Evaluating Sources of Pharmacokinetic Variability Using Breath Testing and Microdialysis

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Title:
Evaluating Sources of Pharmacokinetic Variability Using Breath Testing and Microdialysis
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english
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Gonzalez, Daniel
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University of Florida
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Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Pharmaceutical Sciences, Pharmaceutics
Committee Chair:
Derendorf, Hartmut C
Committee Members:
Palmieri, Anthony
Frye, Reginald F
Dennis, Donn M
Rand, Kenneth H

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Subjects / Keywords:
adherence -- ceftriaxone -- diclofenac -- microdialysis -- pharmacokinetics
Pharmaceutics -- Dissertations, Academic -- UF
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Pharmaceutical Sciences thesis, Ph.D.
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theses   ( marcgt )
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Abstract:
Pharmacokinetic variability may be caused by genetic and non-geneticfactors. Genetic differences can impact drug disposition by affecting theconcentrations of drug metabolizing enzymes and/or transporters critical fordrug distribution and elimination. Demographic variables, disease states, anddrug-drug interactions are known non-genetic causes which may also play a role.Pharmacokinetic variability can lead to differences in drug response. Anotherimportant variable leading to variability in drug response is medicationadherence. Available methods to measure medication adherence are frequentlyimprecise or impractical. The availability of a breath test to measuremedication adherence in real-time may facilitate medical decision making,improve therapeutic outcomes, and can be used to account for differences indrug response in clinical trials. Pharmacokinetic analyses were performed toevaluate the time-course of breath concentrations for a plethora of volatilemarkers used to measure definitive adherence. Microdialysis is another samplingtechnique which uses a semi-permeable membrane to measure free drugconcentrations in vitro and in vivo. In vitro, the technique was applied tostudy the impact of varying albumin concentrations on the protein binding andantimicrobial efficacy of ceftriaxone. In vivo, one potential application ofmicrodialysis is to evaluate topical bioequivalence. With the exception of thevasoconstrictor assay for topical corticosteroids, there is no widely acceptedtechnique which may be used to evaluate bioequivalence for topically appliedproducts. Since microdialysis allows for measurement of free drugconcentrations below the skin, it may be a useful technique to compare twoproducts and assess variability in drug absorption. In vitro and in vivostudies were performed to evaluate the feasibility of using the technique toevaluate bioequivalence using the transdermal patch Flector®.
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Statement of Responsibility:
by Daniel Gonzalez.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
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Adviser: Derendorf, Hartmut C.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-08-31

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1 EVALUATING SOURCES OF PHARMACOKINETIC VARIABILITY USING BREATH TESTING AND MICRODIALYSIS By DANIEL GONZALEZ A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMEN TS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Daniel Gonzalez

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3 To my mom, dad, sister, and wife

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4 ACKNOWLEDGMENTS I would like to thank my major advisor, Dr. Hartmut Derendorf, for giving me the opportunity to b e a part of his research group; my committee members, Drs. Anthony Palmieri III, Reginald Frye, Kenneth Rand, and Donn Dennis for their guidance ; and Drs. Rajendra Pratap Singh, Ravishankar Singh, Sreedharan Sabarinath, Julie Johnson, and Timonthy E. Morey for their continued support I would like to acknowledge the staff of the Clinica l Research Center who helped with our clinical research study, a critical component of this dissertation. Also, I would like to thank all the interns who worked with me duri ng the last four years; I could not have accomplished all this work without the help of Almuth Kaune, Amelia Tucker, Sebastian Wicha, Theresa Mueller, Isabelle Frensch, N ina Deppisch, Annette Raab, Julie Straub Almut Glinzer, Mareike Mieroff, Johannes Mer kle, Emilien Folzer, Johanna Kurre, Marina Weissenborn, Claudia Al Karawi, Eva Mok, Lieth Quffa, and Brandon Wat Thank you for teaching me the value of mentoring students and for always having such a positive attitude. Last, I would like to say thank you to my family, for their constant love, motivation and support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTE R 1 IMPACT AND SOURCES OF VARIABILITY IN PHARMACOKINETICS ................ 16 Impact of Variability in Pharmacokinetics ................................ ................................ 16 Important Sources of Pharmacokinetic Variability ................................ ................... 19 Pharmacokinetic Processes ................................ ................................ ............. 19 Absorption ................................ ................................ ................................ .. 19 Distribution ................................ ................................ ................................ 20 Metabolism ................................ ................................ ................................ 21 Excretion ................................ ................................ ................................ .... 22 2 BREATH TESTING TO ASSESS DEFINITIVE ADHERENCE TO VAGINAL AND ORAL MEDICATIONS ................................ ................................ .................... 23 Introduction ................................ ................................ ................................ ............. 23 Methods ................................ ................................ ................................ .................. 25 Vaginal Adherence Studies ................................ ................................ .............. 25 Oral Adherence Studies ................................ ................................ ................... 27 Result s ................................ ................................ ................................ .................... 29 Vaginal Adherence Studies ................................ ................................ .............. 29 Oral Adherence Studies ................................ ................................ ................... 31 Disc ussion ................................ ................................ ................................ .............. 33 3 INFLUENCE OF VARYING PROTEIN CONCENTRATIONS ON THE ANTIMICROBIAL EFFICACY OF CEFTRIAXONE ................................ ................. 54 Introduction ................................ ................................ ................................ ............. 54 Methods ................................ ................................ ................................ .................. 55 Protein Binding Studies ................................ ................................ .................... 55 Chemicals and equipment ................................ ................................ .......... 55 Reagent preparation ................................ ................................ .................. 55 Sample preparation ................................ ................................ .................... 56 Recovery experiments ................................ ................................ ............... 57

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6 Microdialysis experiments ................................ ................................ .......... 58 Microbiological Experiments ................................ ................................ ............. 58 Chemic als and equipment ................................ ................................ .......... 58 Determination of minimum inhibitory concentration ................................ ... 59 Bioanalytical method development and validation ................................ ..... 60 Static bacterial time kill curves ................................ ................................ ... 61 Microdialysis experiments ................................ ................................ .......... 63 P K/PD modeling ................................ ................................ ......................... 63 Results ................................ ................................ ................................ .................... 65 Protein Binding Studies ................................ ................................ .................... 65 Microbiol ogical Experiments ................................ ................................ ............. 65 Discussion ................................ ................................ ................................ .............. 66 4 DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS TO QUANTIFY DICLOFENAC CONCENTRATIONS IN MICRODIALYSIS AND PLASMA SAMPLES ................................ ................................ ............................... 83 Methods ................................ ................................ ................................ .................. 84 Chemicals and Equipment ................................ ................................ ................ 84 Microdialysis Samples ................................ ................................ ...................... 85 Reagent preparation ................................ ................................ .................. 85 Method conditions ................................ ................................ ...................... 86 Stability studies ................................ ................................ .......................... 87 Plasma Samples ................................ ................................ .............................. 88 Reagent preparation ................................ ................................ .................. 88 Stability s tudies ................................ ................................ .......................... 90 Results ................................ ................................ ................................ .................... 91 Discussion ................................ ................................ ................................ .............. 91 5 IN VITRO STUDIES TO EVALUATE THE FEASIBILITY OF USING FLECTOR IN A CLINICAL MICRODIALYSIS STUDY TO EVALUATE TOPICAL BIOEQUIVALENCE ................................ ................................ .............. 106 Introduction ................................ ................................ ................................ ........... 106 Methods ................................ ................................ ................................ ................ 108 In Vitro Microdialysis Studies ................................ ................................ .......... 108 Reagent preparation ................................ ................................ ................ 108 Study protocol ................................ ................................ .......................... 109 Dissolution Studies ................................ ................................ ......................... 109 Chemicals and equipment ................................ ................................ ........ 10 9 Experimental setup ................................ ................................ .................. 110 Study protocol ................................ ................................ .......................... 110 Residual Content Studies ................................ ................................ ............... 111 Results ................................ ................................ ................................ .................. 112 Discussion ................................ ................................ ................................ ............ 113

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7 6 USE OF MICRODIALYSIS TO EVALUATE THE EFFECT OF SKIN PROP ERTIES AND APPLICATION SITE ON THE TOPICAL BIOEQUIVALENCE OF DICLOFENAC: A FEASIBILITY PILOT STUDY ............. 118 Introduction ................................ ................................ ................................ ........... 118 Methods ................................ ................................ ................................ ................ 118 Volunteers ................................ ................................ ................................ ...... 118 Inclusion criteria ................................ ................................ ....................... 119 Exclusion criteria ................................ ................................ ...................... 119 Prohibitions and restrictions ................................ ................................ ..... 119 Screening ................................ ................................ ................................ 120 Recovery assess ment ................................ ................................ .............. 120 Study protocol ................................ ................................ .......................... 121 Drug Analysis ................................ ................................ ................................ 122 Results ................................ ................................ ................................ .................. 122 Discussion ................................ ................................ ................................ ............ 123 7 USE OF MICRODIALYSIS TO EVALUATE THE EFFECT OF SKIN PROPERTIES AND APPLICATION SITE ON THE TOPICAL BIOEQUIVALENCE OF DICLOFENAC: THE MAIN STUDY ................................ 132 Introduction ................................ ................................ ................................ ........... 132 Methods ................................ ................................ ................................ ................ 134 Volu nteers ................................ ................................ ................................ ...... 134 Inclusion criteria ................................ ................................ ....................... 134 Exclusion criteria ................................ ................................ ...................... 134 Prohibi tions and restrictions ................................ ................................ ..... 135 Screening ................................ ................................ ................................ 135 Recovery Assessment ................................ ................................ .................... 136 Study Protocol ................................ ................................ ................................ 136 Drug Analysis ................................ ................................ ................................ 138 8 DISCUSSION ................................ ................................ ................................ ....... 139 LIST OF R EFERENCES ................................ ................................ ............................. 141 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 150

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8 LIST OF TABLES Table page 2 1 Pharmacokinetic parameters for the parent taggants (2 butyl acetate or 2 pentyl acetate) in human breath (n=8) f ollowing vaginal administration ............. 35 2 2 Pharmacokinetic parameters for the alcohol metabolites (2 butanol or 2 pentanol) in human breath (n=8) f ollowing vaginal administration ...................... 36 2 3 Pharmacokinetic parameters for the ketone metabolites (2 butanone and 2 pentanone) in human breath (n=8 ) following vaginal administration.. ................. 37 2 4 Pharmacokinetic parameters for the appearance 2 pentyl acetate, 2 pentanol, and 2 pentanone in human breath (n=13) fol lowing vaginal administration ................................ ................................ ................................ .... 38 2 5 Pharmacokinetic parameters for the appearance 2 butyl aceate in human breath (n=1 3) following condom application ................................ ....................... 39 2 6 Pharmacokinetic parameters for 2 butanone following oral administration (n=5, 6 replicates each). ................................ ................................ ..................... 40 2 7 Pharmacokinetic parameters for 2 pentanone following oral administration (n=5, 6 re plicates each). ................................ ................................ ..................... 40 2 8 Population parameter estimates from the final model and bootstrap analysis. ... 41 2 9 Pharmacokinetic para meters for exhaled 2 butanone from subjects (n=7) after orally consuming 2 butanol (40 mg). ................................ .......................... 42 3 1 Effect of human serum albumin on the minimum inhibitory concentration (MIC) of ceftriaxone a gainst E. coli (ATCC 25922) ................................ ............ 69 3 2 Parameter and relative standard error estimates for an effect compartment model developed to describe bacterial time kill curve data. ............................... 70 4 1 Calibration curves for microdialysis samples analyzed validation Days 1 3. ...... 92 4 2 Quality control samples analyzed on validation Days 1 and 2. ........................... 93 4 3 Quality control samples analyzed on validation Day 3. ................................ ....... 94 4 4 Inter day precision for microdialysis samples analyzed validation Days 1 3. ..... 95 4 5 Short term stability studies of diclofenac in microdialysis samples stored in an auto sampler for 24 hours ................................ ................................ ................. 96

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9 4 6 Long term stability studies of diclofenac in microdialysis samp les stored at 70C for 30 days ................................ ................................ ................................ 97 4 7 Freeze thaw stability studies of diclofenac in microdialysis samples fro zen at 70C. ................................ ................................ ................................ ................. 98 4 8 Primary stock stability studies of diclofenac in microdialysis samples stored in at 2 to 8C for 7 days. ................................ ................................ ......................... 99 4 9 Calibration curves for plasma samples analyzed on validation Days 1 3. ........ 100 4 10 Precision accuracy (PA) batches for plasma samples analyzed on validation Days 1 3. ................................ ................................ ................................ .......... 101 4 11 Inter day precision for plasma samples analyzed on validation Days 1 3. ....... 102 4 12 Short term stability studies of diclofenac in plasma sam ples stored in an auto sampler for 24 hours ................................ ................................ ............... 103 4 13 Freeze thaw studies of diclofenac in plasma samples. ................................ ..... 104 5 1 Recovery re sults for extraction efficiency and retrodialysis experiments. Each round was performed in triplicate. ................................ ................................ ..... 115 6 1 Time and events table for the pilot study. ................................ ......................... 124 6 2 Demographic characteristics for subjects participating in the pilot study. ......... 125 6 3 Measurement of probe depth in three healthy subjects. ................................ ... 125 6 4 Recovery determination for Subject 01. ................................ ............................ 126 6 5 Recovery determination for Subject 02. ................................ ............................ 127 6 6 Recovery determination for Subject 03. ................................ ............................ 128 6 7 Diclofenac washout period in Subject 01. ................................ ......................... 129 6 8 Diclofenac washout period in Subject 02. ................................ ......................... 130 6 9 Diclofenac washout period in Subject 03. ................................ ......................... 131

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10 LIST OF FIGURES Figure page 2 1 Concentration versus time plot for 2 pentyl acetate following vaginal administration (n=13).. ................................ ................................ ........................ 43 2 2 Concentration versus time plot for 2 penta nol following vaginal administration (n=13). ................................ ................................ ................................ ................ 44 2 3 Concentration versus time plot for 2 pentanone following vaginal administration (n=13).. ................................ ................................ ........................ 45 2 4 Concentration versus time plot for 2 butyl acetate following ap plication using a condom (n=13) ................................ ................................ ................................ 46 2 5 Mean concentration (g/mL) versus time (minutes) profile by subject for 2 butanone (top) and 2 pentanone (bottom) ................................ .......................... 47 2 6 Goodness of fit plots for 2 butanone and 2 pentanone concentrations. ............. 48 2 7 Conditional weighted residuals (CWRES) versus time. ................................ ...... 49 2 8 Individual concentration versus time plots for 2 butanone (5 subjects with 6 replicates each). ................................ ................................ ................................ 50 2 9 Individual concentration versus time plots for 2 pentanone (5 subjects with 6 replicates each). ................................ ................................ ................................ 51 2 10 Visual pr edictive check for 2 butanone. ................................ ............................. 52 2 11 Visual p redictive check for 2 pentanone ................................ ............................ 53 3 1 Protein binding experiments using human serum albumin and pooled human plasma ................................ ................................ ................................ ................ 71 3 2 Bacterial time curves in the absence of human serum albumin.. ........................ 72 3 3 Bacterial time curves in the presence of 2 g/dL o f human serum albumin.. ........ 73 3 4 Bacterial time curves in the presence of 4 g/dL of human serum albumin.. ........ 74 3 5 Follow up bacte rial time curves performed to study the development of drug resistance to ceftriaxone in the absence of human serum albumin.. .................. 75 3 6 Free ceftriaxone concentrations in the absence of human se rum albumin and measured using microdialysis.. ................................ ................................ ........... 76

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11 3 7 Free ceftriaxone concentrations in the presence of 2 g/dL of human serum albumin and measured using microdialysis. ................................ ....................... 77 3 8 Free ceftriaxone concentrations in the presence of 4 g/dL of human serum albumin a nd measured using microdialysis ................................ ....................... 78 3 9 Observed data and 9 5% pr ediction intervals in the absence of human serum albumin. ................................ ................................ ................................ .............. 79 3 10 Observed data and 95% pr ediction intervals in the presence of 2 g/dL human serum albumin.. ................................ ................................ ................................ .. 80 3 11 Observed data and 95% p rediction intervals in the presence of 4 g/dL human serum albumin. ................................ ................................ ................................ ... 81 3 12 Observed data and 95% pr ediction intervals for ba cterial time kill curves performed to evaluate resistance development. ................................ ................. 82 4 1 Percentage recovery and matrix effect following diclofenac extraction from plasma. ................................ ................................ ................................ ............. 105 5 1 Mean dissolution profile for experiments performed using six Flector patches.. ................................ ................................ ................................ ........... 116 5 2 Mean results obtained after cutting a Flector patch into si xteen pieces and extracting the drug content using methanol.. ................................ .................... 117

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12 LIST OF ABBREVIATION S ADME Absorption, Distribution, Metabolism, and Excretion AUC Area Under Concentration versus Time C urve AUC 0 LAST AUC from Zero t o Last Time Point AUC 0 AUC from Zero to Infinity 2 BA 2 Butyl Acetate BQL Below Quantification Limit C e Effect Compartment Concentration C MAX Maximal Drug Concentration CRC Clinical Research Center CS Calibration Standard CV Coefficient of Variation DME Drug Metabolizing Enzymes E. Coli Escherichia coli EC 50 Concentration at Half Maximal Effect EE Extraction Efficiency FDA Food and Drug Administration HEC Hydroxyethylcellulose HIV Human Immunodeficiency Virus HQC High Quali ty Control concentration IS Internal Standard IOV Inter occasion Variability k D Death Rate Constant k MAX Maximum Kill Rate Constant k S Synthesis Rate Constant

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13 k SR Transfer Rate Constant from Susceptible to Resting Populations LAMBDA_Z First order Elimination Rate Constant LLOQ Lower Limit of Quantification LOD Limit of Detection LQC Low Quality Control concentration MIC Minimum Inhibitory C oncentration MQC Medium Quality Control concentration MRT Mean Residence Time NONMEM No nlinear Mixed Effects Modeling NSAID Non Steroidal Anti Inflammatory Drug 2 PA 2 Pentyl Acetate PA Precision Accuracy batch PK Pharmacokinetics PD Pharmacodynamics PPB Parts per Billion QC Quality Control RPM Revolutions per Minute RSE Relative Stand ard Error RT Retrodialysis SD Standard Deviation SE Standard Error T MAX Time at Maximal Drug Concentration DWS Diclofenac Working Stock WS Internal Standard Working Solution

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14 Abstract of Dissertation Presented to the Graduate School of the University of Fl orida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EVALUATING SOURCES OF PHARMACOKINETIC VARIABILITY USING BREATH TESTING AND MICRODIALYSIS By Daniel Gonzalez August 2012 Chair: Hartmut Derendorf Maj or: Pharmaceutical Sciences Pharmacokinetic variability may be caused by genetic and non genetic factors. Genetic differences can impact drug disposition by affecting the concentrations of drug metabolizing enzymes an d /or transporters critical for drug di stribution and eliminati on. D emographic variables, disease states, and drug drug interactions are known non genetic causes which may also play a role Pharmacokinetic variability can lead to differences in drug response. Another important variable leading to variability in drug response is medication adherence. Available methods to measure medication adherence are frequently imprecise or impractical. The availability of a breath test to measure med ication adherence in real time may facilitate medical decisi on making, improve therapeutic outcomes and can be used to account for differences in drug response in clinical trials Pharmacokinetic analyses were performed to evaluate the time course of breath concentrations for a plethora of volatile markers used to measure definitive adherence. Microdialysis is a nother sampling technique which uses a semi permeable membrane to measure free drug concentrations in vitro and in vivo In vitro the technique was applied to study the impact of v arying albumin concentrati ons on the protein binding and antimicrobial efficacy of ceftriaxone. In vivo one potential

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15 application of microdialysis is to evaluate topical bioequivalence. With the exception of the vasoconstrictor assay for topical corticosteroids, there is no widely accepted technique which may be used to evaluate bioequivalence for topically applied products. Since microdialysis allows for measurement of free drug concentrations below the skin, it may be a useful technique to compare two products and assess variabil ity in drug absorption. In vitro and in vivo studies were performed to evaluate the feasibility of using the technique to evaluate bioequivalence using the transdermal patch Flector

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16 CHAPTER 1 IMPACT A ND SOURCES OF VARIABILITY IN PHARMACOKINETICS Impact of Variability in Pharmacokinetics Pharmacokinetics (PK) is a discipline whose aim is to describe the time course of drug concentrations in the body; in particular, it focuses on absorption, distribution, metabolism and excretion (ADME) processes, all of which play a critical role in dictating how much and how often a drug needs to be administered. PK variability is a common phenomenon which explains, at least in part, why patients respond differently to the same medication PK variability can be caused by various factors; including differences in genetic and demographic variables (e.g. age, race, and gender ), drug drug interactions with co administered medications, formulation characteristics pathological conditions, and circadian rhythms The impact of P K variability will depend on the therapeutic index of the drug. For drugs with a narrow therapeutic index, careful dose selection and monitoring is needed. For example, tacrolimus an immunosuppressive medication with a narrow therapeutic index can exhibi t significant PK variability as a result of differences in the extent of drug absorption and drug metabolism 1 One study sought to identify sources of PK variability using a population PK analysis conducted with data from 83 adult kidney transplant patients 2 Using a total of 1,589 trough concentrations, a one compartment model with first order absorption and elimination was fitted to the data. Two covariates were found to contribute significant ly t o the post transplantation and the dosage of a co administered medication, prednisone. It was noted that clearance values reached 50% of the maximal value after 3.8 ( 0.5 ) days and a 1.6 fold incre ase in clearance occurred with prednisone doses greater than

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17 25 milligrams. This example exemplifies how kidney transplantation and co administration of other medications can affect drug clearance and contribute to variability in drug concentrations. PK v ariability observed with medications used in the treatment of human immunodeficiency virus (HIV) has also received significant attention due to the implications that such variability can have on drug efficacy. The importance of identifying sources of varia bility was shown in a study of 275 HIV positive patients prescribed nevirapine 3 Nevirapine is a non nucl eoside reverse transcriptase inhibitor which is administered orally and metabolized by CYP3A4 and CYP2B6. Similar to the tacrolimus example described above a nonlinear mixed effects modeling approach using the software NONMEM was used to model the popu lation PK of nevirapine. In addition to collecting PK data, the authors genotyped all subjects for genetic variations in CYP2B6 (516 G>T and 983 T>C) ; some of which could contribute to variability in the heterozygosity were shown to increase in clearance for every 10 kg increase in weight. In this example data on genetic variants and weight helped explain variability in a nevirapine PK PK variability is known to occur with most drugs; however, determining its impact on drug efficacy will depend on multiple factors. Although not an exhaustive list, here are some factors which one should consider. First, an importan t variable is therapeutic index. If at therapeutic concentrations, the risk for toxicity is low and a drug displays a wide therapeutic index then higher drug concentrations caused by inter or intra individual variability will place less of a rol e. Similarly, if despite significant

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18 variability, drug concentrations remain above some minimal level needed for efficacy, then such variability would be less significant. Second, the drug class and desired efficacy endpoint should be considered For examp le, significant PK variability may be more important for an HIV drug as compared to a medication used in the treatment of nausea; since the risk of adverse effects caused by sub or supra therapeutic drug concentrations is greater with the former example. Third, clearly the extent of the variability and the PK properties of the drug are critical factors. For example, for an orally administere d drug, inter individual variability in the extent of a bsorp tion is more likely to reach clinical significance for a drug a low oral bioavailability; since small changes can have a profound effect. Last, the time course of the variability may be important (i e short lived vs. constant). If a variable contributing to PK variability is short lived, then it may be of less er importance Referring back to the tacrolimus and nevirapine examples, significant PK variability can impact drug efficacy. For tacrolimus, one study showed that patients with organ rejection were more likely to have low trough concentration s ; with a 55 % rejection rate for subjects with trough concentrations between 0 and 10 ng/mL and no rejection episodes observed in patients with a levels of 10 15 ng/m L 4 For nevirapine, simulations were performed to evaluate the impact of genetic variability in CYP2B6 and weight, on trough concentrations 3 The simulations showed that individuals with a greater body weight would benefit from a t wice daily regimen versus once daily due to a greater risk of sub therapeutic concentrations with the latter This increased risk of sub therapeutic concentrations was negated in subjects who were 516T homozygous or 893C heterozygous due to a lower clearan ce.

