© 2015 Alexander Voelkner

PHARMACOKINETIC AND PHARMACODYNAMIC CHARACTERIZATION OF THE PLEUROMUTILIN ANTIBIOTIC RETAPAMULIN

By

ALEXANDER VOELKNER

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

2015

© 2015 Alexander Voelkner

To my parents

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ACKNOWLEDGMENTS

I would like to express my sincere gratitude to Dr. Hartmut Derendorf to give me

this opportunity, but also for his guidance and support throughout my doctoral studies. I

truly enjoyed my time at his lab and working for Dr. Derendorf has been an

extraordinary learning experience. I would like to thank my supervisory committee

members, Dr. Christoph Seubert, Dr. Sihong Song, Dr. Anthony Palmieri III and Dr.

Kenneth Rand, for their support and guidance. Moreover, I would like to thank the

administrative staff of the Department of Pharmaceutics, Pat Khan, Kimberly Howell,

Vivian Lantow and Sarah Foxx and the staff at UF’s Clinical Research Center for their

support. I also had the great privilege to work with my interns Maurice, Laura, Justus,

Trang, Sebastian, Theresa, Jacqueline and Rosalie. They did an outstanding job and

without their help, I would not have been able to complete my research.

Finally, I would like to thank all the graduate students and post-docs in the

Department of Pharmaceutics, especially Frederico Martins and my wife Nivea, who

helped tremendously with completing my in vivo study. Last but not least, special thanks

go out to my parents and my German-Brazilian family for their support.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 9

LIST OF FIGURES ........................................................................................................ 11

LIST OF ABBREVIATIONS ........................................................................................... 13

ABSTRACT ................................................................................................................... 16

CHAPTER

1 INTRODUCTION .................................................................................................... 18

Significance and Background ................................................................................. 18 Hypothesis and Specific Aims ................................................................................. 22

Specific Aims .................................................................................................... 22

Aim 1. Development and validation of a bioanalytical method to quantify retapamulin ............................................................................................. 22

Aim 2. In vitro microdialysis recovery determination of retapamulin ........... 22

Aim 3. Clinical microdialysis feasibility study.............................................. 23 Aim 4. In vivo microdialysis study .............................................................. 23

Aim 5. In vitro time-kill experiments ........................................................... 23

Aim 6. Development of a PK/PD model ..................................................... 24

2 LC-MS/MS METHOD DEVELOPMENT AND VALIDATION TO DETERMINE RETAPAMULIN IN NORMAL SALINE .................................................................... 25

Objective ................................................................................................................. 25 Experimental Procedure ......................................................................................... 25

Laboratory and Study Equipment ..................................................................... 25

Test article ................................................................................................. 25 Reagents.................................................................................................... 25 Equipment and disposables ....................................................................... 25

Reagent Preparation ........................................................................................ 26

Normal saline ............................................................................................. 26 Retapamulin stock solution ........................................................................ 27 Retapamulin working solutions .................................................................. 27 Tiamulin stock solution ............................................................................... 27 Tiamulin working solutions ......................................................................... 27

Retapamulin calibration standards ............................................................. 27 Tiamulin internal standard solution ............................................................ 28

Retapamulin quality control samples ......................................................... 28

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LC-MS/MS mobile phase ........................................................................... 28

LC-MS/MS washing solution ...................................................................... 28 Sample Preparations ........................................................................................ 28

LC-MS/MS Conditions ...................................................................................... 29 Quantification and QC ...................................................................................... 29 Assay Validation ............................................................................................... 30 Data Analysis ................................................................................................... 30

Results .................................................................................................................... 31

Linearity, Accuracy, Precision and Lower Limit of Quantification ..................... 31 Intra- and Inter-day Variability for Quality Control Samples .............................. 31 Freeze-thaw, Short-term, Long-term and Stock Solution Stability .................... 31 Dilution Integrity ................................................................................................ 32

Summary ................................................................................................................ 32

3 IN VITRO MICRODIALYSIS EXPERIMENTS OF RETAPAMULIN ........................ 37

Objective ................................................................................................................. 37 Experimental Procedure ......................................................................................... 37

Laboratory and Study Equipment ..................................................................... 37 Test article ................................................................................................. 37 Reagents.................................................................................................... 37

Equipment and disposables ....................................................................... 37 Reagent Preparation ........................................................................................ 39

Normal saline ............................................................................................. 39 Retapamulin stock and working solution .................................................... 39 Retapamulin calibration standards and quality controls ............................. 39

Tiamulin internal standard solution ............................................................ 39 LC-MS/MS mobile phase and washing solution ......................................... 39

Microdialysis Experiments ................................................................................ 40 Microdialysis calibration solutions .............................................................. 40

Microdialysis system .................................................................................. 40 Extraction efficiency method (EE) .............................................................. 40 Retrodialysis method (RD) ......................................................................... 41

Sample Preparation and Analysis .................................................................... 41 Results .................................................................................................................... 42

Calibration Curve and QCs ............................................................................... 42 In Vitro Microdialysis ........................................................................................ 42

Summary ................................................................................................................ 43

4 IN VITRO ANTIBACTERIAL ACTIVITY OF RETAPAMULIN .................................. 46

Objective ................................................................................................................. 46 Material and Methods ............................................................................................. 46

Antimicrobial Agent .......................................................................................... 46

Microbial Strains ............................................................................................... 46 Reagents .......................................................................................................... 46

Equipment and disposables ............................................................................. 47

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Preparation of Solutions and Broth ................................................................... 48

Sterile saline .............................................................................................. 48 Mueller-Hinton broth II ............................................................................... 48

Retapamulin primary and secondary stock solutions ................................. 48 MIC Determination ........................................................................................... 48 Retapamulin Adsorption on 24 Well Plate ........................................................ 49 Static Time-kill Curve ....................................................................................... 50 Retapamulin Stability in Mueller-Hinton Broth .................................................. 51

Results .................................................................................................................... 51 MIC Determination ........................................................................................... 51 Retapamulin Adsorption on 24 Well Plate ........................................................ 51 Retapamulin Stability in Mueller-Hinton Broth .................................................. 52 Static Time-kill Curves ...................................................................................... 52

Data Analysis ................................................................................................... 52 Summary ................................................................................................................ 52

5 RETAPAMULIN FEASIBILITY STUDY ................................................................... 59


Retapamulin Solution ....................................................................................... 59

Materials ........................................................................................................... 59 Equipment and Disposables ............................................................................. 59

Clinical Feasibility Study ................................................................................... 60 Results .................................................................................................................... 62

Patient Demographics and Baseline Characteristics ........................................ 62 In Vivo Recovery .............................................................................................. 62

Washout Period ................................................................................................ 63

Safety Assessment ........................................................................................... 63 Summary ................................................................................................................ 63

6 IN VIVO PHARMACOKINETICS OF RETAPAMULIN ............................................ 67


Antimicrobial Agents ......................................................................................... 67 Materials ........................................................................................................... 67 Equipment and disposables ............................................................................. 68 In Vivo Pharmacokinetic Study ......................................................................... 69

Anesthetization .......................................................................................... 69 Microdialysis probe implantation ................................................................ 70 Microdialysis probe stabilization ................................................................. 70 Retrodialysis .............................................................................................. 71 Baseline sample collection ......................................................................... 71

Topical administration of ointment ............................................................. 71 IV bolus administration ............................................................................... 72

Microdialysis and blood sample collection ................................................. 72

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Protein binding and Centrifree recovery ..................................................... 72

Sample preparation and analysis ............................................................... 73 Results .................................................................................................................... 74

Plasma Protein Binding and Recovery ............................................................. 74 Retapamulin Concentrations in Plasma and Skin ............................................. 74

IV bolus ...................................................................................................... 74 Topical application after tape-stripping ...................................................... 75 Topical application on intact skin ............................................................... 75

Summary ................................................................................................................ 75

7 PHARMACOKINETIC AND PHARMACODYNAMIC ANALYSIS ............................ 82


Non-compartmental Analysis ............................................................................ 82 Population Pharmacokinetic Analysis ............................................................... 83

Semi-mechanistic Pharmacodynamic Model .................................................... 83 Allometric Scaling ............................................................................................. 84

Results .................................................................................................................... 84 Non-compartmental Analysis ............................................................................ 84 Population Pharmacokinetic Analysis ............................................................... 85

Semi-mechanistic Pharmacodynamic Model .................................................... 88 Allometric Scaling ............................................................................................. 89

Summary ................................................................................................................ 92

8 DISCUSSION AND CONCLUSION ...................................................................... 106

LIST OF REFERENCES ............................................................................................. 113

BIOGRAPHICAL SKETCH .......................................................................................... 129

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LIST OF TABLES

Table page 2-1. Working solutions retapamulin ............................................................................... 33

2-2. Working solutions tiamulin ..................................................................................... 33

2-3. Calibration standards retapamulin .......................................................................... 33

2-4. Quality control samples retapamulin ...................................................................... 33

2-5. Mass transition parameters .................................................................................... 34

2-6. Intra-day variability of retapamulin ......................................................................... 34

2-7. Inter-day variability of retapamulin ......................................................................... 34

2-8. Stability results for retapamulin .............................................................................. 35

3-1. Calibration solutions for in vitro retapamulin microdialysis ..................................... 44

3-2. Calibration curve standards and QCs for in vitro microdialysis .............................. 44

3-3. In vitro microdialysis recovery results for retapamulin ............................................ 44

4-1. Pipetting scheme for retapamulin MIC determination ............................................. 54

4-2. Dilution scheme for retapamulin static time-kill curve experiments against a clinical MSSA isolate .......................................................................................... 54

4-3. Dilution scheme for retapamulin static time-kill curve experiments against MRSA ATCC 43300 ........................................................................................... 54

4-4. Dilution factors for plating the clinical MSSA isolate ............................................... 54

4-5. Dilution factors for plating the MRSA ATCC 43300 strain ...................................... 55

4-6. Contingency table of MICs for retapamulin against a clinical MSSA isolate and MRSA ATCC 4330 ............................................................................................. 55

4-7. Stability test of retapamulin in Mueller-Hinton broth II at 37˚C ............................... 55

5-1. Demographic and Baseline Characteristics ........................................................... 65

5-2. Mean in vivo recovery ............................................................................................ 65

7-1. Non-compartmental pharmacokinetic analysis for retapamulin after IV bolus administration ..................................................................................................... 94

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7-2. Non-compartmental pharmacokinetic analysis for retapamulin after topical application on tape-stripped skin ........................................................................ 94

7-3. Non-compartmental pharmacokinetic analysis for retapamulin after topical application on intact skin .................................................................................... 95

7-4. Population pharmacokinetic parameter estimates.................................................. 95

7-5. Parameter estimates for the MSSA and MRSA PD model ..................................... 96

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LIST OF FIGURES

Figure page 2-1. Mean calibration curve of retapamulin ................................................................... 36

2-2. Representative chromatogram of retapamulin ....................................................... 36

3-1. Mean recoveries of retapamulin at different concentrations using extraction efficiency and retrodialysis ................................................................................. 45

4-1. Labeling-scheme for the 24-well plate used for retapamulin MIC-determination. ... 56

4-2. Spot inoculation on sheep-blood agar plates for colony enumeration after serial dilution ................................................................................................................ 56

4-3. MIC frequency distribution of retapamulin against the tested MSSA and MRSA strains. ................................................................................................................ 57

4-4. Stability of retapamulin in Mueller-Hinton broth II at 37˚C ...................................... 57

4-5. Retapamulin time-kill curves against the tested MSSA and MRSA strains ............ 58

5-1. In vivo recovery of retapamulin determined by retrodialysis ................................... 66

5-2. Mean (SD) skin concentrations of retapamulin during the washout period ............ 66

6-1. Plasma protein binding in humans and rats ........................................................... 77

6-2. Unbound plasma and skin ISF concentration profiles of retapamulin after intravenous administration .................................................................................. 78

6-3. Unbound plasma and skin ISF concentration profiles of retapamulin after tape-stripping and topical application.......................................................................... 79

6-4. Unbound skin ISF concentration profiles of retapamulin after topical application on intact skin....................................................................................................... 80

6-5. Flowchart with time and events of the in vivo PK study .......................................... 81

7-1. Scheme of the final population pharmacokinetic model ......................................... 96

7-2. Individual unbound concentration profiles in plasma .............................................. 97

7-3. Individual unbound concentration profiles in skin ................................................... 98

7-4. Prediction-corrected VPCs for iv bolus administration ........................................... 99

7-5. Prediction-corrected VPCs for topical route of administration ................................ 99

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7-6. Basic goodness-of-fit plots ................................................................................... 100

7-8. Basic goodness-of-fit plots from the MSSA PD model ......................................... 101

7-9. VPCs for the MSSA PD model stratified for MICs ................................................ 102

7-10. Basic goodness-of-fit plots from the MRSA PD model ....................................... 103

7-11. VPCs for the MRSA PD model stratified for MICs .............................................. 104

7-12. Simulated human PK and PD profiles for retapamulin. ...................................... 105

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LIST OF ABBREVIATIONS

AIC Akaike information criterion

ANCOVA Analysis of covariance

ANOVA Analysis of variance

AUC Area under the curve

AUC0-t Area under the curve from time zero to time t

AUC0-∞ Area under the curve from time zero to infinity

BA Bioavailability

BE Bioequivalence

BSA Body surface area

CDC Center for Disease Control and Prevention

CFU Colony forming units

CI Confidence interval

CLSI Clinical and Laboratory Standards Institute

CL Clearance

CLtot Total clearance

Cmax Peak plasma concentration

Cmax/MIC Peak plasma concentration over minimum inhibitory concentration

Css Concentration at steady state

CV Coefficient of variation

dg Delay in onset of growth

dgs Delay in onset of growth of susceptible bacteria

dk Delay in onset of kill

dks Delay in onset of kill of susceptible bacteria

EC50 Concentration which induces the half-maximal drug effect

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EE Extraction efficiency

EMA European Medicines Agency

Emax Maximum effect

FDA Food and Drug Administration

fu Fraction unbound

h Hill factor

ISF Interstitial space fluid

IRB Institutional review board

IDSA Infectious Disease Society of America

IV Intravenous

k0 Zero-order absorption rate constant

k12 Transfer-rate constant from central to peripheral compartment

k21 Transfer-rate constant from peripheral to central compartment

k10 Elimination-rate constant

ka First-order absorption-rate constant

kd Death-rate constant

ks Bacterial growth-rate constant

ksr Transfer-rate constant from susceptible to resting state

krs Transfer-rate constant from resting to susceptible state

LC-MS/MS Liquid chromatography coupled with tandem mass spectrometry

MHB Mueller-Hinton broth

MIC Minimum inhibitory concentration

MD Microdialysis

MRSA Methicillin-resistant Staphylococcus aureus

MSSA Methicillin-susceptible Staphylococcus aureus

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MRT Mean residence time

N Number of bacteria

N0 Starting number bacteria

NCA Non-compartmental analysis

OFV Objective function value

PBP2a Penicillin-binding protein 2a

PD Pharmacodynamics

PK Pharmacokinetics

PPB Plasma protein binding

PopPK Population pharmacokinetics

RSE Residual standard error

S. aureus Staphylococcus aureus

SC Stratum corneum

SD Standard deviation

SE Standard error

SSSI Skin and skin structure infection

t1/2 Half-life

TEWL Transepidermal water loss

T>MIC Cumulative percentage of time where plasma concentrations are above the minimum inhibitory concentration

tmax Time of peak plasma concentration

Vc Volume of distribution in the central compartment

Vd Volume of distribution

Vss Volume of distribution at steady state

Vz Volume of distribution in the terminal phase

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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 AND PHARMACODYNAMIC CHARACTERIZATION OF THE

PLEUROMUTILIN ANTIBIOTIC RETAPAMULIN

By

Alexander Voelkner

December 2015

Chair: Hartmut Derendorf Major: Pharmaceutical Sciences

Retapamulin, an antibiotic from the pleuromutilin class, is approved to treat

impetigo, a skin disease frequently caused by Staphylococcus aureus and

Streptococcus pyogenes. It inhibits the bacterial protein synthesis by binding to the 50s

subunit of the bacterial ribosome and has a unique mechanism of action. Currently,

retapamulin is marketed as topical ointment for use in adults and pediatric patients.

