroquinolones, but substantial differences with respect to hydro and lipophilicity may be detected. The plasma protein binding of JNJ Q2 has been demonstrated to be independent from the dr ug concentration similar to other fluoroquinolones in the class [ 97 ] It was m easured in each individual in this study. Peak concentrations were reached at 4.08 ( 1.72 ) hrs which correlated to the expected gastrointestinal symptoms observed in the subjects. It has a moderate half life time of 13.6 hrs. The high volume of distribution suggests a good tissue distribution of JNJ Q2 into ISF which is highly advantageous be cause most infections are usually located here. One potential reason for high Vd is related to the ability of fluoroquinolones to concentrate intracellularly in human cells [ 98 100 ] The concentrations of antimicrobial agents at the site of infection play an important role in the therapeutic efficacy and clinic al outcome of the patient. However, caution must be taken when attempting to predict tissue penetration in patients due to the possibility of pathophysiological changes that may result in altered distribution and penetration. In conclusion, the major advan tage to the microdialysis technique is the ability to measure the free drug at the site of action, such as the ISF of soft tissues in regards to

PAGE 84

84 skin and skin structure infections. JNJ Q2 penetrates well into the ISF for these target soft tissues.

PAGE 85

85 Figure 5 1 Mean JNJ Q2 concentration time profiles in plasma (unbound concentration) and the interstitial fluids of subcutaneous adipose and skeletal muscle f ollowing single oral dose of JNJ Q2 (400 mg) in healthy subjects

PAGE 86

86 Table 5 1. Patient demograp hics: main s tudy Subject Age (years) Sex Weight (kg) 1 26 M 70.3 2 21 M 56.5 3 26 M 90.6 4 24 F 60.1 5 25 M 100 6 44 M 79.5 7 25 F 71.7 8 23 M 87.3 9 36 M 90.6 10 23 F 48 11 43 F 61.5 12 20 F 56.1 Table 5 2. Study procedure chart for pilot study Study Procedures Screening Pilot Study Follow Up ( 21 to Day 1) (Day 1) (Day 7 14) Signing i nformed c onsent x Medical h istory x Physical e xam x x Clinical l ab e valuations x x x 12 Lead ECG x x x Vital s igns x x x Microdialysis probe insertion x JNJ Q2 administration x Dialysate collection x Microdialysis probe removal x AE evaluation x x

PAGE 87

87 Table 5 3. Study procedure chart for main study Study Procedures Screening Main Study Follow Up ( 21 to Day 1) (Day 1) (Day 7 14) Signing i nformed c onsent x Medical h istory x Physical e xam x x Clinical l ab e valuations x x x 12 Lead ECG x x x Vital s igns x x x Microdialysis probe insertion x JNJ Q2 administration x Dialysate collection x Microdialysis probe removal x Plasma sample collection x Protein Binding sampling x Pharm a cogenomic sampling x AE evaluation x x x Table 5 4. Mean (%CV) perfusate concentrations, cumulative dialysate concentrations, and the percent recovery of JNJ Q2 in interstitial fluids of adipose and skeletal muscle during the pilot study Matrix Perfusate Conc. (g/mL) Dialysate Conc (g/mL) Recovery (%) Adipose n=3 1.77 (11.2) 0.515 (18.3) 70.8 (6.7) Muscle n=2 2.06 (10.3) 0.547 (25.6) 73.7 (5.5) Table 5 5. Mean (%CV) perfusate concentrations, cumulative dialysate concentrations, and the percent recovery of JNJ Q2 in adipose and skeletal muscle in the main study Tissue Perfusate Conc. Dialysate Conc. Recovery (g/mL) (g/mL) (%) Adipose 1.54 0.634 56.5 n=11 (20.9) (28.3) (29.3) Muscle 1.48 0.456 68.1 n=12 (20.6) (25.2) (13.8)

PAGE 88

88 Table 5 6. Mean (SD ) pharmacokinetic parameters of JNJ Q2 in plasma, adipose, and muscle following a single oral dose of JNJ Q2 (400 mg) in healthy subjects Parameter Total plasma a Free plasma a Muscle a Adipose b Cmax (g/mL) 1.95 ( 0.45 ) 0.67 ( 0.14 ) 0 .89 ( 0.32 ) 0.83 ( 0.39 ) t max (h) 4.08 ( 1.72 ) 4.08 ( 1.72 ) 4.81 ( 1.02 ) 4.80 ( 1.11 ) AUC 0 12 (h.g/mL) 14.6 ( 2.79 ) 5.09 ( 1.05 ) 5.80 ( 1.97 ) 5.43 ( 2.29 ) AUC 0 24 (h.g/mL) 23.1 ( 4.29 ) 8.07 ( 1.78 ) NA NA t (h) 13.6 ( 5.48 ) 13.6 ( 5.50 ) NA NA V d /F (L) 223.9 ( 42.8 ) 648.6 ( 137.4 ) NA NA CL / F (L/h) 12.5 ( 3.87 ) 36.0 ( 10.4 ) NA NA a n=12, b n=11 NA: Not Applicable Table 5 7. Mean (%CV) ratio of tissue to free and total plasma JNJ Q2 concentrations Ratio C max g/mL AUC 0 12 h h. g/mL Adipose/Total Plasma 0.425 (47.2) 0.372 (49.0) Muscle/Total Plasma 0.472 (34.8) 0.406 (38.1) Adipose/Free Plasma 1.20 (43.1) 1.06 (44.7) Muscle/Free Plasma 1.33 (27.0) 1.15 (30.8)

PAGE 89

89 CHAPTER 6 PHARMACOKINETIC/PHAR MACODYNAMIC MODELING Pharmacokinetic Modeling Population pharmacokinetics (POP PK) is the study of the sources and correlates of variability in drug concentrations among individuals who are the target population receiving clinically relevant doses of a drug of interest [ 101 ] Sheiner and Beal [ 102 ] introduce d this approach approximately 30 years ago which then gained momentum not until the late 1980s and the early 1990s. Today many pharmaceutical companies use this approach routinely, to differing extents, during their drug development process. Advocacy by th e FDA for PK screening is an important factor in the widespread adoption of this approach. FDA has a guidance document for the industry on population pharmacokinetics which serves as a standard in the drug development process [ 103 ] POP PK seeks to identify the measurable pathophysiologic factors that cause changes in dose concentration relationship and the extent of these changes so that, if such changes are associated with clinically significant shifts in the therapeutic index, dos age can be appropriately modified. POP PK analysis is an approach which can be used to obtain important PK and PD information not just from sparse data sets but also from relatively dense data or a combination of spare and dense data [ 104 ] In contrast to the traditional PK evaluation, the POP PK approach collects relevant PK info rmation in patients who are representative of the target population to be treated with the drug. Also identifies and estimates the magnitude of explained and unexplained variability in target population during evaluation. Residual variation includes intra individual variability occasion variability

PAGE 90

90 concentration measurement error, and model misspecification errors. These variations arise because all mathematical calculations for estimating or predicting parameter values are oversimplifications of reality [ 104 ] This is another reason the POP PK approach has gained popularity due to the realization that the approach can be cost effective in revealing clinically important information about the determinants of inter patient PK and PD variability in target population s. Sources of variation that contribute to differences between expectation and outcome are usually categorized as inter individual and residual in nature. Good therapeutic practice should always be based on an understanding of PK variability. POP PK ensure s that dosage adjustments can be made to accommodate differences in PK due to genetic, environmental, physiological or pathological factors. Success has been measured in terms of the increased efficiency in dosage adjustment, usually based on a subsequ ent Bayesian feedback procedure [ 105 ] Recognition of the importance of developing optimum dosing strategies has led to a surge in use of POP PK approach in drug development and regulatory process. Strategies involving specifically the population approach, which are based on statistical methods such as nonlinear mixed effects modeling (NONMEM) have been advocated for investigating PK and PD variability as well as dose concentration effect relationships. Population approaches, if designed carefully and early, as part of the planning of the drug development program, are expected to play a significant role in contr ibuting to drug development [ 106 ] Population based studies require fewer design criteria than other methods and are adaptable to the clinical setting. Individual based PK studies can be divided into 2 types