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19 Thus predicting the impact of PK variability is complex as it depends on a and making dosing recommendations, not only PK but also sources of pharmacodynamic (PD) variability must be considered. Important Sources of Pharmacokinetic Variability Pharmacokinetic Processes G enetic and non genetic causes may be responsible for variability in absorption, distribution, metabolism and excretion of drug molecules The imp act of variability in each of these processes will depend on the route of administration and the PK properties of the individual compound. Absor ption For drugs administered ext ra vascularly variability in the rate and/or extent of absorption can alter th e time course of local and systemic concentrations Usually a change in the extent of absorption is more likely to reach clinical significance, but this is not necessarily the case when a rapid effect is desired (e.g., analgesics). Following oral administ ration a drug molecule needs to be in solution before it can be absorbed. For passive diffusion to occur, a molecule must also be unionized. As a result formulation and drug specif ic factors which can affect drug dissolution and ionization are important 5 erratic; partly due to variability in gastric pH. 6,7 An extended release formulation of dipyridamole and aspirin, uses tartaric acid to provide an acidic local medium and increase the extent of drug absorption. Ketoconazole and itraconazole are also known to exhibit pH dependent absorption. 8

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20 Si nce most drug absorp tion in the gastrointestinal tract will occur in the small intestine, the rate of gastric emptying, which is altered by food and disease states, will impact the rate of drug absorption. Once at the site of absorption the physicochemical properties of a dr ug molecul e will dictate its permeability; both across (transcellular) an d between (paracellular) cells. 9 Drug transporters (e.g. p glycoprotein) can contribute to PK va riability; often with drugs which exhibit poor permeability acr oss enterocytes. P glycoprotein is the most widely studied effl ux transporter, and genetic variability in its expression has been shown to play an important role in the PK of some drugs (e.g. digoxin) 10 12 In addition to transporters, first pass metabolism, either in the gut or liver can decrease the extent of drug absorption and thu s its oral bioavailability. Such metabolism is also subject to significant inter individual variability as a result in variable expression of enzymes, demographic characteristics drug drug interactions, and disease states 13 Distribution A d rug distribution in the body is dictated by various factors; namely, protein binding, transporter activity, physicochemical pro perties, and tissue specific properties (e.g. weight, blood flow, c omposition) 14 Changes in any of these variables can impact tissue distribution and possibly the extent to which a drug molecule reaches its site of action The impact of protein binding changes is a fre quent source of debate 15 24 Its significance will depend on the extent of the binding and a dr PK properties. 19,24 For drug molecules which bind extensively to plasma and/or tissue proteins both changes in protein concentrations an d binding affinity can result in changes in the unbound drug concentrations U nbound, free concentrations are

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21 respon pharmacological activity thus these changes can have a profound impact on drug efficacy Disease states, including critical illnesses, are known to contribute to v ariable protein concentrations. 14 For example, renal failure, hepatic insufficiency, and HIV have all been shown to result in changes in albumin or alpha 1 acid glycoprotein concentrations. 25 31 Other factors which may contribute to variable protein concentrations include pregnancy, age, and malnutrition 32 Alterations in binding affinity may be observed with various disease states, including renal insufficiency, independent of decreases in protein concentr ations. T his is likely a result of accumulation of various byproducts which are not eliminated properly or a change in the protein structure 14,32 Metabolism Most drugs undergo some degree of biotrans formation Although the liver is the most common site of metabolism, various other organs may be involved (e.g. GI tract, kidneys). In the liver, an alteration in drug metabolism can occur as a result of changes the amount of free drug concentration flowi ng through the liver liver perfusion, and/or The importance of each of these factors to the hepatic clearance of a drug will depend on the hepatic extraction ratio of the drug. Fo r drugs with a high hepatic extraction ratio ( e.g., E H >0.7), clearance is highly dependent on liver blood flow, and thus changes in protein binding are unlikely to affect hepatic clearance. In contrast, for drugs with a low extraction ratio ( e.g., E<0.3), hepatic clearance is dependent on the fraction of total drug which is unbound and the intrinsic clearance of a drug. Thus, low extraction drugs would be susceptible to both protein binding alterations and changes in the intrinsic clearance.

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22 Disease states may be responsible for changes in any of these three factors. For example, hepatic impairment can alter all three variables, while cardiovascular failure could impact drug clearance mostly through a decrease in kidney and liver perfusion. 33 Aside from disease states drug drug interactions may also be responsible for changes in intrinsic activity through either inhibition or induction of drug metabolizing enzymes (DME) Also, inter individual variability in drug metabolism may be caused by genetic differences in key DME. Excretion Frequently drugs undergo biotransformation to a more hydrophilic metabolite, which is then eliminated via the kidneys. Alternatively, a drug can be eliminated unchanged in the ur ine. For either case the kidneys are a vital organ involved in drug clearance. Renal insufficiency can b e an important source of PK variability as it can directly impact the elimination of parent compounds and/or their metabolites. Such variability in re nal clearance makes dosing of renally eliminated drugs difficult, and increases the risk that a patient will experience adverse effects without proper monitoring.

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23 CHAPTER 2 BREATH TESTING TO AS SESS DEFINITIVE ADHE RENCE TO VAGINAL AND ORAL MEDICATIONS Int roduction Medication adherence refers to the extent to which a patient follows instructions dictated by their physician with regards to a prescribed medication. Many factors can contribute to poor medication adherence; for example, frequency of administrat ion, adverse effects, social status, and poly pharmacy can all play a role. 34,35 One systematic review of the literature found that patients took between 51 and 71% of their doses; with poorer adherence observed as the total number of daily doses is increased. 36 Another longitudinal study of patients taking antihypertensive medications found that about half of patients had taken a drug holida y (3 or more days off) during the previous year. 37 Moreover, several studies have shown that medication adherence is time dependent, with a longer treatment duration having inferior outcomes. 38,39 Poor medication ad herence can impact the results and conclusions obtained from PK analyses 40 From a clinical perspective, t here are well known consequences of poor medication adherence; which may include poor therapeutic outcom es, increased health care costs, and possibly unwanted side effects. For some conditions (e.g., HIV), near perfect adherence is needed in order to reach favorable outcomes. Ultimately the impact of poor adherence will depend on the interplay of the PK and PD characteristics of a drug. With regards to the PK drugs with a longer half life of elimination will be less impacted by a missed dose as compared to ones with short half lives. 41 life has been referred to as the medication noncompliance impact factor. 41 The smaller the noncompliance impact factor, the less impact a missed do Unfortunately most

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24 drugs hav e half lives less than 12 hours. 42 As a result, d epending on the dosing frequency, drug concentrations can quickly decrease to sub therapeutic levels within one day The impact of a reduction in drug concentrations will depend on the time course of drug effects. For some drugs, a drug effect quickly dissipates once drug concentrations fall below some therapeutic range (e.g., analgesics); whereas, for others, drug effects can still be observed despite disappearance of drug concentrations from plasma (e.g., clopidogrel). In 42,43 Mathematically this can be described as the difference between the post dose duration of drug effect (D) and the prescribed dosing interval (I). 42 Thus a long forgiveness can be a result of a long half life and/or a prolonged PD effect. Ideally the forgiveness is large, reducing the impact of poor adherence Drugs with a long forgiveness are less susceptible to the adverse effects caused by poor medication compliance. In practice, obtaining an accurate asse ssment of medication adherence is difficult and frequently employed measures (e.g., prescription fills, medication diaries) have numerous drawbacks. Methods available to determine definitive adherence (e.g., directly observed therapy) are costly and imprac tical for most disease states. The availability of a breath test to measure medication adherence in real time, either at home or in a clinic, may facilitate medical decision making and improve therapeutic outcomes. The goal of the analyses described herein was to characterize the PK of two volatile markers and their metabolites. The information gained from these analyses

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25 would aid in the development of a breath test which can be used to assess definitive adherence to oral and vaginal medications. Methods S everal pilot studies were conducted in order to evaluate the feasibility of using a breath test to assess definitive adherence for vaginal and oral products. In these studies we sought to characterize the PK for two volatile metabolites, 2 pentyl acetate a nd 2 butyl acetate, as well as their volatile metabolites; namely, 2 pentanol, 2 butanol, 2 pentanone, and 2 bu tanone. These metabolites are produced in a sequential fashion, where the parent compound is converted to the alcohol metabolite (i.e., 2 pentano l and 2 butanol) and then to ketone metabolite (i.e., 2 pentanone and 2 butanone) Vaginal Adherence Studies Two pilot studies were conducted to evaluate the use of a breath test to evaluate adherence to a vaginal product. S tudy one was conducted at Unive rsity of Florida and was designed to measure the concentrations of volatile markers followi ng application in a vaginal gel. 44 The ester taggants 2 pentyl acetate ( 2 PA) and 2 butyl acetate ( 2 BA) were formulated in two types of gel, hydroxyethylcellulose (HEC) and tenofovir (TNV) placebo gel. While two additional esters were tested (isopropyl butyrate and 2 pentyl butyrate), these could not be q uantified in breath and thus are not described further The HEC gel was selected because it is commonly used in the formulation of vaginal gels while the TNV placebo gel was selected to assess adherence for a microbicide formulation containing no active d rug. Eight volunteers completed a tot al of eight 1 hour visits; where 8 separate vaginal formulations (4 esters, 2 formulations) were applied. Following application breath samples were collected at 0, 1, 2 3, 4, 5, 7.5, 10, 20, 30, 40, 50, and 60 minutes using a 5 L Tedlar bag and analyzed using a miniature gas

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26 chromatograph and/or gas chromatography mass spectroscopy. Ester, alcohol, and ketone concentrations were quantified. S tudy two was conducted at University of California San Francisco. This study was a double blind randomized study which enrolled 13 subjects. Again 2 PA and 2 BA were added to a TNV placebo gel (with no active ingredient) and HEC placebo gel, respectively. The TNV placebo gel was applied to the vagina using a 5 mL syringe applicato r. The HEC gel was used as a lubricant on a condom and applied into the vagina with a dildo (15 thrusts). Subjects were randomized to tagged or untagged products (5:1). Each subject came in for two visits ( at least 1 day apart); where TNV placebo gel (with or without 2 PA) and HEC gel (with or without 2 BA) were applied, respectively. Sequential breath samples were collected for 75 minutes. For both studies, a nalyte breath concentrations reported in parts by billion (ppb) were converted to ng/ml by multiply ing each concentration by the molecular weight of the respective molecule. Once all data analysis was completed, results were reported in ppb units. A noncompartmental PK analysis was conducted using WinNonlin (Version 5.2; Pharsight Corporation, St. Louis MO). Estimates were generated for the following PK parameters: first order elimination rate constant (Lambda_Z, minutes 1 ), half life of elimination (Half life, minutes), maximal drug concentration (C MAX ppb ), time at maximal drug concentration (T MAX m inutes), area under concentration versus time curve from zero to the last time point (AUC 0 LAST min*ppb ), area under concentration versus time curve from zero to infinity (AUC 0 min*ppb ), percentage of area under concentration versus time curve from zero to infinity which is extrapolated from AUC 0 t (% AUC Extrap) and the mean residence time (MRT, minutes). The area under concentration

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27 versus time curve (AUC) was calculated u sing the linear trapezoidal rule. All values are reported as meanSD. For the first study, statistical analyses were conducted using SAS (Version 9.2; Cary, NC). Since all subjects received each treatment, a paired t test was used to compare differences b etween groups. First, we evaluated whether there were any significant differences between the HEC and TNV gel, for each respective molecule. Second, we evaluated whether there were any differences in the PK parameters between the two taggants, for each res pective gel type. The latter comparison was conducted in an effort to determine which molecule would be more favorable for use in future studies. An alpha level of 0.05 was used to evaluate statistical significance. All plots were generated using the packa ges lattice and grid in R (Version 2.12 .2 ). 45 Oral Adherence Studies Two studies, denoted as studies three and four, were conducted at University of Florida and sought measure the levels of these flavorants and their metabolites following oral administration. In study three f ive fasting, healthy subjects were administered a size zero h ard gel capsule (Capsugel, Inc., Greenwood, SC) containing 2 butanol (60 mg), 2 pentanone (60 mg), and L carvone (30 mg) on six different occasions ( i.e., replicates). Subjects directly exhaled into a miniature gas chromatograph (Xhale, Inc., Gainesville, FL), which requires only 10 mL of human breath for analysis. Breath concentrations of 2 butanone and 2 pentanone were determined at 0, 5, 10, 15, 20, 30, 45, and 60 minutes post ingestion of the capsule. First, a non compartmental PK analysis was conducte d as previously described. Second, a population pharmacokinetic analysis was conducted to describe the inter and intra individual variability, as well as the inter occasion variability (IOV) using the

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28 software NONMEM (Version 7.2, Icon Development Soluti ons, Ellicott City, Maryland). All models were fitted using a first order conditional estimation method (FOCE) with interaction and the subroutine ADVAN2 TRANS1. Inter individual variability was incorporated using an exponential function, whereas an additi ve error model was used for the residual error. It was assumed that the IOV did not vary between visits. During model development, initially no IOV was added, and then it was added to each parameter in a step wise fashion. The following equation was used t o model the inter individual and IOV for two parameters denoted as K1 and K2: i which is used to describe the inter individual variability, is a normally distributed 2 Perl speaks NONMEM (PsN, Version 3.4.2) was used for NONMEM submission, while Wings for NONMEM (Version 720) facilitated bootstrapping of the final model. 4 6,47 O ne thousand bootstrap runs were performed and 95% confidence intervals were calculated using the 2.5 th and 97.5 th percentile as the lower and upper bound of the bootstrap distribution, respectively. Last, the lattice and Xpose (Version 4.3.3) packag es in the software R (Version 2.12.2) were used for generation of graphics and for model diagnostic purposes. 48 All collected data was included during model development. Nested models were compared using the objective function values, goodness of fit plots, and visual predictive checks. Using a chi square distribution, a decrease in the objective function value of 10.83 (P<0.001, 1 degree of freedom) was used to assess statistical significance.

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29 Parameter estimates were obtained for the first order absorption rate constant (minutes 1 ), first order elimination rate constant (minutes 1 ), and the volume of distribution (L). Although the latter value was estimated, because the concentrations were measured in breath, the parameter estimate has no real physiological meaning. The two first order rate constants were denoted as K1 and K2 and not assigned to absorption or elimination in the results section because without intravenous data it is and measured in breath directly (i.e., no metabolic conversion is needed); while for butanone, 2 butanol was administered. Since only 2 butanon e and 2 pentanone levels were quantified and the conversion to the ketone occurs very quickly (~5 minutes), ketone concentrations were modeled independently. The oral bioavailability for both compounds was assumed to be 100%. In study four seven subje cts consumed a gelatin capsule (size 0, Capsugel, Inc., Greenwood, SC) containing 2 bu tanol (40 mg); then breath samples were collected at 0, 0.5, 2, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 17.5, 20, 25, 30, 45 and 60 minutes following ingestion. Butanone concentr ations were quantified using gas chromatography mass spectrometer. A noncompartmental PK analysis was conducted using WinNonlin (Version 5.2; Pharsight Corporation, St. Louis, MO). Estimates for all relevant parameters were calculated. Results Vaginal Adh erence Studies For study one t he PK parameters generated for 2 BA and 2 PA are shown in Table 2 1. When comparing the HEC and TNV gels, for each respective molecule, significant differences were observed in the C MAX for BA and in the elimination rate

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30 cons tant (Lambda_Z) for 2 PA When the parameters for 2 BA and 2 PA were compared, within the same gel type, several significant differences were observed. For the HEC containing gels, the elimination rate constant, C MAX AUC 0 and AUC 0 LAST were higher for 2 BA whereas the mean residence time (MRT) were significantly higher for 2 PA Similar significant differences were also observed with the TNV containing gels, and in addition, the half life of elimination was significant ly longer for 2 PA The PK parameters for the alcohol and ketone metabolites of 2 BA and 2 PA are shown in Tables 2 2 and 2 3. When comparing the differences between the HEC and TNV gels for 2 butanol, half life was significantly greater in the TNV contai ning gels, whereas C MAX was greater with the HEC gel. For 2 pentanol, AUC 0 LAST was significantly higher for the HEC gel. T MAX the time at maximal concentration, was the only parameter which differed between 2 butanol and 2 pentanol (significantly greater for 2 pentanol in HEC gel). There were no significant differences between 2 butanone and 2 pentanone, regardless of the gel type. For 2 butanone specifically, C MAX and AUC 0 LAST were higher for the HEC gel. Similarly, for 2 pentanone, AUC 0 LAST was signif icantly greater for the HEC gel. For the second study, e stimates of the PK parameters for 2 PA 2 pentanol, and 2 pentanone are shown in Table 2 4 Figures 2 1, 2 2, and 2 3 depict the breath concentration versus time profile s for each of these three mole cules, respectively. For 2 PA in most subjects, breath concentrations reached a maximum within 15 minutes and rapidly declined with a half life of 25.2 ( 8.9) minutes. In general, the concentrations of the two metabolites, 2 pentanol and 2 pentanone, wer e lower and persisted for a longer period of time than the parent compound; although the short

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31 sampling time increases the variability in the estimates for these two molecules. PK parameter estimates for 2 BA measured in breath following application throug h a condom are shown in Table 2 5 ; while the breath concentration versus time profile is depicted in Figure 2 4. For all subjects, 2 BA was rapidly absorbed and concentrations were detectable in breath within 5 minutes. Upon reaching a maximum, 2 BA concen trations declined rapidly with a half life of approximately 6 ( 4.7) minutes. Concentrations of 2 butanol and 2 butanone were not detected for any subject following condom application Oral Adherence Studies In study three, all five subjects successfully completed a total of six independent studies (i.e replicates). For all 30 visits, 2 butanone and 2 pentanone could be quantified in breath using the miniature gas chromatograph The concentration versus time plots for each respective molecule stratified by subject are depicted in Figure 2 5 The PK parameter estimates from the non compartmental analysis for 2 butanone and 2 pentanone are shown in Tables 2 6 and 2 7 respectively. In most cases, 2 butanone and 2 pentanone concentrations could be quantif ied in breath within 5 minutes post ingestion and were still detectable at 60 minutes. When the data for all 30 studies is averaged, the elimination half life for 2 butanone and 2 pentanone is 21.6 (8) and 23.7 (9.1) minutes, respectively. Average C MAX a nd T MAX were 1376 ( 819.8) ppb and 17.8 ( 8.8) minutes for 2 butanone; and similar values were observed for 2 petanone (1,424.2 ( 741.3) ppb and 14.8 ( 8) minutes). Within each subject, when the six replicates were averaged, 2 butanone AUC 0 LAST varied between 9,582.4 and 68,147.9 min*ppb Similar variability in drug exposure (AUC 0 LAST ) was observed for 2 pentanone (13,782.3 68,007.7 min*ppb ).

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32 In the population PK analysis, a one compartment body model with first order absorption described the PK of both 2 butanone and 2 pentanone. The goodness of fit plot s showing the individual and population predicted values showed no obvious trend for either molecule (Figure 2 6 ). The conditional weighted residuals were within an acceptable range ( 2 to 2) for 95 % of the poi nts for both molecules (Figure 2 7 ). Individual plots stratified by both subject and replicate are shown in Figures 2 8 and 2 9 Visual predictive checks for both molecules helped verify that a one compartmental body model described the data re asonably well (Figures 2 10 and 2 11). For 2 butanone specifically, the typical model estimates for the two first order rate constants (K1 and K2) were 0.129 and 0.034 minutes 1 respectively (Table 2 8 ). Inter individual variability was moderate and more pronounced for K1 (73.7% K1 and 39% K2); whereas the opposite was true for the IOV (27.7% K1 and 62.2% K2). For 2 pentanone, the typical model estimates for K1 and K2 were 0.078 and 0.061 minutes 1 respectively. A similar pattern was observed with the in ter individual and IOV. Inter individual variability was more pronounced for K1 (56.5% K1 and 38.7% K2), but the opposite was true for the inter occasion variability (36.3% K1 and 85.6% K2). For the second oral adherence study, PK parameter estimates for 2 buta none are shown in Table 2 9 Breath concentrations of 2 butanone were detectable at 5 minutes for most subjects and the time at maximal concentration (T MAX ) was 6.57 (1.51) minutes. The half life of the molecule is 10.9 (9.61) minutes; thus demonstra ting a rapid elimination from breath, with undetectable levels observed at 60 minutes in four subjects. The mean residence time for a 2 butanone molecule is estimated to be 12.89 (5.95) minutes.