Pharmacokinetic data on retapamulin is limited due to low systemic exposure

after topical application. No pharmacokinetic parameters have been established for

retapamulin and concentrations at the site of action have yet to be evaluated. To

characterize the pharmacokinetics, dermal concentrations of retapamulin where

quantified by microdialysis and compared to plasma concentrations.

The pharmacodynamics of retapamulin were assessed using in vitro time-kill

curve experiments against methicillin-susceptible Staphylococcus aureus (MSSA).

Although retapamulin is indicated to treat MSSA infections only, the activity against

methicillin-resistant Staphylococcus aureus (MRSA) was also investigated and

compared to that of MSSA.

17

Pharmacokinetics were evaluated with non-compartmental and compartmental

analysis and integrated with a semi-mechanistic pharmacodynamic model to quantify

and predict retapamulin’s concentration-effect relationship under different dosing

regimens.

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CHAPTER 1 INTRODUCTION

Significance and Background

Bacterial infections of the epidermis, dermis and hypodermis are defined as skin

and skin structure infections (SSSIs). They comprise a wide range of clinical

manifestations and can be broadly classified as uncomplicated and complicated.

Uncomplicated SSSIs (uSSSIs) include superficial abscesses, erysipelas, cellulitis,

furuncles and impetiginous lesions. In contrast, complicated SSSIs (cSSSIs) involve

deep tissue infection, such as necrotizing fasciitis and myositis, or require significant

surgical intervention, e.g. ulcers, burns and major abscesses1–9. Staphylococcus aureus

(SA), a gram-positive bacteria, is the most common pathogen in SSSIs with regional

differences and varying prevalence (34.9%10, 44.6%11, 81%12). Nearly half of those SA

isolates were methicillin-resistant S. aureus (MRSA)11,12.

An estimated 7-10% of hospitalizations were due to SSSIs13–15. SA-SSSI hospital

admissions increased by 29%16 from 2000 to 2004 and by 123% between 2001 and

200917. Annual hospitalization costs due to SA-SSSI in 2008 were $4.84 billion and the

average associated cost in 2009 was $11,62217. Moreover, the number of annual

ambulatory care visits from 2001 to 2003 were 11.4 million18 and 34.8 million between

2006 and 201019. It was also reported that outpatient visits increased by 50% in from

1997 to 200520.

Retapamulin, a pleuromutilin antibiotic, is approved to treat superficial

uncomplicated skin infections caused by S. aureus and Streptococcus pyogenes21–23. It

is marketed as topical ointment (Altabax™ in USA and Altargo™ in Europe) for use in

adults and pediatric patients older than nine months. Retapamulin has a unique

19

mechanism of action. It binds to the ribosomal 50S subunit, inhibits peptidyl transferase

activity and interferes with the binding of the initiator tRNA substrate to the ribosomal P-

site24,25. The drug is bacteriostatic against S. aureus at concentrations ranging from

0.03-0.25 mg/L26,27 and lacks clinically relevant target-specific cross-resistance24,28. No

resistance was observed during clinical trials following five days of treatment29–31.

Since retapamulin is applied topically, systemic exposure is low and availability of

pharmacokinetic data is limited6,32. After single topical application on 800 cm2 of intact

skin in healthy adults, systemic concentrations were very low and only 3% of the plasma

samples were above the limit of quantification (LOQ 0.5 ng/mL). On the other hand,

median Cmax in plasma was 3.5 ng/mL (range 1.2-7.8 ng/mL) after seven days of

application and 82% of the samples were quantifiable. The median Cmax in plasma

following a single dose and 7 days of application to 200 cm2 of abraded skin was 11.7

ng/mL (range 5.6-22.1 ng/mL) and 9.0 ng/mL (range 6.7-12.8 ng/mL) and 97-100% of

the samples were above LOQ25.

In another trial, plasma samples were obtained from 380 adults and 136 pediatric

patients who received Altabax™ twice daily. The median concentration was 0.8 ng/mL

and 11% of the plasma samples were quantifiable. The maximum concentrations were

10.7 ng/mL in adults and 18.5 ng/mL in pediatric patients25.

Plasma clearance was reported to be high in rats, dogs and monkeys (1-2 h).

Bioavailability was low (1-2%) following oral administration in rats. Retapamulin showed

extensive biliary excretion and plasma levels in rats and monkeys were reported to be in

the high ng/mL after oral administration of 450 mg/kg1. The drug distributed rapidly

following IV administration of radiolabelled (14C)-retapamulin in rats. Protein binding was

20

reported to be 75-94% in the order monkey<rat<human1. Volume of distribution and

clearance in humans has not been determined25. In vitro human microsome studies

suggested that the major metabolizing enzyme of retapamulin is cytochrome P450 3A4.

The major metabolic pathways were mono and di-oxygenation and N-demethylation25.

Retapamulin is heavily metabolized and about 100 metabolites were detected in

plasma, urine, faeces and bile of rats1.

Microdialysis use dates back to the early 1960s, where it was used to measure

neurotransmitter release in rodents. It was used in preclinical studies and eventually

adopted for clinical use, with first human PK studies in the early 1990s. Nowadays, it

allows to continuously measure unbound drug concentrations in virtually every tissue,

with minimal trauma33. The method itself involves the implantation of a small

microdialysis probe into the tissue, which is then constantly perfused to sample

exogenous or endogenous solutes from the extracellular space34. Solute exchange is

driven by passive diffusion against a concentration gradient and depends on perfusion

flow-rate, pore diameter of the membrane, membrane surface area, temperature, nature

of the tissue and physicochemical properties of the analyte35. Dialysate can be collected

at specific time intervals to determine the analyte concentration. Due to the constant

flow of perfusate, a complete equilibrium between perfusate and extracellular fluid

cannot be achieved, thus analyte recoveries are less than 100%. Therefore, the

microdialysis probe recovery needs to be assessed. Several calibration methods can be

used, such as low-flow-rate method, no-net-flux, dynamic no-net-flux and retrodialysis

by drug or calibrator33,36–38. For topically applied drugs, where systemic exposure is

limited, microdialysis can be useful to determine drug concentrations at the site of

21

action. Dermal microdialysis is a semi-invasive technique, which can directly quantify

free, unbound drug concentrations within the dermis. The method is also suited for

evaluating the local exposure of topical products in the skin, subcutaneous tissue and

muscle under various test conditions.39–41

To optimize antimicrobial therapy and select appropriate dosing regimens, the

PKPD relationship of anti-infectives needs to be well understood. The minimum

inhibitory concentration (MIC) has been used as a PD marker to guide dosing and drug

selections. The quantitative relationship between the concentrations of an anti-infective

drug and its antibacterial effect is labelled as PK/PD index42. The resulting PK/PD

indices are the area-under-the-curve over MIC (AUC/MIC), maximum concentrations

over MIC (Cmax/MIC) and time above MIC (T>MIC)43,44 are frequently used as target

parameters for dosing decisions. However, the MIC is a static parameter and provides

no information about the time course of an antibacterial drug as it only determines the

number of bacteria at a single time point. Furthermore, the MIC approach represents a

threshold concentration, which implies the maximum antibacterial effect at the MIC and

no effect below the MIC. PK/PD models based on time-kill curves evaluate the

antibacterial activity and bacterial growth as a function of time and antibiotic

concentration45. They provide a better understanding of the processes within the

bacterial system and help describe the killing kinetics, bacterial susceptibility, adaptation

and resistance development46–48. Subsequently, these PK/PD models can be useful in

making informed dosing decisions and therefore help to optimize antibacterial therapy

and is also acknowledged by regulatory agencies49,50.

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Hypothesis and Specific Aims

Retapamulin ointment is efficacious to treat uncomplicated skin and skin

structure infections caused by S. aureus. It penetrates the skin and elicits a

bacteriostatic effect at the target site. The main objective of the proposed research was

to evaluate the PK of retapamulin using microdialysis and investigate its PD against S.

aureus with time-kill curve experiments. A mathematical model, integrating

concentration and pharmacological response over time helps to optimize drug therapy

and predict the antibiotic effect at the site of infection.

Specific Aims

Aim 1. Development and validation of a bioanalytical method to quantify retapamulin

Analytical methods are critical for the successful conduct of preclinical and/or

clinical pharmacology studies. To quantify in vitro and in vivo retapamulin samples, a

sensitive LC-MS/MS method will be developed and validated. Chromatographic and

mass spectrometry conditions will be optimized for retapamulin and an internal

standard. As defined in the FDA guidance, the method will be tested for selectivity,

accuracy, precision, linearity in a given concentration range, sensitivity, reproducibility

and stability. Acceptance criteria will be the same as defined in the FDA guidance.

Aim 2. In vitro microdialysis recovery determination of retapamulin

In vitro recovery experiments should be performed prior to any in vivo studies.

These experiments are essential, as they will determine if a drug is dialyzable, exhibit

any non-specific binding to the probes and has similar recovery rates at various

concentrations. All experiments will be conducted with a linear microdialysis catheter in

triplicates. The dialysis and retrodialysis methods will be employed in order to determine

23

non-specific binding of retapamulin. Recovery will be tested at low, medium and high

concentrations, which will cover the expected in vivo concentration range. Additives

may be tested and added to improve recovery.

Aim 3. Clinical microdialysis feasibility study

Three subjects will be enrolled to investigate the feasibility of using microdialysis

as a technique to determine skin concentrations of retapamulin in healthy volunteers.

The in vivo recovery as well as the washout period of retapamulin from skin will be

assessed. This study will determine if the compound is a candidate for this technique

and will help to finalize the study design and procedures for a future microdialysis study

in humans with retapamulin.

Aim 4. In vivo microdialysis study

Fifteen Wistar rats will be used to determine the pharmacokinetics of retapamulin

in plasma and in skin. Three treatment groups, with five animals in each, will be

investigated. For the first groups, an intravenous bolus will be administered to

investigate plasma pharmacokinetics and drug distribution into the skin. In the second

and third group, retapamulin ointment will be applied to intact skin and onto strip

abraded skin. Recovery will be measured prior to drug administration and microdialysis

samples collected to describe the pharmacokinetics of retapamulin in skin tissue.

Aim 5. In vitro time-kill experiments

The pharmacodynamic time course of retapamulin, at different concentrations,

against S. aureus, will be investigated. First, the MIC of retapamulin will be evaluated

using the broth microdilution technique. Then, time-kill experiments will be conducted

with drug concentrations ranging from 0.125-16x MIC. Blood agar plates will be

incubated and cell forming colonies (CFUs) counted.

24

Aim 6. Development of a PK/PD model

The pharmacokinetic (PK) and pharmacodynamic (PD) data will be analyzed

using the non-linear mixed effect modeling software NONMEM® 7.3 (ICON

Development Solutions) and the statistical computation software R version 3.1. PK

parameters, such as drug clearance, apparent volume of distribution, maximum

concentration, time to maximum concentration and area-under-the-concentration curve

(AUC) will be established. A model to describe the drug response vs. time will be

developed, as well, and integrated into the PK model.

25

CHAPTER 2 LC-MS/MS METHOD DEVELOPMENT AND VALIDATION TO DETERMINE

RETAPAMULIN IN NORMAL SALINE

Objective

The objective was to develop and validate a sensitive bioanalytical assay to

quantify retapamulin in normal saline. The assay was used to determine retapamulin

concentrations for the in vitro and in vivo microdialysis experiments. Method

development and validation was performed in accordance with the FDA guidance

((CVM), 2013) and using good laboratory practices (GLP).

Experimental Procedure

Laboratory and Study Equipment

Test article

Micronized Retapamulin (lot #WG0223980-140411001) was obtained from Ark

Pharm, Inc. The compound was stored at room temperature and protected from light.

Reagents

Unless otherwise stated, all reagents were LC-MS grade and obtained from the

specified sources.

Tiamulin, Sigma-Aldrich #34044

Acetonitrile (ACN), Fisher #A998-4

Dimethylsulfoxide (DMSO), Fisher #BP231-1

Methanol (MeOH), Fisher #A456-1

Triple distilled water (TDW), in house, Pharmaceutics Department

Sodium Chloride (NaCl), Fisher #S271-500

Formic Acid (FA), Fisher #A119P-1

Equipment and disposables

Eppendorf Research Pipettes, volume: 2-20 µL, 10-100 µL, 20-200 µL, 100-1000 µL

Eppendorf Research Plus Pipette, volume: 0.1-10 µL

26

Fisherbrand Pipet Tips

Fisherbrand microcentrifuge tubes, 0.5 mL amber vials


Corning 15 mL centrifuge tubes


Sun Sri Autosampler Vial 12x32 mm, 300 µL, conical

Sun Sri 11 mm aluminum seals

Perkin Elmer 1.8 mL Screw Thread Vials 12x32 mm

Pyrex Media-lab bottles, 500 mL

Pyrex volumetric flask, 1,000 mL

Millipore nylon membrane filter type 0.2 µm

Fisher Scientific Hotplate

Fisher Scientific Mini Vortexer

Mettler Toledo AB104 balance

Perkin Elmer Series 200 Pump

Perkin Elmer Series 200 Autosampler

AB Sciex API 4000 LC/MS/MS

Waters Atlantis T3 2.1x150 mm HPLC column (S/N013333145140)

Waters Atlantis T3 2.1x10 mm Guard Cartridge

Reagent Preparation

Normal saline

Normal saline (0.9% m/V NaCl) was prepared by weighing out 9 g of NaCl and

dissolving it in 1,000 mL of triple distilled water in a volumetric flask. The solution was

27

then sonicated for 10 minutes and filtered through a Millipore nylon membrane filter (0.2

µm).

Retapamulin stock solution

The stock solution was prepared by weighing out 10 mg of retapamulin and

dissolving it in 10 mL DMSO, to obtain a 1 mg/mL solution. It was stored in a freezer at -

80˚C for not longer than 60 days.

Retapamulin working solutions

Working solution were prepared from the 1 mg/mL stock solution and methanol,

using serial dilutions (Table 2-1). All retapamulin working solutions were stored in a

freezer at -80˚C until needed.

Tiamulin stock solution

Tiamulin primary stock solution was prepared by adding 410 µL of DMSO into a

glass vial of 100 mg of tiamulin (specific gravity ~ 1.1 g/cm3). The glass vial was

vortexed. Then 50 µL of the tiamulin-DMSO solution (200 mg/mL) were mixed with 950

µL DMSO in a 1.5 mL amber micro centrifuge vial, to yield a 10 mg/mL tiamulin stock

solution. It was then stored in a refrigerator at 2-8˚C.

Tiamulin working solutions

The 10 mg/mL stock solution was used to prepare tiamulin working solutions

using serial dilutions (Table 2-2). Methanol was used as diluent and all working

solutions were stored at 2-8˚C.

Retapamulin calibration standards

Calibration standards were freshly prepared, in normal saline, on the day of

analysis from the retapamulin working solutions (Table 2-3). During method

development, a loss of signal and non-linear behavior was observed when calibration

28

standards were prepared in normal saline by serial dilution. However, when calibration

standards were prepared from the working solutions, signal response was linear.

Tiamulin internal standard solution

The internal standard solution was prepared freshly on the day of the

experiments. To yield a 25 ng/mL internal standard solution, 25 µL of a 1 µg/mL tiamulin

working solution was diluted with 975 µL of methanol in a 1.5 mL amber micro

centrifuge vial.

Retapamulin quality control samples

Quality control solutions (QCs) were obtained by diluting retapamulin working

solutions with normal saline (Table 2-4). QCs for lower limit of quantification (LLOQ; 0.5

ng/mL), low quality control (LQC; 1.5 ng/mL), medium quality control (MQC; 75 ng/mL)

and high quality control (HQC; 200 ng/mL) were prepared fresh daily.