PAGE 91

91 with respect to their evaluation: compartmental and non compartmental investigations. The latter type of study was originally thought to require fewer assumptions than the former but subsequently it has been sho wn that non compartmental analyses are more restrictive and are basically compartmental in their approach. These studies estimate parameters which the compartmental investigation does not usually consider such as area under the moment curve and mean reside nce time [ 107 ] Pharmacodynamic Modeling With a good understanding of the exposure response relationship, or PD, it may be possible to identify a quantitative link between the dose and drug eff ect For antibiotics, this link has been established by correlating PK parameters that are based on free ( f ) plasma or serum concentrations to the MIC of the respective pathogen. For fluoroquinolones, the parameter that better correlates to therapeutic outcome is f AUC 24/ MIC ratio [ 44 46 108 ] However, the MIC as single point in time estimate is n ot capable of characterizing the time course of neither growth nor antibiotic induced kill or the antibiotic effect at concentrations besides the MIC [ 109 ] In addition, the methodology to determine the actual MIC value has not yet been internationally standardized and is a source of variability between different MIC determination methods [ 110 ] To overcome these limitations, other susceptibility breakpoints, such as, the EC50 have been suggested as the PD input for PK/PD indices Additionally, to truly characterize the antimicrobial effect over time, time kill experiments should be performed. Th e FDA has recognized that model based drug development provides an opportunity to streamline the drug development process. There have been numerous cases in which model based development has saved money and time by aiding in dose

PAGE 92

92 selection, clinical trial design, or support of a given dosing regimen Therefore, this chapter will present the PK/PD techniques used to evaluate the currently recommended dosing regimen of JNJ Q2, 400mg administered orally as a single dose. This will be done by developing a POP P K model to simultaneously fit concentration time curves in three different tissues, i.e. plasma, skeletal muscle ISF, and s.c. adipose tissue ISF, based on plasma and microdialysis data. This will be then used to predict the target attainment rate using tr adi tional endpoints, the f AUC 24 /MIC and time kill curves. Materials and Methods Population Pharmacokinetic Model Development The PK data was obtained from a clinical microdialysis study, details of which are presented in chapter 5. Plasma data from the microdialysis experiment were adjusted for individual protein binding and compiled with the microdialysis data, which represents free concentrations, and used as full data set. A model was developed using NONMEM VI I software with the ADVAN 4 subroutine usin g First Order Conditional Estimation To model the concentrations in plasma and tissues, a compartmental model was developed. Inter individual variability was included in the model using exponential error model (Equation 6 7) An additive error model was u sed to describe the residual error (Equation 6 8) A covariate analysis was done by assessing the impact of covariates on inter individual errors and parameters. D ue to the fact that this population is comprised of twelve individuals, who 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, adipose and muscle compartment, respectively. Th e fits of the model for the plasma conce ntrations are shown in Figures 6 2 and 6 3 The fits for the tissue compa rtments

PAGE 93

93 are shown in Figures 6 4 to 6 7 The fits for plasma tissue are generally better than those for the free tissues. Model fit was assesse d using visu al inspection of the diagnostic (Figure 6 1, 6 8 and 6 9 ). Model fit was assessed by visual inspection. Lower concentrations, particularly in the absorption phase are not very well predicted by the model. PK/PD Analysis AUC/ MIC r atio I n vitro MIC values for various isolates of pathogens were taken from Morrow et al [ 28 ] study. The f AUC 24 values were taken from the PK analysis of JNJ Q2 after a single oral dose of 400mg (see Chapter 5) To calculate a clinically relevan t target the f AUC 24 was divided by MIC for the different isolates. Time kill curve a pproach Data for time kill analyses performed at 6 time points when exposed to JNJ Q2 were taken from the study by Morrow et al [ 28 ] for the strains MRSA OC 8525, MRSA OC 11696 and MRSA OC 2838 An Emax model has been successfully applied to describe the relationship between concentration and effect [ 36 53 ] The study used a modified Emax model to evaluate antimicrobial ac tivity using Scientist software [ 54 ] The MIC for the MRSA strains is 0 .25 ug/mL. Results Population Pharmacokinetic Modeling A two compartment body model with elimination from the central compartment was a ble to accurately fit the data [Figure 6 1] This model was developed using

PAGE 94

94 concentrations from plasma and both tissues The deferential equations for this model are as follows: (6 1) (6 2 ) (6 3) In the above equations, K a and K e represent the absorption and elimination rate constants K12 and K21 are transfer rate constants. Also, s ince volumes in ISF of tissues may not be equal an additional parameter was added to the model to account for this, termed the distribution factor for muscle or adipose (FM and FA). Therefore, the equations for the concentration in plasma, skeletal muscle, and s.c. adipose tissue respectively are: ( 6 4 ) (6 5 ) ( 6 6 ) The inter subject variability was modeled assuming exponential error as follows: (6 7) where P pop 2 ) is a normally distributed inter individual random effect The residual variability was modeled using an additive error: (6 8) Where C obs the observed concentration, C pred the predicted concentration by individual parameter, i ~ 2 ) is the residual random effect. Individual patient PK parameters

PAGE 95

9 5 for which random effects were included in the model were calculated using the posterior conditional estimation technique using first order conditional es timation. Diagnostic plots and objective function value s were used to check for model fitting and selection Model stability was confirmed by 800 successful non parametric bootstrap replicates which generated 95% confidence intervals which were then compared with the original estimates (Table 6 1) AUC/MIC I ndex Table 6 2 lists the f AUC 24 /MIC ratio for JNJ Q2 against different strains of bacteria f AUC 24 /MIC ratios for G ram negative pathogens, Pseudomonas aeruginosa Klebsiella pneumonia and Haemophilus influenzae were 4.04, 32.3 and 538 respectively. Gram positive isolates of c iprofloxacin resistant S. pneumonia e MRSA, c iprofloxacin resistant MRSA, c iprofloxacin resistant MRSE and Streptococcus pyogens had f AUC 24 /MIC ratio of 32.3 and for S. pneumonia e was 67.3 Literature suggests AUC 24 /MIC ratio of 100 to 125 correlates with optimal clinical and microbiological outcomes in seriou sly ill patients infected with G ram negative pathogens and Pseudomonas aeruginosa an d is said to be much lower for G ram positive b acteria [ 111 ] However, there has been considerable controversy as to whether or not this PD target applies to all patient populations and all organisms. Data from in vitro and animal models of infection that hav e recently emerged suggest that, for S pneumoniae the optimal AUC 24 /MIC ratio is much lower than 100 to 125 I n a study by Lister and Sanders 24 /MIC ratios of 32 to 64 were associated with eradication of S. pneumoni ae from an in vitro model of infection [ 112 ] Also other studies in animal models for pneumococcal infection had AUC 24 /MIC ratios