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33 Discussion The results of these pilot studies demonstrate tha t 2 butyl acetate, 2 pentyl acetate, and their metabolites can be measured in breath following vaginal and oral administration. In most cases, detectable concentrations were observed as early as 5 minutes and up to 60 minutes. The availability of a validat ed method which uses a portable device for detection of these volatile markers can allow for a real time assessment of medication adherence. Considerable PK variability was observe d during each study, which could b e attributed to various factors. For examp le there is inherent variability in PK processes between individuals. In this study, variability can result from differences in drug absorption, dis tribution to the lungs, and elim ination from the gas phase of exhaled breath. Second, there can be variabil ity caused by the technique itself. Last, a ssay variability can be another source of variability in the concentrations measured. In the study conducted in five subjects with six replicates each (study three) for the non compartmental analysis, the observ ed variability in drug exposure is likely a result of differences in both clearance and absorption. When comparing the results from the noncompartmental and population PK analyses for 2 butanone, one could conclude that the estimate for K1 likely represent s first order absorption rate constant, whereas K2 is the first order elimination rate constant. If this is true, then for 2 butanone, the results suggest that a larger portion of the inter individual variability is caused by differences in absorption, whe reas there is greater IOV in the elimination. The extent of the variability was similar for 2 pentanone, although estimates for the two first order rate constants were almost the same.

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34 Despite the observed PK variability, in all 30 visits, 2 pentanone and 2 butanone concentrations could be quantified. For the development of a medication adherence system, documenting the presence of one of these two exogenous molecules would suffice to document definitive adherence to a medication.

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35 Table 2 1 Pharmac okinetic parameters for the parent taggant s (2 butyl acetate or 2 pentyl acetate) in human breath (n=8) following vaginal administration. The taggants were formula ted in either hydroxyethylcellul ose (HEC) or tenofovir placebo (Placebo) gels. Parameter 2 b utyl acetate 2 pentyl acetate HEC Placebo HEC Placebo LAMBDA Z (min utes 1 ) 0.05 (0.03) b 0.04 (0.01) b 0.02 (0.01) 0.02 (0.01) a Half life (min utes ) 18.0 (7.1) 19.4 (4.5) 61.8 (50.3) 33.7 (15.5) b C MAX (ppb) 590.3 (261.2) a,b 381.6 (105.0) b 100.4(49.6) 94. 23 (39.5) T MAX (min utes ) 8.1 (2.6) 8.4 (4.8) 9.4 (4.2) 8.8 (4.4) AUC 0 LAST (min utes *ppb) 15011.0 (7540.9) b 10782.8 (2821.8) b 3540.8 (2053.4) 3220.8 (1954.5) AUC 0 (min utes *ppb) 17033.3 (8229.3) b 12420.7 (3071.0) b 7216.3 (5595.0) 4968.5 (4010.7) % AUC Extrap 11.4 (7.4) 13.4 (7.2) 45.0 (19.3) a,b 28.3 (12.5) b MRT (min utes ) 20.5 (4.0) 22.6 (1.7) 24.8 (2.5) b 25.3 (3.2) b The data is reported as the mean with standard deviation in parentheses. P<0.05 was used to evaluate statistical significance. a HEC com pared to placebo gel. b 2 butyl acetate and 2 pentyl acetate for a respective gel.

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36 Table 2 2 Pharmacokinetic parameters for the alcohol metabolites ( 2 butanol or 2 pentanol ) in human breath (n=8) following vaginal administration. The taggants were formulated in either hydroxyethylc ellul ose (HEC) or tenofovir placebo (Placebo) gels. Parameter 2 butanol 2 pentanol HEC Placebo HEC Placebo LAMBDA Z (min utes 1 ) 0.03 (0.03) 0.02 (0.01) 0.02 (0.018) 0.02 (0.02) Half life (min utes ) 29.4 (12.9) 54.1 (23. 8) a 61.9 (36.4) 57.0 (35.4) C MAX (ppb) 42.3 (18.5) a 21.8 (12.7) 36.5 (22.9) 27.3 (29.0) T MAX (min utes ) 9.4 (3.2) 24.4 (21.9) 19.4 (12.1) b 21.4 (18.4) AUC 0 LAST (min utes *ppb) 1279.7 (878.8) 804.3 (431.0) 1460.9 (799.9) a 954.4 (742.7) AUC 0 (min utes *ppb) 2052.7 (1557.2) 1858.1 (974.6) 3788.8 (3106.0) 3623.9 (4223.1) % AUC Extrap 35.9 (11.6) 53.5 (15.4) a 52.0 (21.0) 46.8 (28.2) MRT (min utes ) 23.7 (6.2) 28.4 (5.9) 29.5 (3.1) 29.9 (4.7) The data is reported as the mean with standard devi ation in parentheses. P<0.05 was used to evaluate statistical significance. a HEC compared to placebo gel. b 2 butanol and 2 pentanol for a respective gel type

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37 Table 2 3. Pharmacokinetic parameters for the ketone metabolites (2 butanone and 2 pentanone ) in human breath (n=8) following vaginal administration. The taggants were formula ted in either hydroxyethylcellul ose (HEC) or tenofovir placebo (Placebo) gels. Parameter 2 butanone 2 pentanone HEC Placebo HEC Placebo LAMBDA Z (min utes 1 ) 0.02 (0.02) 0 .01 (0.01) 0.01 (0.01) 0.02 (0.01) Half life (min utes ) 56.9 (33.8) 64.5 (27.6) 328.9 (466.8) 68.9 (50.1) C MAX (ppb) 52.1 (21.1) a 28.1 (15.8) 48.6 (35.5) 33.4 (30.8) T MAX (min utes ) 18.8 (16.9) 31.3 (24.9) 26.3 (6.9) 38.8 (22.0) AUC 0 LAST (min utes *ppb) 2 204.4 (1108.6) a 1165.6 (631.9) 2111.7 (1511.5) a 1271.9 (1048.6) AUC 0 (min utes *ppb) 4916.7 (3231.5) 3419.9 (2590.4) 16920.0 (19512.2) 2442.7 (1879.2) % AUC Extrap 44.5 (20.6) 57.1 (11.5) 72.1 (20.4) 50.6 (21.8) MRT (min utes ) 28.6 (4.0) 31.4 (2.6) 32.4 (2.8) 33.6 (4.3) The data is reported as the mean with standard d eviation in parentheses. P<0.05 was used to evaluate statistical significance. a HEC compared to placebo gel.

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38 Table 2 4 Pharmacokinetic parameters for the appearance 2 pentyl acetate, 2 pentanol, and 2 pentanone in human brea th (n=13 ) following vaginal administration. The taggant was formulated in tenofovir placebo (Placebo) gel 2 pentyl acetate 2 pentanol 2 pentanone LAMBDA_Z (min utes 1 ) 0.031 (0.012) 0.008 (0.006) 0.009 (0.007) Half life (min utes ) 25.2 (8.9) 195.2 (254.9) 155.9 (159.2) C MAX (ppb) 270.4 (218.2) 56.7 (28.4) 66.6 (41.8) T MAX (min utes ) 14.4 (12.1) 23.3 (7.9) 46.7 (15.8) AUC 0 LAST (min utes *ppb) 7881.2 (4757.1) 3140.2 (1494.1) 3673.5 (2239.6) AUC 0 (min utes *ppb) 10047.3 (6558.4) 15927.8 (23291.5) 18669.5 (17266.9) % AUC Extrap 18.6 (8.9) 61.2 (23.4) 63.4 (23.6) MRT 0 last (min utes ) 30.1 (4.3) 37.0 (2.0) 42.4 (2.7) The d ata is reported as the mean with standard deviation in parenthes e s.

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39 T able 2 5 Pharmacokinetic parameters for the appearance 2 butyl aceate in human brea th (n=13 ) following condom application The taggant was formulated in a hydroxyethylcellul ose (HEC) gel 2 Butyl Acetate LAMBDA_Z (min utes 1 ) 0.156 (0.079) Half life (min utes ) 6.0 (4.7) C MAX (ppb ) 49.9 (49.9) T MAX (min utes ) 6.4 (3.2) AUC 0 LAST (min utes *ppb) 4 12.4 (445.1) AUC 0 (min utes *ppb) 499.3 (523.4) % AUC Extrap 19.1 (15.1) MRT 0 last (min utes ) 8.0 (2.3) The d ata is reported as the mean with standard deviation in parenthes e s.

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40 Table 2 6 Pharmacokinetic parameters for 2 butanone following oral admi nistration (n=5 6 replicates each ) Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 LAMBDA_Z (minutes 1 ) 0.033 (0.008) 0.034 (0.005) 0.024(0.008) 0.043 (0.010) 0.046 (0.017) Half life (minutes) 22.6 (6.1) 20.6 (3.2) 30.7 (9.0) 16.4 (3.4) 17.5 (8.9) C MAX (ppb ) 1296.6 (450.7) 1926.2 (239.9) 1020.7 (710.0) 2238.2 (708.6) 398.0 (257.9) T MAX (minutes) 21.7 (10.3) 20.8 (4.9) 11.7 (10.8) 18.3 (8.2) 16.7 (7.5) AUC 0 LAST (minutes*ppb ) 45297.5 (16577.2) 66465.5 (5168.5) 19607.5 (11598.9) 68147.3 (18757.5) 95 81.2 (7641.1) AUC 0 (minutes*ppb ) 59105.5 (19880.7) 82627.9 (10044.4) 25623.4 (15158.8) 77592.6 (19556.2) 10998.5 (8575.8) % AUC Extrap 22.6 (11.0) 19.2 (3.5) 23.2 (4.7) 12.58 (3.3) 12 (10.5) MRT 0 last (minutes) 29.3 (5.9) 27.7 (1.6) 22.7 (5.5) 26.2 (3 .2) 22.9 (3.6) The data is reported as the mean with standard deviation in parentheses. Table 2 7 Pharmacokinetic parameters for 2 pentanone following oral administration (n=5, 6 replicates each) Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 L AMBDA_Z (minutes 1 ) 0.029 (0.007) 0.030 (0.004) 0.023 (0.009) 0.040 (0.08) 0.042 (0.015) Half life (minutes) 24.8 (5.6) 23.2 (2.6) 34.6 (13.3) 17.9 (3.5) 17.8 (5.2) C MAX (ppb ) 1328.2 (395.9) 1849.5 (180.0) 1334.0 (859.2) 2072.4 (696.6) 536.4 (290.3) T MA X (minutes) 20.0 (9.5) 15.8 (4.9) 10.0 (10.0) 15.0 (7.7) 13.3 (6.1) AUC 0 LAST (minutes*ppb ) 49094.4 (17665.2) 68007.7 (6248.7) 31020.6 (18913.3) 66163.9 (17201.9) 13782.7 (9108.3) AUC 0 (minutes*ppb ) 65626.4 (21988.9) 87240.2 (9621.5) 45946.8 (34359.7) 76449.6 (19188.4) 15806.3 (10707.1) % AUC Extrap 24.5 (9.7) 21.9 (2.6) 27.1 (9.1) 13.6 (2.7) 11.1 (5.5) MRT 0 last (minutes) 28.4 (4.9) 27.3 (1.4) 23.7 (4.6) 25.6 (2.6) 22.2 (3.4) *The data is reported as the mean with standard deviation in parentheses.

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41 Table 2 8 Population parameter estimates from the final model and bootstrap analysis 2 Butanone 2 Pentanone Parameter And Model Estimate (SD Scale) S E From Final Model Bootstrap Estimate (95% CI ) a Estimate (SD Scale) S E From Final Model Bootstrap E stimate (95% CI ) b Structural Model K1 (m in utes 1 ) 0.13 0.04 0.13 (0.07 0.25) 0.08 0.02 0.08 (0.05 0.11) K2 (m in utes 1 ) 0.04 0.01 0.04 (0.01 0.06) 0.06 0.02 0.07 (0.04 0.11) V ( L ) c 0.15 0.02 0.18 (0.12 0.33) 0.19 0.04 0.20 (0.16 0.27) Variance Model 2 ( K1 ) 0.54 (73.7%) 0.31 0.55 (0 0.89) 0.32 (56.5%) 0.24 0.48 (0 0.70) 2 ( K2 ) 0.15 (39.0%) 0.14 0.46 (0 1.63) 0.15 (38.7%) 0.12 0.27 (0 0.60) IOV (K1) 0.08 (27.7%) 0.64 0.28 (0.11 0.55) 0.13 (36.3%) 0.10 0.37 (0.20 0.54) IOV (K2) 0.39 (62 .2%) 0.20 0.71 (0.39 1.44) 0.73 (85.6%) 0.29 0.86 (0.40 1.53) Residual Error (p pb) 264.87 61.02 256.55 (147.0 368.88) 222.92 35.06 218.27 (146.29 282.13) a 2 Butanone : 1,000 runs; 98.8% of runs with successful minimization; 53.5% of runs with successful covariance step. b 2 Pentanone : 1,000 runs; 98.3% of runs with successful minimization; 64.3% of runs with successful covariance step c No physiological meaning as drug concentrations were measured in breath.

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42 Table 2 9 Pharmacokinetic paramete rs for exhaled 2 butanone from subjects (n=7) after orally consuming 2 butanol (40 mg). Parameter Value LAMBDA_Z (min utes 1 ) 0 21 ( 0.26) Half life (min utes ) 10 9 ( 9.6) C MAX (ppb) 548 ( 235) T MAX (min utes ) 6 6 ( 1.5) AUC 0 LAST (min utes *ppb) 5227 ( 3858) AUC 0 (min utes *ppb) 5585 ( 3976) % AUC Extrap 6 69 ( 9.18) MRT 0 last (min utes ) 12 9 ( 6.0) The d ata is reported as the mean with standard deviation in parenthes e s.

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43 Figure 2 1. Concentration versus time plot for 2 pentyl acetate following vaginal adm inistration (n=13) The taggant was formulated in tenofovir placebo (Placebo) gel. The dark line signifies median values for each time point, while the shaded region represents the 5th and 95th percen tiles.

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44 Figure 2 2 Concentration versus time plot for 2 pentanol following vaginal administration (n=13) The taggant was formulated in tenofovir placebo (Placebo) gel. The line signifies median values for each time point, while the shaded region represents the 5th and 95th percen tiles.

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45 Figure 2 3 Concen tration versus time plot for 2 pentanone following vaginal administration (n=13) The taggant was formulated in tenofovir placebo (Placebo) gel. The line signifies median values for each time point, while the shaded region represents the 5th and 95th perce n tiles.

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46 Figure 2 4 Concentration versus time plot for 2 butyl acetate following application using a condom (n=13) The taggant was formulated in hydroxyethylcellulose (HEC) gel. The line signifies median values for each time point, while the shaded re gion represents the 5th and 95th percen tiles.

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47 Figure 2 5 Mean concentration (g/ mL ) versus time (minutes) profile by subject for 2 butanone (top) and 2 pentanone (bottom). Individual replicates (n=6) are plotted for each subject (Replicate 1: open circ les; Replicate 2: open triangles; Replicate 3: crosses; Replicate 4: closed squares; Replicate 5: closed circles; Replicate 6: closed triangles).

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48 Figure 2 6 Goodness of fit plots for 2 butanone and 2 pentanone concentrations.

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49 Figure 2 7 Conditi onal weighted residuals (CWRES) versus time.

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50 Figure 2 8 Individual concentration versus time plots for 2 butanone (5 subjects with 6 replicates each)

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51 Figure 2 9 Individual concentration versus time plots for 2 pentanone (5 subjects with 6 replic ates each)

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52 Figure 2 10 Visual predictive check for 2 butanone. The shaded area represents the prediction interval based on 1,000 simulations; whereas the dashed and solid lines represent the 2 .5 50 and 97 .5 th percentile s for the observed data.

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53 Figure 2 11 V isual predictive check for 2 pen tanone. The shaded area represents the prediction interval based on 1,000 simulations; whereas the dashed and solid lines r epresent the 2.5 50 and 97.5 th percentiles for the observed data.

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54 CHAPTER 3 INF LUENCE OF VARYING PR OTEIN CONCENTRATIONS ON THE ANTIMICROBIAL EFFICACY OF CEFTRIAX ONE Introduction Ceftriaxone is a third lactam antibiotic which displays broad bactericidal activity against gram negative bacteria, such as Haemophilus influenza or Neisseria meningitides as well as coverage against gram positive pathogens such as Strep tococcus pneumonia. Due to its high plasma protein binding (up to 98%), ceftriaxone displays a significantly longer half life (~ 7 8 hours) than other beta lactam antibio tics. 49 Since only the unbound drug is responsible for the antibacterial activity, protein binding must be accounted for when determining an appropriate dosing scheme. As a result, investigators often add a protein supplement or plasma in vitro in an effort to elucidate the role of protein binding on antibacterial activity. There are several factors which must be considered when studying the impact of protein binding in an in vitro setting. These include the type of protein supplement u sed, the technique used to measure the degree of protein binding, and th e interpretation of the results 23 O ne study reported that available protein supplements may differ in their impact on protein binding and antimicrobial efficacy 50 Aside from the type of protein supplement utilized, the protein conc entration used may also be important Frequently a protein concentration of 4 g/dl is used, although this may not be appropriate for all drugs. We sought to evaluate the impact of adding varying protein concentrations on the protein binding and antimicrobi al efficacy of ceftriaxone using microdialysis and bacterial time kill curve experiments, respectively Microdialysis is a versatile tool which m ay be used to measure free drug concentrations both in an in vitro and in vivo setting With microdialys is a probe is

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55 placed into the tissue of interest (when used in vivo ) and perfuse d with a physiological solution commonly referred to as the perfusate. Due to diffusion processes, drug flows down its concentration gradient and is taken up into the perfus ate The collected sample (i.e., dialysate) contains a fraction of the drug which is in the surrounding medium. Equilibrium between the surrounding medium and perfusate is not established, and thus the fraction of drug diffusing through the membrane has to be determined using recovery exp eriments (e.g., extraction efficiency, retrodialysis). Since only the free unbound drug can pass through the membrane of the probe, microdialysis can be used in vitro to evaluate the binding of drugs to plasma proteins. M ethods Protein Binding Studies Chemicals and equipment A Metler balance (AB104) was used to weigh all chemicals. Vortexing and pH measurement was done using a Kraft Apparatus ( model PV 5) and a Corning pH meter ( model 430), respectively. An Agilent 1100 se ries HPLC and Restek C 18 column (Pinnacle DB 5 m) was used for analysis of the collected samples. A Harvard apparatus 22 syringe pump was used for infusion of the drug solution while the CMA 60 microdia lysis probe was used to measure free drug concentra tions in all studies Ceftriaxone (Sigma C 5793), hexadecyl trimethyl ammonium bromide (HDTA, Fisher Scientific 03042), human serum albumin ( HSA, Calbiochem CAS 70024 90 7), and on. Reagent preparation Ceftriaxone calibration samples (31.25, 62.5, 125, 250, 500, and 1000 ng/ mL )

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56 0.2 liters of 7.5 mM KH 2 PO 4 (MW 136.09 g/mol) dissolved in water (204 mg KH 2 PO 4 ) with 0.8 liters of HPLC grade methanol. Then the ion pairing agent, HDTA (MW 364.46 g/mol), was added at a concentration of 1.2 mM (455 mg HDTA). The pH of the mobile phase was adjusted to 8.8. T he mobile phase was filtered and sonicated for 20 minutes. Sample preparation probe (M Dialysis, Solna, Sweden) For the extraction efficien cy (EE) experiments where the drug is diluted in the surrounding media, ceftriaxone using three concentrations (50, 100, mL ) prepared in lactated ringer s solution. Fo r the retrodialysis (RT) experiments where the drug is diluted in the perfusate, similarly three concentrations were prepared mL ) by ution. During preparation of drug solutions, exposure to light was minimized. Using the same drug concentrations tested in the recovery experiments, protein binding experiments were conducted using varying concentrations of human serum albumin. On the firs t day, mL drug solutions containing varying concentrations of human serum albumin (no protein, 0.5, 1.5, 2.5, 3.5, 4.5, or 5.5 g/dl) were prepared in triplicate. On the second and third day, the same protein concentrations were tested using the 100 and 1 50 mL drug concentrations respectively pooled human plasma in an effort to evaluate the impact of varying protein concentration s in plasma. For the experiments with diluted plas ma, undiluted plasma was diluted by one half and one fourth using lactated solution. Actual protein

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57 concentrations were measured using an albumin assay kit ( BioChain BCG Albumin Assay Kit Hayward, CA ). Recovery experiments Before any protein bi nding experiments could be conducted the recovery of the drug was determined EE and RT experiments were conducted on separate days. For the EE experiments, a 5 mL syringe containing lactated ringer s solution was used as the perfusate. The probe was plac ed in a drug solution containing 50, 100, or 150 mL of ceftriaxone. L actated ringer s solution was infused through the probe at flow rate of 1.5 l/min for a period of 15 minutes (equilibration period). Then dialysate samples were collected for a period of 20 minutes each (collection period). A total of three separate samples were collected; each twenty minutes apart. The recovery (R %) was calculated according to Equation 3 1; using the measured concentration in the dialysate (C dialysate ) and the known concentration in the perfusate (C perfusate ). 3 1 In the RT experiments, the drug is infused in the perfusate solution) Syringes containing 50, 100, or 150 mL of ceftriaxone were fastened to the syringe pump. First the drug solution was allowed to equilibrate through the probe for a period of 15 minutes. Next there was a 20 mi n ute sample collection period A total of three separate samples were collect ed; each twenty minu tes apart. The recovery in the RT experiments was calculated using equation 3 2. 3 2

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58 Microdialysis experiments Using microdialysis the protein binding of ceftriaxone (50, 100, and 150 g/ mL ) was teste d using varying concentrations of human serum albumin (no protein, 0.5, 1.5, 2.5, 3.5, 4.5, or 5.5 g/dl) During the p rotein bindin g experiments, first the lowest protein concentration (i.e., no protein) was tested and in a stepwise fashion increasing prot ein concentrations were evaluated Briefly the pro be was placed in a drug solution and then there was a 15 minute equilibration period where lactated ringer s solution was perfused through the probe. Once the equilibration period was complete, there was a 20 minute sample col lection period. T his process was repeated for each protein concentration. Samples were analyzed immediately upon collection. All experiments were conducted in triplicate. A similar procedure was followed for protein binding experiment s conducted using undiluted and diluted plasma. On three separate days, the protein binding for one ceftriaxone concentration (50, 100, or 150 g/ mL ) was measured in undiluted plasma as well as plasma diluted by one half and one each case there was a 30 minute equilibration period followed by a 20 minute sample collection period. In addition, there was a 15 minute flushing period in between each run Samples were analyzed immediately upon collection. All experiments were conducted in triplicate. Microbiological Experiments Chemicals and equipme nt A Metler balance (AB104) was used to weigh all chemicals. The A JUST (Abbott Lab) Turbidmeter and McFarland Equivalence Turbidity Standards were used to prepare bacterial dispersions. A Harvard apparatus 22 syringe pump was used for