LC-MS/MS mobile phase

Reservoir A (0.1% formic acid in H2O) was prepared by mixing 500 mL of triple

distilled water with 570 µL of 88% formic acid. Reservoir B (0.1% formic acid in ACN)

was obtained by adding 570 µL formic acid to 500 mL ACN. Mobile phases were filtered

through a Millipore nylon membrane filter (0.2 µm) and subsequently degassed with

helium for 10 min.

LC-MS/MS washing solution

375 mL of ACN and 125 mL of MeOH were mixed, filtered and degassed to

obtain the washing solution.

Sample Preparations

Frozen samples were thawed and allowed to equilibrate at room temperature.

Samples were then vortexed and an aliquot of 20 µL pipetted into a Sun Sri

29

Autosampler Vial to which 10 µL of internal standard were added. Prior to injection into

LC-MS/MS, all samples were vortexed again and checked for air bubbles.

LC-MS/MS Conditions

The LC system consisted of a Perkin Elmer Series 200 autosampler and a Perkin

Elmer Series 200 pump. Separation was performed using a Waters Atlantis T3 2.1x150

mm HPLC column, coupled with a Waters Atlantis T3 2.1x10 mm Guard Cartridge. The

mobile phases were delivered under isocratic conditions, with a ratio of 65:35 of mobile

phase A (0.1% FA in H2O) and B (0.1%FA in ACN), at a flow rate of 0.3 mL/min.

Injection volume was 10 µL.

The LC system was coupled with an AB Sciex API 4000 triple-quadrupole mass

spectrometer. Analyte and IS were ionized in positive electrospray mode and quantified

using multiple reaction monitoring (MRM). Optimized mass spectrometric parameters

were as follows: dwell time collision gas flow 5 psig, curtain gas flow 20 psig, IonSpray

nebulizer gas 35 psig, TurboIonSpray heater gas 20 psig, TurboIonSpray probe voltage

5,500 V and temperature 550˚C. Mass transitions from the protonated precursors to the

ion products are listed in Table 2-5. Hardware management and data acquisition was

handled using the Analyst Software version 1.4.

Quantification and QC

Peak area ratios of analyte and IS were used for quantification. Calibration

curves were calculated using linear least-square regression analysis and a 1/X2

weighting. Duplicates of LQC, MQC and HQC were placed within a sample batch for

quality control. Analytical runs were not rejected if at least five out of six calibration

standards, including the LLOQ and the highest concentration, where within ±15% of

30

their nominal concentration and if at least one QC at each concentration and not less

than 75% of all the QCs were within ±15% of their nominal levels.

Assay Validation

Linearity over the inspected concentration range was assessed based on

correlation coefficient (r2 ≥0.9). Intra-day and inter-day accuracy and precision were

investigated on three different days. Calibration curves and five replicates of LLOQ,

LQC, MQC and HQC were analyzed on each day. Accuracy and precision of the

standards had to be within ±15% of their nominal concentration, except for the lower

limit of quantification (LLOQ) with an allowance of ±20% deviation and if at least five out

of six standards, including the LLOQ and the highest concentration, met the criteria.

Short-term, freeze thaw, long-term and stock solution stability was determined.

For all stability tests, three replicates of each LQC and HQC were analyzed and

compared with the results of freshly prepared samples. For stock solution stability, three

aliquots of each retapamulin and tiamulin stock were compared with those made from

fresh stock solution. Samples were considered stable if the deviation was ≤10% from

their nominal concentration.

Dilution integrity was evaluated with analyte concentrations above the ULOQ

(250 ng/mL) and at the HQC level. Three replicates of each 4xULOQ and HQC

concentration were then analyzed after ten-fold dilution and compared against their

nominal concentrations.

Data Analysis

Microsoft Excel 2013, R version 3.1.2, RStudio version 0.98.1087, and the

ggplot2 package were used for data collection, statistical analysis and plot generation.

31

Results

Linearity, Accuracy, Precision and Lower Limit of Quantification

All three calibration curves were linear over the tested concentration range 0.5-

250 ng/mL (Figure 2-1). The mean slope±SE of the linear regression equation was

0.0221±0.0006, with a mean intercept±SE of -0.0018±0.0006 and a good coefficient of

determination (r2=0.997). The average±SE retention times were 3.44±0.02 minutes for

retapamulin and 3.41±0.02 minutes for tiamulin, respectively. Calibration standard’s

accuracy (%) and precision (CV %) were 92.7-102.9% and 1.5-7.8%, respectively. The

lower limit of quantification was 0.5 ng/mL. A representative chromatogram for

retapamulin and tiamulin is depicted in Figure 2-2.

Intra- and Inter-day Variability for Quality Control Samples

Intra-day accuracy and precision was ranging from 90.0-114.6% and 1.0-13.1%,

respectively (Table 2-6). Inter-day accuracy and precision were 100.4-103.7% and 7.1-

13.2% (Table 2-7).

Freeze-thaw, Short-term, Long-term and Stock Solution Stability

Retapamulin was stable at room temperature for 18h and for 86 days when

stored at -80˚C. After three freeze-thaw cycles, the concentrations at both LOQ and

HQC were still measured within ±10% of their nominal concentrations. For stock

solution stability, the area under the curve of fresh prepared retapamulin and tiamulin

stock solutions were compared with those stored for 60 days at -80˚C. All stability

results are compiled in Table 2-8.

32

Dilution Integrity

The samples used to test dilution integrity were within the acceptance range of

±15%. The accuracies after ten-fold dilution of 4xULOQ (100 ng/mL) and HQC (20

ng/mL) were 101.5% and 98.9%; precision was 1.2% and 2.8%, respectively.

Summary

A selective and sensitive bioanalytical method for the quantitative determination

of retapamulin using LC-MS/MS was developed and evaluated as per FDA guidance.

The method was reproducible and suitable to analyze retapamulin samples in saline,

ranging from 0.5 to 250 ng/mL. Retapamulin was stable under the tested conditions.

Overall, the developed method provides the means to carry out in vitro microdialysis

experiments and in vivo pharmacokinetic sample analysis.

33

Table 2-1. Working solutions retapamulin Starting Concentration (µg/mL)

Starting Volume (mL) Final Concentration (µg/mL)

Total Volume (mL)

1,000 0.1 100 1 100 0.1 10 1 10 0.1 1 1 1 0.025 0.25 1

Table 2-2. Working solutions tiamulin Starting Concentration (µg/mL)

Starting Volume (mL) Final Concentration (µg/mL)

Total Volume (mL)

10,000 0.1 1,000 1 1,000 0.1 100 1 100 0.01 1 1

Table 2-3. Calibration standards retapamulin Standard Name

Starting Concentration (µg/mL)

Starting Volume (mL)

Final Concentration (ng/mL)

Total Volume (mL)

R6 100 0.025 250 1 R5 100 0.01 100 1 R4 10 0.05 50 1 R3 10 0.01 10 1 R2 1 0.04 1 1 R1 1 0.02 0.5 1

Table 2-4. Quality control samples retapamulin QC Name Starting

Concentration (µg/mL)

Starting Volume (mL)

Final Concentration (ng/mL)

Total Volume (mL)

HQC 100 0.02 200 1 MQC 10 0.075 75 1 LQC 1 0.06 1.5 1 LLOQ 1 0.02 0.5 1

34

Table 2-5. Mass transition parameters MRM

Transition m/z

Dwell Time (ms)

Collision Energy (V)

Declustering Potential (V)

Entrance Potential (V)

Collision Exit Potential (V)

Retapamulin 518.3 → 216.1

500 37 60 15 13

Tiamulin 494.5 → 192.2

500 29 60 15 13

Table 2-6. Intra-day variability of retapamulin (n=5) LLOQ

(0.5 ng/mL) LOQ (1.5 ng/mL)

MQC (75 ng/mL)

HQC (200 ng/mL)

Day 1 Mean 0.45 1.50 85.36 201.20 Accuracy (%) 90.0 100.0 113.8 100.6 CV (%) 13.1 8.2 3.5 12.5 Day 2 Mean 0.50 1.53 75.80 207.60 Accuracy (%) 100.3 102.1 101.1 103.8 CV (%) 4.7 5.9 3.0 2.3 Day 3 Mean 0.57 1.49 72.14 197.60 Accuracy (%) 114.6 99.2 96.2 98.8 CV (%) 8.2 9.2 10.7 1.0

Table 2-7. Inter-day variability of retapamulin (n=15) LLOQ

(0.5 ng/mL) LOQ (1.5 ng/mL)

MQC (75 ng/mL)

HQC (200 ng/mL)

Mean 0.51 1.51 77.77 202.13 Accuracy (%) 101.6 100.4 103.7 101.1 CV (%) 13.2 7.4 9.5 7.1

35

Table 2-8. Stability results for retapamulin Storage condition Level Mean stability

sample (ng/mL) CV (%) Change (%)

Short-term 18h room temperature

LQC 1.32 4.5 -6.1

HQC 220.5 1.1 2.0 Freeze-thaw Three cycles at -

80˚C LQC 1.6 5.5 0.4

HQC 219 3.6 5.12 Long-term 86 days at -80˚C LQC 1.5 13.2 -6.4 HQC 210.5 5.2 -3.9 Stock 60 days at -80˚C Retapamulin 1.13x106* 4.5 6.9 Tiamulin 4.69x105* 8.4 6.3

* Area under the curve

36

Figure 2-1. Mean calibration curve of retapamulin (n=3). A linear regression model is

used with 1/X2 as weighting factor. Linearity was displayed across the tested concentration range (r2=0.997).

Figure 2-2. Representative chromatogram of retapamulin (50 ng/mL, left panel) and the

internal standard tiamulin (right panel).

0

2

4

6

0 50 100 150 200 250

Retapamulin Concentration (ng/mL)

Inte

nsity

37

CHAPTER 3 IN VITRO MICRODIALYSIS EXPERIMENTS OF RETAPAMULIN

Objective

The aim of this experiment was to evaluate the in vitro microdialysis recovery of

retapamulin under various conditions. In addition, these in vitro experiments also help to

determine the feasibility of an in vivo study. Two techniques, extraction efficiency (EE)

and retrodialysis (RD), were used to measure recovery and binding characteristics of

retapamulin.

Experimental Procedure

Laboratory and Study Equipment

Test article



Reagents

Unless otherwise stated, all reagents were LC-MS grade and obtained from the

specified sources.







Equipment and disposables




38




5 mL BD Luer-Lok syringe, non-sterile



Pyrex petri dish

Pyrex volumetric flasks, volume: 10 mL, 200 mL



Harvard Apparatus Model 22 Multiple Syringe Pump

µdialysis 66 Linear Catheter









Parafilm

39

Reagent Preparation

Normal saline

9 g of NaCl were dissolved in 1,000 mL triple distilled water to obtain normal

saline (0.9% m/V). After sonication for 10 min, the solution was filtered.

Retapamulin stock and working solution

Stock solution was prepared as described in chapter 2, by weighing out 10 mg of

retapamulin and dissolving it in 10 mL DMSO. Then, 100 µL of the retapamulin stock

solution (1 mg/mL) were diluted with 900 µL of methanol, to yield a 100 µg/mL working

solution.

Retapamulin calibration standards and quality controls

Six calibration standards were freshly prepared from the retapamulin working

solutions according to table 2-3. The quality control solutions LQC (1.5 ng/mL), MQC

(75 ng/mL) and HQC (200 ng/mL) were prepared using the dilution scheme described in

table 2-4.

Tiamulin internal standard solution

Internal standard solution was prepared freshly on the day of the experiments as

described in chapter 2. 25 µL of a 1 µg/mL tiamulin working solution was diluted with

975 µL of methanol in a 1.5 mL amber micro centrifuge vial, to obtain internal standard

solution (25 ng/mL).

LC-MS/MS mobile phase and washing solution

Reservoir A (0.1% formic acid in H2O) and reservoir B (0.1% formic acid in ACN)

were obtained by adding 570 µL formic acid to 500 mL triple distilled water and ACN,

respectively. Washing solution was made by mixing 375 mL of ACN with 125 mL of

40

MeOH. Before degassing with helium, the fresh prepared mobile phases and washing

solution were filtered through a 0.2 µm Millipore nylon membrane filter.

Microdialysis Experiments

Microdialysis calibration solutions

The in vitro microdialysis recovery of retapamulin was determined by extraction

efficiency (EE) and retrodialysis (RD) at three different concentrations (5, 50 and 100

ng/mL). Preparation of the calibration solutions was done in a 200 mL volumetric flask

by adding working solution to 200 mL of normal saline (Table 3-1).

Microdialysis system

A µdialysis 66 linear mircrodialysis catheter, with 30 mm membrane length and

20kDa cut-off, was used for the in vitro experiments. The probe was connected to a 5

mL BD Luer-Lok syringe. Perfusion rate was kept at 1.5 µL/min using a Harvard

Apparatus Model 22 multiple syringe pump. The three different retapamulin

concentrations, ranging from 5 ng/mL to 100 ng/mL, were tested in ascending order. All

in vitro microdialysis experiments were carried out at 37°C and run in triplicates.

Extraction efficiency method (EE)

For recovery determination using extraction efficiency, the 5 mL syringe was

filled with blank saline and connected to the microdialysis catheter. Prior to the start of

the experiments, the probe was flushed for 20 min to remove air bubbles and check

proper functioning. Then it was placed into a petri dish, filled with 30 mL of retapamulin

solution (reservoir) and covered with parafilm to prevent evaporation. After a 30 min

equilibration period, approximately 45 µL dialysate was collected in a microcentrifuge

tube for 30 mins. Two 45 µL aliquots from the reservoir were also taken at the beginning

and at the end of the experiment.

41

Recovery determined by extraction efficiency was calculated by the equation:

𝑅% = 𝐶𝑑𝑖𝑎𝑙𝑦𝑠𝑎𝑡𝑒

𝐶𝑟𝑒𝑠𝑒𝑟𝑣𝑜𝑖𝑟∗ 100% (3-1)

where R% is recovery in percent, Cdialysate is the retapamulin concentration in the

dialysate and Creservoir is the average drug concentration in the reservoir.

Retrodialysis method (RD)

In the retrodialysis experiment, the 5 mL syringe was filled with retapamulin

solution and connected to the microdialysis catheter. The probe was flushed for 20 min

before the start of the experiment to remove air bubbles and check functionality. It was

then placed into a petri dish, filled with 30 mL of blank saline and covered with parafilm

to prevent evaporation. Similar to the EE experiment, the probe was equilibrated for 30

min and approximately 45 µL dialysate was collected in a microcentrifuge tube over 30

mins. Before the start and after the experiment was finished, two 45 µL aliquots were

taken from the 5 mL drug-containing syringe.

Retrodialysis recovery was calculated by the equation:

𝑅% = 100% ∗ (1 −𝐶𝑑𝑖𝑎𝑙𝑦𝑠𝑎𝑡𝑒

𝐶𝑠𝑦𝑟𝑖𝑛𝑔𝑒) (3-2)

where R% is recovery in percent, Cdialysate is the retapamulin concentration in the

dialysate and Csyringe is the average drug concentration in the syringe.

Sample Preparation and Analysis

Samples were analyzed on the same day of the in vitro experiments. The

microcentrifuge tubes containing the samples were vortexed and 20 µL pipetted into

Sun Sri Autosampler vials. Next, 10 µL of IS was added, the autosampler vials capped

and vortexed again. Double blank, blank, six calibration standards and duplicates of

LQC, MQC and HQC were prepared and placed throughout the run. Analytical runs

42

were not rejected if at least five out of six calibration standards were within ±15% (±20%

for LLOQ) and if four out of six QCs, with at least one QC at each level, were within

±15% of their nominal concentrations. LC-MS/MS conditions were the same as

specified in chapter 2. In order to determine the actual drug concentration at the

sampling site, Ctissue, the concentration in the dialysate, Cdialysate, has to be back-

calculate using the following equation:

𝐶𝑡𝑖𝑠𝑠𝑢𝑒 = 100% ∗ (𝐶𝑑𝑖𝑎𝑙𝑦𝑠𝑎𝑡𝑒

𝑅%) (3-3)

Microsoft Excel 2013, R version 3.1.2, RStudio version 0.98.1087, and the

ggplot2 package were used for data collection, statistical analysis and plot generation.