PAGE 96

96 of 25 to 34 for [ 111 ] Like all fluoroquinolone s JNJ Q2 belongs to the class of concentration dependent antibiotics, therefore the ratio AUC 24 /MIC is the most relevant PK or pharmacodynamic (PD) parameter to determine antibacterial effects [ 113 ] and to predict clinical efficacy [ 22 ] I t needs to be pointed out that breakpo int values are generally based on total drug concentrations, whereas free concentrations of JNJ Q2 in tissues and plasma were used for PK and PD calculations in the present study. Based on PK and PD calculations, it is tempting to speculate that JNJ Q2 wil l effectively combat bacteria in the extracellular space fluid in soft tissues. However, tissue PK data should also be derived from patients with cSSSIs because tissue penetration might be significantly altered by infection. Therefore, though this index m ay not be solely used to base dosing decisions on but can be combined with stochastic modeling techniques to predict efficacious breakpoints for fluoroquinolones. Modeling of Time Kill Curve s A modified Emax model was deve loped to describe the bactericidal properties of JNJ Q2 over time. Parameters such as the maximum number of bacteria (Nmax) at the end of the growth phase, adaptati on rate term ( x ) and Hill factor (h) have been included in a PK/PD model to optimize the mod els (6 7) (6 8 )

PAGE 97

97 The results are shown in Table 6 3 and in Figures 6 10 to 6 1 2 The model predicted the kill and growth curves well. It was observed that the killing effect is more pronounced with increasing the concentration. No re growth was seen up to 8 hours i n all the strains Model selection was based on the model selection criterion provided by the program, as well as visual inspection of the fitted curves. The parameters generated from the PD model were successfully used to simulate the different MIC streng ths ranging from 0.25 up to 16 times MIC. No re growth is seen at 4 x MIC strength for MRSA OC 8525 and at 2 x MIC for MRSA OC 11696. A concentration of 16 x MIC is needed to avoid re growth of bacteria for MRSA OC 2838 strain. The minimum MIC strength to prevent bacterial re growth is between the range 2 16 for the 3 MRSA strains simulated (Figures 6 1 3 6 1 4 6 1 5 ) No re growth was observed with the different simulated doses of 200mg, 300mg, 400mg and 500mg for MRSA OC 8525 a nd 11696 strains but re growth seen in MRSA 2838. At the 400 mg once a day oral dosing regimen a bacterial kill of 3 log units is observed at 24 hrs for MRSA OC 11696 and a 2 log kill for MRSA OC 8525 strain. For MRSA OC 2838 re growth is seen after 10 hrs and hence a higher dose ( greater than 5 00mg) may be needed to see an y effect ( Fig ures 6 16, 6 17, 6 18) 400mg oral dose once a day seems to be an adequate dosing regimen for certain clinical strains of MRSA.. It should be stressed, that the resulting simu lations are valid only for expected outcome in the in vitro system but do not necessarily reflect therapeutic outcome in vivo for the same concentrations

PAGE 98

98 Figure 6 1. Observations versus population and individual predictions for all compartments Predicted concentrations ( g/mL) : x axis and o bserved concentrations ( g/mL) : y axis

PAGE 99

99 Figure 6 2 Observation versus population and individual predictions for plasma concentrations Predicted concentrations ( g/mL): x axis and obser ved concentrations ( g/mL): y axis

PAGE 100

100 Figure 6 3. Individual fits for plasma Time (hr): x axis and o bserved / p redicted c oncentrations ( g/mL) : y axis

PAGE 101

101 Figure 6 4 Observations versus population and individual predictions for adipose tissue Predicted concentrations ( g/mL): x axis and observed concentrations ( g/mL): y axis

PAGE 102

102 Figure 6 5. Individual fits for adipose tissue T ime (hr): x axis and o bserved / p redicted c oncentrations ( g/mL) : y axis

PAGE 103

103 Figure 6 6 Observations versus population and indiv idual predictions for muscle Predicted concentrations ( g/mL): x axis and observed concentrations ( g/mL): y axis.

PAGE 104

104 Figure 6 7. Individual fits for muscle T ime (hr): x axis and o bserved / p redicted c oncentrations ( g/mL ) : y axis

PAGE 105

105 Figure 6 8 Weighted residuals plot versus time for all compartments

PAGE 106

106 Figur e 6 9. Weighted residuals versus population predictions for all compartments

PAGE 107

107 Figure 6 10. Curve fit for MRSA OC 8525

PAGE 108

108 Figure 6 11 Curve fit for MRSA OC 11696

PAGE 109

109 Figure 6 12 Curve fit for MRSA OC 2838

PAGE 110

110 Figure 6 13. Simulated curves for bacterial kill over a range of MIC strengths for MRSA OC 8525 & once a day oral administration of 400mg JNJ Q2

PAGE 111

111 Figure 6 1 4 Simulated curves for bacterial kill over a range of MIC strength s for MRSA OC 11696 & once a day oral administration of 400mg JNJ Q2

PAGE 112

112 Figure 6 1 5 Simulated curves for bacterial kill over a range of MIC strengths for MRSA OC 2838 & once a day oral a dministration of 400mg JNJ Q2

PAGE 113

113 Figure 6 1 6 Simulated PK/PD of JNJ Q2 against MRSA OC 8525 after once daily oral administration of 200mg, 300mg, 400mg and 500mg JNJ Q2

PAGE 114

114 Figure 6 1 7 Simulated PK/PD of JNJ Q2 against MRSA OC 11696 after once daily oral administration of 200mg, 300mg, 400mg and 500mg JNJ Q2

PAGE 115

115 Figure 6 1 8 Simulated PK/PD of JNJ Q2 against MRSA OC 2838 after once daily oral administration of 200mg, 300mg, 400mg and 500mg JNJ Q2

PAGE 116

116 Table 6 1. Estimated PK m odel p arameters with b ootstrap 95% c onfidence intervals Parameter Estimate Bootstrap 95 % CI Vc 138 95.2 168 Vp 5.2 5.2 9.2 CL 16.8 14.55 19.4 Q 6.27 5.16 7.95 KA 0.3 4 0.21 0.48 FA 0.3 7 0.28 0.47 FM 0.42 0.35 0.51 Btwn. Subj. Variability Vc 0.283 0.008 0.42 Btwn. Subj. Variability Vp 0. 166 0.003 0.38 Btwn. Subj. Variability CL 0. 224 0.13 0.31 Btwn. Subj. Variability Q 0. 262 0.0003 0.36 Btwn. Subj. Variability KA 0. 511 0.002 0.65 Btwn. Subj. Variability FA 0. 436 0.21 0.59 Btwn. Subj. Variability FM 0. 316 0.15 0.42 Residual Variability 0. 158 0.12 0.19

PAGE 117

117 Table 6 2 f AUC 24 /MIC ratio for JNJ Q2 Pathogen f AUC 24 /MIC ratio Type S. pneumoniae all isolates 67.3 Gram p ositive Ciprofloxacin resistant S. pneumoniae 32.3 Gram positive MRSA 32.3 Gram positive Ciprofloxacin resistant MRSA 32.3 Gram positive Ciprofloxacin resistant MRSE 32.3 Gram positive Streptococcus pyogens 538 Gram positive Haemophilus influenzae 538 Gram negative Klebsiella pneumoniae 32.3 Gram negative Pseudomonas aeruginosa 4.04 Gram negative *isolates collected from 2004 2006 Table 6 3 Parameter estimates for 3 strains of MRSA Strain / Parameter OC 852 5 OC 11696 OC 2838 k (hr 1 ) 0.17 0.20 0.26 Nmax (CFU/mL ) 10^11.3 10^11.6 10^9.6 Emax (hr 1 ) 1.12 0.74 0.32 EC50 ( g/mL ) 0.95 0.68 0.44 x (hr 1 ) 0.09 0.06 0.12 h 1.8 1.6 0.69