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59 infusion of the drug solution during microdialysis experiments, while the CMA 20 and 60 microdialysis probe s were utilized for determination of free drug concentrations during the bacterial time kill curves Ceftriaxone (Sigma C 5793), hexadecyl trimethyl ammonium bromid e (HDTA, Fisher Scientific 03042), human serum albumin (Calbiochem CAS 70024 90 reagent and sample preparation. All microdialysis samples were analyzed using a PE200 series pump and autosampler (Per kin Elmer, Norwalk, CT, USA); as well as a triple quadruple API 4000 mass spectrometer (Applied Biosystems, Carlsbad, CA, USA) equipped with an electrospray ion source. The column of choice was a Symmetry C 18 column (4.6 x 500 mm, 3.5 m particle size; W aters, Dublin, Ireland). Corning 24 and 96 well plates (#3524), 50 mL tissue culture flasks, and Remel 5% sheep blood agar plates were used during bacterial time kill curves. Determination of minimum inhibitory concentration The minimum inhibitory concen tration of ceftriaxone against E. coli (ATCC 25922) was determined in the presence and absence of human serum albumin. For the former case, f irst, the broth used in the experiments was prepared. For determination in triplicate, 20 mL of Mueller Hinton brot h (Becton Dickenson, BBL 211443) was prepared by suspending 440 mg o f the powder in 20 mL of purified water. The broth was m ix ed thoroughly, heat ed with frequent agitation, and allowed to b oil for 1 minute to completely dissolve the powder. T he broth was a utoclave d at 121C for 15 minutes. Last, 500 of the prepared broth was added into each of the 24 wells on the cell culture plate. Second, p rimary and secondary stock solutions (2 and 0.1 mg/ mL ) of ceftriaxone were prepared. Then 10 l of the secondary s tock was added to the first well and serial dilutions were performed to obtain the following concentrations: 0.0078, 0.0157, 0.0313,

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60 0.0625, 0.125, 0.25, 0.5, and 1 g/ mL Third, a dispersion of bacteria containing 1.5 x 10 8 CFU/ mL was prepared. Of this ba cterial dispersion, 10 was added to each well. Negative (containing no drug or bacteria) and positive (containing no drug but bacteria) control samples were prepared. For the negative control, 500 of broth was dispensed into each well; and no bacter ia were added. For the positive controls, 500 L of broth and 10 L of 1.5 x 10 8 CFU/ mL E. coli dispersion were added into each well. All well plates were incubated at 37C for 20 hours. The minimum inhibitory concentration was determined as the ceftriaxon e concentr ation where no visible growth was observed. The minimum inhibitory concentration was then determined in the presence of human serum albumin. Broth was prepared in a similar fashion as described above except that albumin was added. To prepare bro th containing 2 and 4 g/dl of human serum albumin, 0.4 and 0.8 grams of human serum albumin was each added to 20 mL of broth. All other procedures were performed in a similar fashion. These experiments were performed on two separate dates. Bioanalytical method development and validation Following completion of the minimum inhibitory concentration experiments, static bacterial time kill curve experiments were performed. Durin g these experiments, one objective was to evaluate the feasibility of using microd ialysis to quantify free drug concentrations directly in the culture flasks containing the bacteria, broth, and drug solution. Before this objective could be tested development and validation of a method to detect ceftriaxone in broth samples was complete d For preparation of calibration sta ndards and quality controls (QC) a CMA 20 probe was placed in a broth solution and dialysate was collected for a period of 12

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61 hours. The dialysate was then mixed one to one with a cefazolin solution (2000 ng/ mL inter nal standard); cen trif uged at 10 000 rpm for 5 minutes; and the supernatant was working solution, calibration standards were prepared ranging from 7.81 1000 ng/ mL In addi tion, QC samples (8, 20, 100, and 800 ng/ mL ) were also prepared with the working solution. During analysis, the p recursor and product ion transitions monitored were m/z 555.2/396.2 and m/z 555.2/167.0 for ceftriaxone and 455.1/323.0 for cefazolin. The mob ile phase consisted of methanol (75%) and 10 mM ammonium acetate in wat er (pH 7.2). A flow rate and inj ection volume of 0.45 mL /min and 10 L was used respectively The method was validated according to the guidelines set forth by the United States Food and Drug Administration (FDA) Static bacterial time kill curves Bacterial time kill curves were conducted to evaluate the effect of varyi ng protein concentrations on the antimicrobial efficacy of ceftriaxone against E. coli (ATCC 25922). To achieve this goal three scenarios were evaluated. First, the time kill profile of ceftriaxone was evaluated in the absence of human serum albumin. Then it was evaluated in the presence of 2 and 4 g/dl of human serum albumin separately Each scenario was conducted in triplicate. In each case, seven different ceftriaxone concentrations were evaluated, each representing multiples of the MIC (0.25*MIC, 0.5*M IC, MIC, 2*MIC, 4*MIC, 8*MIC,16*MIC); where the MIC was determined to be 0.06 g/ mL in the previously described experiments. In addition to these seven concentrations, negative and growth controls were also included.

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62 For each scenario, nine 50 mL culture flasks were filled with 29.6 mL of Mueller Hinton broth. Then, with the exception of the negative control, 100 L of a bacteria dispersion containing 1.5 x 10 8 CFU/ mL was added. Flasks were incubated for 2 hours at 37C. A primary stock solution (4.8 mg/ mL ) was prepared by adding 48 mg of ceftriaxone to 10 mL of triple deionized water. Then a secondary stock was prepared (480 g/ mL ), and serially diluted, to obtain concentrations between 0.015 0.96 g/ mL ; each concentration corresponding to an MIC multiple. After two hours of incubation, flasks were taken out of the incubator and a 20 L aliquot was removed from each flask for spot inoculation. Before being returned to incubator, 300 L of a drug solution was added to each flask but the positive and negat ive controls. For the spot inoculation a 10 fold serial dilution scheme was performed. Briefly, 180 L of sterile saline was added to each well and a 20 l sample was taken out of each flask Serial dilutions were performed by taking a 20 l from the prev ious well. Last, 10 L was s potted five times per dilution on one fourth of each 5% sheep blood agar plate. Plates were incubated for 20 to 24 hours at 37C and then viable counts were determined. Upon completion of the three scenarios described above (i .e., no protein, 2 g/dl, and 4 g/dl), additional bacterial time kill curves were performed to study the bacterial regrowth observed when no human serum albumin was added. In these experiments, 0.5*MIC, MIC, 1.5*MIC, 2*MIC, 2.5*MIC, 3*MIC, and 3.5*MIC were studied. In addition, negative and growth controls were performed.

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63 Microdialysis experiments During bacterial time kill curve experiments, a 2.5 mL sample of culture media was drawn out of each flask at 2, 6, 8, 10, 14, 18, and 24 hours for measurement of free drug concentrations. One of three probes was placed in a 5 mL glass tube containing 2.5 mL of the removed aliquot. After a 15 minute equilibration period, a dialysate sample was collected for 20 minutes In between each time point there was at leas t 5 minutes of flushing to prevent potential clogging of the microdialysis probe. Each sample was stored at 70C until the time of analysis. Additional experiments were performed to During the foll ow up experiments to study bacterial regrowth, microdialysis experiments were not performed. PK/PD modeling The data from the static time kill curves was analyzed in NONMEM version 7.2 using the first order conditional estimation method algorithm with AD VAN9. Perl speaks NONMEM was used for generation of visual predictive checks (PsN, Version 3.4.2) while the xpose4 and lattice packages in the software R were used for data visualization. 46 48 NONMEM workflow was managed using Piraa 51 A semimechanistic PK/PD model previously developed and validated by Neilsen et al. was fitted to the data. 52,53 In this model, the total bacterial population is divided into tw o subpopula tions; one which is growing (S) and another which is in a resting phase (R). Movement from the former to the latter is dictated by a rate constant, k SR which is dependent on the total bacterial concentration (i.e., S+R) as shown in Equation 3 3 The parameters k S and k D represent bacterial synthesis and death rate constants.

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64 3 3 For the PK model, drug concentrations measured using microdialysis were included in the data analysis. For time points wh ere free concentrations were not measured, the average of the previo us and later time points was used. To describe a delay in drug effect, an effect compartment (C e ) was added to the PK model, where movement between compartments is dictated by the rate con stant k e0 The drug effect was modeled using a sigmoidal E MAX model (Equation 3 4) where parameters represe nting the maximal drug effect (k MAX ) and the concentration at which half maximal effect is observed (EC 50 ) are estimated. To account for the observed bacterial regrowth, an adaption function was included to account for a change in the EC 50 w ith time. 54,55 As shown in equation 3 5 this adaptation function is a function of a maximal adaption () ). 3 4 3 5 Thus, the bacterial concentration in the growing and resting subpopulation s is dictated by equation s 3 6 and 3 7 3 6 3 7 Since experiments were performed in triplicate, three data points were available for each time point. The lower limit of detection (LOD) for the bacterial counts was set to

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65 20 CFU/ ML For b acterial counts below the LOD, 10 CFU/ ML was entered as first value in consecutive series while all other LOD data was omitted 56 Results Protein Binding Studies Ceftriaxo EE and RT was determined to be 75.2% and 82.1% respectively. The results of the protein binding studies are shown in Figure 3 1. In both the experiments with human serum albumin and pooled human plasma, as the protein concentration is increased Slightly greater binding is observed with pooled human plasma as compared to human serum albumin. With human plasma, a slight increase in the fraction unbound is observed as the drug concent ration is increased. Microbiological Experiments When determining the minimum inhibitory concentration, addition of human serum albumin resulted in a high er drug concentration needed to inhibit visible growth (Table 3 1) No difference was noted when co mparing 2 and 4 g/dl of human serum albumin. In the bacterial time kill curves, in the absence of human serum albumin, 4*MIC, 8*MIC, and 16*MIC resulted in no visible growth after 8 10 hours (Figure 3 2) For 2*MIC and MIC, regrowth was observed after 10 and 6 hours, respectively. With all other MIC multiples, bacterial growth was observed immediately (i.e., 2 hours). In the presence of 2 g/dl of human serum albumin, due to lower free concentrations, only the 16*MIC multiple resulted in no observable growt h after 10 hours (Figure 3 3) For 8*MIC, regrowth occurred after 6 hours of sampling. For all other multiples, bacterial growth occurred immediately. When 4 g/dl of human serum albumin was added, only

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66 16*MIC prevent ed regrowth; while regrowth was observed after 4 hours for the 8*MIC concentration (Figure 3 4) For all other MIC multiples, regrowth occurred after 2 hours. In the follow up experiments to study bacterial regrowth, only the highest drug concentration (i.e., 3.5*MIC) resulted in visible regrow th (Figure 3 5) Although the time to regrowth varied (3*MIC 20 hours, 2.5*MIC 10 hours, 2*MIC 8 hours, 1.5*MIC 8 hours, MIC 6 hours, 0.5*MIC 2 hours), for all other MIC multiples bacterial regrowth was observed As expected the addition of HSA impacted the antimicrobial efficacy of ceftriaxone. This reduced efficacy is to a decreased amount of free drug available in each flask. Mean free drug concentrations observed for the six highest drug concent rations are shown in Figures 3 6, 3 7, and 3 8 A s the p rotein concentration is increased there is an observable reduction in free ceftriaxone concentrations respectively. Parameter estimates for the PK/PD model describing the b acterial time kill curve data are shown in Table 3 2. Visual predictive checks for each scenario are shown in Figures 3 9 3 12 With the exception of the highest drug concentration in the presence of 4 g/dl HSA; it appears that the model results in a rea sonable fit of the observed data. Discussion The goal of the described experiments was to evaluate the role of variable protein binding on the antimicrobial efficacy of ceftriaxone using microdialysis. In the protein binding experiments, as the human ser um albumin concentration was increased, an increase in the protein binding of ceftriaxone was observed. When pooled human plasma was diluted, a similar relationship was observed; with slightly greater binding

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67 observed when compared to the human serum album in experiments. This difference can be explained by the availability of additional proteins in plasma which can interact with and bind to ceftriaxone. The observed decrease in free drug concentrations as protein concentrations were increased resulted in an resulted in a 2 4 fold increase in the MIC of the drug. No difference in the MIC was observed when comparing the 2 and 4 g/dl experiments. A lack of a difference may be attributed to the exper imental setup. Although the MIC provides a rough estimate of the concentration needed to inhibit visible growth, it may be difficult to observe the effect of relatively small differences in free drug concentration. Moreover, the tec hnique itself is subject to investigator bias as determination of this concentration is subjective Bacterial time kill curves were performed to account for changes in growth with time. Addition of HSA to the culture flasks resulted in an immediate growth or regrowth for all dru g concentrations minus the highest MIC multiple (i.e., 16*MIC). For the 2 and 4 g/dl scenarios, the major difference was faster regrowth in the 8*MIC flasks. Additional follow up experiments were then performed to evaluate the range of drug concentrations which resulted in bacterial regrowth (0.5*MIC 3.5*MIC). Regrowth was observed at all concentrations except 3.5*MIC. As the drug concentration was increased, the time to regrowth was prolonged. The observed regrowth is likely a result of resistance developm ent to ceftriaxone ; although this was not tested directly An additional goal of these studies was to evaluate the feasibility of using microdialysis to measure free drug concentrations in culture flasks during bacterial time kill curve experiments. Micro dialysis allowed for a measurement of free drug

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68 concentrations at the site of action; data which could then be correlated with the PD data using PK/PD modeling. A noticeable decease in free drug concentrations was observed as the protein concentration was increased. Significant variability was observed in the 4 g/dl scenario and it was difficult to differentiate culture flasks on the basis of free drug concentrations. In addition, when protein was added to the flasks, a noticeable increase in free drug conc entrations occurred at approximately 14 hours. Although follow up experiments have not been performed to study the mechanism of this change; one potential mechanism may be a conformational change in the structure of the protein which occurs halfway through the experiments.

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69 Table 3 1 Effect of human serum albumin on the minimum inhibitory concentration (MIC) of ceftriaxone against E. coli (ATCC 25922) Replicate Human Serum Albumin Concentration No Protein 2 g/dL 4 g/dL Trial 1 (01/21/2010) MIC ( mg /mL ) 0.125 0.25 0.25 MIC ( mg/mL ) 0.125 0.5 0.25 MIC ( mg/mL ) 0.125 0.25 0.25 Mode 0.125 0.25 0.25 Trial 2 (07/30/2010) MIC ( mg/mL ) 0.0625 0.25 0.5 MIC ( mg/mL ) 0.125 0.5 0.25 MIC ( mg/mL ) 0.0625 0.25 0.25 Mode 0.0625 0.25 0.25

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70 Table 3 2 Par ameter and relative standard error estimates for an effect compartment model developed to describe bacterial time kill curve data. Parameter Estimate RSE (%) k max (h ours 1 ) 1.19 14.6 EC 50 (g/ mL ) 0.04 31.6 k S (h ours 1 ) 4.38 6.8 N MAX (cfu/ mL ) 5.2 X 10 8 19.5 K d (h ours 1 ) 1.63 4.1 0.91 26.8 K e0 (h ours 1 ) 100 FIX 17.5 14.3 (L*g/ mL ) 0.08 20.9 Residual Error (LN cfu/ mL ) 2.54 7.3

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71 Figure 3 1. Protein binding experiments using human serum albumin and pooled human plasma. Mean fraction unbound is reported (n=3).

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72 Figure 3 2 Bacterial t ime curves in the absen ce of human serum albumin. The data plotted is the mean of experiments performed in triplicat e.

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73 Figure 3 3 Bacterial time curves in the presence of 2 g/dL of human serum albumin. The data plotted is the mean of experiments performed in triplicate.

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74 Figure 3 4 Bacterial time curves in the presence of 4 g/dL of human serum albumin. The data plotted is the mean of experiments performed in triplicate.

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75 Figure 3 5 Follow up bacterial time curves performed to stu dy the development of drug resistance to ceftriaxone in the absence of human serum albumin. The data plotted is the mean of expe riments performed in triplicate.

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76 Figure 3 6 Free ceftriaxone concentrations in the absence of human serum albumin and measur ed using microdialysis. The data plotted is the mean of experiments performed in triplicate. Expected concentrations for each flask were: C1 960 ng/ mL ; C2 480 ng/ mL; C3 240 ng/mL ; C4 120 ng/ mL ; C5 60 ng/ mL ; C6 30 ng/ mL

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77 Figure 3 7 Free ceftriaxone con centr ations in the presence of 2 g/dL of human serum albumin and measured using microdialysis. The data plotted is the mean of experiments performed in triplicate.

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78 Figure 3 8 Free ceftriaxone concentr ations in the presence of 4 g/dL of human serum alb umin and measured using microdialysis. The data plotted is the mean of experiments performed in triplicate

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79 Figure 3 9 Observed data (open circles) and 95% prediction intervals (shaded area) in the absence of human serum albumin. Individual plots repr esent the various MIC multiples.

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80 Figure 3 10 Observed data (open circles) and 95% prediction intervals (shaded area) in the presence of 2 g/dL human serum albumin. Individual plots represent the various MIC multiples.

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81 Figure 3 1 1 Observed data (op en circles) and 95% prediction intervals (shaded area) in the presence of 4 g/dL human serum albumin. Individual plots represent the various MIC multiples.

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82 Figure 3 12 Observed data (open circles) and 95% prediction intervals (shaded area) for bacteria l time kill curves performed to evaluate resistance development.

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83 CHAPTER 4 DEVELOPMENT AND VALI DATION OF ANALYTICAL METHODS TO QUANTIFY DICLOFENAC CONCENTRA TIONS IN MICRODIALYS IS AND PLASMA SAMPLE S Introduction Diclofenac sodium (2 (2 (2 ,6 dichloro phenylamino)phenyl)acetic acid) is a nonsteroidal anti inflammatory drug (NSAID) administered either locally or systemically for its analgesic and anti inflammatory properties It is commercially available as a sodium and potassium salt. Diclofenac is a we ak acid with pKa of 4 and a partition coefficient in n octanol/water of 13.4. 57 Follo wing a 50 mg oral dose of diclofenac, a maximal concentration of 7.8 g/ mL were observed ; whereas much lower peak plasma concentrations (0.7 6 ng/ mL ) were observed following administration as a transdermal patch (Flector) 58 Following oral administration, diclofenac has a half life of elimination and an oral bioavailab ility of approximately 1.8 hours and 50%, respectively. 58 The low oral bioavailability is largely a result of significant first pass metabolism. Several publications have described the development of bioanalytical methods to quantify diclofenac concentrations in plasma, urine, and microdialysis samples using high perfor mance liquid chromatography (HPLC) and liquid chrom atography mass spectroscopy (LC/ MS). 59 67 One study compar ed the use of an HPLC UV method and tandem mass spectrometry methods for detection of diclofenac in microdialysis samples. 60 A lower limit of quantification was observe d with the LC/MS method (10 vs. 1 ng/ mL ). In addition, a higher number of false positives and negative values were observed with the HPLC UV assay when analyzing biological samples collected in a clinical microdialysis study performed using Voltaren Emulg el. The major objectives of the studies described herein was to develop and validate two analytical methods in accordance to published guidelines set forth by the U.S.

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84 FDA 68 These methods would be used to quantify diclofenac concentrations in microdialysis and plasma samples collected from a clinical study in which a single dose of Flector is applied. Methods Chemicals an d Equipment The following chemicals and reagents were used: Water, double distilled, Corning AG 3 Methanol, Fis her, #A452 4 Hexane, Acros, #26836 0025 0.9% Sodium Chloride Injection USP (Saline), Premixed Bag, 500 mL Exp. January 2012, Baxter LOT C817619 Ammonium acetate, Fisher, #A639500 Acetic Acid, Glacial, Fisher, #A 38 O phosphoric acid 85%, Fisher, #A260 500 Aldrich D6899 10G, 127K1220 Indomet h Aldrich 17378 5G, 088K0666 The following equipment was used: Eppendorf Research (adjustable) pipettes, capacity of 20, 200 & 1000 l, Eppendor f Eppendorf Centrifuge, 5810R, Eppendorf Jouan Centrifuge Evaporator RC10 10 and Edwards RV5 Pump epT.I.P.S., 200 & 1000 l tips for Eppendorf Research pipettes, Eppendorf Vortex Maxi Mix II, Thermo Scientific Microcentrifuge Tubes with Flat Top Cap, 1.5 mL Fisher LOT 10430508 Centrifuge Tubes, 15 mL Corning 430055

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85 Centrifuge Tubes, 50 mL Corning 430828 Borosilicate Culture Tubes, 13 x 100 mm, Fisher Vial, Crimp/Snap, 12x32, 300L, PP, Concial, Sun Sri TM 500 102, LOT 00117973 Seal, AL Crimp, 11mm, TFE/Re d Rubber, Silver, Sun Sri TM 200 100, LOT 00118189 PE200 series pump Autosampler, Perkin Elmer, Norwalk, CT, USA Symmetry C 18 column (4.6 x 50 mm, 3.5 m particle size), Waters, Dublin, Ireland, Part No. WAT200625, Lot No. 0196302231 0.2 m filter, Milli pore, Cork, Ireland Triple quadrupole API 4000 mass spectrometer, Applied Biosystems, Carlsbad, CA, USA Leybold TW 700 turbo molecular pump, Oerlikon Leybold, Cologne, Germany Microdialysis Samples Reagent preparation A primary stock diclofenac solution (1 mg/ mL ) was prepared by dissolving 10 mg of diclofenac sodium in 10 mL of a one to one mixture containing methanol and deionized water. Similarly, a primary stock solution of indomethacin (1 mg/ mL ), an internal standard in our method was prepared by mix ing 10 mg of indo methacin with 10 mL of methanol. Then a secondary indomethacin stock solution was prepared by mixing 100 L of the primary stock solution with 9.9 mL of a one to one mixture con taining methanol and sodium chloride 0.9% (WS) of indomethacin was prepared by adding 200 L of the secondary stock with 39.8 mL of a one to one mixture con taining methanol and 0.9% sodium chloride

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86 This WS containing indomethacin was then used to dilute the primary diclofenac solution. A secondary diclofenac study was prepared by adding 10 L of diclofenac primary stock with 990 L of WS Then a third diclo fenac stock was prepared by adding 100 L of the secondary diclofenac stock to 990 L of WS Calibration and QC samples were prepared as described below. Double Blank: 100 l MeOH: Saline (1:1) (double blank) Blank: 100 l working solutio n (blank) Standard 1 (100 ng/ mL ): Third Stock (100 L) is added to 900 L WS (C1) Standard 2 (50 ng/ mL ): Standard 1 (500 L) is added to 500 L WS (C2) St andard 3 (25 ng/ mL ): Standard 2 (500 L) is added to 500 L WS (C3) Standard 4 (12.5 ng/ mL ): Standard 3 (500 L) is added to 500 L WS (C4) Standard 5 (6.25 ng/ mL ): Standard 4 (500 L ) is a dded to 500 L W S (C5) Standard 6 (3.13 ng/ mL ): Standard 5 (500 L) is added to 500 L WS (C6) Standard 7 (1.56 ng/ mL ): Standard 6 (500 L) is added to 500 L WS (C7) Standard 8 (0.78 ng/ mL ): Standard 7 (500 L) is added to 500 L WS (C8) Standard 9 (0.39 ng/ mL ): Standard 8 (500 L) is added to 500 L WS (C9) QC1 (80 ng/ mL ): Third Stock (80 L) is added to 920 L of WS (HQC) QC2 (10 ng/ mL ): Quality Control 1 (125 L) is added to 875 L of WS (MQC1) QC 3 (4 ng/ mL ): Quality Control 2 (400 L) is added to 600 L of WS (MQCC2) QC 4 (1 ng/ mL ): Quality Control 2 (100 L) is added to 900 L of WS (LQC) QC5 (0.39 ng/ mL ): Standard 8 (100 L ) is added to 100 L of WS (LLOQ) During the method validation process, at least one calibration curve and three sets of quality co ntrol samples were prepared and assessed for accuracy and precision. Following preparation in micro centrifuge tubes, 100 L of each sample was transferred to 300 L vials. Method conditions As the mobile phase, HPLC grade methanol and 10 mM ammonium acet ate buffer adjusted to pH 4.2 was used at a constant ratio of 75:25, respectively. The ammonium acetate buffer was prepared by weighing 385.5 mg of HPLC grade ammonium acetate and dissolving it in 500 mL of deionized water. The pH was adjusted to 4.2 using acetic

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87 acid. The buffer was filtered and sonicated for 15 minutes. A flow rate of 0.45 mL /min was used. All samples were analyzed using a triple quadrupole API 4000 mass spectrometer. The system was operated in multiple reaction monitoring (MRM) mode and was operated using the Analyst software version 1.4.2 (MDS Sciex, Toronto, Canada). P recursor and product ions were monitored for a dwell time of 250 ms at m/z 296.1/215 m/z 296.1/249.9, and m/z 358.2/139 in positive ion mode For each of these precursor and product ion combinations, the declustering potential (DP) was set to 35, 35, and 65 volts; while the collision energy was 29, 19, and 25 volts. A run time and injection volume of 6 minutes and 10 L was used, respectively. Stability studies Stability samples were conducted to evaluate the stability of diclofenac in a working solution containing a one to one mixture of methanol and 0.9% sodium chloride. Short and long term, freeze thaw, and prima ry stock stability studies were conducted. For the short term stability studies, stability was evaluated following 24 hours of storage in the auto sampler. Long term stability was assessed following 30 days of storage at 70C. Samples were exposed to thre e freeze thaw cycles were they were frozen at 70C, the n thawed and analyzed after three cycle s Primary stock stability was assessed by diluting th e primary stock to 100 ng/ mL and then comparing the observed concentrations on day 1 and day 8. Except for the primary stock stability, all stability studies were also studied with addition of dextran 3% Dextran may be added to the perfusate in clinical microdialysis studies in order to prevent ultrafiltration.