Results

Calibration Curve and QCs

The slope of the calibration curve after linear regression, with 1/X2 weighting,

was 0.0208±0.0006 (estimate±SE) with an intercept of 0.0138±0.0006 (estimate±SE)

and a coefficient of correlation r2=0.996. Accuracy of calibration standards was between

91.5% and 109%. The accuracy of the QCs was 98-108.3% and precision at the LQC,

MQC and HQC level was 4.6%, 5.1% and 7.8%, respectively. Inclusion criteria

(accuracy and precision within ±15% and ±20% for LLOQ) were met for all calibration

standards and QCs (Tables 3-2).

In Vitro Microdialysis

The recovery results from the in vitro microdialysis experiments determined by

the extraction efficiency and retrodialysis method are summarized in Table 3-3.

Recovery for the three concentrations tested ranged from 83.8-96.5 for EE and 95.0-

97.7% for RD, respectively. Overall recoveries were 90.1% for the extraction efficiency

43

method and 96.0% for the retrodialysis method, suggesting that there is no difference

between the two methods (ANCOVA, p=0.0711) and that retapamulin can freely pass

the membrane.

Summary

The in vitro microdialysis experiments confirmed that retapamulin was dialyzable

in saline. Recovery was high for both extraction efficiency and retrodialysis method and

no significant difference was found between the methods. Therefore, microdialysis can

be used as a sampling technique to obtain pharmacokinetic profiles of retapamulin.

44

Table 3-1. Calibration solutions for in vitro retapamulin microdialysis Starting Concentration (µg/mL)

Starting Volume (mL) Final Concentration (ng/mL)

Total Volume (mL)

100 0.2 100 200 100 0.1 50 200 100 0.01 5 200

Table 3-2. Calibration curve standards and QCs for in vitro microdialysis Name Concentration

(ng/mL) Calculated concentration (ng/mL)

Accuracy (%)

R1 0.5 0.502 100.4 R2 1 0.915 91.5 R3 10 9.92 99.2 R4 50 50.7 101.4 R5 100 109 109 R6 250 243 97.2 LQC 1.5 1.48 98.7 LQC 1.5 1.58 105.3 MQC 75 75.6 100.8 MQC 75 81.2 108.3 HQC 200 196 98.0 HQC 200 207 103.5

Table 3-3. In vitro microdialysis recovery results for retapamulin (n=3) EE RD Concentration (ng/mL)

R% SD% CV% R% SD% CV%

5 83.8 8.1 9.7 95.2 1.3 1.4 50 96.5 6.8 7.1 97.7 0.8 0.8 100 90.0 8.4 9.3 95.0 2.8 2.9 Total 90.1 8.7 9.7 96.0 2.1 2.1

45

Figure 3-1. Mean recoveries of retapamulin at different concentrations using extraction

efficiency and retrodialysis.

0

10

20

30

40

50

60

70

80

90

100

110

5 50 100

Concentration (ng/mL)

Me

an

Re

co

ve

ry (

%)

Method

Extraction efficiency

Retrodialysis

46

CHAPTER 4 IN VITRO ANTIBACTERIAL ACTIVITY OF RETAPAMULIN

Objective

The goal of this study was to assess the in vitro antibacterial effect of retapamulin

against a clinical methicillin-susceptible Staphylococcus aureus (MSSA) isolate and to

test if the drug is also effective against the methicillin-resistant Staphylococcus aureus

(MRSA) ATCC 43300 strain. Retapamulin’s pharmacodynamics were investigated by

minimum inhibitory concentration (MIC) and static time-kill curve experiments, at varying

concentrations, for both strains.

Material and Methods

Antimicrobial Agent



Microbial Strains

Methicillin-susceptible Staphylococcus aureus (MSSA), clinical isolate provided by UF Shands microbiology lab

Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, in house collection

Cryopreserved bacterial cultures were activated by incubating them on sheep-

blood agar plates for three days. The same subcultured isolate was used throughout the

MIC and time-kill curve experiments.

Reagents






Muller-Hinton broth II (Becton Dickinson, #212322)

47

Equipment and Disposables

Remel sheep-blood agar plates





Labline Model 460 CO2 culture incubator

Steris Amsco Renaissance 3021 autoclave

Fisher culture flasks (50 mL, vented caps)

Fisher glass tubes


Abbott Lab A-JUST turbidimeter

Remel Microbiolgy Products McFarland equivalence turbidity standard

Corning 24- and 96-well plates

Fisher sterile disposable loops










48




Parafilm

Preparation of Solutions and Broth

Sterile saline

Sterile saline was prepared by dissolving 9 g of NaCl in 1,000 mL of triple

distilled water. The solution was then autoclaved for 30 minutes at 121˚C.

Mueller-Hinton broth II

Mueller-Hinton broth II (cation-adjusted) was prepared according to the

manufacturer’s instructions: 22 g of the powder were suspended into 1 L of triple

distilled water, mixed thoroughly and heated with frequent agitation. It was then

autoclaved at 121˚C for 30 minutes.

Retapamulin primary and secondary stock solutions

Primary stock solution (1 mg/mL) was prepared by weighing out 10 mg of

retapamulin and dissolving it in 10 mL of DMSO. The suspension was vortexed until

drug was completely dissolved. Secondary stock solution (50 µg/mL) was made by

mixing 250 µL of primary stock solution with 4.75 mL triple distilled water. The

secondary stock solution was sterilized by filtration (filter pore size 0.22 µm).

MIC Determination

All experiments were conducted according to the Clinical and Laboratory

Standards Institute’s M07-A9 method (“Methods for Dilution Antimicrobial Susceptibility

Tests for Bacteria That Grow Aerobically”). MICs of both strains were determined in

duplicates on three different days.

49

A bacterial dispersion, containing approximately 1.5 x 108 CFU/mL, was prepared

by dispensing overnight S. aureus colonies into sterile saline. It was then adjusted to a

0.5 McFarland standard using the turbidimeter.

Next, a 24-well plate was labeled according to Figure 4-1. 920 µL broth and 80

µL retapamulin secondary stock solution were pipetted into well C1 and mixed.

Subsequently, 500 µL of broth were added to wells C2 to C8 and retapamulin

concentrations were prepared by serial two-fold dilutions, ranging from 0.016 µg/mL to 2

µg/mL (Table 4-2). After serial dilution, 500 µL of C8 were discarded and wells C1 to C8

filled again with 490 µL of broth. Then, 10 µL of bacterial dispersion were added to each

well to yield an approximate starting inoculum of 106 CFU/mL. Negative control (NC,

containing no bacteria and no drug) and growth control (GC, containing no drug but

bacteria) were also included. For the growth control, 990 µL of broth and 10 µL bacterial

dispersion were added to well GC. The negative control contained 1,000 µL of broth.

The well plates were wrapped in parafilm immediately, to prevent evaporation,

and placed in an incubator at 37˚C for 20h. Following the incubation under constant

shaking, plates were removed from the incubator and the least concentration with no

visible growth indicated the MIC. Growth control and negative control were also

checked to ensure viability of the bacterial isolate and sterility of the broth.

Retapamulin Adsorption on 24 Well Plate

During the development of the bioanalytical assay we encountered loss of signal

of the calibration standards and nonlinear calibration curves when serial dilutions of

retapamulin were employed. As MICs were determined with serial two-fold dilution, we

wanted to investigate if the same behavior was seen throughout the MIC experiments.

50

To examine linearity of the MICs, broth was added to eight wells of a 24 well

plate and diluted as described in Table 4-2. However, the 10 µL of bacterial dispersion

were replaced with broth. Broth with drug concentrations ranging from 0.016-2 µg/mL

were incubated at 37˚C and constantly shaken for 20h. Thereafter, 20 µL were removed

from each well, pipetted into 0.5 mL microcentrifuge vials, mixed with 180 µL of ACN

and centrifuged at 12,000 rpm for 10 minutes. 20 µL of the supernatant were transferred

into autosampler vials and spiked with 10 µL of IS. Analysis was done using the LC-

MS/MS method described in Chapter 2.

Static Time-kill Curve

The modified droplet method51 was used and time-kill curve experiments were

performed in triplicates for each strain. The flasks were filled with 19.9 mL of MHBII and

0.1 mL of bacterial suspension, yielding a starting inoculum of approximately 106

CFU/mL. Prior to adding any drug the culture flasks were incubated at 37˚C, under

constant shaking, for 2h. This was done to ensure that bacteria were in their exponential

growth phase. Retapamulin was then added and concentrations for the clinical MSSA

isolate were 0.25-16xMIC and for the MRSA ATCC 43300 strain 0.125-16xMIC,

respectively. Dilution schemes for retapamulin are shown in Table 4-3. A growth control,

without antimicrobial agent, and a negative control, with broth only, were also included.

The flasks were incubated for 24 h at 37˚C. Sampling times were 0, 2, 4, 6, 8, 10,

12, 16 and 24 h and 20 µL aliquots were taken from each flask. Appropriate serial ten-

fold dilutions, ranging from 1:10 to 1:108, were performed with sterile saline in 96-well

plates (Tables 4-4 and 4-5). Five 10 µL aliquots from the diluted samples were plated in

duplicates on sheep-blood agar plates (Figure 4-2). After incubation for 16-20 h at 37˚C,

51

colonies were counted in all readable plates (≤200 colonies). Bacterial count was

transformed into CFU/mL by the following equation:

𝐶𝐹𝑈

𝑚𝐿= (𝐶𝐹𝑈𝑐𝑜𝑢𝑛𝑡𝑒𝑑 ∗ 10𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛) ∗

10

𝑚𝐿 (4-1)

Retapamulin Stability in Mueller-Hinton Broth

Drug stability was evaluated at three different concentrations over 24 h. Sampling

times for stability tests were 0, 8, 16 and 24 h at concentrations 0.25x, 1x and 4xMIC.

100 µL of broth was removed from the respective flasks. Then, 200 µL of ACN was

added, the mix centrifuged at 3,000 g for 15 minutes and the supernatant stored at -80

˚C until analysis. Samples were vortexed, thawed and 20 µL pipetted into autosampler

vials and spiked with 10 µL internal standard. The samples were analyzed with the

developed LC-MS/MS method and concentrations at times 8, 16 and 24 h were

compared with the initial concentrations

Results

MIC Determination

The MIC values of retapamulin for both strains were 0.125-0.25 µg/mL. The

higher MIC was detected in 66.7% of the observations for the MSSA strain, while it was

found in 50% of the experiments for the MRSA ATCC 43300 strain. The results are

summarized in Table 4-6 and shown in Figure 4-3.

Retapamulin Adsorption on 24 Well Plate

Retapamulin displayed linearity for the tested concentrations and the coefficient

of correlation was r2=0.996. Hence, adsorption on the 24-well plate was not observed

and did not interfere with MIC determination.

52

Retapamulin Stability in Mueller-Hinton Broth

Retapamulin was stable in MHBII at 37˚C for 24 h (Figure 4-4) and no

degradation was detected (ANCOVA p=0.533). Concentrations after 24 h were 92.3%

(0.25xMIC), 99.4% (1xMIC) and 99.0% (4xMIC) of the initial concentrations. Results are

summarized in Table 4-7.

Static Time-kill Curves

An MIC of 0.25 µg/mL was assumed for the experiments. The higher MIC was

selected because of its frequency and the fact that MICs may be anywhere between the

last dilution inhibiting growth and the first dilution not inhibiting growth52. Time-kill curve

results are presented in Figure 4-5. Both curves looked similar. Initially, the drug effect

was delayed during the first 2 h, followed by a rapid killing and a slower rate of

elimination after 6 h. The bacterial effect was dose dependent until 1xMIC (0.25 µg/mL)

was reached. Concentrations greater than 1xMIC did not increase the antimicrobial

effect. For concentrations ≥1xMIC, a 2-log10 reduction (99% bacteria killed) from the

initial inoculum was observed after 24 h, indicating a bacteriostatic effect of

retapamulin53. The time-kill curves also showed biphasic behavior suggesting the

existence of susceptible and persistent bacterial cells48,54,55

Data Analysis

Data was entered in Microsoft Excel 2013, statistical analysis was done in R

version 3.1.2 and RStudio version 0.98.1087, and plots were generated using the

ggplot2 package.

Summary

Retapamulin was shown to be active against the tested MSSA and MRSA

isolates. MICs for both strains were 0.125 µg/mL and 0.25 µg/mL and well within the

53

range of literature reports26,27. Time-kill curves were also similar which supports

previous findings that retapamulin has in vitro activity against certain MRSA

strains27,56,57. The drug showed a delayed onset of action and a bacteriostatic,

concentration-dependent effect. This was consistent with literature reports of

concentration-dependent inhibition of ribosomal subunit assembly in S. aureus cells58.

However, drug concentrations ≥0.25 µg/mL did not further increase the bacterial kill. A

biphasic kill was observed for retapamulin suggesting growing susceptible and dormant,

non-susceptible bacterial cells48.

54

Table 4-1. Pipetting scheme for retapamulin MIC determination C1 C2 C3 C4 C5 C6 C7 C8


2 1 0.5 0.25 0.125 0.063 0.031 0.016

Volume solution (µL) 80 2nd stock

500 of C1

500 of C2

500 of C3

500 of C4

500 of C5

500 of C6

500 of C7

Broth (µL) 920 500 500 500 500 500 500 500 Broth to add (µL) 490 490 490 490 490 490 490 490 Bacterial dispersion (µL)

10 10 10 10 10 10 10 10

Final volume (µL) 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000

Table 4-2. Dilution scheme for retapamulin static time-kill curve experiments against a clinical MSSA isolate

MIC 0.25 0.5 1 2 4 16


0.063 0.125 0.25 0.5 1 4

Volume 2nd stock (µL)

25 50 100 202 408 1,740

Table 4-3. Dilution scheme for retapamulin static time-kill curve experiments against MRSA ATCC 43300

MIC 0.125 0.25 0.5 1 2 4 16


0.031 0.063 0.125 0.25 0.5 1 4

Volume 2nd stock (µL)

12.5 25 50 100 202 408 1,740

Table 4-4. Dilution factors for plating the clinical MSSA isolate Time (h) Growth

control 0.25xMIC 0.5xMIC 1xMIC 2xMIC 4xMIC 16xMIC

0 3-4 3-4 3-4 3-4 3-4 3-4 3-4 2 4-5 3-4 3-4 3-4 3-4 3-4 3-4 4 5-6 4-5 3-4 2-3 2-3 2-3 2-3 6 6-7 4-5 3-4 2-3 2-3 2-3 2-3 8 6-7 4-5 3-4 2-3 2-3 2-3 2-3 10 6-7 4-5 3-4 2-3 1-2 1-2 2-3 12 6-7 5-6 3-4 2-3 1-2 1-2 1-2 16 7-8 5-6 3-4 2-3 1-2 1-2 1-2 24 7-8 5-6 4-5 1-2 1-2 1-2 1-2

55

Table 4-5. Dilution factors for plating the MRSA ATCC 43300 strain Time (h)

Growth control

0.125xMIC 0.25xMIC 0.5xMIC 1xMIC 2xMIC 4xMIC 16xMIC

0 3-4 3-4 3-4 3-4 3-4 3-4 3-4 3-4 2 4-5 4-5 4-5 3-4 3-4 3-4 3-4 3-4 4 5-6 4-5 3-4 3-4 2-3 2-3 2-3 2-3 6 6-7 4-5 3-4 3-4 2-3 2-3 2-3 2-3 8 6-7 4-5 3-4 2-3 2-3 2-3 2-3 2-3 10 6-7 5-6 4-5 2-3 2-3 2-3 2-3 2-3 12 6-7 5-6 4-5 2-3 1-2 1-2 1-2 1-2 16 6-7 5-6 4-5 2-3 1-2 1-2 1-2 1-2 24 7-8 5-6 4-5 2-4 1-2 1-2 1-2 1-2

Table 4-6. Contingency table of MICs for retapamulin against a clinical MSSA isolate and MRSA ATCC 4330

MSSA MRSA

MIC 0.125 µg/mL 2 3 MIC 0.25 µg/mL 4 3

Table 4-7. Stability test of retapamulin in Mueller-Hinton broth II at 37˚C. Results are

shown as percentage of starting concentration 0 h 8 h 16 h 24 h

0.25xMIC (0.063 µg/mL) 100 % 104.3 % 94.9 % 92.3 % 1xMIC (0.25 µg/mL) 100 % 100.3 % 100.3 % 99.4 % 4xMIC (1 µg/mL) 100 % 99.2 % 111.8 % 99.0 %

56

Figure 4-1. Labeling-scheme for the 24-well plate used for retapamulin MIC-determination.