PAGE 118

118 CHAPTER 7 CONCLUSION JNJ Q2 is a novel fluorinated 4 quinolone oral antimicrobial agent, with activity against key pathogens associated with CAP and cSSSIs including diabetic foot infection. They target the DNA topoisomerases showing potent activ ity against G ram negative and positive bacteria including MRSA strains. It is important, therefore, to assess the in vivo penetration of JNJ Q2 into ISF of soft tissues, such as subcutaneous adipose tissue and skelet al muscles. The selection of the correc t dose and dosing regimen is a fundamental step for therapeutic success with any pharmacological agent. In the past, pharmacokinetic and pharmacodynamic assessment of antimicrobials were based on measuring plasma concentrations, however, most infections oc cur at tissue sites. Microdialysis is currently the most appropriate sampling technique that can provide continuous sampling of free drug in tissues to estimate active drug profiles at site of action. For antimicrobial agents, the selection of the best dru g and dosing scheme for a specific pathogen not only increases the chances of cure while preventing toxic side effects, but it has been shown that suboptimal dosing can lead to bacterial resistance development and should be avoided. One approach to make an educated dose selection is PK/PD approach PK/PD modeling has become an important tool to streamline drug development and is encouraged by regu latory agencies such as the FDA. The traditional PK/PD model for dose selection in antimicrobial drug development was based on the relationship of drug kinetics to m inimum inhibition concentration, from which the maximum efficacy can be classified into either time dependent or exposure (AUC or Cmax) dependent. PK/PD characterization of antimicrobial agents has allowed

PAGE 119

119 a better understanding of why a particular dosing regimen achiev es clinical success or failure. Over the years, d ose selection has become a much more sophisticated process over previous empiric methods. In vitro and in vivo experiments are use d to define a relationship between drug concentration (PK) and effect (PD) and allow for a clear target to be identified so that efficacy is achieved in the clinical setting. The PK/PD indices typically used for fluoroquinolones include the ratio f AUC 24 /MI C which is based on a direct comparison of plasma concentrations of the antibiotic and MIC of the respective bacteria. The efficacy of the antibiotic can be predicted by this ratio where higher the value greater the efficacy The in vivo PK parameters are usually determined from plasma drug profiles while in vitro PD parameters commonly use culture media drug concentrations. Fluoroquinolones which display concentration dependent killing produce an increasing effect as the concentration increases as seen in case of JNJ Q2. This project explored the usefulness of PK/PD relationships to support drug development and selection of a dosage regimen for JNJ Q2. The in vivo PK study conducted was a single center, open label, one arm, nonrandomized study in healthy men and women. The study was designed to assess the in vivo penetration of JNJ Q2 into ISF of soft tissues, such as s.c. adipose tissue and skeletal muscles, after administration of 400 mg single oral dose, since unbound concentration of an antimicrobial a gent in the ISF at target site is responsible for its efficacy. F ollowing single oral 400 mg JNJ Q2 administration, unbound JNJ Q2 was rapidly distributed and equilibrated to target sites, s.c. adipose and muscle tissues, in healthy subjects. Exposures in ISF of target tissues were slightly greater than that of unbound drug in plasma. Single oral dose of 400 mg JNJ Q2 was well tolerated in

PAGE 120

120 volunteers. The peak plasma levels were reached within 4 5 hours post dose and p rotein binding in plasma for JNJ Q2 assessed was found to be consistent with other fluoroquinolones in the class. I t may be reasonable to conclude that free, unbound plasma concentrations can be used as surrogate for unbound concentrations in ISF of s.c. a dipose and skeletal muscle tissues. The data from this study was used to develop a POP PK model so that the concentration in all three tissues could be predicted simultaneously. It was determined that a two compartment body model with elimination from the central compartment fit the data well. Additionally, due to the fact that free concentrations in the ISF were not equal to free plasma concentrations, a distribution factor for each soft tissue was included in this model. For fluoroquinolones, the PK/PD parameter with the best correlation to clinical outcome is f AUC 24 /MIC ratio. The high ratio value beyond the accepted break points for JNJ Q2 indicates the high efficacy of bacterial kill in vitro This information combined with the high penetration of JNJ Q2 into ISF where infections are expected give us a good heads up on the promising of use of this antibiotic. The major PD parameter used for antimicrobials is the MIC. This parameter, however, has some limitations i.e. it does not show the antimicrobial activity over time. Time kill curves whereas, display the change in the number of bacteria over time offering a more dynamic effect profile than the static MIC parameter. Data was taken f or the i n vitro PD studies for JNJ Q2 at different concentrations of the MIC against 3different strains of MRSA from t he work done by Morrow et al. [ 28 ] By developing a mathematical model corresponding to the change in bacteria over time given the

PAGE 121

121 concentration a much more precise pharmacodynamic pictur e can be obtained than with the MIC PD parameter. A modified Emax model was developed t o define the antibacterial activity over time and the pharmacodynamic parameters were calculated. The PD parameters for JNJ Q2 derived from time kill model fit was comb ined with in vivo PK data in an integrated PK/PD model that best describe d as a function of time and concentration and different dosing regimens simulated In conclusion PK/PD characterization of JNJ Q2 allow ed a better understanding of dosing regimen of 400 mg currently recommended for use This can be further used in optimizing the dosing regimen to have an efficacious drug in the treatment of CAP, cSSSIs and other infections.

PAGE 122

122 APPENDIX A REAGENTS AND E QUIPMENT FOR LC MS/MS STUDY Reagents Triple distilled water Filtered in house by Corning AG 3 Methanol Fisher Scientific A452 4 Formic Acid Fisher Scientific A118 P 100 Saline solution Baxter 2B2323 Ammonium Acetate Fisher Scientific A639 500 Equipment and Disposables Weighing Balance Mettler AE240 Vortex Kraft Apparatus Inc. Model PV 5 Micropipettes Eppendorf Research Pipette tips (1 200 L) Fisherbrand 22 707 500 Pipette tips (100 1000 L) Fisherbrand 21 197 8F Autosampler vials Sun Sri 200 046 Aluminum seals Sun Sri 200 100 Bath Sonicator Fisher Scientific FS110H 15mL Centrifuge tubes Corning 430052 50 mL Centrifuge Tubes Corning 430828 Microcentrifuge tubes Fisherbrand 05 408 129 Work Station Dell Precision 390 LC MS/MS system API 4000 LC MS/MS SYSTEM, J1940112 Column Symmetry C18 3.5 m, 4.6mm x 50mm, SNo. 016835292102; 019138233136 Analytical software Analyst Software 1.4.2 for LC MS/MS Systems Autosampler Elmer Series 200, 293N5020903 Pump Perkin E lmer Series 200, 291N7100502A Manual Crimpers Fisher 03 375 7 Volumetric flask Pyrex 5640 Microsyringes Tyco Healthcare 8881501400 Plastic Cannula BD 303345 Graduated Cylinder Pyrex 2982

PAGE 123

123 APPENDIX B REAGENTS AND EQUIPMENTS FOR IN VITRO MICRODIAL YSIS STUDY Reagents Triple distilled water Filtered in house by Corning AG 3 Methanol Fisher Scientific A452 4 Formic Acid Fisher Scientific A118 P 100 Saline solution Baxter 2B2323 Ammonium Acetate Scientific A639 500 Equipment Weighing Balance Mettler AE240 Vortex Kraft Apparatus Inc. Model PV 5 Micropipettes Eppendorf Research Pipette tips (1 200 L) Fisherbrand 22 707 500 Pipette tips (100 1000 L) Fisherbrand 21 197 8F Autosampler vials Sun Sri 200 046 Aluminum seals Sun Sri 200 100 Bath Sonicator Fisher Scientific FS110H 15mL Centrifuge tubes Coning 430052 50 mL Centrifuge Tubes Corning 430828 Syringes Becton Dickinson 309603 Needle Becton Dickinson 305167 Microcentrifuge Tubes 1.5m L Fisherbrand 05 408 129 Microcentrifuge Tubes 0.5mL Fisherbrand 05 408 120 Syringe Pump Harvard Apparatus Model 55 4150 Heated Stir Plate Fisherbrand Isotemp Thermometer Fisherbrand 76mm Immersion 14 997 Microdialysis Probes CMA 60 P000002 Amber IV Tubing Cover Heal th Care Logistics 7617 Mass Spectroscopy: LC MS/MS API 4000 LC MS/MS SYSTEM, J1940112 Perkin Elmer HPLC : (1) Autosampler Perkin Elmer Series 200, 293N5020903 ; (2) Pump Perkin Elmer Series 200, 291N7100502A ; (3) Work Station Dell Precision 390 ; (4) Column Symmetry C18 3.5 m, 4.6mm x 50mm, WAT200625 ; (5) Analytical software Analyst Software 1.4.2 for LC MS/MS Systems