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88 Plasma Samples Reagent preparation The mobile ph ase consisted of HPLC grade methanol and 10 mM ammonium acetate adjusted to pH 4.2. Preparation of the ammonium acetate buffer was previously described. Indomethacin was again used as the internal standard in this method. First, (1 mg/ mL ) was prepared by dissolving 10 mg of indomethacin in 10 mL of (10 mL ) was prepared by adding 100 L of IS Stock 1 to 9.9 mL methano mL ) was prepared using 150 L of IS Stock 2 and 1.85 mL of c old methanol. Diclofenac stock solutions were prepared. A diluent of deionized water and methanol in a one to one mixture was prepared. Then primary and secondary drug stocks were prepared similar to indomethacin using a one to one mixture of deionized w ater and methanol. A third drug stock (1000 ng/ mL ) was prepared using 1 mL of the secondary stock and 9 mL of diluent. Next, diclofenac working stocks ( D WS) were prepared as sh own below. D WS1 (1000 ng/ mL ): 4 mL of third stock D WS2 (500 ng/ mL ): 2 mL WS1 pl us 2 mL diluent D WS3 (250 ng/ mL ): 2 mL WS2 plus 2 mL diluent D WS4 (125 ng/ mL ): 2 mL WS3 plus 2 mL diluent D WS5 (62.5 ng/ mL ): 2 mL WS4 pus 2 mL diluent D WS6 (31.25 ng/ mL ): 2 mL WS5 plus 2 mL diluent D WS7 (15.63 ng/ mL ): 2 mL WS6 plus 2 mL diluent Using thes e working stocks, calibration standards and QC samples were prepared as follows:

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89 Double Blank: 2 00 l p lasma Blank: 2 00 l p lasma is added to 10 l IS WS Standard 1 (100 ng/ mL ): 20 L DWS1 added to 18 0 L plasma and 10 L IS WS Standard 2 (50 ng/ mL ): 20 L DWS2 added to 18 0 L plasma and 10 L IS WS Standard 3 (25 ng/ mL ): 20 L DWS3 added to 18 0 L plasma and 10 L IS WS Standard 4 (12.5 ng/ mL ): 20 L DWS4 added to 18 0 L plasma and 10 L IS WS Standard 5 (6.25 ng/ mL ): 20 L DWS5 added to 18 0 L plasma and 10 L IS WS Standard 6 (3.13 ng/ mL ): 20 L DWS6 added to 18 0 L plasma and 10 L IS WS Standard 7 (1.56 ng/ mL ): 20 L DWS7 added to 18 0 L plasma and 10 L IS WS HQC (80 ng/ mL ): 16 L D WS 1 is added to 184 L plasma and 10 L IS WS MQC (5 0 ng/ mL ): 20 L D WS2 is added to 18 0 L plasma and 10 L IS WS LQC (5 ng/ mL ): 20 L QC2 is added to 18 0 L plasma and 10 L IS WS LLOQ (1 .56 ng/ mL ): 20 L D WS7 is added to 18 0 L plasma and 10 L IS WS QC samples were prepared in bulk. Then precision accuracy batches (PA) were analyzed on three separate days. For each PA batch, QC samples wer e prepared six times using the extraction process detailed below (steps 1 17). Similarly, for preparation of calibration standards (CS), Steps 3 17 were followed. Step 1: Remove 200 L out of each bulk six times (HQC, MQ C, LQC, LLOQ) Step 2: Add 10 L of IS (35 ng/ mL ) to each sample Step 3 : Vortex for 10 seconds Step 4 : Add 5 0 L of 1:1 dilution of 85% o phosphoric Acid Step 5 : Vortex each tube for 30 seconds Step 6 : Add 2 mL of h exane Step 7 : Vortex for 2 min utes Step 8 : Centrifuge at 2000xg for 10 min utes Step 9 : Separate all supernatant with glass Pasteur pipettes Step 10 : Evaporate all samples to dryness under vacuum Step 11 : Add 2 mL of h exane Step 12 : Vortex for 2 min utes Step 13 : Centrifuge at 2000xg f or 10 min utes Step 14 : Separate all supernatant with glass Pasteur pipettes Step 15 : Evaporate all samples to dryness under vacuum Step 16 : Reconstitute in 100 l of diluent (TDW: MeOH, 50:50) Step 17: Place 100 l in a vial for analysis.

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90 The impact of the matrix was evaluated by preparing QC samples in diluent and spiking each QC in blank plasma processed as shown above. M atrix effect QC samples were prepared as follows: HQC (80 ng/ mL ): 16 l WS1 is added to 74 L diluent and 10 L IS WS MQC (5 0 ng/ mL ): 20 l WS2 is added to 70 L diluent and 10 L IS WS LQC (5 ng/ mL ): 20 l MQC is added to 70 L diluents and 10 L IS WS L LOQ (1.56 ng/ mL ): 20 l WS7 is added to 70 L diluent and 10 L IS WS These samples were then added to plasma processed as follows : Step 1: 200 L of blank human p lasma is placed in 24 glass tubes Step 2 : Add 5 0 l of 1:1 dilution of 85% o phosphoric Acid Step 3 : Vortex for 30 seconds Step 4 : Add 2 mL of h exane Step 5 : Vortex for 2 min utes Step 6 : Centrifuge at 2000xg for 10 min utes Step 7 : Separate all supernatant with glass Pasteur pipettes Step 8 : Evaporate all samples to dryness under vacuum Step 9 : Add 2 mL of h exane Step 10 : Vortex for 2 min utes Step 11 : Centrifuge at 2000xg for 10 min utes Step 12 : Separate all supernatant with glass Pasteur pipettes Step 13 : Evaporate all samples to dryness under vacuum Step 14 : Reconstitute in 100 L of each QC sample Step 15 : Place 100 l of each sample in a vial for injection Last, to calculate the recovery of the drug following this extraction process, quality control samples were prepared in diluents as described above and injected directly Stability studies Studies were conducted to evaluate the short term and freeze thaw stability of diclofenac in plasma samples. For the short term stability studies, stability was evaluated following 24 hours of storage in the auto sampler. Samples were exposed to three freeze thaw cycles were they were frozen at 70C, then thawed and analyzed after three cycles.

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91 Results During validation of an analytical method to quantify diclofenac in microdialysis samples, accuracy and precision were assessed. A ll ca libration standards were within 15% accuracy on three separate days (Table 4 1). Three sets of quality controls were analyzed on each validation day. Of these samples, no more than one quality control sample per day failed to meet accuracy standards (Table s 4 2 and 4 3). Inter day precision was less than 15% for all quality controls (Table 4 4) Short and long term, freeze thaw, and primary stock stability studies resulted in acceptable accuracy and precision values (Tables 4 5 4 8). An analytical metho d was also developed to quantify diclofenac concentrations in plasma samples. Again accuracy and precision values for the calibration standards and quality controls met acceptability criteria set forth by the U.S. FDA ( Tables 4 9, 4 10, and 4 11). Diclofen ac appeared stable in plasma following 24 hours of storage in an autosampler and 3 freeze thaw cycles (Tables 4 12 and 4 13). The average recovery of the analyte was approximately 62% ; an average matrix enhacement of 39% was observed (Figure 4 1). Greater matrix enhancement was observed at lower diclofenac concentrations. Discussion The two developed analytical methods met acceptable standards during three days of validation. Although the recovery values were relatively low, they were reproducible. Signif icant matrix enhancement was observed ; although it was more pronounced at low diclofenac concentrations.

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92 Table 4 1 Calibration curves for microdialysis samples analyzed validation Days 1 3 Sample Name Sample Type Calculated Concentration (ng/mL) Accuracy (%) Day 1 Double Blank Double Blank N/A N/A Blank Blank N/A N/A 0.390 Stand ard 0.387 99.3 0.781 Standard 0.817 105. 1.562 Standard 1.55 99.0 3.125 Standard 2.81 89.9 6.25 Standard 6.12 98.0 12.5 Standard 12.7 101. 25 Standard 25.9 104. 50 Standard 50.2 100. 100 Standard 104. 104. DAY 2 Double Blank Double Blank N/A N /A Blank Blank N/A N/A 0.390 Standard 0.411 105. 0.781 Standard 0.739 94.6 1.562 Standard 1.47 94.3 3.125 Standard 3.03 96.9 6.25 Standard 6.19 99.1 12.5 Standard 12.5 100. 25 Standard 25.3 101. 50 Standard 50.8 102. 100 Standard 103. 103. DAY 3 Double Blank Double Blank N/A N/A Blank Blank N/A N/A 0.390 Standard 0.339 87.0 0.781 Standard 0.684 87.6 1.562 Standard 1.61 103. 3.125 Standard 2.84 90.7 6.25 Standard 5.84 93.4 12.5 Standard 12.0 96.2 25 Standard 24.1 96.3 50 Standard 46.6 93.2 100 Standard 96.5 96.5

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93 Table 4 2 Quality control samples analyzed on validation D ays 1 and 2. Sample Name Sample Type Calculated Concentration (ng/mL) Accuracy (%) Day 1 LLOQ Quality Control 0.401 103. LQC Quality Control 1. 33 133. MQC2 Quality Control 4.29 107. MQC1 Quality Control 10.8 108. HQC Quality Control 88.6 111. LLOQ Quality Control 0.416 107. LQC Quality Control 1.03 103. MQC2 Quality Control 4.24 106. MQC1 Quality Control 10.8 108. HQC Quality Control 90.2 113. LLOQ Quality Control 0.433 111. LQC Quality Control 1.13 113. MQC2 Quality Control 4.29 107. MQC1 Quality Control 11.4 114. HQC Quality Control 86.0 108. Day 2 LLOQ Quality Control 0.408 105. LQC Quality Control 0.958 95.8 MQC2 Quality Co ntrol 3.76 94.1 MQC1 Quality Control 10.1 101. HQC Quality Control 78.7 98.4 LLOQ Quality Control 0.397 102. LQC Quality Control 0.982 98.2 MQC2 Quality Control 4.07 102. MQC1 Quality Control 10.3 103. HQC Quality Control 81.3 102. LLOQ Quality Con trol 0.390 100 LQC Quality Control 1.01 101 MQC2 Quality Control 3.25 81.3 MQC1 Quality Control 9.07 90.7 HQC Quality Control 86.1 108

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94 Table 4 3 Quality control samples analy zed on validation D ay 3. Sample Name Sample Type Calculated Concentration (ng/mL) Accuracy (%) Day 3 LLOQ Quality Control 0.444 114. LQC Quality Control 0.931 93.1 MQC2 Quality Control 3.83 95.6 MQC1 Quality Control 9.74 97.4 HQC Quality Control 79.9 99.9 LLOQ Quality Control 0.436 112. LQC Quality Control 1.03 103. MQC2 Quality Control 3.98 99.4 MQC1 Quality Control 9.63 96.3 HQC Quality Control 80.9 101. LLOQ Quality Control 0.372 95.3 LQC Quality Control 1.01 101. MQC2 Quality Control 3 .96 98.9 MQC1 Quality Control 9.68 96.8 HQC Quality Control 81.1 101.

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95 Table 4 4 Inter day precision for microdialysis samples analyzed validation Days 1 3. Coefficient of Variation [%] Day 1 Day 2 Day 3 LLOQ 3.84 8.54 10.11 LQC 13.13 5.53 7.48 MQC2 0.68 9.11 3.22 MQC1 3.15 5.65 2.56 HQC 2.40 3.32 5.59

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96 Table 4 5 Short term stability studies of diclofenac in microdialysis samples stored in an auto sampler for 24 hours. Each concentration was prepared in triplicate. Sample Name Observed Concentration (0 hours) Observed Concentration (24 hours) No Dextran ST LQC a 0.851 0.948 ST LQC b 0.894 1.02 ST LQC c 0.813 1.07 ST HQC a 76.3 75.5 ST HQC b 77.9 76.6 ST HQC c 78.8 79.0 Mean L 0.85 1.01 SD 0.04 0.06 Precision 4.75 % 6.06 % Accuracy 85.27 % 101.27 % Mean H 77.67 77.03 SD 1.27 1.79 Precision 1.63 % 2.32 % Accuracy 97.08 % 96.29 % Dextran Added ST LQC a D 0.95 0.826 ST LQC b D 0.999 0.96 ST LQC c D 1.09 0.832 ST HQC a D 78.0 82.7 ST HQC b D 85.9 89.1 ST HQC c D 81.6 87.2 Mean L 1.01 0.87 SD 0.07 0.08 Precision 7.01 % 8.67 % Accuracy 101.30 % 87.27 % Mean H 81.83 86.33 SD 3.96 3.29 Precision 4.83 % 3.81 % A ccuracy 102.29 % 107.92 %

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97 Table 4 6 Long term stability studies of diclofenac in microdialysis samples stored at 7 0C for 30 days Each concentration prepared in triplicate. Sample Name Observed Concentration (0 hours) Observed Concentration (24 hours) No Dextran LT LQC a 1.03 0.88 LT LQC b 1.03 0.871 LT LQC c 1.12 0.973 LT H QC a 77.3 75.9 LT HQC b 77.0 76.7 LT HQC c 79.0 75.1 Mean L 1.06 0.91 SD 0.05 0.06 Precision 4.90 % 6.22 % Accuracy 106.00 % 90.80 % Mean H 77.77 75.90 SD 1.08 0.80 Precision 1.39 % 1.05 % Accuracy 97.21 % 94.88 % Dextran Added LT LQC a D 0.9 43 1.07 LT LQC b D 0.857 1 LT LQC c D 0.722 0.808 LT HQC a D 81 87.2 LT HQC b D 84 94.1 LT HQC c D 86.6 97.5 Mean L 0.90 0.96 SD 0.06 0.14 Precision 6.76 % 14.14 % Accuracy 90.00 % 95.93 % Mean H 83.87 92.93 SD 2.80 5.25 Precision 3.34 % 5.65 % Accuracy 104.83 % 116.17 %

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98 Table 4 7 Freeze thaw stability studies of diclofenac in mi crodialysis samples frozen at 7 0C. Sample Name Observed Concentration (0 hours) Observed Concentration (cycle 3) No Dextran FT LQC a 1.16 0.883 FT LQC b 0.99 7 0.971 FT LQC c 0.942 0.783 FT HQC a 81.4 73.1 FT HQC b 86.0 75.5 FT HQC c 80.7 76.9 Mean L 1.03 0.88 SD 0.11 0.09 Precision 10.97 % 10.70 % Accuracy 103.30 % 87.90 % Mean H 82.70 75.17 SD 2.88 1.92 Precision 3.48 % 2.56 % Accuracy 103.38 % 93 .96 % Dextran Added FT LQC a D 1.25 1.44 FT LQC b D 1.13 1.02 FT LQC c D 1.01 0.907 FT HQC a D 79.1 67.4 FT HQC b D 81.1 68.3 FT HQC c D 81.9 70.2 Mean L 1.13 1.12 SD 0.12 0.28 Precision 10.62 % 25.02 % Accuracy 113.00 % 112.23 % Mean H 80.70 68.63 SD 1.44 1.43 Precision 1.79 % 2.08 % Accuracy 100.88 % 85.79 %

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9 9 Table 4 8 Primary stock stability studies of diclofenac in micr odialysis samples stored in at 2 to 8 C for 7 days. Sample Name Observed Concentration (0 hours) Observed Concentra tion (24 hours) PS a 112.00 104.00 PS b 112.00 108.00 PS c 115.00 113.00 Mean 113.00 108.33 SD 1.73 4.51 Precision 1.53 % 4.16 % Accuracy 113.00 % 108.33 %

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100 Table 4 9 Calibration curves for plasma samples analyzed on validation Days 1 3. Sample Name Sample Type Calculated Concentration (ng/mL) Accuracy (%) Day 1 Double Bl ank Double Blank N/A N/A Blank Blank N/A N/A 1.562 Standard 1.62 104 3.125 Standard 2.98 95.4 6.25 Standard 5.56 89 12.5 Standard 14.1 113 25 Standard 24.1 96.6 50 Standard 47.9 95.8 100 Standard 126 126 DAY 2 Double Blank Double Blank N/A N/A Blank Blank N/A N/A 1.562 Standard 1.41 90.5 3.125 Standard 3.6 115 6.25 Standard 6.98 112 12.5 Standard 11.7 93.9 25 Standard 24 95.9 50 Standard 33 66.1 100 Standard 97 97 DAY 3 Double Blank Double Blank N/A N/A Blank Blank N/A N/A 1.562 Standard 1.71 110 3.125 Standard 3.21 103 6.25 Standard 6.27 100 12.5 Standard 11.9 95 25 Standard 25.3 101 50 Standard 54.3 109 100 Standard 107 107

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101 Table 4 10 P recision accuracy (PA) batches for plasma samples analyzed on validation Days 1 3. LLOQ LQC MQC HQC 1.56 5 50 80 [DRUG] ACCURACY (%) [DRUG] ACCURACY (%) [DRUG] ACCURACY (%) [DRUG] ACCURACY (%) PA 1 1.85 118.60 5.12 102.47 42.22 84.44 69.01 86.26 2 .40 153.84 3.51 70.22 40.00 80.01 65.76 82.20 1.60 102.25 4.36 87.20 40.40 80.81 66.27 82.84 1.81 116.27 4.25 85.04 42.59 85.17 66.07 82.59 1.74 111.50 4.54 90.71 55.18 110.37 81.37 101.71 1.50 96.20 5.16 103.22 55.69 111.39 78.65 98.31 PA 2 1.12 71.75 5.77 115.50 46.24 92.48 80.23 100.28 1.42 91.01 3.64 72.78 50.55 101.09 83.99 104.99 1.33 85.36 4.49 89.75 39.86 79.71 99.52 124.40 1.30 83.56 4.29 85.77 44.09 88.17 80.21 100.26 1.37 87.99 4.60 91.95 42.16 84.33 81.50 101.87 1.59 101.88 4. 34 86.82 37.58 75.16 78.51 98.14 PA 3 1.64 105.43 4.52 90.30 51.47 102.93 69.43 86.78 1.67 107.20 4.90 97.90 40.92 81.84 69.60 87.00 2.64 169.30 4.10 82.02 50.01 100.02 69.12 86.41 1.56 100.28 5.09 101.74 49.68 99.35 68.00 85.00 1.58 101.10 4.70 9 3.91 45.93 91.85 71.77 89.71 1.80 115.36 3.89 77.71 51.01 102.02 NA NA N 18.00 18.00 18.00 18.00 18.00 18.00 17.00 17.00 MEAN (ng/mL) 1.66 106.60 4.51 90.28 45.87 91.73 75.24 94.04 SD (ng/mL) 0.37 23.65 0.56 11.28 5.58 11.16 8.93 11.16 CV (%) 22.18 2 2.18 12.49 12.49 12.17 12.17 11.87 11.87

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102 Table 4 11 Inter day precision for plasma samples analyzed on validation Days 1 3. Coefficient of Variation [%] Day 1 Day 2 Day 3 LLOQ 17. 34 11. 34 22. 71 LQC 13. 68 15. 46 10. 22 MQC 16. 02 1. 69 8. 42 HQC 9. 81 9. 31 1. 97