Figure 4-2. Spot inoculation on sheep-blood agar plates for colony enumeration after serial dilution. Five 10 µL spots in duplicates were plated for each dilution (n and (n+1)).

C1 C2 C3 C4 C5 C6

C7 C8 C1 C2 C3 C4

C5 C6 C7 C8 NC GC

10-n

10-n 10-(1+n)

10-(1+n)

57

Figure 4-3. MIC frequency distribution of retapamulin against the tested MSSA and

MRSA strains.

Figure 4-4. Stability of retapamulin in Mueller-Hinton broth II at 37˚C.

33.3

50

66.7

0.125 0.25

MIC (µg/mL)

Fre

qu

en

cy (

%)

Strain

MRSA

MSSA

0

20

40

60

80

100

0 8 16 24

Time (h)

Dru

g R

em

ain

ing

(%

)

MIC

0.25

1

4

58

Figure 4-5. Retapamulin time-kill curves against the tested MSSA and MRSA strains.

59

CHAPTER 5 RETAPAMULIN FEASIBILITY STUDY

Objective

Microdialysis allows to measure drug concentrations in skin after topical

application. The rationale of the feasibility study was to assess the in vivo recovery and

washout time following perfusion of retapamulin in healthy volunteers. Secondary

objectives were to investigate the safety and tolerability of the microdialysis technique.


Retapamulin Solution

Sterile, 50 ng/mL retapamulin solution in normal saline was provided by DaVita

Clinical Research (Minneapolis, MN).

Materials

1% Lidocaine solution

8.4% Sodium bicarbonate solution

10.5 mL ChloraPrep applicator

Normal saline

Ultrasound gel


Sterile drapes

BD 27g needles

BD 19g needles

BD 20g spinal needles

Latex biogel gloves

Luer lock injection ports

Surgical clamps


60




10 mL BD Syringe, sterile


BD Microtainer PST tubes with Li-Heparin

3M Surgical Clipper Kit

Surgical marker








AB Sciex QTrap 5500 LC/MS/MS






Clinical Feasibility Study

The study protocol and informed consent form (ICF) was approved by the

institutional review board (IRB) in accordance with the International Conference on

61

Harmonisation of Technical Requirements for Registration of Pharmaceuticals for

Human Use (ICH), Good Clinical Practice (GCP) and United States (US) 21 Code of

Federal Regulations (CFR) 312.3(b) for constitution of independent ethics committees.

The study was conducted in accordance with ICH GCP and the ethical principles of the

Declaration of Helsinki.

This study was a single session, open-label, single-center study and consisted of

a screening phase (up to 30 days prior to Study day), treatment phase (study day) and

a follow-up phone call (within 72 h after discharge). After screening and obtaining

written consent, eligible subjects were admitted to the clinical research center.

On the day of dosing, the thigh of the subjects was locally anesthetized with

lidocaine solution and three microdialysis probes were placed into the skin. Probe depth

was measured using ultrasound. After a 30 min equilibration period with normal saline,

the retapamulin solution was perfused through each probe at a flow rate of 1.5 µL/min

for 90 minutes. Samples for recovery determination were collected for the last 30

minutes of the retapamulin perfusion. Then, saline was perfused for four hours and

dialysate samples were collected every 30 minutes to determine the washout period.

Subjects were discharged from the research center following completion of the study

procedures and a follow-up phone call occurred 1-3 days after the last dose of study

medication.

All samples were stored at -80˚C and analyzed using the validated LC-MS/MS

method. The sample analysis, however, was performed on an AB Sciex QTrap 5500

system, which was more sensitive with an LLOQ of 0.25 ng/mL. Samples were thawed,

vortexed and transferred into autosampler vials. Samples containing less than 10 µL of

62

dialysate were not able to be analyzed. Calibration curve standards along with quality

controls (QCs) at three different concentration levels (duplicates) were prepared.

Analytical runs were accepted if at least two-thirds of the QCs were within ±15% of their

nominal range and not more than one QC at each level outside the ±15% concentration

interval.

Adverse events (AEs) were collected from the start of the study treatment until

the follow-up phone call. Clinical laboratory tests and vital signs were measured at

screening and on the study day. Electrocardiograms and physical examinations were

performed during screening only.

Results

Patient Demographics and Baseline Characteristics

Two male and one female subject were enrolled into the study. Their age ranged

from 24 to 55 years (mean±SD 34.7±17.6 years). Their average±SD weight was

79.8±16.1 kg with a mean±SD BMI of 26.7±3.8 kg/m2. All baseline characteristics are

summarized in Table 5-1.

In Vivo Recovery

All subjects received 50 ng/mL of retapamulin solution through the implanted

microdialysis probes. Three catheters were inserted into the thigh of subjects 1 and 2,

while subject 3 only had two probes inserted. Recovery percentage was calculated

using equation 3-2.

In vivo recovery ranged from 77.49% to 94.36%, with an overall recovery of

88.22%. The lowest recovery was observed in the only female of the study (subject 1).

Variability was also higher if compared to the two male study participants. Results are

summarized in Table 5-2 and Figure 5-1.

63

Washout Period

Washout sample concentrations (mean±SD) for each subject are displayed in

Figure 5-2. Because dialysate samples were collected over a period of 30 minutes, the

corresponding time point of a concentration was the mean of this time interval.

The first washout time point (2 to 2.5 h) for subject 2 yielded an insufficient

volume, hence no concentrations were determined. The next time point (2.5 to 3 h) had

only one measurable sample, therefore SD estimation was not applicable. All mean

concentrations and SD for subject 3 were estimated based on two dialysate samples at

each time point.

Retapamulin dialysate concentrations were rapidly decreasing during the first

hour of the washout (2 to 3 h). Drug clearance then slowed down and appeared to be

steady at a concentration close to the limit of quantification for subjects 2 (0.39 ng/mL)

and 3 (0.27 ng/mL). Subject 1, however, had higher dialysate concentrations in

comparison to subjects 2 and 3 and a slower rate of elimination of the drug. After four

hours of washout, the tissue dialysate concentrations were 0.99 ng/mL.

Safety Assessment

Two subjects each reported one mild adverse event after being discharged from

the clinical research center. One experienced a headache which resolved on the same

day, while the other reported AE was a mild pain in the extremity that resolved four days

after follow-up. All reported AEs were non serious and might be related to study

treatment.

Summary

Three healthy subjects completed the microdialysis feasibility study. Retapamulin

is dialyzable in vivo and exhibited good probe recovery, with a mean recovery rate of

64

88.22±11.59% at a flow rate of 1.5 µL/min. The results were in agreement with recovery

rates determined by in vitro experiments. Skin concentrations after four hours of

retapamulin washout were ranging from 0.27 to 0.99 ng/mL. Although retapamulin

concentrations were detectable, a four hour washout period may be sufficient

considering that concentrations in the skin will likely be higher after topical application.

In order to have an antimicrobial effect, skin concentrations should be in the magnitude

of the MIC of retapamulin (e.g. 0.03-0.25 µg/mL for S. aureus). Thus, remaining

retapamulin in the skin should be negligible.

Occurred adverse events were of mild intensity and not serious. Overall, the

microdialysis method was tolerable and safe.

65

Table 5-1. Demographic and Baseline Characteristics Demographics

Age in Years [Mean (SD)] 34.7 (17.6) Sex [n (%)] Female 1 (33.3) Male 2 (66.7) BMI (kg/m2) [Mean (SD)] 26.7 (3.8) Height (cm) [Mean (SD)] 172.7 (12.5) Weight (kg) [Mean (SD)] 79.8 (16.1) Ethnicity [n (%)] Hispanic or Latino 1 (33.3) Not Hispanic or Latino 2 (66.7) Race [n (%)] Asian – Central/South Asian Heritage 1 (33.3) White – White/Caucasian/European Heritage 2 (66.7)

Table 5-2. Mean in vivo recovery percentages, sample size (N), standard deviation (SD)

and coefficient of variation (CV) for retapamulin after retrodialysis Subject Mean recovery (%) N SD (%) CV (%)

1 77.49 3 14.71 18.98

2 92.81 3 3.48 3.75

3 94.36 2 2.00 2.65

Overall 88.22 8 11.59 13.14

66

Figure 5-1. In vivo recovery of retapamulin determined by retrodialysis (mean±SD).

Figure 5-2. Skin concentrations (mean±SD) of retapamulin during the washout period.

The concentration at 1.75 h corresponds to the retrodialysis sample.

0

20

40

60

80

100

1 2 3Subject

%R

ecovery

0

1

2

3

4

5

6

7

8

9

1.75 2.25 2.75 3.25 3.75 4.25 4.75 5.25 5.75

Time (hours)

Co

nce

ntr

atio

n (

ng

/mL

)

Subject

000001

000002

000003

67

CHAPTER 6 IN VIVO PHARMACOKINETICS OF RETAPAMULIN

Objective

Since retapamulin is applied topically, systemic exposure is low and availability of

pharmacokinetic data is limited. Pharmacokinetic parameters for retapamulin have not

been established yet25; in vitro protein binding is 75-94%1. No information on unbound

concentrations at the target site or tissue distribution of retapamulin was available prior

to the start of the study.

The objective was to investigate the pharmacokinetics of retapamulin in plasma

and in skin in healthy Wistar rats.


Antimicrobial Agents

Micronized Retapamulin (lot #WG0223980-140411001) was purchased from Ark

Pharm, Inc. Altabax™ (1% retapamulin ointment 15 g) was obtained from a local

pharmacy. The drug powder and the ointment were stored at room temperature and

protected from light.

Materials


Acetic Acid, Fisher #A35-500


Ethanol USP (EtOH), Aaper #05A25GA


Isoflurane, Piramal Healthcare


Normal saline (0.9%), Ricca #2502970

Isopropanol (70%)

Chlorhexidine (2%)

Virkon disinfectant (1%)

Heparin 210 U/mg, Affymetrix #4229340

68










1 mL BD Syringe, sterile


BD 21g needle

BD 27g needle

BD Microtainer PST tubes with Li-Heparin

3M Surgical Clipper Kit

Delfin VapoMeter

Surgical marker

Millipore Centrifree







69









In Vivo Pharmacokinetic Study

A total of 15 male, pathogen-free Wistar rats, weighing between 280-310 g, were

randomly assigned to three treatment groups. Two groups received 1% retapamulin

ointment after topical administration on either intact or tape-stripped skin. The

administered dose was 0.1 mg/cm2 (approximately 0.525 mg retapamulin). The third

group received 5 mg/kg retapamulin after an IV bolus. The study was approved by the

IACUC and the protocol adhered to the “Guide for the Care and Use of Laboratory

Animals”1. A flow chart of study procedures is shown in Figure 5-5.

Anesthetization

On the day of the experiment, rats were anesthetized with an isoflurane

vaporizer. Isoflurane inhalation was performed as follows: the rat was placed in an

induction chamber under the chemical fume hood, which was supplied with an air-

isoflurane (4%) mixture at a flow rate of 2,000 mL/min. Loss of consciousness took 2-3

minutes, and surgical stage of anesthesia, with loss of reflexes and muscular relaxation,

was examined by pinching the tail and ears. Once under anesthesia, a mask was

placed onto the nose of the animal and the isoflurane concentration was decreased to

70

approximately 1.5%. Then, the animal was placed on a temperature-controlled heating

pad in a dorsal position, with the tail toward the investigator. Body temperature was

maintained at 37°C.

Microdialysis probe implantation

Two µdialysis 66 Linear Microdialysis Catheters were used and the inlet tube of

the probes were connected to a Harvard Apparatus 22 microinjection.

The probe insertion site (abdominal region of the rat) was clipped and disinfected

by swiping the area with 2% chlorhexidine and 70% isopropanol. For the tape-stripping

group, adhesive tapes were applied and pressed onto the skin, to remove the stratum

corneum before probe insertion. This procedure was repeated 20 times59,60. Probe

insertion site and drug absorption window (approximately 35x15 mm) was marked with

a surgical marker. An introducer needle was carefully inserted intradermally and the

outlet of the microdialysis probe was pushed through the needle end. Then, the probe

was held in place and the introducer needle was withdrawn carefully. The probe was

positioned in such a way that the membrane was within the marked area and probe

position was secured with tape.

Microdialysis probe stabilization

Two 5 mL syringes with 0.9% normal saline were connected with the probe’s

inlets. The pump was started, flow rate set to 5 µL/min, and the outlet tubing was

observed to see if perfusate was flowing. Flushing of the probes continued for another

five minutes in order to remove air bubbles from the system.

Flow rate was then lowered to 1.5 µL/min and the probes were perfused for 30

minutes, to equilibrate the system and allow the skin to recover from the insertion

trauma.

71

Retrodialysis

A 50 ng/mL retapamulin solution for in vivo microdialysis calibration using

retrodialysis61–64 was prepared on the study day. Briefly, 10µL of a 1 mg/mL primary

retapamulin stock solution were diluted with 990 µL of ethanol to yield a 10 µg/mL

secondary stock solution. Subsequently, 25 µL of the secondary stock solution were

mixed with 4,975 µL of sterile normal. The retrodialysis solution was dispensed in two 5

mL syringes. Drug samples were taken and the syringes were connected with the inlets

of the probes. Again, the pump was started at a flow rate set to 5 µL/min for five minutes

and then lowered to 1.5 µL/min. An equilibration period of 30 minutes was allowed,

followed by a 30 minutes sampling period of the dialysate to calculate microdialysis in

vivo recovery. Probes were disconnected and drug was sampled from each syringe

again.

Baseline sample collection

Syringes were changed and replaced with syringes containing normal saline.

After flushing for five minutes at a flow rate of 5 µL/min, the flow rate was changed back

to 1.5 µL/min and a 30 minutes washout sample was collected from both catheters. The

syringes and the flow rate were not changed for the remainder of the study.

Topical administration of ointment

After 30 minutes washout period and baseline sample collection, transepidermal

water loss (TEWL) was measured in triplicates and 1% retapamulin ointment was

applied on the abdominal skin at a dose of 0.1 mg/cm2 (approximately 0.525 mg

retapamulin). In the tape-stripped skin group ointment was wiped off 3 h after

application to capture the distribution and elimination phase.

72

IV bolus administration

Solution for IV bolus was fresh prepared on the study day. 10 mg of micronized

retapamulin were dissolved in 2 mL of 0.1% acetic acid in normal saline (approximate

pH=3.2) and filtered through a 0.22 µm Millipore filter. An IV bolus of 5 mg/kg was

injected via the lateral tail vein.

Microdialysis and blood sample collection

Microdialysis samples were collected every 30 minutes for 6 h and stored at -

80˚C until analysis.

Arterial blood (200 µL) was collected at pre-dose, 0.5, 1, 2, 3, 4, 5 and 6 h when

drug was administered topically and at pre-dose, 5, 15 and 30 minutes and 1, 2, 3, 4, 5

and 6 h for IV bolus administration. Lithium-Heparin tubes were used for blood

collection and the samples were centrifuged at 10,000 rpm for 12 minutes. The plasma

was transferred into microcentrifuge vials and stored at -80˚C.