PAGE 124

124 APPENDIX C STUDY CRITERIA Inclusion c riteria: Men or Women between 18 and 55 years of age, inclusive BMI between 18.5 and 32 kg/m2, inclusive [BMI = weight (kg)/height (m2)] Non smoker (not smoked for 3 months prior to screening) and have a urine cotinine level indicative of a non smoker Healthy on the basis of physical examination, medical history, vital signs, and 12 lead ECG performed at screening. Healthy on the basis of clinical laboratory tests performed at screening Women must be : (1) Postmenopausal (defined as having had the last menstrual period greater than 12 months prior to screening, or greater than 6 months prior to screening with a serum follicle stimulating hormone (FSH) concentration greater than 40 mIU/mL) or surgically sterilized (defined as being at least 6 weeks post surgical bilateral oophorectomy, bilateral tubal ligation, or hysterectomy), and (2) Women of childbearing potential must be practicing an effective method of birth sterilization) at least 3 months before the Screening visit, and (3) Non pregnant based on a negative serum beta HCG) test analysis at screening and a ne gative serum or urine HCG test at admission Men must a gree to use a double barrier method of birth control and to not donate sperm during the study and for 3 months after receiving the last dose of study drug. Agree not to consume any products containing q uinine, grapefruit juice, sugar substitutes or Seville oranges, within 2 days prior to admission until discharge from the unit. Agree not to consume any methylxanthine products (such as tea, coffee, cola and chocolate, cocoa) within 2 days prior to admiss ion until discharge from the unit. Agree not to consume any alcohol within 2 days prior to admission to the clinical research unit and until discharge from the unit. No clinically significant medical history according to the investigator. Subjects must agr ee to abstain from any strenuous exercise that is more excessive than normal routine, 72 hours prior to the first dose until the completion of the study.

PAGE 125

125 Willing/able to adhere to the prohibitions and restrictions specified in this protocol Subjects must h ave signed an informed consent and are willing to participate in the study. Exclusion c riteria: Clinically significant abnormal values for hematology, clinical bio chemistry, coagulation, hematology, or urinalysis at screening. History of or current sign ificant medical illness including (but not limited to) cardiac arrythmias or other cardiac disease, hematological disease, lipid abnormalities, bronchospastic respiratory disease, diabetes mellitus, renal or hepatic i nsufficiency, thyroid disease, Parkinso that the Investigator considers should exclude the subject. Known allergies, hypersensitivity, or intolerance to JNJ 32729463 or its excipients Clinically significant abnormal physical examination, vital signs (e.g. SBP >140 mmHg, DBP >90 mmHg, heart rate >100 bpm and <45 bpm) or 12 lead ECG (e.g. QTc >450 msec) at screening or prior to first dose of study drug. Family history of long QT or short QT syndrome Received an investigational drug (including vaccines) or used an investigational medical device within 30 days before the pl anned start of treatment or are currently enrolled in an investigational study Pregnant or breast feeding Recent history of surgery; within the past 3 months prior to screening. Sexually active men, who have not been surgically sterilized, and who are unwilling to use a condom during intercourse or to refrain from sexual intercourse through the study and for up to 3 months after the last dose of study medication. Serology positive for hep atitis B surface antigen, hepatitis C antibodies or HIV antibodies. Positive urine screen for drugs of abuse. Positive alcohol breath test. Recent history (within previous 6 months) of alcohol or drug abuse. Drinks, on average, more than 8 cups of tea/coff ee/cocoa/cola per day. Clinically significant acute illness within 7 days prior to study drug administration.

PAGE 126

126 History of drug and/or food allergies. Subjects who developed SAEs or experienced intolerance following administration of quinolone antibiotics D onation of 1 or more units (approximately 450 mL) of blood or acute loss of an equivalent amount of blood within 90 days prior to study drug administration. Exposure to any new investigational agent within 90 days prior to study drug administration. Use of any prescription or over the counter (OTC) medication, herbal medication, herbal teas, vitamins, or mineral supplements within 14 days prior to study drug administration (not including paracetamol). Psychological and/or emotional problems, which would ren der the informed consent invalid, or limit the ability of the subject to comply with the study requirements. Any condition that, in the opinion of the investigator, would compromise the well being of the subject or the study or prevent the subject from mee ting or performing study requirements 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

PAGE 127

127 LIST OF REFERENCES 1. Zhanel GG, Noreddin AM: Pharmacokinetics and pharmacodynamics of the new fluoroquinolones: focus on respiratory infections. Curr Opin Pharmacol 2001; 1:459 463. 2. Hooper DC: New uses for new and old quinolones and the challenge of resistance. Clin Infect Dis 2000; 30:243 254. 3. Zhanel GG, Ennis K, Vercaigne L, Walkty A, Gin AS, Embil J, Smith H, Hoban DJ: A critical review of the fluoroquinolones: focus on respiratory infections. Drugs 2002; 62:13 5 9. 4. Bolon MK: The newer fluoroquinolones. Infect Dis Clin North Am 2009; 23:1027 1051, x. 5. Oliphant CM, Green GM: Quinolones: a comprehensive review. Am Fam Physician 2002; 65:455 464. 6. Ambrose PG, Owens RC, Jr., Quintiliani R, Nightingale CH: New ge nerations of quinolones: with particular attention to levofloxacin. Conn Med 1997; 61:269 272. 7. Turnidge J: Pharmacokinetics and pharmacodynamics of fluoroquinolones. Drugs 1999; 58 Suppl 2:29 36. 8. Nightingale CH: Moxifloxacin, a new antibiotic designed to treat community acquired respiratory tract infections: a review of microbiologic and pharmacokinetic pharmacodynamic characteristics. Pharmacotherapy 2000; 20:245 256. 9. Allen A, Bygate E, Clark D, Lewis A, Pay V: The effect of food on the bio availability of oral gemifloxacin in healthy volunteers. Int J Antimicrob Agents 2000; 16:45 50. 10. Fish DN, Chow AT: The clinical pharmacokinetics of levofloxacin. Clinical Pharmacokinetics 1997; 32:101 119. 11. Shimada J, Nogita T, Ishibashi Y: Clinical pharmacokinetics of sparfloxacin. Clin Pharmacokinet 1993; 25:358 369. 12. Rodvold KA, Neuhauser M: Pharmacokinetics and pharmacodynamics of fluoroquinolones. Pharmacotherapy 2001; 21:233S 252S. 13. Walker RC: The fluoroquinolones. Mayo Clin Proc 1999; 74 :1030 1037.