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103 Table 4 12 Short term stability studies of diclofenac in plasma samples stored in an auto sampler for 24 hours. Each concentration prepared in triplicate. Sample Name Observed Concentration (0 hours) Observed Concentration (24 hours) Sampl e Name Observed Concentration (0 hours) Observed Concentration (24 hours) LLOQ 1 1.85 1.69 MQC 1 42.20 43.80 LLOQ 2 2.40 2.24 MQC 2 39.90 42.60 LLOQ 3 1.59 1.48 MQC 3 40.50 38.40 LLOQ 4 1.81 1.83 MQC 4 42.50 42.70 LLOQ 5 1.74 1.73 MQC 5 55.20 51.60 LLOQ 6 1.50 1.59 MQC 6 55.60 55.80 Mean (ng/mL) 1.82 1.76 Mean (ng/mL) 45.98 45.82 SD (ng/mL) 0.32 0.26 SD (ng/mL) 7.36 6.52 CV (%) 17.40 15.00 CV (%) 16.01 14.22 Accuracy (%) 116.35 112.82 Accuracy (%) 91.97 91.63 LQC 1 5.11 4.59 HQC 1 68.90 61.90 L QC 2 3.51 4.23 HQC 2 65.60 64.00 LQC 3 4.35 4.36 HQC 3 66.10 61.00 LQC 4 4.24 4.48 HQC 4 66.10 69.70 LQC 5 4.53 3.96 HQC 5 81.50 78.00 LQC 6 5.16 5.08 HQC 6 78.50 78.80 Mean (ng/mL) 4.48 4.45 Mean (ng/mL) 71.12 68.90 SD (ng/mL) 0.61 0.38 SD (ng/mL) 7 .04 7.96 CV (%) 13.67 8.49 CV (%) 9.90 11.55 Accuracy (%) 89.67 89.00 Accuracy (%) 88.90 86.13

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104 Table 4 13 Freeze thaw studies of diclofenac in plasma samples. Sample Name Observed Concentration for PA 1 (ng/mL) Observed Concentration After 3 F /T Cycles (ng/mL) Sample Name Observed Concentration for PA 1 (ng/mL) Observed Concentration After 3 F/T Cycles (ng/mL) LQC 1 (F/T) 5.12 5.21 HQC 1 (F/T) 69.01 82.50 LQC 2 (F/T) 3.51 5.36 HQC 2 (F/T) 65.76 92.40 LQC 3 (F/T) 4.36 5.87 HQC 3 (F/T) 66.27 75.80 LQC 4 (F/T) 4.25 5.69 HQC 4 (F/T) 66.07 90.90 LQC 5 (F/T) 4.54 4.77 HQC 5 (F/T) 81.37 62.60 LQC 6 (F/T) 5.16 5.55 HQC 6 (F/T) 78.65 71.80 Mean (ng/mL) 4.49 5.41 Mean (ng/mL) 71.19 79.33 SD (ng/mL) 0.61 0.39 SD (ng/mL) 6.99 11.52 CV (%) 13.68 7. 22 CV (%) 9.81 14.52 Accuracy (%) 89.81 108.17 Accuracy (%) 88.99 99.17 MQC 1 (F/T) 42.22 43.10 MQC 2 (F/T) 40.00 48.60 MQC 3 (F/T) 40.40 62.60 MQC 4 (F/T) 42.59 47.40 MQC 5 (F/T) 55.18 46.00 MQC 6 (F/T) 55.69 55.70 Mean (ng/mL) 4 6.02 50.57 SD (ng/mL) 7.37 7.23 CV (%) 16.02 14.31 Accuracy (%) 92.03 101.13

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105 Figure 4 1. Percentage recovery and matrix effect following diclofenac extraction from plasma.

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106 CHAPTER 5 IN VITRO STUDIES TO EVALUATE THE FEASIBI LITY OF USI NG FLECTOR IN A CLINICAL MICRODIALYS IS STUDY TO EVALUATE TOPICAL BIOEQUIVALENCE Introduction Microdialysis is a technique used to quantify free drug concentrations in both in vitro and in vivo settings. 69 73 When used in vivo concentration s of endogenous and exogenous compounds can be quantified in most tissues 74 79 T hus the technique is commonly used for assessment of drug delivery, distribution kinetics and determination of drug concentrations in tissue fluids. Microdialysis may be applied in drug discovery, preclinical, and clinical stages of drug development In an in vivo microdialysis study, a probe with a semi permeable membrane is placed in the tissue. A syringe pump delivers the perfusate to the probe at a constant flow rate where i t enters through an inlet and travels down the probe allowing for passive diffusion to occur Dialysate samples are collected at pre defined intervals. During passive diffusion an exchange between the peri probe fluid (fluid immediately surrounding the p robe) and perfusate occurs. However, at commonly used flow rates ( ~ 1.5 L/min) and membrane lengths (3 10 nm), an equilibrium between the extracellular fluid and the perfusate does not occur. In other words, the analyte concentration inside and outside the probe will not be the same in most cases. The concept of recovery is used to relate these two concentrations and tissue concentration. In vitro microdialysis experiments are usually done beforehand in order to determine the feasibilit y of performing the experiments in vivo These experiments are especially important when you are dealing with a novel compound whose recovery and binding characteristics with a microdialysis probe are unknown. Also, since the analyte

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107 recovery may be studied. This includes the effects that flow rate, perfusate, drug, and membrane characteristics may have on relative recovery. RT and EE are frequently used to determine in vitr o recovery. During in vitro EE experiments, the drug of interest is dissolved in a physiological media which surrounds the probe. The goal is these experiments is to mimic what occurs in vivo where a microdialysis probe is placed in a tissue containing a n analyte of interest. The percent recovery (R %) is calculated as the ratio between the drug concentration measured in the dialysate and the known concentration in the physiological medium surrounding the probe (Equation 5 1). 5 1 For RT experiments drug is dissolved in the perfusate and infused through the probe at a cons tant flow rate. This method is typically used in vivo to calculate recovery. The percent recovery (R %) is calculated as the ratio of drug concen tration in the dialysate and the known concentrat ion in the perfusate (Equation 5 2 ). 5 2 The goal of the studies described herein was to study the in vitro recovery and dissolution profile of dicofenac sodium, a non st eriodal a nti inflammatory drug (NSAID), prior to conducting a clinical microdialysis study using the product Flector. This product is a transdermal patch containing 180 mg of diclofenac epolamine and is indicated for the treatment of minor strains, sprain s, and contusions 80 In addition to

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108 studying the dissolution profile of a Flector patch, a method was developed to quantify the residual cont ent in used Flector patches Methods In Vitro Microdialysis Studies Six rounds of recovery experiments were performed. For each round, the recovery of diclofenac using a CMA 66 microdialysis probe was determined in triplicate using both EE and RT experim ents. Dextran was added in varying concentration to prevent ultrafiltration across the membrane. Reagent preparation For the first three rounds, EE and RT experiments were performed using diclofenac solutions containing 5, 10, and 25 ng/ mL These concentr ations were attained by preparing a 1 mg/ mL drug solution in methanol and water mixture (50:50); and 10 g/ mL an d 1 g/ mL drug solutions in sodium chloride 0.9% For the EE experiments, t he perfusate was made up of dextran ( 3%, low fraction) solution in so dium chloride 0.9% For the RT experiments, the three desired drug con centrations were prepared by diluting the 1 g/ mL dru g solution with a solution of dextran 3% in sodium chloride 0.9% Rounds four through six were performed with diclofenac concentrati ons of 2 15, and 40 ng/ mL Dextran concentrations were also varied between 1 3%. The desired drug concentrations were prepared by diluting a 1 g/ mL diclofenac drug solution in sodium chloride 0.9% for the EE experimen ts and a dextran containing sodium ch loride 0.9% solution for RT.

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109 Study protocol For EE evaluation, the probe is placed in a solution containing diclofenac dissolved in sodium chloride 0.9% ; while a perfusate (dextran in sodium chloride 0.9% ) is infused at 1.5 l/min. In round 1, an equilibr ation period of 15 minutes was performed, while 30 minutes was used for all other rounds. Following the equilibration period, two dialysate samples were collected, each 20 minutes apart. Once sample collection is complete, the probe is transferred to the n ext highest drug concentration, and another 30 minute equilibration period is performed before beginning sample collection. Once all three concentrations have been evaluated, the experiments are repeated two additional times. In RT scenario, the same proce dure was followed, except that drug syringes were switched for each drug concentration. In addition, the pr obe was placed in a sodium chloride 0.9% solution following completion of each concentration. Dissolution Studies Chemicals and equipment The following chemicals and reagents were used: Water, double distilled, Corning AG 3 Methanol, Fis her, #A452 4 Ammonium acetate, Fisher, #A639500 Aldrich D6899 10G, 127K1220 Indomet h Aldrich 17378 5G, 088K0666 7 Flector patches, King Pharmaceuticals, Inc The following equipment was used: Vanderkamp 600, six spindle disso lution tester, QC 0372 Sonicater Fisher Scientific, FS 110H 3 White Back Red Spirit Lab Thermometers, Fisherbrand 305 mm Bulb pipette 10 mL 20 mL 25 mL Microcentri fuge Tubes with Flat Top Cab, 1. 5 mL Fisher Centrifuge Tubes 15 mL Corning 430055 Researc h adjustable pipettes, capacity of 20,200 & 1000 l, Eppendorf

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110 Redi Tip TM general purpose pipet tips 200 &1000l, Fisher Scientific Vortex Maxi Mix II, Thermo Scientific Vial, Crimp/Snap 12x32, 300l,PP ,Concial, Sun Sri TM 500 102 Seal, AL Crimp, 11 mm, T FE/ Red Rubber, Silver Sri TM 200 100 Aluminum screening Plastic fishing line, diameter 0.11 mm 35 Blue Vinyl Electrical Tape (19 mm) Experimental setup Each of the six vessels in the Vanderkamp 600 was filled with 1 L of heated double distilled water fol lowing placement of a modified patch holder A constant temperature of 32C wa s maintained in each vessel. A modified patch holder was created in order to accommodate the siz e of each vessel. Briefly, a rectangular piece of aluminum window screen (18x65 cm ) was cut and sown together in a circular fashion using plastic finishing line. For each vessel, a Flector patch was opened and the drug containing side of the patch was affixed to the circular aluminum mesh using electrical tape. The modified patch holde r assembly was then placed in the vessel. Study protocol Once the patch holder was in place and the vessel filled with water, the power was switched on; the paddle speed was adjusted to 50 rpm ; and the paddle lowered so that it is approximately halfway d mL aliquots were taken at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 hours. The samples were frozen and analyzed at a later date using the previously described LC/MS method. Before analysis, the samples were diluted in an effort to obtain drug concentrations in the range of the validated method. The results were plotted using the lattice and ggplot 2 packages in the statistical software R. 45,81

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111 Residual Content Studies The goal of these studies was to develop a method to quantify diclofenac concentrations in unused and used Flector patches. Four separate methods were identified and tested. 82 85 These methods differed in the extracting agent and extraction time used. Method A used 0.1 M hydrochloric acid in m ethanol and a 2 hour extraction time; Methods B and C both used methanol and 1 and 2 hour ext raction times respectively ; Method D used 2 p ropanol and an extraction time of 16 hours. Three patches were cut into 16 pieces and all four methods were tested. The method tested on each piece was pre defined in an effort to test each method on different corners of the patch. Each piece was placed in a 50 mL centrifuge tube; 30 mL of extracting agent was added; and each tube was placed in a s haker ( Eberbach Corporation, Ann Arbor, Michigan ) for the pre defined extraction time. When complete, a 1 mL aliquot was taken from each tube a nd centrifuged ( Marathon 16KM, Fisher Scientific ) at 12,000 rpm for 10 minutes. Samples were then frozen until the time of analysis. Before analyzing, samples were diluted so that the analyzed concentrations were within the limits of the validated method. Based on preliminary studies, method C (methanol, 2 hour extraction time) was chosen for further development and validation. In the validation studies, three unused patches were processed and 10 mL of extracting agent was used. In an effort to study the e hicken breast skin for 12 hours, and then the extraction process using methanol and a 2 hour extraction time was performed.

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112 Results Recovery experiments were performed in vitro to evalu ate the feasibility of using diclofenac as a model drug in a clinical microdialysis study. These experiments were performed in six rounds, each in triplicate, where the drug and dextran concentrations were varied in an effort to evaluate their impact on th e recovery of the study drug (Table 5 1) using EE and RT experiments In all scenarios studied, the recovery of the study drug exceeded 75% ; ranging between 77.6 99.6%. When comparing the results of the EE and RT experiments within a round, a slightly hig her recovery was observed with RT. High recovery values were obtained across a range of diclofenac concentrations (2 40 ng/ mL ) Dextran concentration did not appear to have a large impact on the observed recovery value. Studies were performed to study the rate of dissolution of the product Flector. After a period of 24 hours, the average amount of diclofenac released was 110.1 ( 10.8) mg of diclofenac was released (Figure 5 1) An increase in variability around the me an was observed at the last three tim e points (i.e., 10, 12, and 24 hours). A method was developed and validated to quantify the amount of diclofenac in new and used Flector patches. Four methods which differed in the solvent and extraction time were tested. Of these four methods, the use o f methanol with a 2 hours extraction time, proved to be most reproducible. Validation results for this method are shown in Figure 5 2. On average, three unused Flector patches (n=3) cut into 16 pieces each resulted in diclofenac concentrations reasonably close to expected value s (i.e., 40.5 ng/ mL per piece). The average diclofenac content for an en tire Flector patch was 123 mg. When four unused patches were applied to chicken breasts for a period of

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113 12 hours, the mean residual content was 82.4 mg; corresp onding to 36.6% of drug content released. Discussion exceeded 85% when dextran (3%) was added to the perfusate. As expected, only minor differences were observed in the measured recovery when compari ng the values observed using EE and RT. The probe manufacturer recommends that dextran 3% be added to the perfusate to prevent ultrafiltration across the membrane ; with results of these experiments supporting use of this agent. The high in vitro recovery v alue s observed using a linear microdialysis probe supported further study in humans. The dissolution experiments performed helped to describe the rate at which diclofenac is released from the product Flector in vitro The expected amount of pure diclofen ac in an unused Flector patch is 129.6 mg. After a period of 24 hours, an average of 110.1 ( 10.8) mg was released. The greatest variability was observed with later time points (i.e., 10, 12, and 24 hours); possibly due to the patches swelling to a great er extent over time. In vitro experiments were performed to d evelop and validate a method which can be used to quantify residual diclofenac content in used Flector patches. First, we tested various methods which varied in the extracting agent and the ext raction time used. Second, the selected method was validated by processing three unused Flector patches. Last, four patches were applied to chicken breast purchased at a local supermarket. After 12 hours of application, the patches were processed accordin g to the validated method and percentage of diclofenac released from each patch was calculated. Of the four methods tested, methanol and a 2 hour extraction time resulted

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114 in the most reproducible results. Upon validation with three patches, a mean of 123 m g was extracted. This value is reasonably close to the expected 129.6 mg of pure diclofenac one would expect. After application to chicken skin, an average of 36.6% of the patch content was released. When comparing the amount of diclofenac released after 1 2 hours for the dissolution and chicken breast experiments; 79.7 mg and 47.2 mg were observed. This difference in the amount released is not surprising since greater release was observed when the patch was placed in water; whereas with the latter scenario the patch was applied directly on chicken breast.

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115 Table 5 1 Recovery results for extraction efficiency and retrodialysis experiments. Each round was performed in triplicate. Round Dextran Concenration Diclofenac Concentration E E Mean (SD ) R T Mean (SD ) 1 3% 5, 10 and 25 ng/ml 85.7 (18.2) 97.2 (3.7) 2 3% 5, 10 and 25 ng/ml 88.7 (10.8) 96.1 (6.6) 3 3% 5, 10 and 25 ng/ml 98.2 (11.7) 99.6 (0.7) 4 3% 2, 15 and 40 ng/ml 87.7 (16.1) 95.3 (5.3) 5 1% 2, 15 and 40 ng/ml 94.1 (12.0) 93.8 (7.8) 6 2% 5, 15 and 40 ng/ml 77.6 (11.4) 87.4 (12.9)

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116 Figure 5 1. Mean dissolution profile for experiments performed using six Flector patches. E rror bars represent the standard deviation

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117 Figure 5 2 Mean results obtained after cutting a Flector patch into sixteen pieces and extracting the drug content using methanol. The solid line represents the expected concentration for each piece.

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118 CHAPTER 6 USE OF MICRODIALYSIS TO EVALUATE THE EFFE CT OF SKIN PROPERTIE S AND APPLICATION SITE ON THE TOPICAL BIOEQUI VALENCE OF DICLOFENA C: A FEASIBILITY PILOT ST UDY Introduction Evaluating the recovery of a study drug in vivo is a key objective of all clinical microdialysis studies The recovery value should be determined for each subject and microdialysis probe. Severa l microdialysis studies have been performed to evaluate the tissue distribution of diclofenac in vivo 59,86 90 In one study, the recovery was reported to be 66%12% (mean SE) for su perficial layers of the skin; where as a slightly lower value was reported for deep layers ( 63%15% ) 59 Another study reported an in vivo recovery value o f 70.5%22.9% (mean SD) for diclofenac 88 We sought to conduct a pilot study to aid in preparation for a larger pivotal study. The objectives of the pilot study were to evaluate the feasibility of using a linear in vivo ; and determine the length of time needed for all diclofenac to be washed out following infusion of a low concentration administered for recovery determination. The me thods and results described herein pertain to a pilot study conducted in three healthy subjects. Methods Volunteers Three healthy male or female subjects between 18 and 55 years old (inclusive) provided written informed consent and underwent a screening examination prior to study enrollment. The study was approved by the Institutional Review Boards at the University of Florida and FDA

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119 Inclusion criteria Body mass index (BMI) of 18.5 and 32 kg/m 2 ( inclusive ) Non smoker status for at least 12 months prio r to study entry Healthy on the basis of physical examination, medical history, and vital signs. Normal results for clinical laboratory tests performed at screening. Female subjects must be postmenopausal, on proper contraception or abstinent A lcohol may not be consumed 72 hours prior to study admission D iclofenac containing medications will be avoided while enrolled in the study. Subject must adhere to prohibitions an d restrictions detailed in the protocol. Subjects must provide written informed co nsent. Exclusion criteria Clinically si gnificant abnormal values in the performed laboratory tests Significant medical illness precluding participation in this clinical study. History of atopic eczema, dry skin or ichtyosis. Excessive hair at the site of application Known allergies or hypersensitivity to diclofenac containing products. Abnormal physical examination of vital signs. Subject administered an investiga tional drug within 60 days Pregnant or breast feeding. Recent history of surgery (withi n 3 months prior to screening). Clinically significant acute illness within 7 days prior to study drug administration. Recent acute blood loss or donation of blood. Employees of th e investigator or study center. Over the counter or prescription use of o ther NSAID products. Prohibitions and restrictions Subjects will avoid strenuous exercise for 48 hours prior to the study visit. There will be no alcohol consumption for at least 72 hours prior to the study visit. Smoking is prohibited during study par ticipation. Blood and/or plasma donation is prohibited during study participation. Subjects will inform the study team if they become pregnant during study participation. Subjec ts will refrain from using topical moisturizers for 48 hours prior to the st udy participation

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120 Screening The follow ing screening procedures were performed for all subject s prior to study entry : 1) m edical history was evalu ated and a physical exam was performed; 2) i nclusio n and exclusion criteria were verified; 3) h eight, body we ight a nd body mass index (BMI) were determined; 4) v ital signs (systolic/diastolic blood pressure, pulse rate and temperature) following 5 minute s in the supine position were recorded; 5) c linical laboratory testing was performed ( comprehensive metabolic p ane l and complete blood count ) ; 6) a serum pregnancy test was performed during the i nitial screening procedure. 7) a urine pregnancy test was p erformed upon admission to the Clinical Research C enter (CRC) Recovery assessment During microdialysis studies, recovery determination is a critical study component in order to correctly calculate tissue concentrations of a desired analyte The RT method was used to evaluate probe recovery. With the RT method, a known drug concentration is infused through the probe prior to administration of the study drug. The change in analyte concentration in the perfusate is then compared with the co ncentration in the dialysate and the recovery of the probe is calculated using the equation shown below. 6 1 In this pilot study, a diclofenac concentration of 25 ng/ mL was selected for re covery determination using the RT method. This concentration was selected based on several factors. First, published microdialysis studies evaluating the local pene tration of diclofenac (other formulations) were considered. 59,86,88 In these studies, mean maximum concentrations of diclofenac in subcutaneous tissue varied between 13.1 ng/ mL a nd 5

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121 mL following administration of diclofenac as a spray gel (4%) and Voltaren Emulgel (6% and 300 mg/100 cm 2 ). Second, we evaluated data reported in the NDA package for Flector in order to determine what range of tissue concentrations would be likely f or this product. Although local concentrations of Flector have not been studied in humans, based on plasma concentrations (0.7 6 ng/ mL 10 20 hours after single application), we felt that a concentration of 25 ng/ mL was reasonable. 80 Third, we considered the total dose of diclofenac delivered using this concentration. For a 25 ng/ mL concentration infused f or 1.5 hours at /min ute a total of 3.375 ng of diclofenac would be delivered. We felt that this was reasonable given what is considered to be a therapeutic dose. Last, we considered what concentration would be appropriate given the lower limit of quantifi cation for our analytical method. We feel confident that we will be able to quantify diclofenac concentrations in the dialysate using a concentration of 25 ng/ mL Study protocol In the feasibility study each subject visited the CRC on two separate outpat ient visits; the first for screening purposes and the second for determination of probe recovery. Once a subject provided written informed consent, he/she was screened by a study physician to verif y that all inclusion criteria were met Any subjects meetin g any of the e xclusion criteria were not allowed to participate. Once a subject was enrolled in the study, they were asked to visit the clinical research center (CRC) between 8 10 AM on Day 1. On Day 1, upon admission to the CRC a study physician inserte d three microdialysis probes (CMA 66, M Dialysis, Solna, Sweden) into sub cutaneous tissue in the abdomen and t he recovery of the probe was determined using the RT metho d

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122 (Table 6 1) L idocaine 1% was used during probe insertion to minimize the discomfort t o the subject Next, there was a 30 minute equilibration period where sodium chloride 0.9% solution (containing dextran 3%) was ute Probe recovery was then determined using the RT method. A drug solution containing 25 ng/ mL was infused through each pr /min ute for 60 minutes in order to reach a steady state with the tiss ue. Then a dialysate sample was collected for 30 minutes. Post retrodialysis, sodium chloride 0.9% (containing dextran 3%) was i nfused through the probes, and dialysate samples were collected every 30 minutes for 3.5 hours in order to determine the length of time needed until disappearance of the drug in the dialysate. Th e recovery experiments lasted approximately 6 hours. A follo w up phone call will be performed within 72 hours of the subject being discharged. Subjects who participated in the feasibility study were not required to participate in the main study (the two studies will be treated separately). Drug Analysis The LC/MS m ethod described in the previous chapter was used for quantification of diclofenac in microdialysis samples. Results No adverse events occurred during the three study visit s. S ubject s reported minimal discomfort during probe insertion. Duri ng the follow up phone call, two subject s reported minor bruising around the needle insertion site, but stated that there was no pain or tenderness. No other compl aints were reported The demographic characteristics of all three subjects are shown in Table 6 2. For each s ubject, measurements of probe depth were obtained for each probe (Table 6 3).