Protein binding and Centrifree recovery

Plasma protein binding (PPB) in Wistar rats and humans was examined by the

ultrafiltration technique65. It is reported that plasma protein binding of retapamulin is not

concentration dependent1,25. A mass balance approach was used to calculate PPB and

recovery from the Centrifree devices66. A 10 µg/mL retapamulin solution was prepared

by mixing 10 µL primary stock with 990 µL saline. 30 µL of this solution was added to

1,170 µL of pooled plasma, vortexed and three 50 µL aliquots were immediately taken

out for measurement of initial concentrations (C1). The plasma-drug mix (250 ng/mL)

was incubated for 30 minutes at 37˚C. Top and bottom of the Centrifree device were

weighed before addition of plasma-drug mix and after ultrafiltration (10 min at 1,000 g)

to obtain the volume of retinate (V2) and ultrafiltrate (V3). Aliquots of 50 µL were

73

removed from the top (C2) and bottom (C3) of the Centrifree device. Experiments were

conducted in triplicates.

Protein precipitation of all samples was done prior to analysis with 100 µL of a

1:1 ACN:IS mixture and centrifugation. Analyte/IS response area after LC-MS/MS

analysis was used for PPB and recovery calculations. The percentage of protein binding

was calculated by:

𝑃𝑃𝐵% =(𝐶2−𝐶3)∗𝑉2

(𝐶2∗𝑉2+𝐶3∗𝑉3)∗ 100% (6-1)

Recovery in percent was calculated by:

𝑅% =(𝐶2∗𝑉2+𝐶3∗𝑉3)

(𝐶1∗𝑉1)∗ 100% (6-2)

Sample preparation and analysis

Samples were thawed at room temperature. Plasma proteins were removed

using Centrifree ultrafiltration devices to obtain free unbound drug67–69. Briefly, plasma

samples were incubated for 30 minutes at 37˚C in a water bath, then Centrifree devices

were loaded with 100 µL plasma and centrifuged at 1,000 g for 10 minutes. Free plasma

concentrations were corrected for ultrafiltration recovery.

For analysis with LC-MS/MS, 20 µL of dialysate and ultrafiltrate was spiked with

10 µL of IS. LC-MS/MS conditions were the same as specified in chapter 2. Calibration

standards, double blank, blank and QCs (5% of the number of samples analyzed or a

total of six, whichever was greater) were prepared on the day of analysis. Analytical

runs were accepted if more than 75% of the calibration standards were within ±15%

(±20% for LLOQ) and if more than 67% of the QCs, with at least 50% at each level,

were within ±15% of their nominal concentrations. In vivo microdialysis recovery R% for

each animal was calculated using equation 3-2, measured retapamulin skin

74

concentrations were divided by R% (see equation 3-3) and the mean free

concentrations of both probes used. Since samples were collected over a period of 30

minutes, the corresponding time point of a concentration was the mean of this time

interval (e.g. time point was 2.25 h for a collection interval from 2 to 2.5 h).

Data collection, analysis and plotting was performed using in Microsoft Excel

2013, R version 3.1.2, RStudio version 0.98.1087 and the ggplot2 package.

Results

Plasma Protein Binding and Recovery

Plasma Protein binding was higher in humans if compared to Wistar rats (90.8%

vs. 81.0%) and in accordance with literature reports1.Recovery form the Centrifree

devices were 88.6±4.5% (mean±SD) from human plasma and 93.9±7.5% (mean±SD)

from rat plasma.

Retapamulin Concentrations in Plasma and Skin

IV bolus

Five animals received an IV bolus of retapamulin via the tail vein. Dosing was 5

mg/kg, however, one animal (ID1) received 1 mg/kg due to dilution error and another

animal (ID4) received only approximately 32% of the total dose, because the needle

slipped out of the tail vein. Total administered retapamulin dose was ranging from 0.30-

1.64 mg. Distribution of retapamulin from plasma into peripheral compartments was

rapid and appeared to follow a three-compartmental disposition model (Figure 6-2).

Free plasma and free skin concentrations were in equilibrium after 3 hours.

Microdialysis probe recovery was 92.2±2.8% (mean±SD).

75

Topical application after tape-stripping

Five animals received Altabax™ topically after tape-stripping of the skin. Total

applied retapamulin dose was 0.60±0.04 mg (mean±SD). TEWL after tape-stripping

varied from 26.5-187 g*m-2*s-1 with a mean of 110.1±72.5 g*m-2*s-1.

Drug absorption into the skin seemed to be faster during the first 1.25 h and

slowed down afterwards (Figure 6-2). After ointment removal (3 h), drug concentrations

declined. Contrary to the observations from the IV bolus group, distribution and

elimination from the skin was slower and biphasic.

Free plasma concentrations were much lower after topical application if

compared to IV bolus. Probe recovery was 91.3±5.4% (mean±SD).

Topical application on intact skin

A total of four animals received Altabax™ topically on intact skin. Total

retapamulin dose was 0.56±0.06 mg (mean±SD) and was 6.8±0.8 g*m-2*s-1 (mean±SD).

Compared to the tape-stripped group, drug absorption into the skin was very slow and

delayed (Figure 6-3). Only three out of 32 analyzed plasma samples had quantifiable

drug concentrations (>0.5 ng/mL). Since the ointment was not removed from the skin,

no distribution and absorption phase was captured.

Summary

Retapamulin concentrations were determined in plasma and skin ISF of Wistar

rats. In vivo microdialysis recovery from animal skin was high and comparable to the

results from the in vitro and the clinical feasibility study. After IV administration,

retapamulin showed fast elimination and rapid distribution from plasma into peripheral

tissues. High variability in retapamulin skin concentrations was observed following

topical application of ointment on tape-stripped skin. Percutaneous drug absorption was

76

slightly delayed and maximum skin concentrations were achieved before the removal of

the ointment. Average maximum skin concentrations were also above the MIC (0.125-

0.25 µg/mL). Free plasma concentrations were quantifiable but low. When retapamulin

was applied on intact skin, percutaneous absorption was very slow and maximum

concentrations were observed at the end of the study. However, maximum skin

concentrations were very low and would most likely not elicit an antibacterial effect.

Additionally, systemic exposure was low too and only a few plasma samples were

quantifiable.

77

Figure 6-1. Plasma protein binding (mean±SD) in humans and rats.

0

10

20

30

40

50

60

70

80

90

100

Human Rat

Species

Me

an

PP

B (

%)

78

Figure 6-2. Unbound plasma and skin ISF concentration profiles of retapamulin after intravenous administration. In the upper panel concentrations are shown on a normal scale, while on the lower panel concentrations are displayed on a log-normal scale

79

Figure 6-3. Unbound plasma and skin ISF concentration profiles of retapamulin after

tape-stripping and topical application. The upper panel displays concentrations on normal scale, whereas the lower panel shows concentrations on a log-normal scale

80

Figure 6-4. Unbound skin ISF concentration profiles of retapamulin after topical

application on intact skin. Concentrations in the upper panel and lower panel are plotted on a normal scale and log-normal scale, respectively. Plasma concentrations were below the LOQ and therefore not shown.

81

Time (h) 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8

Saline

Retapamulin

Blood X X X X X X X X

RD Sample X

MD Sample X X X X X X X X X X X X X

Dosing X

Figure 6-5. Flowchart with time and events of the in vivo PK study.

82

CHAPTER 7 PHARMACOKINETIC AND PHARMACODYNAMIC ANALYSIS

Objective

The aim was to quantitatively describe the disposition and elimination of

retapamulin in plasma and skin using a non-compartmental and population

pharmacokinetic approach. Additionally, a semi-mechanistic pharmacodynamic model

was developed for the tested S. aureus strains and integrated with the PK model, to

guide development and help optimizing the pharmacotherapy of retapamulin. Lastly,

allometric scaling was used to predict the human exposure-response relationship.


Non-compartmental Analysis

Non-compartmental analysis (NCA) was performed with Phoenix WinNonlin

version 6.4. Free plasma concentrations were adjusted based on the plasma protein

binding to obtain total plasma concentrations. The area-under-the-concentration curve

(AUC0-t) was calculated using the trapezoidal rule. AUC0-∞ was the sum of AUC0-t and

the extrapolated AUCt-∞, calculated by Clast/ke. Elimination rate constant ke was

estimated based on the best fit of concentration-time points in the terminal phase. In

addition, extrapolated concentration at time zero (C0), total clearance (CL), observed

maximal concentration (Cmax), terminal half-life (t1/2), mean residence time (MRT), time

at Cmax (Tmax), volume of distribution in steady-state (Vss) and volume of distribution in

the terminal phase (Vz) were calculated. AUCt-∞ and Cmax were also normalized for

dose. For topical application, apparent clearance (CL/F) and apparent volume of

distribution in the terminal phase (Vz/F) were estimated. The distribution between skin

and plasma was calculated by the ratio of free plasma AUC0-∞ (fAUCplasma) and skin ISF

83

AUC0-∞ (AUCISF) from each animal. Difference between plasma and skin PK parameters

was evaluated by one-way analysis of variance (ANOVA) at a significance level of

α=5%.

Population Pharmacokinetic Analysis

Population PK (PopPK) analysis was performed with the non-linear mixed effects

modeling software NONMEM version 7.3 and the integrated modeling and simulation

workbench Pirana version 2.9, PSN version 4.2.0 and Xpose version 4.570. Plots were

generated with R version 3.1.2, RStudio version 0.98.1087 and Xpose. Different

disposition models were tested, consisting of two and three compartment mammillary

models, with plasma as central compartment. Percutaneous absorption, with or without

lag-time, was investigated by testing zero-order absorption, single/parallel first-order

absorption, sequential/parallel/linked mixed zero-order and first-order absorption, transit

compartment and Weibull absorption models71–77. Interindividual variability was

assumed to be log-normal distributed, with a mean θ and variance ω2. Residual

unexplained variability was examined (additive, proportional and combined error

models). All models were parameterized as a set of ordinary differential equations

(ODE) using the ADVAN13 subroutine. PK parameters were estimated with the Monte-

Carlo importance sampling (IMP) method and optimized estimation options78,79. Model

selection was based on the Akaike information criterion (AIC)80, parameter estimation

precision, goodness-of-fit plots, ill conditioning81 and prediction corrected visual

predictive checks (pcVPC)82.

Semi-mechanistic Pharmacodynamic Model

PD model development was also performed in NONMEM 7.3. The ADVAN13

subroutine with natural log transformed data was used and PD parameter estimates

84

were obtained with the iterative two stage (ITS) method. A model with two bacterial

subpopulations, including susceptible and persistent cells, was evaluated48,54,83. Drug

effect was modeled as sigmoidal Emax model and delay in growth and/or delay of drug

onset were also investigated51,84. Model selection was based on AIC, goodness-of-fit

plots and VPCs.

Allometric Scaling

To predict human concentration time-profiles in plasma and skin, the animal

profiles were normalized. Time was normalized with the MRT and concentrations with

dose/Vss85. Micro rate constants obtained from the PopPK model were converted into

hybrid rate constants. Estimation of human clearance was based on scaling from one

species86 and human Vss was predicted the Øie-Tozer equation87. Human equivalent

dose was calculated based on body surface area88. Predicted human hybrid constants

were then back transformed into micro rate constants and retapamulin concentration

profiles in human skin were simulated using a typical subject. The simulated

concentration profiles were then linked with the PD models of the MSSA and MRSA

strains to predict the time-effect course of retapamulin in human skin.

Results

Non-compartmental Analysis

The parameter estimates from the non-compartmental analysis after IV bolus and

topical application are summarized in Table 7-1, Table 7-2 and Table 7-3, respectively.

Half-life of retapamulin after IV bolus administration was comparable in plasma

and skin (2.2 vs 2.0 h, p>0.05). Dose normalized fAUC0-∞ was larger in plasma than in

skin ISF (126.0 vs 69.4 ng/mL*h, p<0.05) and the free plasma fraction distributing into

the skin ISF was 57%. The dose-normalized maximum skin concentration was

85

approximately 25% of the free observed plasma concentration. MRT in the skin was

longer but did not differ if compared to plasma (2.8 vs 1.9 h, p>0.05). Clearance of

retapamulin from the plasma was fast and exceeded total hepatic blood flow (55

mL/min/kg)89–91, suggesting extrahepatic clearance mechanisms. Terminal volume of

distribution of retapamulin (Vz 15,470 mL/kg) was greater than the total body water of a

rat (670 mL/kg)90 indicating extensive distribution into peripheral tissues.

After topical application of Altabax™ ointment on tape-stripped skin, retapamulin

exposure was significantly greater in skin ISF compared to plasma (AUC0-∞ 1,049 vs

20.5 ng/mL*h, p<0.05) and distribution from skin into plasma was 3.9%. Maximum

unbound drug concentrations in skin ISF were 303.5 ng/mL observed at 2.5 h and in

plasma 4.2 ng/mL observed at 2.2 h, respectively. Half-life in plasma was longer than in

skin (2.7 vs 1.3 h) but not different if compared to t1/2 after IV bolus administration (2.7

vs 2.2 h, p>0.05). MRT resembled the mean residence time after intravenous

administration and duration of ointment application.

For topical application on intact skin, only Cmax, AUC0-t and Tmax were

determined. Maximum concentrations in skin ISF were achieved at 5.6 h and were 8.9

ng/mL. AUC0-t was 15.9 ng/mL*h and only 1.7% of the tape-stripped skin exposure.

Population Pharmacokinetic Analysis

Plasma concentrations were adjusted for protein binding and compiled with

microdialysis data. IV and topical route of administration (tape-stripped group only) was

modeled simultaneously. The final model comprised a two-compartment body model,

with elimination from the central compartment and zero-order percutaneous absorption

with lag time for topical application (Figure 7-1). Residual variability was explained by a

combined error model. A distribution factor DF was added to account for the difference

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between free plasma and free skin ISF concentrations. The residual unexplained

variability was

The ordinary differential equations describing the change of drug amounts over

time (t) in the plasma [A(1)] and skin [A(2)] compartments after IV administration were

described as follows:

𝑑𝐴(1)

𝑑𝑡= −(𝑘𝑒 + 𝑘12) ∗ 𝐴(1) + 𝑘21 ∗ 𝐴(2) (7-1)

𝑑𝐴(2)

𝑑𝑡= 𝑘12 ∗ 𝐴(1) − 𝑘21 ∗ 𝐴(2) (7-2)

where ke is the elimination rate constant and k12 and k21 are the transfer rate

constants.

Concentrations in plasma and skin were obtained by scaling drug amounts with

volumes. Volume of distribution in the central compartment Vc was estimated and

volume of distribution in the skin compartment Vs was parameterized as k12/k21*Vc, since

k12*Vc = k21* Vs at steady state. Because concentrations were measured in ng/mL and

dose was given in mg, a conversion factor of 1000 was included. Hence, concentrations

in plasma and skin were:

𝐶𝑝𝑙𝑎𝑠𝑚𝑎 =𝐴(1)

𝑉𝑐∗1000 (7-3)

𝐶𝑠𝑘𝑖𝑛 =𝐴(2)∗𝑘21∗𝐷𝐹

𝑘12∗𝑉𝑐∗1000 (7-4)

The best model fit for topical application was obtained using a zero-order

absorption process with lag time. k0 was the zero-order drug input constant, duration

was fixed to three hours and the change of amount over time in skin was described as:

𝑑𝐴(2)

𝑑𝑡= 𝑘0 + 𝑘12 ∗ 𝐴(1) − 𝑘21 ∗ 𝐴(2) (7-5)

87

Concentrations in plasma after topical application were modeled as described in

equation 7-3. For skin concentrations, a scaling factor S was included for the skin

volume of distribution (equation 7-6). The skin volume where the microdialysis probes

were placed, and ISF concentrations measured, only reflected a fraction of the total

skin, therefore the skin volume of distribution differs from that of the intravenous route of

administration.