PAGE 128

128 14. Schuler P, Zemper K, Borner K, Koeppe P, Schaberg T, Lode H: Penetration of sparfloxacin and ciprofloxacin into alveolar macrophages, epithelial lining fluid, and polymorphonuclear leucocytes. European Respiratory Journal 1997; 10:1130 1136 15. Vance Bryan K, Guay DR, Rotschafer JC: Clinical pharmacokinetics of ciprofloxacin. Clin Pharmacokinet 1990; 19:434 461. 16. Wise R, Jones S, Das I, Andrews JM: Pharmacokinetics and inflammatory fluid penetration of clinafloxacin. Antimicrob Agents Ch emother 1998; 42:428 430. 17. Wise R, Andrews JM, Ashby JP, Marshall J: A study to determine the pharmacokinetics and inflammatory fluid penetration of gatifloxacin following a single oral dose. J Antimicrob Chemother 1999; 44:701 704. 18. Fish DN, Chow AT : The clinical pharmacokinetics of levofloxacin. Clin Pharmacokinet 1997; 32:101 119. 19. Wise R, Andrews JM, Marshall G, Hartman G: Pharmacokinetics and inflammatory fluid penetration of moxifloxacin following oral or intravenous administration. Antimicro b Agents Chemother 1999; 43:1508 1510. 20. Johnson JH, Cooper MA, Andrews JM, Wise R: Pharmacokinetics and inflammatory fluid penetration of sparfloxacin. Antimicrob Agents Chemother 1992; 36:2444 2446. 21. Muller M, Stass H, Brunner M, Moller JG, Lackner E, Eichler HG: Penetration of moxifloxacin into peripheral compartments in humans. Antimicrob Agents Chemother 1999; 43:2345 2349. 22. Saravolatz LD, Leggett J: Gatifloxacin, gemifloxacin, and moxifloxacin: the role of 3 newer fluoroquinolones. Clin Infect Dis 2003; 37:1210 1215. 23. Gatifloxacin and moxifloxacin: two new fluoroquinolones. Med Lett Drugs Ther 2000; 42:15 17. 24. Sarkozy G: Quinolones: a class of antimicrobial agents. Vet Med Czech 2001; 46:257 274. 25. Chien SC, Chow AT, Natarajan J, Willia ms RR, Wong FA, Rogge MC, Nayak RK: Absence of age and gender effects on the pharmacokinetics of a single 500 milligram oral dose of levofloxacin in healthy subjects. Antimicrob Agents Chemother 1997; 41:1562 1565. 26. Craig WA: Pharmacokinetic/pharmacodyn amic parameters: rationale for antibacterial dosing of mice and men. Clin Infect Dis 1998; 26:1 10; quiz 11 12.

PAGE 129

129 27. Lode H, Borner K, Koeppe P: Pharmacodynamics of fluoroquinolones. Clinical Infectious Diseases 1998; 27:33 39. 28. Morrow BJ, He WP, Amsler KM, Foleno BD, Macielag MJ, Lynch AS, Bush K: In Vitro Antibacterial Activities of JNJ Q2, a New Broad Spectrum Fluoroquinolone. Antimicrob Agents Ch 2010; 54:1955 1964. 29. Investigator's Brochure. In. Edited by Development JJPR, 01 edn; 2007. 30. Niederm an MS: Challenges in the management of community acquired pneumonia: the role of quinolones and moxifloxacin. Clin Infect Dis 2005; 41 Suppl 2:S158 166. 31. Mandell LA, Bartlett JG, Dowell SF, File TM, Jr., Musher DM, Whitney C: Update of practice guidelin es for the management of community acquired pneumonia in immunocompetent adults. Clin Infect Dis 2003; 37:1405 1433. 32. Johnson AP, Sheppard CL, Harnett SJ, Birtles A, Harrison TG, Brenwald NP, Gill MJ, Walker RA, Livermore DM, George RC: Emergence of a f luoroquinolone resistant strain of Streptococcus pneumoniae in England. J Antimicrob Chemother 2003; 52:953 960. 33. Ho PL, Yung RW, Tsang DN, Que TL, Ho M, Seto WH, Ng TK, Yam WC, Ng WW: Increasing resistance of Streptococcus pneumoniae to fluoroquinolone s: results of a Hong Kong multicentre study in 2000. J Antimicrob Chemother 2001; 48:659 665. 34. Andes D, Anon J, Jacobs MR, Craig WA: Application of pharmacokinetics and pharmacodynamics to antimicrobial therapy of respiratory tract infections. Clin Lab Med 2004; 24:477 502. 35. Treyaprasert W, Schmidt S, Rand KH, Suvanakoot U, Derendorf H: Pharmacokinetic/pharmacodynamic modeling of in vitro activity of azithromycin against four different bacterial strains. Int J Antimicrob Agents 2007; 29:263 270. 36. d e la Pena A, Grabe A, Rand KH, Rehak E, Gross J, Thyroff Friesinger U, Muller M, Derendorf H: PK PD modelling of the effect of cefaclor on four different bacterial strains. Int J Antimicrob Agents 2004; 23:218 225. 37. Drusano G, Labro MT, Cars O, Mendes P, Shah P, Sorgel F, Weber W: Pharmacokinetics and pharmacodynamics of fluoroquinolones. Clin Microbiol Infect 1998; 4 Suppl 2:S27 S41. 38. Dudley MN: Pharmacodynamics and pharmacokinetics of antibiotics with special reference to the fluoroquinolones. Am J Med 1991; 91:45S 50S.

PAGE 130

130 39. Derendorf H, Meibohm B: Modeling of pharmacokinetic/pharmacodynamic (PK/PD) relationships: concepts and perspectives. Pharm Res 1999; 16:176 185. 40. Preston SL: The importance of appropriate antimicrobial dosing: pharmacokinetic and pharmacodynamic considerations. Ann Pharmacother 2004; 38:S14 18. 41. MacGowan AP: Role of pharmacokinetics and pharmacodynamics: Does the dose matter? Clinical Infectious Diseases 2001; 33:S238 S239. 42. Vinks AA: The application of population pharma cokinetic modeling to individualized antibiotic therapy. Int J Antimicrob Agents 2002; 19:313 322. 43. Hochhaus G, Barrett JS, Derendorf H: Evolution of pharmacokinetics and pharmacokinetic/dynamic correlations during the 20th century. J Clin Pharmacol 200 0; 40:908 917. 44. Sanchez Navarro A, Sanchez Recio MM: Basis of anti infective therapy: pharmacokinetic pharmacodynamic criteria and methodology for dual dosage individualisation. Clin Pharmacokinet 1999; 37:289 304. 45. Sanchez Recio MM, Colino CI, Sanch ez Navarro A: A retrospective analysis of pharmacokinetic/pharmacodynamic indices as indicators of the clinical efficacy of ciprofloxacin. J Antimicrob Chemother 2000; 45:321 328. 46. Schentag JJ: Pharmacokinetic and pharmacodynamic surrogate markers: stud ies with fluoroquinolones in patients. Am J Health Syst Pharm 1999; 56:S21 24. 47. Preston SL, Drusano GL, Berman AL, Fowler CL, Chow AT, Dornseif B, Reichl V, Natarajan J, Corrado M: Pharmacodynamics of levofloxacin: a new paradigm for early clinical tria ls. JAMA 1998; 279:125 129. 48. Forrest A, Nix DE, Ballow CH, Goss TF, Birmingham MC, Schentag JJ: Pharmacodynamics of intravenous ciprofloxacin in seriously ill patients. Antimicrob Agents Chemother 1993; 37:1073 1081. 49. Craig WA: Does the dose matter? Clinical Infectious Diseases 2001; 33:S233 S237. 50. Lacy MK, Lu W, Xu X, Tessier PR, Nicolau DP, Quintiliani R, Nightingale CH: Pharmacodynamic comparisons of levofloxacin, ciprofloxacin, and ampicillin against Streptococcus pneumoniae in an in vitro mode l of infection. Antimicrob Agents Chemother 1999; 43:672 677.