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123 There was some inter individual variability in the degree of probe depth, likely a result of difference s in body weight. Within each individual, there were only small differences in the probe depth. For all three subjects the calculated recovery by loss was approximately 100% (Tables 6 4 6 6) For Subject 3, one probe did not function properly, as noted by the lack of dialysate during collection. A major objective of this feasi bility was to evaluate the time necessary for complete washout of diclofenac following recovery determination. For Subject 1, diclofenac concentrations were below the limit of quantification (BQL) 30 minutes after recovery determination. For Subjects 2 and 3, diclofenac concentrations could still be quantified 3.5 and 3 hours following infusion of diclofenac for recovery determination (Tables 6 7 6 9) Discussion In each of the three subjects, three microdialysis probes were inserted, an ultrasound measu rement of probe depth was obtained, and the probe recovery was determined. No major adverse events related to probe insertion or removal were reported. With the exception of one probe in Subject 3, all probes worked properly and dialysate samples could be collected. It is not clear what resulted in the poor functionality of this single probe. Recovery by loss was approximately 100% in all subjects. The time to washout for diclofenac differed between the three subjects; varying between 0.5 3.5 hours. This va riability in the necessary washout period may be explained (at least in part) due to differences in body weight. The results of this study recovery would not be a limiting f actor in vivo

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124 Table 6 1. Time and events table for the pilot study. Phase Screening Recovery Experiments (N=3) Study Day Prior to Study Entry Day 1 Time 0 0.5 h 0.5 1.5 h 1.5 5.5 h Residence in Clinic Outpatient Visit Medical history X Inclus ion/Exclusion Criteria X Physical examination X Weight, height and BMI X Vital signs/oral temperature X X Clinical Lab Testing X Serum Pregnancy Test X Urine Pregnancy Test X Diclofenac probe calibration (3.5 hours) EQUILI BRATI ON (Saline) EQUILI BRATION (Drug Solution) Sample Collection (every 30 minutes) AE Reporting Continuous

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125 Table 6 2. Demographic characteristics for subjects participating in the pilot study Variable Subject 1 Subject 2 Subject 3 Age (years) 28 45 2 0 Height (cm) 182.7 169 172.7 Weight (kg) 101.1 90.5 56.7 BMI 29 31.7 19 Table 6 3. Measurement of probe depth in three healthy subjects. Variable Subject 1 Subject 2 Subject 3 Probe 1 8 mm 10 mm 5 mm Probe 2 9 mm 11 mm 5 mm Probe 3 10 mm 12 mm 5 mm

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126 Table 6 4 Recovery determination for Subject 01. Sample Type Perfusate Probe Number Diclofenac Concentration (ng/m L ) Sample From Diclofenac Syringe 1 D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 1 17.18 Sample From Diclofenac Syringe 2 D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 2 17.36 Sample From Diclofenac Syringe 3 D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 3 14.92 Sample From Placebo Syringe 1 0.9% Saline With 3% Dextran 40 1 BQL Sample From Placebo Syringe 2 0.9% Saline With 3% Dextran 40 2 BQL Sample From Placebo Syringe 3 0.9% Saline With 3% Dextran 40 3 BQL Retrodialysis (1.5 2 Hours) D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 1 BQL Retrodialysis (1.5 2 Hours) D iclofenac 25 ng/mL In 0.9% S aline With 3% Dextran 40 2 BQL Retrodialysis (1.5 2 Hours) D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 3 BQL Relative Recovery 1 100% 2 100% 3 100% *BQL = Be low Quantification Limit (0.39 n g/ mL )

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127 Table 6 5 Recovery determination for Sub ject 02. Sample Type Perfusate Probe Number Diclofenac Concentration (ng/m L ) Sample From Diclofenac Syringe 1 D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 1 17.52 Sample From Diclofenac Syringe 2 D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextra n 40 2 18.78 Sample From Diclofenac Syringe 3 D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 3 13.58 Sample From Placebo Syringe 1 0.9% Saline With 3% Dextran 40 1 BQL Sample From Placebo Syringe 2 0.9% Saline With 3% Dextran 40 2 BQL Sample Fro m Placebo Syringe 3 0.9% Saline With 3% Dextran 40 3 BQL Retrodialysis (1.5 2 Hours) D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 1 BQL Retrodialysis (1.5 2 Hours) D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 2 BQL Retrodialysis (1 .5 2 Hours) D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 3 BQL Relative Recovery 1 100% 2 100% 3 100% *BQL = Be low Quantification Limit (0.39 n g/ mL )

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128 Table 6 6 R ecovery determination for S ubject 03. Sample Type Perfusate Probe Nu mber Diclofenac Concentration (ng/m L ) Sample From Diclofenac Syringe 1 D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 1 17.56 Sample From Diclofenac Syringe 2 D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 2 19.48 Sample From Diclofenac Sy ringe 3 D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 3 19.56 Sample From Placebo Syringe 1 0.9% Saline With 3% Dextran 40 1 BQL Sample From Placebo Syringe 2 0.9% Saline With 3% Dextran 40 2 BQL Sample From Placebo Syringe 3 0.9% Saline With 3% Dextran 40 3 BQL Retrodialysis (1.5 2 Hours) D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 1 BQL Retrodialysis (1.5 2 Hours) D iclofenac 25 ng/mL In 0.9% Saline With 3% Dextran 40 2 NA a Retrodialysis (1.5 2 Hours) D iclofenac 25 ng/mL In 0. 9% Saline With 3% Dextran 40 3 B QL Relative Recovery 1 100% 2 NA a 3 100% *BQL = below quantification limit (0.39 ng/ m L ) a N o dialysate obtained through this probe.

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129 Table 6 7. Diclofenac washout period in Subject 01. Sample Type Perfusate Probe Numb er Diclofenac Concentration (ng/m L ) Post RT (2 2.5 Hours) 0.9% Saline With 3% Dextran 40 1 B QL Post RT (2 2.5 Hours) 0.9% Saline With 3% Dextran 40 2 BQL Post RT (2 2.5 Hours) 0.9% Saline With 3% Dextran 40 3 16.78 Post RT (2.5 3 Hours) 0.9% Sa line With 3% Dextran 40 1 BQL Post RT (2.5 3 Hours) 0.9% Saline With 3% Dextran 40 2 BQL Post RT (2.5 3 Hours) 0.9% Saline With 3% Dextran 40 3 BQL Post RT (3 3.5 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (3 3.5 Hours) 0.9% Saline Wit h 3% Dextran 40 2 BQL Post RT (3 3.5 Hours) 0.9% Saline With 3% Dextran 40 3 BQL Post RT (3.5 4 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (3.5 4 Hours) 0.9% Saline With 3% Dextran 40 2 BQL Post RT (3.5 4 Hours) 0.9% Saline With 3% Dex tran 40 3 BQL Post RT (4 4.5 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (4 4.5 Hours) 0.9% Saline With 3% Dextran 40 2 BQL Post RT (4 4.5 Hours) 0.9% Saline With 3% Dextran 40 3 BQL Post RT (4.5 5 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (4.5 5 Hours) 0.9% Saline With 3% Dextran 40 2 BQL Post RT (4.5 5 Hours) 0.9% Saline With 3% Dextran 40 3 BQL Post RT (5 5.5 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (5 5.5 Hours) 0.9% Saline With 3% Dextran 40 2 BQL P ost RT (5 5.5 Hours) 0.9% Saline With 3% Dextran 40 3 BQL *BQL = Be low Quantification Limit (0.39 n g/ mL )

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130 Table 6 8. Diclofenac washout period in Subject 02. Sample Type Perfusate Probe Number Diclofenac Concentration (ng/m L ) Post RT (2 2.5 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (2 2.5 Hours) 0.9% Saline With 3% Dextran 40 2 BQL Post RT (2 2.5 Hours) 0.9% Saline With 3% Dextran 40 3 BQL Post RT (2.5 3 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (2.5 3 Hours) 0.9% Sal ine With 3% Dextran 40 2 BQL Post RT (2.5 3 Hours) 0.9% Saline With 3% Dextran 40 3 BQL Post RT (3 3.5 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (3 3.5 Hours) 0.9% Saline With 3% Dextran 40 2 BQL Post RT (3 3.5 Hours) 0.9% Saline With 3% Dextran 40 3 BQL Post RT (3.5 4 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (3.5 4 Hours) 0.9% Saline With 3% Dextran 40 2 1.894 Post RT (3.5 4 Hours) 0.9% Saline With 3% Dextran 40 3 1.938 Post RT (4 4.5 Hours) 0.9% Saline With 3% Dextran 40 1 5.1 Post RT (4 4.5 Hours) 0.9% Saline With 3% Dextran 40 2 1.888 Post RT (4 4.5 Hours) 0.9% Saline With 3% Dextran 40 3 BQL Post RT (4.5 5 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (4.5 5 Hours) 0.9% Saline With 3% Dextra n 40 2 BQL Post RT (4.5 5 Hours) 0.9% Saline With 3% Dextran 40 3 2.9 Post RT (5 5.5 Hours) 0.9% Saline With 3% Dextran 40 1 8.76 Post RT (5 5.5 Hours) 0.9% Saline With 3% Dextran 40 2 BQL Post RT (5 5.5 Hours) 0.9% Saline With 3% Dextran 40 3 2.96 *BQL = Be low Quantification Limit (0.39 n g/ mL )

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131 Table 6 9 Diclofenac washout period in Subject 03. Sample Type Perfusate Probe Number Diclofenac Concentration (ng/m L ) Post RT (2 2.5 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (2 2.5 Hours) 0.9% Saline With 3% Dextran 40 2 NA Post RT (2 2.5 Hours) 0.9% Saline With 3% Dextran 40 3 BQL Post RT (2.5 3 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (2.5 3 Hours) 0.9% Saline With 3% Dextran 40 2 NA Post RT (2.5 3 Hours) 0. 9% Saline With 3% Dextran 40 3 0.894 Post RT (3 3.5 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (3 3.5 Hours) 0.9% Saline With 3% Dextran 40 2 NA Post RT (3 3.5 Hours) 0.9% Saline With 3% Dextran 40 3 0.85 Post RT (3.5 4 Hours) 0.9% Sal ine With 3% Dextran 40 1 BQL Post RT (3.5 4 Hours) 0.9% Saline With 3% Dextran 40 2 NA Post RT (3.5 4 Hours) 0.9% Saline With 3% Dextran 40 3 BQL Post RT (4 4.5 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (4 4.5 Hours) 0.9% Saline With 3% Dextran 40 2 NA Post RT (4 4.5 Hours) 0.9% Saline With 3% Dextran 40 3 BQL Post RT (4.5 5 Hours) 0.9% Saline With 3% Dextran 40 1 1.66 Post RT (4.5 5 Hours) 0.9% Saline With 3% Dextran 40 2 NA Post RT (4.5 5 Hours) 0.9% Saline With 3% Dextra n 40 3 BQL Post RT (5 5.5 Hours) 0.9% Saline With 3% Dextran 40 1 BQL Post RT (5 5.5 Hours) 0.9% Saline With 3% Dextran 40 2 NA Post RT (5 5.5 Hours) 0.9% Saline With 3% Dextran 40 3 BQL *BQL = Be low Quantification Limit (0.39 n g/ mL )

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132 CHAPTER 7 USE OF MICRODIALYSIS TO EVALUATE THE EFFE CT OF SKIN PROPERTIE S AND APPLICATION SITE ON THE TOPICAL BIOEQUIV ALENCE OF DICLOFENAC : THE MAIN STUDY Introduction For most topically applied products, clinical end point studies must be conducted to establish bioe quivalence between two products. 91 The need for these trials may increase drug development costs and limit the availability of cheaper generic drugs which are applied topically. The only widely accepted method by the U.S. FDA is the vasoconstrictor assay used to establish topical bioequivalence with topical glucocorticoids. 92 In 2007, the FDA acknowledged the need for the need for further research to identify techniques to evaluate bioequivalence for topical dermatological products. 93,94 In this report, four techniques were acknowledged; pharmacokinetic studies, skin stripping, microdialysis, and near infrared spectroscopy. Each of these techniques has advantages and disadvantages which could potentially limit their use. In vitro tests are frequently performed using excised human/animal skin, although the lack of live tissue and circulation, among oth er factors, has limited its applicability to evaluating scale up or post approval changes for a product 91 Pharmacokinetic studies are of limited usefulness unless concentrations are sufficiently high in the systemic circulation to allow detection and if they reflect delivery to the site of action. 94 Skin stripping is a technique by which drug con tent is quantifi ed in the strat um corneum This technique may be particularly useful for drugs whose site of action is the stratum corneum. 91,95 97 For other topical drugs, the measured amount is assumed to provide a reflection of what is occurring in lower layers of the skin. Although significant research has shown the val ue of the technique for bioequivalence

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133 determination, one of its limitations is the lack of standardization in the study design Near infrared spectroscopy is an imaging technique which may be useful for drug molecules with spect ral characteristics necess ary for detection. 91,98,99 Dermal microdialysis is a technique under investigation for use in the evaluation of bioequivalence for topically applied products. Microdialysis allows for the continuous monitoring of drug concentrations in the desired tissue la yer ; an advantage it provides over other available techniques. One paper sought to compare microdialysis and the tape stripping method in healthy volunteers. 100 When comparing lidocaine cream a nd ointment products, both methods reached the same conclusion; 3 5 fold greater penetration was obtained with the cream formulation. In this study, four microdialysis probes were inserted in two penetration areas. The authors reported a 19% intrasubject v ariability between probes and 20% for the two penetration areas. The current study described herein sought to evaluate the use of microdialysis for the evaluation of topical bioequivalence using the transdermal patch Flector. The main study will enroll a sufficient number of subjects in order to complete all treatments in six healthy subjects. In an effort to evaluate the sensitivity of the microdialysis technique, t wo different batches of Flector will be evaluated. Moreover, to evaluate the impact of ad ministration site, both test and reference products will be compared after a single dose is applied in the abdomen and thigh. These sites were selected because they are vely mobile throughout the study. The study design is described below.

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134 Methods Volunteers Six healthy male or female subjects between 18 and 55 years old (inclusive) will provide writte n informed consent and undergo a screening examination prior to enroll ment. Subjects who participated in the pilot study could participate in the main study, but were not obliged to do so. The study was approved by the Institutional Review Boards at the University of Florida and FDA Inclusion criteria Body mass index (BMI) of 18.5 and 32 kg/m 2 ( inclusive ) Non smoker status for at least 12 months prior to study entry Healthy on the basis of physical examination, medical history, and vital signs. Normal results for clinical laboratory tests performed at screening. Female s ubjects must be postmenopausal, use proper contraception or practice abstinence A lcohol may not be consumed 72 hours prior to study admission D iclofenac containing medications will be avoided while enrolled in the study. Willing to adhere to the prohi bitions and restrictions specified in this protocol. Subjects must provide written informed consent. Subjects should be willing avoid the use of body oils, creams, lotions, or powders to the test areas for a period of 48 hours before the application of pa tches. Exclusion criteria Clinically significant abnormal values in the clinical laboratory tests Significant medical illness precluding participation in this clinical study. History of atopic eczema, dry skin or ichtyosis. Excessive hair at the site o f application Known allergy to Flector Patch and/or diclofenac containing products. Abnormal physical examination of vital signs.

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135 Subject administered an investiga tional drug within 60 days Pregnant or breast feeding. Recent history of surgery (within 3 months prior to screening). Clinically significant acute illness within 7 days prior to study drug administration. Recent acute blood loss or donation of blood. Employees of th e investigator or study center. Over the counter or prescription use of oth er NSAID products. Prohibitions and restrictions Subjects will avoid strenuous exercise for 48 hours prior to the study visit. There will be no alcohol consumption for at least 72 hours prior to the study visit. Smoking is prohibited during study parti cipation. Blood and/or plasma donation is prohibited during study participation. Subjects will inform the study team if they become pregnant during study participation. Subjec ts will refrain from using topical moisturizers for 48 hours prior to the stud y participation Screening The follow ing screening procedures are performed for all subject s prior to study entry : 1) m edical history is evalu ated and a physical exam performed; 2) i nclusio n and exclusion criteria are verified; 3) h eight, body weight a nd body mass index (BMI) are determined; 4) v ital signs (systolic/diastolic blood pressure, pulse rate and temperature) following 5 minute s in the supine position are recorded; 5) c linical laboratory tests are performed ( comprehensive metabolic pane l and comp lete blood count ) ; 6) a serum pregnancy test is performed during the i nitial screening procedure. 7) a urine pregnancy test is performed upon admission to the CRC ; 8) evaluation of skin type using the Fitzpatrick skin type scale and a measurement of the t r ansepidermal water loss (TEWL).

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136 Recovery Assessment Similar the pi lot study, recovery is determined using RT with a diclofenac 25 ng/ mL drug solution prepared in 0.9% sodium chloride containing dextran 3%. The recovery will be calculated using Equation 7 1 7 1 Study Protocol Upon providing informed consent, enrolled subjects undergo an outpatient screening at the CRC. This screening will be performed by a study physician prior to study entry. F or the first treatment pha e is performed The evaluation of the skin type includes use of the Fitzpatrick skin type scale and a measurement of the transepidermal water loss (TEWL); this information will not be used for screening pur poses, but these measures may be included as covariates during data analysis. On the evening of Day 1 (1 day before probe placement), the subject is admitted to the CRC, where he/she will stay s overnight. Upon admission, vital signs, s kin and oral tempe rature are recorded, a urine pregnancy test is completed, and the subject is offered a dinner meal. On the morning of Day 1 subjects receive a standardized breakfast 30 minutes prior to dosing. Between 8:00 10:00 AM, a study physician inserts three mi crodialysis probes (CMA 66, CMA Microdialysis, Inc.) into the subcutaneous tissue of the abdomen or thigh based on a predetermined ra ndomization scheme L idocaine 1% is used during probe insertion to minimize discomfort to the study subject

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137 Prior to the start of the main study t he recovery of the probe is determined. First, there is a 30 minute equilibration period where sod ium chloride 0.9% (containing dextran 3% ) is infused through each probe at a rate of 1 in ute Dextran is added to the s odium chloride 0.9% to prevent ultra filtration over the membrane; a technique which is recommended by the manufacturer. Second, probe recovery is determined using the RT method; where a drug solution containing 25 ng / mL (and 3% Dextran) is infused through each pr /min ute for 60 minutes in order to reach a steady state with the tissue. Then a dialysate sample is collected for 30 minutes. Before the patch is applied there is a 4 hour wash out period where sodium chloride 0.9% (containing dextran 3% ) is infused through the probe. Then the study patch is applied and sample collection begins During the sample collection period, sodium chloride 0.9% (containing dextran 3%) is infused continuou sly at a rate of 1.5 L/min. For all treatments, t he room temperature is maintained at 70 75F. The pat ch is removed 12 hours following appli cation. No other patches are applied (single dose only). In total, sample collection continue s for a total of 24 ho urs Blood samples (about 3 mL each) for analysis of diclofen ac plasma concentrations are collected immediately before application of the study patch (i.e., 0 hours) and at 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 12 hours following application. The patch is remo ved at 12 hours. B lood samples are co llected at 14, 16, and 24 hours; all time points occurring after patch removal. Additional blood samples (5 mL each) are collected at 2 and 12 hours to Microdialysis samples for an alysis of d iclofenac concentrations are collected at 20 minute intervals for 8 hours.