𝐶𝑠𝑘𝑖𝑛 =𝐴(2)∗𝑘21∗𝐷𝐹

𝑘12∗𝑆∗𝑉𝑐∗1000 (7-6)

Individual and population predictions described the observed plasma data

adequately (Figure 7-2). IDs 6 to 10 received retapamulin ointment and deviation from

the individual predictions were greater. However, it should be noted that the

measurements were at the lower end of the linearity range of the assay and that they

also mirror the greater variability observed in the skin.

Compared to IV bolus, variability of skin concentrations after topical application

was large (Figure 7-3) with greater than ten-fold difference in maximum concentrations.

TEWL was tested as covariate to account for variability, but was not significant.

Prediction-corrected VPCs are displayed in Figure 7-4 (IV bolus), Figure 7-5

(topical application) and basic goodness-of-fit plots are depicted in Figure 7-6,

respectively. Although, variability was inflated by the skin observations from the group

treated with ointment, the overall fit was adequate and the model predicted the

observed data reasonably.

The population PK parameter estimates are summarized in Table 7-4. All

estimates had reasonable precision. Higher inter-individual variability was observed for

the scaling factor S and bioavailability F. Clearance calculated as CL = ke*Vc was

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greater than the CL obtained from NCA (1.96 vs 1.57 L/h), while Vss, calculated as Vc +

Vp, was virtually identical (2.7 vs 2.7 L). The transfer rate constants indicated a fast

distribution from the central to the skin compartment. The distribution factor DF was

0.68 and slightly larger if compared with NCA (0.57). Bioavailability after topical

application was 23.4%.

Semi-mechanistic Pharmacodynamic Model

The PD final model included a susceptible and persistent bacterial

subpopulation. The susceptible subpopulation S included a first-order rate constant for

bacterial multiplication kg and a first-order rate for natural death kd. The susceptible

subpopulation was also able to switch into a resting state R. While in the resting state,

bacteria was not able to replicate itself, but also was not susceptible to the antimicrobial

effect of retapamulin. Figure 7-7 illustrates the semi-mechanistic model. Since growth

and death rate constants could not be estimated separately, knet, a composite of growth

and death rate constant, was used (knet = kg – kd)47. Bacterial growth was modeled by

applying a logistic growth function92–94. Delay in bacterial growth and onset of drug was

also included. The differential equations for the change in the number of susceptible

and resting bacteria over time was described as follows:

𝑑𝑆

𝑑𝑡= [𝑘𝑛𝑒𝑡 ∗ (1 − 𝑒−𝑑𝑔𝑠∗𝑡) ∗ (1 −

𝑆+𝑅

𝑁𝑚𝑎𝑥) − 𝐸𝑓𝑓 ∗ (1 − 𝑒−𝑑𝑘𝑠∗𝑡)] ∗ 𝑆

−𝑘𝑆𝑅 ∗ 𝑆 + 𝑘𝑅𝑆 ∗ 𝑅 (7-7)

𝑑𝑅

𝑑𝑡= 𝑘𝑆𝑅 ∗ 𝑆 − 𝑘𝑅𝑆 ∗ 𝑅 (7-8)

Where ksr and krs are the transfer rate constants, Nmax is the maximum carrying

capacity, Eff is the drug effect and dgs and dks are delay constants for growth and drug

onset. The antimicrobial effect was incorporated as sigmoidal Emax model, with

89

maximum drug effect Emax, retapamulin concentration C, drug concentration EC50 where

the half-maximum antimicrobial effect is achieved and the Hill factor h, which describes

the steepness of the concentration-response curve.

𝐸𝑓𝑓 =𝐸𝑚𝑎𝑥∗𝐶ℎ

𝐸𝐶50+𝐶ℎ (7-9)

A log-additive error model was used for both MSSA and MRSA PD models.

Parameter estimates were reasonable and are summarized in Table 7-5. Goodness-of-

fit plots and VPCs, stratified for MICs, are displayed in Figure 7-8 and Figure 7-9 for

MSSA and in Figure 7-10 and Figure 7-11 for MRSA. Both models described the

bacterial counts quite well. Retapamulin appeared to have a higher maximum effect

against the MRSA ATCC4330 strain when compared to the clinical MSSA isolate (4.8

vs 2.43 h-1). The EC50 was also slightly lower for the MRSA strain (0.147 vs 0.19 mg/L),

yet the concentration-response relationship was steeper against the MSSA isolate (1.35

vs 0.678).

Allometric Scaling

Micro rate constants were converted into hybrid constants A, B, α and β. The

equations were:

𝛼 + 𝛽 = 𝑘𝑒 + 𝑘12 + 𝑘21 (7-10)

𝛼 ∗ 𝛽 = 𝑘𝑒 ∗ 𝑘12 (7-11)

𝛼, 𝛽 =(𝛼+𝛽)±√𝑏(𝛼+𝛽)2−4𝛼∗𝛽

2 (7-12)

𝐴 =𝐷𝑜𝑠𝑒∗(𝛼−𝑘21)

𝑉𝑐∗(𝛼−𝛽) (7-13)

𝐵 =𝐷𝑜𝑠𝑒∗(𝑘21−𝛽)

𝑉𝑐∗(𝛼−𝛽) (7-14)

90

The converted hybrid constants were A=151, B=52.3, α=7.307 and β=0.6026.

Concentration profiles from the animal was normalized according to the equation:

𝐶′ =𝐴

𝐶𝑠𝑠∗ 𝑒(−𝛼∗𝑀𝑅𝑇∗𝑡) +

𝐵

𝐶𝑠𝑠∗ 𝑒(−𝛽∗𝑀𝑅𝑇∗𝑡) (7-15)

To obtain human hybrid constants following equations were used:

𝐴𝑚𝑎𝑛 =𝐶𝑠𝑠,𝑚𝑎𝑛

𝐶𝑠𝑠,𝑎𝑛𝑖𝑚𝑎𝑙∗ 𝐴𝑎𝑛𝑖𝑚𝑎𝑙 (7-16)

𝐵𝑚𝑎𝑛 =𝐶𝑠𝑠,𝑚𝑎𝑛

𝐶𝑠𝑠,𝑎𝑛𝑖𝑚𝑎𝑙∗ 𝐵𝑎𝑛𝑖𝑚𝑎𝑙 (7-17)

𝛼𝑚𝑎𝑛 =𝑀𝑅𝑇𝑎𝑛𝑖𝑚𝑎𝑙

𝑀𝑅𝑇𝑚𝑎𝑛∗ 𝛼𝑎𝑛𝑖𝑚𝑎𝑙 (7-18)

𝛽𝑚𝑎𝑛 =𝑀𝑅𝑇𝑎𝑛𝑖𝑚𝑎𝑙

𝑀𝑅𝑇𝑚𝑎𝑛∗ 𝛽𝑎𝑛𝑖𝑚𝑎𝑙 (7-19)

Human CL and Vss were calculated using scaling form one species and the Øie-

Tozer equation:

𝐶𝐿𝑚𝑎𝑛/𝑘𝑔 = 0.152 ∗ 𝐶𝐿𝑎𝑛𝑖𝑚𝑎𝑙/𝑘𝑔 (7-20)

𝑉𝑠𝑠 = 𝑉𝑝 + (𝑓𝑢𝑝 + 𝑉𝑒) + [(1 − 𝑓𝑢𝑝) ∗ 𝑅𝐸

𝐼

∗ 𝑉𝑃] + 𝑉𝑟 ∗ 𝑓𝑢𝑝/𝑓𝑢𝑡,𝑚𝑎𝑛 (7-21)

where fup is the fraction unbound in plasma, fut is the fraction unbound in tissues,

and RE/I is the extravascular/intravascular ratio of binding proteins and Vp, Ve, and Vr are

the volumes of plasma, extracellular fluid, and remainder fluid.

Equation 7-21 was rearranged to express fut in terms of Vss and fup95:

𝑓𝑢𝑡 =𝑉𝑟∗𝑓𝑢𝑝

[𝑉𝑠𝑠−𝑉𝑝−(𝑓𝑢𝑝∗𝑉𝑒)]−[(1−𝑓𝑢𝑝)∗𝑅𝐸𝐼

∗𝑉𝑝]

(7-22)

Then, fut was estimated using 9.267 L/kg, 0.364 L/kg, 0.0313 L/kg and 0.265

L/kg for animal Vss, Vr, Vp and Ve87,95,96, respectively. Ratio of binding proteins RE/I was

1.495 and fraction unbound fu in plasma was 0.19. Estimated fut was 0.0076. Human Vss

was predicted (5.14 L/kg) with Vr, Vp and Ve at 0.38 L/kg, 0.0436 L/kg and 0.151 L/kg.

91

RE/I and fut were assumed to be equal to the animal values and fu in plasma was 0.1.

The human clearance estimate was 0.99 L/kg. Human Css and MRT were then

calculated using the equations:

𝐶𝑠𝑠,𝑚𝑎𝑛 = 𝐷𝑜𝑠𝑒/𝑉𝑠𝑠 (7-23)

𝑀𝑅𝑇𝑚𝑎𝑛 = 𝑉𝑠𝑠/𝐶𝐿 (7-24)

Human equivalent dose (HED) was based on body surface area and was

calculated from the following formula88:

𝐻𝐸𝐷(𝑚𝑔 𝑘𝑔⁄ ) = 𝑎𝑛𝑖𝑚𝑎𝑙 𝑑𝑜𝑠𝑒(𝑚𝑔 𝑘𝑔⁄ ) ∗𝑘𝑚𝑎𝑛𝑖𝑚𝑎𝑙

𝑘𝑚𝑚𝑎𝑛 (7-25)

where the conversion factors (km) from mg/kg to mg/m2 for rat and human were

6 and 37, respectively. HED, Css and MRT for a typical person weighing 70 kg were 7.9

mg, 22.23 ng/mL and 5.15 h. Hybrid constants for human, estimated with equations 7-

16-19, were A=43, B=14.9, α=1.95 and β=0.16. Back conversion into micro rate

constants and volume of distribution was performed with equations:

𝑘21 =𝐴∗𝛽+𝐵∗𝛼

𝐴+𝐵 (7-26)

𝑘10 =𝛽∗𝛼

𝑘21 (7-27)

𝑘12 = 𝛼 + 𝛽 − 𝑘21 − 𝑘10 (7-28)

𝑉𝑐 =𝐷𝑜𝑠𝑒

𝐴+𝐵 (7-29)

And ke, k21, k12 and Vc were 0.503 h-1, 0.621 h-1, 0.987 h-1 and 137.9 L. Those

constants were used to simulate skin ISF concentrations in humans after topical

application of 10 mg of retapamulin ointment on 100 cm2 (0.1 mg/cm2), twice a day.

Absorption rate constant k0, distribution factor DF and bioavailability were assumed to

be equal to the estimates obtained from rats. The skin volume proportionality factor S

92

was approximated with regards to ointment application area, body surface area (BSA)

and animal scaling factor S. A BSA of 1.73m2 for humans was assumed and animal

BSA was calculated with Meeh’s formula97:

𝐵𝑆𝐴 = 𝑘 ∗ 𝑊2/3 (7-30)

where Meeh’s constant k was 9.8398 and the weight W was 300 g.

Four different scenarios, where ointment stayed on the skin for 3, 4, 6 and 12 h

were simulated. The skin PK profiles were then used to simulate the time course of the

drug against MSSA and MRSA. PK and PD profiles are shown in Figure 7-12. The

antimicrobial effect seemed to be time-dependent with only slight differences between

MSSA and MRSA. Increasing the duration from 3 to 4 hours inhibited bacterial growth

and reduced the bacterial burden. Longer ointment exposure led to further antimicrobial

clearance. In spite of achieving higher steady-state concentrations, the difference of

antibacterial efficacy after 12 h exposure was marginal compared to 6 h exposure.

Summary

Non-compartmental analysis of retapamulin revealed fast plasma clearance and

extensive tissue distribution. The fraction of unbound retapamulin distributing from

plasma into the skin was approximately 57%. A simultaneous population

pharmacokinetic model for IV and topical administration was developed. The change

over time of retapamulin plasma and skin concentrations were best described by a two

compartment body model. Percutaneous absorption followed zero-order kinetics with

lag time. The bacterial system consisted of a susceptible and persistent bacterial

subpopulation with delayed growth. The antimicrobial drug effect was included as a

sigmoidal Emax model with a delayed onset. Concentration and time normalization of the

animal pharmacokinetic data was used to predict plasma and skin concentrations in

93

humans. Clearance was estimated using a one species approach and volume of

distribution calculations were based on physiological parameters from rats and humans.

Simulations of the antibacterial effect of retapamulin following topical application

displayed only minor differences between the tested MSSA and MRSA strains.

94

Table 7-1. Non-compartmental pharmacokinetic analysis for retapamulin after IV bolus administration

Parameters Total Plasma Free Plasma SkinISFa

Mean SD CV% Mean SD CV% Mean SD CV% AUC0-t (ng/mL*h) 635.8 359.9 56.6 120.8 68.4 56.6 65.4 44.7 68.3 AUC0-∞/D (ng/mL/mg*h)

663.1 141.2 21.3 126.0 26.8 21.3 69.4* 8.7 12.5

AUC0-∞ (ng/mL*h) 687.2 369.2 53.7 130.6 70.1 53.7 75.8 44.0 58.1 C0 (ng/mL) 970.2 586.0 60.4 184.3 111.3 60.4 ND ND ND CL (mL/h) 1,569 365.0 23.2 ND ND ND ND ND ND Cmax (ng/mL) 740.6 440.6 59.5 140.7 83.7 59.5 39.3* 30.2 76.9 Cmax/D (ng/mL/mg)

677.5 104.0 15.3 128.7 19.8 15.3 31.5* 12.2 38.6

t1/2 (h) 2.2 1.2 53.4 ND ND ND 2.0 1.0 48.3 MRT (h) 1.9 1.1 57.3 ND ND ND 2.8 1.0 36.5 Tmax (h) 0.1 0.0 0.0 ND ND ND 0.4‡ 0.2 63.9 Vss (mL) 2,780 1,099 39.5 ND ND ND ND ND ND Vz (mL) 4,641 1,675 36.1 ND ND ND ND ND ND AUCISF/fAUCplasma ND ND ND ND ND ND 0.57 0.13 22.2

ND not determined, SD standard deviation, CV coefficient of variation; a statistical difference determined by one-way ANOVA, * p<0.05 compared to free plasma, ‡ p<0.05 compared to total plasma

Table 7-2. Non-compartmental pharmacokinetic analysis for retapamulin after topical

application on tape-stripped skin Parameters Total Plasma Free Plasma SkinISF

a

Mean SD CV% Mean SD CV% Mean SD CV% AUC0-t (ng/mL*h) 77.9 32.4 41.5 14.8 6.2 41.5 944.3* 616.1 65.3

AUC0-∞/D (ng/mL/mg*h) 182.8 97.8 53.5 34.7 18.6 53.5 1,793* 1,202 67.1

AUC0-∞ (ng/mL*h) 108.0 53.3 49.4 20.5 10.1 49.4 1,049* 682.5 65.0

CL/F (mL/h) 7,255 4,836 66.7 ND ND ND ND ND ND

Cmax (ng/mL) 21.9 8.9 40.8 4.2 1.7 40.8 303.5* 208.3 68.6

Cmax/D (ng/mL/mg) 37.1 16.9 45.5 7.1 3.2 45.5 514.0* 350.9 68.3

t1/2 (h) 2.7 0.9 33.1 ND ND ND 1.3‡ 0.4 33.5

MRT (h) 4.8 1.0 20.3 ND ND ND 3.3‡ 0.4 11.5

Tmax (h) 2.2 0.8 38.0 ND ND ND 2.5 0.8 31.0

Vz (mL) 25,392 13,591 53.5 ND ND ND ND ND ND

fAUCplasma/AUCISF ND ND ND 0.039 0.043 109.5 ND ND ND

ND not determined, SD standard deviation, CV coefficient of variation; a statistical difference determined by one-way ANOVA, * p<0.05 compared to free plasma, ‡ p<0.05 compared to total plasma