PAGE 131

131 51. Schentag JJ, Gilliland KK, Paladino JA: What have we learned from pharmacokinetic and pharmacodynamic theories? Clinical Infectious Diseases 2001; 32:S39 S46. 52. Schentag JJ, Meagher AK, Fo rrest A: Fluoroquinolone AUIC break points and the link to bacterial killing rates. Part 2: human trials. Ann Pharmacother 2003; 37:1478 1488. 53. DallaCosta T, Nolting A, Rand K, Derendorf H: Pharmacokinetic pharmacodynamic modelling of the in vitro antii nfective effect of piperacillin tazobactam combinations. Int J Clin Pharm Th 1997; 35:426 433. 54. Delacher S, Derendorf H, Hollenstein U, Brunner M, Joukhadar C, Hofmann S, Georgopoulos A, Eichler HG, Muller M: A combined in vivo pharmacokinetic in vitro pharmacodynamic approach to simulate target site pharmacodynamics of antibiotics in humans. J Antimicrob Chemother 2000; 46:733 739. 55. Schmidt S, Banks R, Kumar V, Rand KH, Derendorf H: Clinical microdialysis in skin and soft tissues: an update. J Clin P harmacol 2008; 48:351 364. 56. Brunner M, Derendorf H: Clinical microdialysis: Current applications and potential use in drug development. Trac Trends in Analytical Chemistry 2006; 25:674 680. 57. Muller M, dela Pena A, Derendorf H: Issues in pharmacokinet ics and pharmacodynamics of anti infective agents: distribution in tissue. Antimicrob Agents Chemother 2004; 48:1441 1453. 58. Benfeldt E, Serup J, Menne T: Effect of barrier perturbation on cutaneous salicylic acid penetration in human skin: in vivo pharm acokinetics using microdialysis and non invasive quantification of barrier function. Br J Dermatol 1999; 140:739 748. 59. Morgan CJ, Renwick AG, Friedmann PS: The role of stratum corneum and dermal microvascular perfusion in penetration and tissue levels o f water soluble drugs investigated by microdialysis. Br J Dermatol 2003; 148:434 443. 60. Benfeldt E, Groth L: Feasibility of measuring lipophilic or protein bound drugs in the dermis by in vivo microdialysis after topical or systemic drug administration. Acta Derm Venereol 1998; 78:274 278. 61. Klimowicz A, Farfal S, Bielecka Grzela S: Evaluation of skin penetration of topically applied drugs in humans by cutaneous microdialysis: acyclovir vs. salicylic acid. J Clin Pharm Ther 2007; 32:143 148.

PAGE 132

132 62. Borg N, Gotharson E, Benfeldt E, Groth L, Stahle L: Distribution to the skin of penciclovir after oral famciclovir administration in healthy volunteers: comparison of the suction blister technique and cutaneous microdialysis. Acta Derm Venereol 1999; 79:274 277. 63. Tegeder I, Brautigam L, Podda M, Meier S, Kaufmann R, Geisslinger G, Grundmann Kollmann M: Time course of 8 methoxypsoralen concentrations in skin and plasma after topical (bath and cream) and oral administration of 8 methoxypsoralen. Clin Pharmacol Th er 2002; 71:153 161. 64. Muller M, Stass H, Brunner M, Moller JG, Lackner E, Eichler HG: Penetration of moxifloxacin into peripheral compartments in humans. Antimicrob Agents Ch 1999; 43:2345 2349. 65. Burkhardt O, Derendorf H, Jager D, Kumar V, Madabushi R, Rohl K, Barth J: Moxifloxacin distribution in the interstitial space of infected decubitus ulcer tissue of patients with spinal cord injury measured by in vivo microdialysis. Scand J Infect Dis 2006; 38:904 908. 66. Dehghanyar P, Burger C, Zeitlinger M, Islinger F, Kovar F, Muller M, Kloft C, Joukhadar C: Penetration of linezolid into soft tissues of healthy volunteers after single and multiple doses. Antimicrob Agents Chemother 2005; 49:2367 2371. 67. Hollenstein U, Brunner M, Mayer BX, Delacher S, Erov ic B, Eichler HG, Muller M: Target site concentrations after continuous infusion and bolus injection of cefpirome to healthy volunteers. Clin Pharmacol Ther 2000; 67:229 236. 68. Muller M: Microdialysis in clinical drug delivery studies. Adv Drug Deliv Rev 2000; 45:255 269. 69. Liu P, Muller M, Grant M, Obermann B, Derendorf H: Tissue penetration of cefpodoxime and cefixime in healthy subjects. J Clin Pharmacol 2005; 45:564 569. 70. Barbour A, Schmidt S, Sabarinath SN, Grant M, Seubert C, Skee D, Murthy B, Derendorf H: Soft tissue penetration of ceftobiprole in healthy volunteers determined by in vivo microdialysis. Antimicrob Agents Chemother 2009; 53:2773 2776. 71. Jones RN, Deshpande LM, Mutnick AH, Biedenbach DJ: In vitro evaluation of BAL9141, a novel p arenteral cephalosporin active against oxacillin resistant staphylococci. J Antimicrob Chemother 2002; 50:915 932.

PAGE 133

133 72. Hebeisen P, Heinze Krauss I, Angehrn P, Hohl P, Page MG, Then RL: In vitro and in vivo properties of Ro 63 9141, a novel broad spectrum c ephalosporin with activity against methicillin resistant staphylococci. Antimicrob Agents Chemother 2001; 45:825 836. 73. Liu P, Muller M, Derendorf H: Rational dosing of antibiotics: the use of plasma concentrations versus tissue concentrations. Int J Ant imicrob Agents 2002; 19:285 290. 74. Joukhadar C, Frossard M, Mayer BX, Brunner M, Klein N, Siostrzonek P, Eichler HG, Muller M: Impaired target site penetration of beta lactams may account for therapeutic failure in patients with septic shock. Crit Care M ed 2001; 29:385 391. 75. Burkhardt O, Brunner M, Schmidt S, Grant M, Tang Y, Derendorf H: Penetration of ertapenem into skeletal muscle and subcutaneous adipose tissue in healthy volunteers measured by in vivo microdialysis. J Antimicrob Chemother 2006; 58 :632 636. 76. Brunner M, Hollenstein U, Delacher S, Jager D, Schmid R, Lackner E, Georgopoulos A, Eichler HG, Muller M: Distribution and antimicrobial activity of ciprofloxacin in human soft tissues. Antimicrob Agents Chemother 1999; 43:1307 1309. 77. Brun ner M, Stabeta H, Moller JG, Schrolnberger C, Erovic B, Hollenstein U, Zeitlinger M, Eichler HG, Muller M: Target site concentrations of ciprofloxacin after single intravenous and oral doses. Antimicrob Agents Chemother 2002; 46:3724 3730. 78. Hollenstein UM, Brunner M, Schmid R, Muller M: Soft tissue concentrations of ciprofloxacin in obese and lean subjects following weight adjusted dosing. Int J Obes Relat Metab Disord 2001; 25:354 358. 79. Schuck EL, Grant M, Derendorf H: Effect of simulated microgravit y on the disposition and tissue penetration of ciprofloxacin in healthy volunteers. J Clin Pharmacol 2005; 45:822 831. 80. Nicogossian AE, Sawin CF, Huntoon CL: Overall physiologic response to space flight. In: Space physiology and medicine. Edited by Nico gossian AE, Sawin CF, Huntoon CL. Philadelphia, PA: Lea & Febiger; 1989: 139 154. 81. Taylor GR, Konstantinova I, Sonnenfeld G, Jennings R: Changes in the immune system during and after spaceflight. Adv Space Biol Med 1997; 6:1 32.