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138 The subject remains one additional night at the CRC and is discharged the next morning following probe removal There is a one week washout period (at least), and then su bject will return for the reference (or test) product. This next study section is done in the same administration site, but the contra lateral side. Then there is another one week washout period (at least) before the next administrati on site is evaluated. T he same flow of events (minus the screening and skin evaluation) described above is repeated for the remaining two treatment phases. Drug Analysis P lasma and microdialysis samples are analyzed using an LC MS assay which was validated according to FDA guide lines and described previously

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139 CHAPTER 8 DISCUSSION P K variability may increase the risk of experiencing a sub or supra therapeutic drug response. This is particularly problematic for drugs with a narrow therapeutic index. For example, inter individu al variability in drug exposure to cytotoxic drugs can vary between 2 10 fold. 101 104 Inter and intra individual variability may be caused by differences or alterations in drug absorption, distribution, metabolism, and excretion of drug molecules. In addition to variability in PK processes poor medication adherence can impact the results obtained in PK analyses; as well as contribute to differences in drug response. Currently available direct and indirect methods of adherence measurement are either imprecise or impractical in most settings. Examples of direct measurement include biologic al assays and directly observed therapy; whereas, pill counts, self reporting, and electronic monitors indirectly document adherence. 105 Breath testing may provide another objective method to document adherence. With breath testing volatile drug molecules may be measured directly Another option would be to measure volatile markers which are s afe and have no impact on a dosage form or the pharmacological response obtained from an active ingredient Pentyl acetate and butyl acetate are two volatile markers, which are metabolized to alcohol and ketone metabolites. Breath concentrations of the parent compounds and/or their metabolites could be measured in breath within minutes of administration via the vaginal or oral route. When evaluating the inter individual and inter occasion variability for these markers, PK variability was observed in both absorption and elimination processes. Despite these results, use of volatile metabolites in various

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140 dosage forms may provide a valuable technique to assess medication adherence in clinical trials and practice. Microdialysis is a versatile technique whic h may aid in a direct assessment of PK variability by measurement free drug concentrations. In vitro the technique was used to assess differences in the protein binding and antimicrobial efficacy of ceftriaxone. When used to measure free drug concentration s in culture flasks, differences in drug concentrations occurring over time were observed Measured concentrations could then be correlated directly with drug effect using a PK/PD modeling approach. The sampling technique may also be used to measure diff er ences in skin absorption and may prov ide a method to evaluate bioequivalence for topically applied products The results of the preliminary studies described herein provide a framework to conduct a clinical microdialysis study using the product Flector. Sensitive analytical methods were developed to quantify diclofenac concentrations in microdialysis and plasma samples. A method was developed and validated which could be used to quantify residual drug content in Flector patches. Data obtained from measu rement of diclofenac in the subcutaneous layers of the skin, plasm a samples, and used patches, may be combined to evaluate the feasibility of using the technique to evaluate topical bioequivalence for the product Flector

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141 LIST OF REFERENCES 1 Staatz CE, Tett SE. Clinical pharmacokinetics and pharmacodynamics of tacrolimus in solid organ transplantation. Clinical Pharmacokinetics 2004; 43 : 623 53. 2 Antignac M, Barrou B, Farinotti R, Lechat P, Urien S. P opulation pharmacokinetics and bioavailability of tacrolimus in kidney transplant patients. British Journal of Clinical Pharmacology 2007; 64 : 750 7. 3 Schipani A, Wyen C, Mahungu T, et al. Integration of population pharmacokinetics and pharmacogenetics: a n aid to optimal nevirapine dose selection in HIV infected individuals. The Journal of Antimicrobial Chemotherapy 2011; 66 : 1332 9. 4 Staatz C, Taylor P, Tett S. Low tacrolimus concentrations and increased risk of early acute rejection in adult renal trans plantation. Nephrology Dialysis Transplantation 2001; 16 : 1905 9. 5 Conrado DJ, Gonzalez D, Derendorf H. Role of drug absorption in the pharmacokinetics of therapeutic interventions for stroke. Annals of the New York Academy of Sciences 2010; 1207 : 134 42. 6 Derendorf H, VanderMaelen CP, Brickl R S, MacGregor TR, Eisert W. Dipyridamole bioavailability in subjects with reduced gastric acidity. Journal of Cinical Pharmacology 2005; 45 : 845 50. 7 Russell T, Berardi R, Barnett J. Upper gastrointestinal pH in se venty nine healthy, elderly, North American men and women. Pharmaceutical Research 1993; 10 : 187 96. 8 Albengres E, Le Lout H, Tillement JP. Systemic antifungal agents. Drug interactions of clinical significance. Drug Safety 1998; 18 : 83 97. 9 Rowland M, Tozer TN. Clinical pharmacokinetics and pharmacodynamics: concepts and applications, 4th Ed. Lippincott Williams & Williams, 2010. 10 Ayrton A, Morgan P. Role of transport proteins in drug absorption, distribution and excretion. Xenobiotica 2001; 31 : 469 97. 11 Kerb R. Implications of genetic polymorphisms in drug transporters for pharmacotherapy. Cancer Letters 2006; 234 : 4 33. 12 Nakamura T, Yamamori M, Sakaeda T. Pharmacogenetics of intestinal absorption. Current Drug Delivery 2008; 5 : 153 69.

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142 13 Gertz M, Houston J. Physiologically based pharmacokinetic modeling of intestinal first pass metabolism of CYP3A substrates with high intestinal extraction. Drug Metabolism and Disposition 2011; 39 : 1633 42. 14 Gonzalez D, Conrado DJ, Theuretzbacher U, Derendorf H. The effect of critical illness on drug distribution. Current pharmaceutical biotechnology 2011; 12 : 2030 6. 15 Berezhkovskiy LM. On the influence of protein binding on pharmacological activity of drugs. Journal of Pharmaceutical Sciences 2010; 99 : 2153 65. 16 Boffito M, Back DJ, Blaschke TF, et al. Protein binding in antiretroviral therapies. AIDS Research and Human Retroviruses 2003; 19 : 825 35. 17 Faed E. Protein binding of drugs in plasma, interstitial fluid and tissues: Effect on pharmacokinetics. E uropean Journal of Clinical Pharmacology 1981; 21 : 77 81. 18 Grandison MK, Boudinot FD. Age related changes in protein binding of drugs: implications for therapy. Clinical Pharmacokinetics 2000; 38 : 271 90. 19 Benet LZ, Hoener B ann. Changes in plasma prot ein binding have little clinical relevance. Clinical Pharmacology and Therapeutics 2002; 71 : 115 21. 20 Kunin CM, Craig W a, Kornguth M, Monson R. Influence of binding on the pharmacologic activity of antibiotics. Annals of the New York Academy of Sciences 1973; 226 : 214 24. 21 Mehvar R. Role of protein binding in pharmacokinetics. American Journal of Pharmaceutical Education 2005; 69 : 1 8. 22 Wise R. The clinical relevance of protein binding and tissue concentrations in antimicrobial therapy. Clinical Phar macokinetics 1986; 11 : 470 82. 23 Zeitlinger M a, Derendorf H, Mouton JW, et al. Protein binding: do we ever learn? Antimicrobial Agents and Chemotherapy 2011; 55 : 3067 74. 24 Schmidt S, Gonzalez D, Derendorf H. Significance of protein binding in pharmacok inetics and pharmacodynamics. Journal of Pharmaceutical Sciences 2010; 99 : 1107 22. 25 Boffito M, Sciole K, Raiteri R, et al. Alpha 1 acid glycoprotein levels in human immunodeficiency virus infected subjects on antiretroviral regimens. Drug Metabolism and Disposition 2002; 30 : 859 60.

PAGE 143

143 26 Hochepied T, Berger FG, Baumann H, Libert C. Alpha(1) acid glycoprotein: an acute phase protein with inflammatory and immunomodulating properties. Cytokine & Growth Factor Reviews 2003; 14 : 25 34. 27 Kaysen G. Biological b asis of hypoalbuminemia in ESRD. Journal of the American Society of Nephrology 1998; 9 : 2368 76. 28 Kremer J, Wilting J, Janssen L. Drug binding to human alpha 1 acid glycoprotein in health and disease. Pharmacological Reviews 1988; 40 : 1 47. 29 Barry M, K eeling PW, Weir D, Feely J. Severity of cirrhosis and the relationship of alpha 1 acid glycoprotein concentration to plasma protein binding of lidocaine. Clinical Pharmacology and Therapeutics 1990; 47 : 366 70. 30 Pedersen L, Bonde J, Graudal N. Quantitati ve and qualitative binding characteristics of disopyramide in serum from patients with decreased renal and hepatic function. British Journal of Pharmacology 1987; 23 : 41 6. 31 Pacifici GM, Viani A, Taddeucci Brunelli G, Rizzo G, Carrai M, Schulz HU. Effect s of development, aging, and renal and hepatic insufficiency as well as hemodialysis on the plasma concentrations of albumin and alpha 1 acid glycoprotein: implications for binding of drugs. Therapeutic Drug Monitoring 1986; 8 : 259 63. 32 Wood M. Plasma pr otein binding: implications for anesthesiologists. Anesthesia and Analgesia 1986; 65 : 786 804. 33 Power BM, Forbes a M, van Heerden PV, Ilett KF. Pharmacokinetics of drugs used in critically ill adults. Clinical Pharmacokinetics 1998; 34 : 25 56. 34 Murray MD, Morrow DG, Weiner M, et al. A conceptual framework to study medication adherence in older adults. The American Journal of Geriatric Pharmacotherapy 2004; 2 : 36 43. 35 Morris AB, Li J, Kroenke K, Bruner England TE, Young JM, Murray MD. Factors associate d with drug adherence and blood pressure control in patients with hypertension. Pharmacotherapy 2006; 26 : 483 92. 36 Claxton a J, Cramer J, Pierce C. A systematic review of the associations between dose regimens and medication compliance. Clinical Therapeu tics 2001; 23 : 1296 310. 37 Vrijens B, Vincze G, Kristanto P, Urquhart J, Burnier M. Adherence to prescribed antihypertensive drug treatments: longitudinal study of electronically compiled dosing histories. BMJ 2008; 336 : 1114 7.

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144 38 de Klerk E, van der Hei jde D, Landew R, van der Tempel H, Urquhart J, van der Linden S. Patient compliance in rheumatoid arthritis, polymyalgia rheumatica, and gout. The Journal of Rheumatology 2003; 30 : 44 54. 39 Simmons MS, Nides M a., Rand CS, Wise R a., Tashkin DP. Trends i n compliance with bronchodilator inhaler use between follow up visits in a clinical trial. Chest 1996; 109 : 963 8. 40 Vrijens B, Goetghebeur E. The impact of compliance in pharmacokinetic studies. Statistical Methods in Medical Research 1999; 8 : 247. 41 Le vy G. A pharmacokinetic perspective on medicament noncompliance. Clinical Pharmacology and Therapeutics 1993; 54 : 242 4. 42 Osterberg LG, Urquhart J, Blaschke TF. Understanding forgiveness: minding and mining the gaps between pharmacokinetics and therapeut ics. Clinical Pharmacology and Therapeutics 2010; 88 : 457 9. 43 Urquhart J. Pharmacodynamics of variable patient compliance: implications for pharmaceutical value. Advanced Drug Delivery Reviews 1998; 33 : 207 19. 44 Morey T, Wasdo S, Wishin J, Quinn B. Fea sibility of a breath test for monitoring adherence to vaginal administration of antiretroviral microbicide gels. Journal of Clinical Pharmacology 2012; Jan 31 45 Sarkar D. Lattice: Multivariate Data Visualization with R., 1st Ed. New York, Springer, 2008. 46 Lindbom L, Pihlgren P, Jonsson EN, Jonsson N. PsN Toolkit -a collection of computer intensive statistical methods for non linear mixed effect modeling using NONMEM. Computer Methods and Programs in Biomedicine 2005; 79 : 241 57. 47 Lindbom L, Ribbing J, Jonsson EN. Perl speaks NONMEM (PsN) -a Perl module for NONMEM related programming. Computer Methods and Programs in Biomedicine 2004; 75 : 85 94. 48 Jonsson EN, Karlsson MO. Xpose -an S PLUS based population pharmacokinetic/pharmacodynamic model building aid for NONMEM. Computer Methods and Programs in Biomedicine 1999; 58 : 51 64. 49 Perry TR, Schentag JJ. Clinical use of ceftriaxone: a pharmacokinetic pharmacodynamic perspective on the impact of minimum inhibitory concentration and serum protein binding. Clinical Pharmacokinetics 2001; 40 : 685 94.

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145 50 Schmidt S, Rck K, Sahre M, et al. Effect of protein binding on the pharmacological activity of highly bound antibiotics. Antimicrobial Agents and Chemotherapy 2008; 52 : 3994 4000. 51 Keizer RJ, van Benten M, Beijnen JH, Schellens JHM, Huitema ADR. Piraa and PCluster: a modeling environment and cluster infrastructure for NONMEM. Computer Methods and Programs in Biomedicine 2011; 101 : 72 9. 52 Nielsen EI, Viberg A, Lwdin E, Cars O, Karlsson MO, Sandstrm M. Se mimechanistic pharmacokinetic/pharmacodynamic model for assessment of activity of antibacterial agents from time kill curve experiments. Antimicrobial Agents and Chemotherapy 2007; 51 : 128 36. 53 Nielsen EI, Cars O, Friberg LE. Pharmacokinetic/Pharmacodyna mic (PK/PD) Indices of Antibiotics Predicted by a Semimechanistic PKPD Model: a Step toward Model Based Dose Optimization. Antimicrobial agents and chemotherapy 2011; 55 : 4619 30. 54 Tam VH, Schilling AN, Nikolaou M. Modelling time kill studies to discern the pharmacodynamics of meropenem. The Journal of Antimicrobial Chemotherapy 2005; 55 : 699 706. 55 Grgoire N, Raherison S, Grignon C, et al. Semimechanistic pharmacokinetic pharmacodynamic model with adaptation development for time kill experiments of cip rofloxacin against Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 2010; 54 : 2379 84. 56 Beal SL. Ways to fit a PK model with some data below the quantification limit. Journal of Pharmacokinetics and Pharmacodynamics 2001; 28 : 481 504. 57 Sal lmann A. The history of diclofenac. The American Journal of Medicine 1986; 80 : 29 33. 58 Willis JV, Kendall MJ, Flinn RM, Thornhill DP, Welling PG. The pharmacokinetics of diclofenac sodium following intravenous and oral administration. European Journal of Clinical Pharmacology 1979; 16 : 405 10. 59 Mller M, Mascher H, Kikuta C, et al. Diclofenac concentrations in defined tissue layers after topical administration. Clinical Pharmacology and Therapeutics 1997; 62 : 293 9. 60 Mayer BX, Namiranian K, Dehghanyar P, Stroh R, Mascher H, Mller M. Comparison of UV and tandem mass spectrometric detection for the high performance liquid chromatographic determination of diclofenac in microdialysis samples. Journal of Pharmaceutical and Biomedical Analysis 2003; 33 : 745 54.

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146 61 Arcelloni C, Lanzi R, Pedercini S, et al. High performance liquid chromatographic determination of diclofenac in human plasma after solid phase extraction. Journal of Chromatography 2001; 763 : 195 200. 62 Borenstein MR, Xue Y, Cooper S, Tzeng TB. S ensitive capillary gas chromatographic mass spectrometric selected ion monitoring method for the determination of diclofenac concentrations in human plasma. Journal of Chromatography 1996; 685 : 59 66. 63 Kang W, Kim E Y. Simultaneous determination of acecl ofenac and its three metabolites in plasma using liquid chromatography tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 2008; 46 : 587 91. 64 Ikeda M, Kawase M, Hiramatsu M, Hirota K, Ohmori S. Improved gas chromatographic method of determining diclofenac in plasma. Journal of Chromatography 1980; 183 : 41 7. 65 Sparidans RW, Lagas JS, Schinkel AH, Schellens JHM, Beijnen JH. Liquid chromatography tandem mass spectrometric assay for diclofenac and three primary metabolites in mouse p lasma. Journal of Chromatography 2008; 872 : 77 82. 66 Zecca L, Ferrario P, Costi P. Determination of diclofenac and its metabolites in plasma and cerebrospinal fluid by high performance liquid chromatography with electrochemical detection. Journal of Chrom atography 1991; 567 : 425 32. 67 Zecca L. Determination of diclofenac in plasma and synovial fluid by high performance liquid chromatography with electrochemical detection. Journal of Chromatography 1989; 495 : 303 8. 68 FDA. Guidance for industry: bioanalyt ical method validation. Rockville, MD: CDER 2001.http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInform ation/Guidances/ucm070107.pdf (accessed 27 Nov2011). 69 de Lange EC, de Boer a G, Breimer DD. Methodological issues in microdialysis sampl ing for pharmacokinetic studies. Advanced Drug Delivery Reviews 2000; 45 : 125 48. 70 Plock N, Kloft C. Microdialysis -theoretical background and recent implementation in applied life sciences. European Journal of Pharmaceutical Sciences 2005; 25 : 1 24. 71 Chaurasia CS. In vivo microdialysis sampling: theory and applications. Biomedical Chromatography 1999; 13 : 317 32.

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147 72 Chaurasia CS, Mller M, Bashaw ED, et al. AAPS FDA workshop white paper: microdialysis principles, application and regulatory perspectives Pharmaceutical Research 2007; 24 : 1014 25. 73 Davies MI, Cooper JD, Desmond SS, Lunte CE, Lunte SM. Analytical considerations for microdialysis sampling. Advanced Drug Delivery Reviews 2000; 45 : 169 88. 74 de Lange EC, Danhof M, de Boer a G, Breimer DD. Critical factors of intracerebral microdialysis as a technique to determine the pharmacokinetics of drugs in rat brain. Brain Research 1994; 666 : 1 8. 75 Verbeeck RK. Blood microdialysis in pharmacokinetic and drug metabolism studies. Advanced Drug Deliver y Reviews 2000; 45 : 217 28. 76 Mantovani V, Kennergren C, Bugge M, Sala A, Lnnroth P, Berglin E. Myocardial metabolism assessed by microdialysis: a prospective randomized study in on and off pump coronary bypass surgery. International Journal of Cardiolo gy 2010; 143 : 302 8. 77 Waelgaard L, Thorgersen EB, Line P D, Foss A, Mollnes TE, Tnnessen TI. Microdialysis monitoring of liver grafts by metabolic parameters, cytokine production, and complement activation. Transplantation 2008; 86 : 1096 103. 78 Awad AS Webb RL, Carey RM, Siragy HM. Renal nitric oxide production is decreased in diabetic rats and improved by AT1 receptor blockade. Journal of Hypertension 2004; 22 : 1571 7. 79 Buerger C, Plock N, Dehghanyar P, Joukhadar C, Kloft C. Pharmacokinetics of unbo und linezolid in plasma and tissue interstitium of critically ill patients after multiple dosing using microdialysis. Antimicrobial Agents and Chemotherapy 2006; 50 : 2455 63. 80 Pfizer. Flector Full Prescribing Information. 2011. 81 Wickham H. ggplot2: e legant graphics for data analysis. New York, Springer, 2009. 82 Marquardt K, Tharratt R. Fentanyl remaining in a transdermal system following three days of continuous use. The Annals of Pharmacotherapy 1995; 29 : 969 71. 83 Van Nimmen NFJ, Veulemans H a F. Validated GC MS analysis for the determination of residual fentanyl in applied Durogesic reservoir and Durogesic D Trans matrix transdermal fentanyl patches. Journal of Chromatography B 2007; 846 : 264 72.

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148 84 Kochak G, Sun J, Choi R. Pharmacokinetic disposi tion of multiple dose transdermal nicotine in healthy adult smokers. Pharmaceutical research 1992; 9 : 1451 5. 85 Marier J F, Lor M, Potvin D, Dimarco M, Morelli G, Saedder EA. Pharmacokinetics, tolerability, and performance of a novel matrix transdermal de livery system of fentanyl relative to the commercially available reservoir formulation in healthy subjects. Journal of Clinical Pharmacology 2006; 46 : 642 53. 86 Brunner M, Dehghanyar P, Seigfried B, Martin W, Menke G, Mller M. Favourable dermal penetrati on of diclofenac after administration to the skin using a novel spray gel formulation. British Journal of Clinical Pharmacology 2005; 60 : 573 7. 87 Mller M, Rastelli C, Ferri P, Jansen B, Breiteneder H, Eichler H. Transdermal penetration of diclofenac aft er multiple epicutaneous administration. Journal of Rheumatology 1998; 25 : 1833 6. 88 Dehghanyar P, Mayer B, Namiranian K. Topical skin penetration of diclofenac after single and multiple dose application. International Journal of Clinical Pharmacology and Therapeutics 2004; 42 : 353 9. 89 Fang JY, Sung KC, Lin HH, Fang CL. Transdermal iontophoretic delivery of diclofenac sodium from various polymer formulations: in vitro and in vivo studies. International Journal of Pharmaceutics 1999; 178 : 83 92. 90 Burian M, Tegeder I, Seegel M, Geisslinger G. Peripheral and central antihyperalgesic effects of diclofenac in a model of human inflammatory pain. Clinical Pharmacology and Therapeutics 2003; 74 : 113 20. 91 Narkar Y. Bioequivalence for topical products -an updat e. Pharmaceutical Research 2010; 27 : 2590 601. 92 Wiedersberg S, Leopold CS, Guy RH. Bioavailability and bioequivalence of topical glucocorticoids. European Journal of Pharmaceutics and Biopharmaceutics 2008; 68 : 453 66. 93 FDA U. Critical Path Opportuniti es for Generic Drugs. 2012; : 1 8. 94 Lionberger R a. FDA critical path initiatives: opportunities for generic drug development. The AAPS Journal 2008; 10 : 103 9. 95 Pershing L, Silver B, Krueger G, Shah V. Feasibility of measuring the bioavailability of t opical betamethasone dipropionate in commercial formulations using drug content in skin and a skin blanching bioassay. Pharmaceutical Research 1992; 9 : 45 51.

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149 96 Pershing LK, Corlett J, Jorgensen C. In vivo pharmacokinetics and pharmacodynamics of topical ketoconazole and miconazole in human stratum corneum. Antimicrobial Agents and Chemotherapy 1994; 38 : 90 5. 97 Alberti I, Kalia YN, Naik A, Bonny JD, Guy RH. In vivo assessment of enhanced topical delivery of terbinafine to human stratum corneum. Journal o f Controlled Release 2001; 71 : 319 27. 98 Mlot M, Pudney PD a, Williamson A M, Caspers PJ, Van Der Pol A, Puppels GJ. Studying the effectiveness of penetration enhancers to deliver retinol through the stratum cornum by in vivo confocal Raman spectroscopy. Journal of Controlled Release 2009; 138 : 32 9. 99 Pudney PD a, Mlot M, Caspers PJ, Van Der Pol A, Puppels GJ. An in vivo confocal Raman study of the delivery of trans retinol to the skin. Applied Spectroscopy 2007; 61 : 804 11. 100 Benfeldt E, Hansen SH, Vlund A, Menn T, Shah VP. Bioequivalence of topical formulations in humans: evaluation by dermal microdialysis sampling and the dermatopharmacokinetic method. Journal of Investigative Dermatology 2007; 127 : 170 8. 101 Undevia SD, Gomez Abuin G, Ratain MJ Pharmacokinetic variability of anticancer agents. Nature reviews Cancer 2005; 5 : 447 58. 102 Evans W. Clinical pharmacokinetics pharmacodynamics of anticancer drugs. Clinical Pharmacokinetics 1989; 16 : 327 36. 103 Freyer G, Ligneau B, Tranchand B, Ardiet C, Serre Debeauvais F, Trillet Lenoir V. Pharmacokinetic studies in cancer chemotherapy: usefulness in clinical practice. Cancer Treatment Reviews 1997; 23 : 153 69. 104 Masson E, Zamboni W. Pharmacokinetic optimisation of cancer chemotherapy. Effect on ou tcomes. Clinical Pharmacokinetics 1997; 32 : 324 43. 105 Kenna L a, Labb L, Barrett JS, Pfister M. Modeling and simulation of adherence: approaches and applications in therapeutics. The AAPS journal 2005; 7 : E390 407.

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150 BIOGRAPHICAL SKETCH Daniel Gonzalez was born in Miami, FL. He has one sibling, Danisa Borges, and is married to Kady Rae Gonzalez. Daniel graduated from Southwest Miami Senior High School in 2002; completed two years of pre pharmacy studies at University of Florida; and 4 years towards his professional degree. In May 2008, he was awarded the Doctor of Pharmacy degree by the University of Florida, and had the highest grade point average in his graduating class. In August 2008, he began his graduate studies at University of Florida; working un der the direction of Professor Hartmut Derendorf in the Department of Pharmaceutics. While in graduate school, Daniel was an active member of the American College of Clinical Pharmacology, American College of Clinical Pharmacy, American Society for Clinica l Pharmacology and Therapeutics, American Association of Pharmaceutical Scientists, and the American Society of Pharmacometrics. He received his Ph.D. from the University of Florida in the summer of 2012.