95

Table 7-3. Non-compartmental pharmacokinetic analysis for retapamulin after topical application on intact skin

Parameters SkinISF

Mean SD CV% AUC0-t (ng/mL*h) 15.9 5.8 36.7 AUC0-∞/D (ng/mL/mg*h) ND ND ND AUC0-∞ (ng/mL*h) ND ND ND CL/F (mL/h) ND ND ND Cmax (ng/mL) 8.9 6.1 68.4 Cmax/D (ng/mL/mg) 16.5 12.0 72.5 t1/2 (h) ND ND ND MRT (h) ND ND ND Tmax (h) 5.6 0.3 4.4 Vz (mL) ND ND ND fAUCplasma/AUCISF ND ND ND

ND not determined, SD standard deviation, CV coefficient of variation

Table 7-4. Population pharmacokinetic parameter estimates

Parameter Estimate RSE% Inter-individual Variability RSE%

Ke (h-1) 1.89 16 16.3 32 Vc (L) 1.04 15 NA NA K12 (h-1) 3.69 28 15.1 47 K21 (h-1) 2.33 15 19.8 43 DF 0.675 11 22 37 S 0.018 48 100.5 32 F 0.234 32 73.3 35 tlag (h) 0.183 6 NA NA Prop. Error (%) 19.5 8 NA NA Add. Error (ng/mL) 0.514 15 NA NA

NA not applicable, RSE residual standard error

96

Table 7-5. Parameter estimates for the MSSA and MRSA PD model

MSSA MRSA

Parameter Mean RSE Mean RSE knet (h-1) 1.85 31.9% 3.09 37.5% Nmax (CFU/mL) 2.0x109 0% 1.1x109 0% Emax (h-1) 2.43 32.3% 4.8 40.4% EC50 (mg/L) 0.19 24.5% 0.147 25% h 1.35 33.7% 0.678 22.7% ksr (h-1) 0.04 49.8% 0.034 22.5% krs (h-1) 0.189 18.5% 0.157 9.1% dgs (h-1) 0.432 44.9% 0.289 53.6% dks (h-1) 0.33 29.8% 0.231 45.5% Interexperimental variability h (%) 12 70.5% 17 68.8% Residual Variability Prop. error (%) 43.6 11.3% 48.8 4.6%

Figure 7-1. Scheme of the final population pharmacokinetic model. Central

compartment refers to plasma. ke is the elimination rate constant and k12 and k21 denote the transfer rate constants from plasma to skin and vice versa. k0 is the zero-order percutaneous absorption rate constant with lag time Tlag.

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Figure 7-2. Individual unbound concentration profiles in plasma. The upper panel refers

to concentration profiles after IV bolus, whereas the lower panel denotes concentration profiles after ointment application. Grey circles display observed data, red solid lines are individual predictions and blue dotted lines represent population predictions.

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Figure 7-3. Individual unbound concentration profiles in skin. Upper panel presents IV

bolus concentration profiles after IV bolus and lower panel refers to concentration profiles after topical application. Grey circles are observed data, red solid lines display individual predictions and blue dotted lines show population predictions.

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Figure 7-4. Prediction-corrected VPCs for IV bolus administration. The open circles

present observed data. The grey shaded area displays the 90% model prediction interval with the solid black line as median.

Figure 7-5. Prediction-corrected VPCs for topical route of administration. Open circles

display observed data, while the grey shaded area presents the 90% model prediction interval.

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Figure 7-6. Basic goodness-of-fit plots. On the upper panels, the black line denotes the

line of unity, while on the lower residual plots, the black line displays the mean zero and the dashed lines ±1.96 standard deviations. Red lines indicate trend lines.

Figure 7-7. Semi-mechanistic PD model for retapamulin against S. aureus.

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Figure 7-8. Basic goodness-of-fit plots from the MSSA PD model. Observed and

predicted concentrations are displayed on natural log scales.

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Figure 7-9. VPCs for the MSSA PD model stratified for MICs. Open circles are

observations and grey shaded area the 90% prediction interval.

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Figure 7-10. Basic goodness-of-fit plots from the MRSA PD model. Observed and

predicted concentrations are displayed on natural log scales.

104

Figure 7-11. VPCs for the MRSA PD model stratified for MICs. Open circles are

observations and grey shaded area the 90% prediction interval.

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Figure 7-12. Simulated human PK and PD profiles for retapamulin.

106

CHAPTER 8 DISCUSSION AND CONCLUSION

Retapamulin is the first drug from the pleuromutilin class which is approved for

clinical use. It is indicated to treat impetigo and uncomplicated superficial skin infections

caused by S. aureus and S. pyogenes. Even though superficial skin infections heal

spontaneously, treatment can help prevent disease spread and reduce the risk of

developing serious complications99–101. Its unique mechanism of action makes

retapamulin a valuable treatment option against pathogens which developed resistance

against mupirocin and fusidic acid31.

Usually, plasma concentrations are used as a surrogate for concentrations of

antimicrobials at the target site. The PK/PD index, derived from these plasma

concentrations, is then used to guide dosing recommendations and evaluate the

antimicrobial activity. For topical antibiotic drugs, however, systemic exposure is low

and the target site is the skin. Therefore, it is pivotal to measure drug concentrations at

the site of infection. Tissue samples can be harvested and concentrations can be

determined by homogenizing or lysing the tissue102. As a result, total tissue

concentrations are measured rather than active unbound concentrations at the site of

infection103–105. Therefore, microdialysis was used to determine the unbound

pharmacologically active retapamulin concentrations. Since there is a constant flow of

perfusate, a true equilibrium between the microdialysis probe and the surrounding

tissue cannot be achieved. The recovery has to be determined, which is dependent on

flow rate, membrane surface area, temperature, physicochemical characteristics of the

investigated drug, perfusion medium and probe membrane and tubing material. These

influencing factors were investigated in vitro and helped to determine the recovery and

107

optimize the microdialysis set up. As in vivo recovery was measured by retrodialysis

(drug loss) but tissue concentrations were measured by extraction efficiency (drug

gain), it was crucial to investigate if there were any differences between the two

methods. Non-specific binding of highly lipophilic drugs on the tubing or membrane

makes quantification difficult and could lead to underestimation of concentrations.

Furthermore, it was important to examine whether there was a concentration-dependent

recovery relationship or not. Overall, the in vitro recovery of retapamulin in saline, at a

flow rate of 1.5 µL/min, was high for both extraction efficiency (90.1%) and retrodialysis

(96.0%) method with no significant differences between the methods no concentration-

dependent recovery.

The clinical feasibility study also confirmed that retapamulin freely crosses the

microdialysis membrane. Overall in vivo drug recovery was 88.22±11.59% and the male

subjects had higher recoveries than the female subject. Retapamulin tissue

concentrations at the end of the 4 hour washout period were 0.39 and 0.27 ng/mL for

the male subjects and 0.99 ng/mL for the female subject. Although the sample size was

too small to draw an inference, the differences in recovery and washout could be due to

differences in body fat composition and blood flow. Retapamulin may retain longer in

adipose tissue due to its lipophilic properties and its extensive tissue distribution.

Increased blood flow can also enhance systemic distribution and elimination from the

skin35,106. Clough et al.107 also reported that changes in blood flow altered analyte

recovery but left drug loss unaffected. In summary, retapamulin is dialyzable in human

skin and a 4 hour washout period may be sufficient.

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In vivo recovery of retapamulin in Wistar rats was comparable to that in humans.

Following IV administration, retapamulin was rapidly eliminated and distributed into the

ISF of the skin, with a distribution factor of 0.57 (AUCISF/fAUCplasma). Plasma half-life,

determined by NCA, was 2.2 h and higher than the reported half-life of 1 h in rats and

monkeys1. Total plasma clearance of retapamulin exceeded the hepatic blood flow of

rats (55 mL/min/kg), hence extrahepatic clearance may play a role in drug elimination.

Retapamulin PK after topical application was examined on intact and tape-stripped skin.

After application of retapamulin on intact skin, percutaneous absorption was very slow.

The outermost skin layer, the stratum corneum (SC), acts as a barrier and provides the

rate-limiting step of drug penetration into the skin. It is composed of corneocytes, fatty

acids, cholesterol and ceramides108. In order to permeate through skin, drugs need to

partition into the lipophilic SC and subsequently, as they permeate further, partition into

the hydrophilic epidermis and dermis109. Due to its lipophilic properties (logP 5)110

retapamulin should partition well into the SC. As a result, retapamulin may form a depot

in the SC, leading to sustained release kinetics and very low dermal concentrations. It is

important to mention that the measured skin Cmax after application on intact skin was

more than 16-fold below the lowest observed MIC. On the other hand, maximum

retapamulin concentrations after application on tape-stripped skin were above the MIC.

Skin infections could impair or even disrupt the skin barrier and lead to an increased

TEWL111,112. Simulating perturbed skin is commonly done by the tape-stripping

method113–116. TEWL increased more than 15-fold after SC removal and variability was

high. Likewise, dermal drug concentrations varied but the average retapamulin

exposure was 59-fold greater compared to intact skin. Although correlation between

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TEWL and percutaneous absorption has been shown,117,118,119, we did not find such a

relationship. Probe depth could also influence skin concentration measurements120,121,

but not all studies show a correlation122–124. We did not measure probe depth with

ultrasound and thus could not investigate a probe depth-skin concentration relationship.

Plasma concentrations were quantifiable for the tape-stripped group and variability was

not as pronounced as the corresponding skin concentrations. This may support the

hypothesis that probe depth matters in dermal microdialysis. If we suppose that

percutaneous absorption was significantly different between the tested animals, than it

should be also reflected in the systemic exposure.

The simultaneous two-compartment PK model was the most stable model. It

should be mentioned that when IV data was modeled alone, without the topical route of

administration, a three-compartment PK model had a lower AIC and described the data

better. However, for the simultaneous approach, a three-compartment model was over-

parameterized and was also not supported by the data following topical application. Our

main goal was to describe and evaluate the time-course of retapamulin in skin after

ointment application. Following model parsimony, we wanted to explain drug disposition

and elimination with as few variables as possible. Because drug was absorbed from the

ointment at a constant rate, and the skin was under sink conditions, a zero-order

absorption process was selected. Zero-order percutaneous absorption has also been

described in other publications125–127. The remaining amount of drug in the ointment,

however, was not determined and therefore only the apparent zero-order rate constant

k0 was obtained.

110

The PD model consisted of a susceptible and persistent bacterial subpopulation

and a logistic growth function. Another way to model self-limiting bacterial growth is to

use a nonlinear function128. A growth delay was also included into the PD model using

an empirical first-order growth delay constant. Different approaches could also describe

the growth delay. For instance, a prebacterial lag compartment, where bacteria

transfers into proliferating, susceptible stage could have been used129,130. Similarly to

the delay in growth, the onset of antimicrobial effect was implemented, but delay of

effect by introducing an effect compartment or by depletion of cell wall constituents have

also been described129,131. Transfer rate constants were used to model the transition

between susceptible and persistent bacterial population. Although this approach was

described in other papers83,132,133, Nielsen et al.48 assumed that the transfer back from

persistent to susceptible stage was negligible.

The MICs indicated activity of retapamulin against both clinical MSSA isolate and

MRSA ATCC43300 strain. The shortcomings of the MIC, i.e. limited information on drug

activity kinetics, two-fold variability and inability to show presence of persistent/resistant

bacteria, warranted the conduct of time-kill curve experiments. Static time-kill curves,

however, do not mimic the in vivo situation where drug concentrations constantly

change over time. As a consequence, dynamic time-kill curves could provide more

meaningful information. Nevertheless, the performed static time-kill curve experiments

confirmed the MIC findings and activity of retapamulin against the tested MRSA strain

was akin to MSSA. In vitro MRSA activity has also been shown in other studies134,135.

Methicillin-resistance of S. aureus is mediated by the acquisition of the

extrachromosomal gene mecA, which encodes penicillin-binding protein 2a

111

(PBP2a)136,137. Beta-lactam antibiotics, such as methicillin, have lower affinity to PBP2a

and as a result, cannot inhibit the transpeptidation reaction and bacterial cell wall

synthesis. Because retapamulin binds to the 50S ribosomal subunit and inhibits protein

synthesis it may not be surprising that it exhibits MRSA activity.

The prediction of human skin ISF concentrations was done by the Css-mean

residence method. Other approaches are the species-invariant time method138 or

physiologically-based pharmacokinetic modeling. The Css-MRT method normalizes

plasma concentration profiles between species and back-transform them using Css and

MRT, which are estimated from Vss and CL, to predict human plasma profiles. The

original method uses rat and dog data to estimate human Vss and CL. Since we only

had data from one species, different Vss87 and CL86 estimation methods were used and

results may deviate from the original method. Usually, simple allometry is used to

predict clearance and volume of distribution. It assumes similar relationships of anatomy

and physiological functions between species. It is simple, widely used and applicable for

compounds with high hepatic clearance, but requires data from multiple species139. CL

prediction performance for biliarily excreted drugs, however, was poor140,141. Prediction

of clearance from one species is applicable for hepatically metabolized compounds and

requires less data, but neglects interspecies differences in metabolism and protein

binding139. Regarding the prediction of volume of distribution, the Øie-Tozer equation

was more accurate compared to simple allometry142, but may be inappropriate for drugs

with nonlinear or complex PK143. The equation also assumes distribution which is driven

by nonspecific binding, rapid equilibrium between blood and tissue, no active transport

and nonsaturating distributional processes144. Drugs with a Vss < 0.6L/kg and logP less

112

than 0 did not obey the Øie-Tozer equation144. Because of retapamulin’s lipophilicity and

high volume of distribution, it was thought to be not a substrate of efflux transporters

and thus, Vss predictions may be reasonable.

Prediction of percutaneous absorption was assumed to be similar between rats

and humans. Relative to human skin, rat skin is observed to be two to five time more

permeable145. An in vitro study146 found no difference in rat and human skin permeation

of salicylic acid. Conversely, Benfeldt et al.117,123,147 reported a 53-fold increase of

salicylic acid penetration in rat skin. Anatomically, rat skin differs from human skin; it

contains less layers of corneocytes and is thinner126. According to Fick’s second law,

diffusion is inversely proportional to the thickness of the diffusion layer. As a

consequence, the rate of permeation may be lower in human skin. Lastly, the difference

in thickness may also change the volume of distribution in the skin. For topical

application, the volume of distribution was modeled as a fraction of the IV volume of

distribution. It was approximately the ratio of application area and total body surface

area of the rat. Similarly, the fraction of skin volume of distribution was adjusted in

humans. Still, this was only a gross approximation and might over-/underestimate the

volume.

In conclusion, showed in vitro activity against the tested MSSA and MRSA

strains. Relative to intact skin, retapamulin permeation was higher in perturbed skin and

concentration levels suggest that the drug may be suited to not only treat superficial

skin diseases. The antibacterial activity of retapamulin seemed to be time-dependent.

Nonetheless, clinical studies are necessary to evaluate our predictions, establish human

PK parameters, refine the model and assess the efficacy against other indications.

113

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BIOGRAPHICAL SKETCH

Alexander Voelkner was born in Zwenkau, Germany. He studied pharmacy at the

Martin-Luther-University in Halle, Germany and earned his degree, B.S. in

pharmaceutical sciences, in November 2009. Upon completion of his practical

pharmaceutical training year he received his license to work as a pharmacist (R.Ph.) in

Germany in April 2011. In January 2012, he was accepted into the Ph.D. program at the

University of Florida, College of Pharmacy, Pharmaceutics Department. Under the

supervision of Dr. Hartmut Derendorf, he focused his research on the pharmacokinetics

and pharmacodynamics of anti-infectives. He has been an active member of the

American Association of Pharmaceutical Scientists (AAPS) and the American College of

Clinical Pharmacology (ACCP), where he also served on their leadership panels. He

received his Ph.D. in pharmaceutical sciences in December 2015.

Documents

© 2015 Alexander Voelkner