PAGE 134

134 82. Chaurasia CS, Muller M, Bashaw ED, Benfeldt E, Bolinder J, Bullock R, Bungay PM, DeLange EC, Derendorf H, Elmquist WF et al: AAPS FDA Workshop White Paper: microdialysis principles, application, and regulatory perspectives. J Clin Pharmacol 2007; 47:589 603. 83. Kreilgaard M: Assessment of cutaneous drug delivery using microdialysis. Adv Drug Deliv Rev 2002; 54 Suppl 1:S99 121. 84. Joukhadar C, Derendorf H, Muller M: Microdialysis. A novel tool for clinical studies of anti infective agents. Eur J Clin Pharmacol 2001; 57:211 21 9. 85. de la Pena A, Liu P, Derendorf H: Microdialysis in peripheral tissues. Adv Drug Deliv Rev 2000; 45:189 216. 86. Schmidt S, Banks R, Kumar V, Rand KH, Derendorf H: Clinical microdialysis in skin and soft tissues: an update. Journal of Clinical Pharma cology 2008; 48:351 364. 87. Chaurasia CS, Muller M, Bashaw ED, Benfeldt E, Bolinder J, Bullock R, Bungay PM, DeLange EC, Derendorf H, Elmquist WF et al: AAPS FDA workshop white paper: microdialysis principles, application and regulatory perspectives. Phar m Res 2007; 24:1014 1025. 88. Muller M, Haag O, Burgdorff T, Georgopoulos A, Weninger W, Jansen B, Stanek G, Pehamberger H, Agneter E, Eichler HG: Characterization of peripheral compartment kinetics of antibiotics by in vivo microdialysis in humans. Antimi crob Agents Chemother 1996; 40:2703 2709. 89. Marchand S, Dahyot C, Lamarche I, Mimoz O, Couet W: Microdialysis study of imipenem distribution in skeletal muscle and lung extracellular fluids of noninfected rats. Antimicrob Agents Chemother 2005; 49:2356 2 361. 90. Liu P, Fuhrherr R, Webb AI, Obermann B, Derendorf H: Tissue penetration of cefpodoxime into the skeletal muscle and lung in rats. Eur J Pharm Sci 2005; 25:439 444. 91. Muller M: Science, medicine, and the future: Microdialysis. BMJ 2002; 324:588 5 91. 92. Barbour A, Schmidt S, Rout WR, Ben David K, Burkhardt O, Derendorf H: Soft tissue penetration of cefuroxime determined by clinical microdialysis in morbidly obese patients undergoing abdominal surgery. Int J Antimicrob Agents 2009; 34:231 235.

PAGE 135

135 93. Traunmuller F, Zeitlinger M, Zeleny P, Muller M, Joukhadar C: Pharmacokinetics of single and multiple dose oral clarithromycin in soft tissues determined by microdialysis. Antimicrob Agents Chemother 2007; 51:3185 3189. 94. Islinger F, Dehghanyar P, Sauer mann R, Burger C, Kloft C, Muller M, Joukhadar C: The effect of food on plasma and tissue concentrations of linezolid after multiple doses. Int J Antimicrob Agents 2006; 27:108 112. 95. Islinger F, Bouw R, Stahl M, Lackner E, Zeleny P, Brunner M, Muller M, Eichler HG, Joukhadar C: Concentrations of gemifloxacin at the target site in healthy volunteers after a single oral dose. Antimicrob Agents Chemother 2004; 48:4246 4249. 96. Bellmann R, Kuchling G, Dehghanyar P, Zeitlinger M, Minar E, Mayer BX, Muller M, Joukhadar C: Tissue pharmacokinetics of levofloxacin in human soft tissue infections. Br J Clin Pharmacol 2004; 57:563 568. 97. Bergogne Berezin E: Clinical role of protein binding of quinolones. Clin Pharmacokinet 2002; 41:741 750. 98. Garcia I, Pascual A, Ballesta S, Joyanes P, Perea EJ: Intracellular penetration and activity of gemifloxacin in human polymorphonuclear leukocytes. Antimicrob Agents Chemother 2000; 44:3193 3195. 99. Pascual A, Garcia I, Ballesta S, Perea EJ: Uptake and intracellular activi ty of moxifloxacin in human neutrophils and tissue cultured epithelial cells. Antimicrob Agents Chemother 1999; 43:12 15. 100. Yang Q, Nakkula RJ, Walters JD: Accumulation of ciprofloxacin and minocycline by cultured human gingival fibroblasts. J Dent Res 2002; 81:836 840. 101. Aarons L: Population pharmacokinetics: theory and practice. Br J Clin Pharmacol 1991; 32:669 670. 102. Sheiner LB, Rosenberg B, Marathe VV: Estimation of population characteristics of pharmacokinetic parameters from routine clinical data. J Pharmacokinet Biopharm 1977; 5:445 479. 103. Samara E, Granneman R: Role of population pharmacokinetics in drug development. A pharmaceutical industry perspective. Clin Pharmacokinet 1997; 32:294 312. 104. Ette EI, Williams PJ: Population pharmacok inetics I: background, concepts, and models. Ann Pharmacother 2004; 38:1702 1706.

PAGE 136

136 105. Whiting B, Kelman AW, Grevel J: Population pharmacokinetics. Theory and clinical application. Clin Pharmacokinet 1986; 11:387 401. 106. Vozeh S, Steimer JL, Rowland M, M orselli P, Mentre F, Balant LP, Aarons L: The use of population pharmacokinetics in drug development. Clin Pharmacokinet 1996; 30:81 93. 107. Powers JD: Statistical considerations in pharmacokinetic study design. Clin Pharmacokinet 1993; 24:380 387. 108. S chuck EL, Dalhoff A, Stass H, Derendorf H: Pharmacokinetic/pharmacodynamic (PK/PD) evaluation of a once daily treatment using ciprofloxacin in an extended release dosage form. Infection 2005; 33:22 28. 109. Mueller M, de la Pena A, Derendorf H: Issues in p harmacokinetics and pharmacodynamics of anti infective agents: kill curves versus MIC. Antimicrob Agents Chemother 2004; 48:369 377. 110. Mouton JW, Dudley MN, Cars O, Derendorf H, Drusano GL: Standardization of pharmacokinetic/pharmacodynamic (PK/PD) term inology for anti infective drugs: an update. J Antimicrob Chemother 2005; 55:601 607. 111. Ambrose PG, Grasela DM, Grasela TH, Passarell J, Mayer HB, Pierce PF: Pharmacodynamics of fluoroquinolones against Streptococcus pneumoniae in patients with communit y acquired respiratory tract infections. Antimicrob Agents Chemother 2001; 45:2793 2797. 112. Lister PD, Sanders CC: Pharmacodynamics of levofloxacin and ciprofloxacin against Streptococcus pneumoniae. J Antimicrob Chemother 1999; 43:79 86. 113. Hyatt JM, McKinnon PS, Zimmer GS, Schentag JJ: The importance of pharmacokinetic/pharmacodynamic surrogate markers to outcome. Focus on antibacterial agents. Clin Pharmacokinet 1995; 28:143 160.

PAGE 137

137 BIOGRAPHICAL SKETCH Runa S. Naik was born in Mangalore, India to Mrs. Prathima Naik and Mr. Shivaram Naik. She graduated with bachelors in pharmacy from Rajiv Gandhi University of Health Sciences from 2000 to 2004 and was registered as a pharmacist by the Karnataka State Pharmacy Council, India in 2004. After a brief experience working in a pharmacy company in Mumbai, she was joined the PhD program in the Department of Pharmaceutical Sciences at Texas Tech Health Sciences Center. After a year and half of successful work under Dr Jochen Klein she tr ansferred to University of Florida to continue her PhD under Dr. Hartmut Derendorf. She completed her graduate work under the tutorship of Dr. Hartmut Derendorf. She graduated in August 2011 with a doctorate of philosophy in pharmaceutical sciences.