Exploring the Effects of Ammi Visnaga L. on Nephrolithiasis Prevention

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Exploring the Effects of Ammi Visnaga L. on Nephrolithiasis Prevention In Vivo Pharmacokinetic and Pharmacodynamic Evaluation of Ammi Visnaga L. Extract and Visnagin
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english
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Haug, Karin G
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Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Pharmaceutical Sciences, Pharmaceutics
Committee Chair:
Derendorf, Hartmut C
Committee Co-Chair:
Butterweck, Veronika D
Committee Members:
Khan, Saeedur R
Winner, Lawrence Herman

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Subjects / Keywords:
ammi-visnaga -- extract -- kidney-stones -- nephrolithiasis -- nonmem -- pharmacokinetics -- prevention -- visnagin
Pharmaceutics -- Dissertations, Academic -- UF
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Pharmaceutical Sciences thesis, Ph.D.
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theses   ( marcgt )
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Abstract:
Kidney stones are common in developed countries. Causes are multifold including genetic predisposition, but dietary habits and lifestyle are most important contributors to nephrolithiasis formation. Over the last 30 years, the prevalence of stone disease in the USA has increased from 3.8 to 8.8%. After the first incidence the recurrence rate within five to ten years is higher than 50%. Usage of current drugs for kidney stone prevention is often limited by side effects and tolerance. Thus, the problem of recurrent stone formation remains. Ammi visnaga L.(syn. Khella, Apiaceae) fruit preparations have traditionally been used in the Middle East for the management of nephrolithiasis. Visnagin, a furanochromone, is one of the main compounds of Ammi visnaga with potential effects on nephrolithiasis prevention. No information is available about the pharmacokinetic (PK) properties of visnagin.Therefore, the PK of visnagin after intravenous (i.v.) bolus injection of visnagin as well as after oral administration of the pure compound and visnagin in form of an aqueous Ammi visnaga extract (AVE) was characterized in rats. Preceding the PK studies, a HPLC-UV method for visnagin quantification in visnagin and AVE solutions and a LC-MS/MS method for visnagin quantification in rat plasma were developed and validated. The i.v. study revealed non-linear elimination kinetics for visnagin and a non-linear mixed effect model that describes the observed data was developed and validated. The oral PK studies demonstrated that average visnagin plasma concentration, exposure (AUC) and residence time were significantly increased when administered in form of AVE. A developed PK model that describes both i.v.and oral PK data suggested that this effect is induced by the AVE compound khellin acting as competitive inhibitor of the saturable elimination process. A pharmacodynamic study was conducted to test if these observed characteristics result in a superior efficacy of AVE compared to an equivalent dose of visnagin. Utilizing a developed method for consistent and reproducible calcium oxalate crystal induction, preventive effects of visnagin and AVE that were demonstrated in previous studies could not be confirmed. Conclusively, the need for research and new substances in the area of kidney stone prevention continues.
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by Karin G Haug.
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Thesis (Ph.D.)--University of Florida, 2013.
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Adviser: Derendorf, Hartmut C.
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Co-adviser: Butterweck, Veronika D.
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1 EXPLORING THE EFFECTS OF AMMI VISNAGA L. ON NEPHROLITHIASIS PREVENTION: IN VIVO PHARMACOKINETIC AND PHARMACODYNAMIC EVALUATION OF AMMI VISNAGA L. EXTRACT AND VISNAGIN By KARIN G HAUG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013

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2 2013 Karin G Haug

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3 To my family and friends

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4 ACKNOWLEDGMENTS First of all I would like to thank my supervisor Dr. Veronika Butterweck for the opportunity to pursue the degree of doctor of philosophy in her working group at the Department of Pharmaceutics at University of Florida. I enjoyed the free d om and trust to guide my research towards scientific areas of personal interest which significantly contributed to my professional intellectual and personal development Many thanks go to Dr. Saeed Khan for his continuous advice regarding the kidney ston e experiments. I want to thank my dissertation committee members Dr. Hartmut Derendorf for his general support and recommendations and Dr. Larry Winner for his suggestions with respect to experimental design and statistical analysis Furthermore I would l ike to acknowledge Dr. Guenther Hochhaus and Yufei Tang for their technical support regarding the LC MS/MS equipment Dr. Salvador Gezan for his suggestions on the subject of statistical questions and Marda Jorgensen for providing histological services I am very grateful for the advice and input I received from Benjamin Weber who introduced me to the idea of pursuing a Ph.D. degree at the Department of Pharmaceutics and who additionally represented a source of support and motivation throughout my time a s graduate student I would like to acknowledge my lab members, Chetan Sampath, Li Li and Xuan Liu as well as the interns Stephanie Hertlein, Maren Krohne, Lena Butz, Silvia Geiser, Eva Fick and Gabriela Turan, for supporting my practical studies. Moreove r, I want to thank friends, colleagues and interns from the College of Pharmacy who enriched my time at University of Florida Finally, special thanks go to my friends and family, who always believed in me and supported me from a distance.

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5 TABLE OF CONTEN TS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 15 ABSTRACT ................................ ................................ ................................ ................... 18 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 20 Numbers and Facts about Kidney Stones ................................ ............................... 20 Pathophysiology of Nephrolithiasis ................................ ................................ ......... 21 Management of Stone Disease Treatment and Prevention ................................ 22 Ammi Visnaga ................................ ................................ ................................ ......... 23 Pharmacokinetics and Pharmacodynamics ................................ ............................ 26 Hypothesis and Specific Aims ................................ ................................ ................. 28 2 CHARACTERIZATION OF AMMI VISNAGA EXTRACTS AND VISNAGIN SOLUTIONS ................................ ................................ ................................ ........... 33 Background ................................ ................................ ................................ ............. 33 Specific Aims ................................ ................................ ................................ .......... 34 Material and Methods ................................ ................................ ............................. 35 Chemicals an d Reagents ................................ ................................ ................. 35 Instrumentation and HPLC UV Conditions ................................ ....................... 35 Preparation of Visnagin and Khellin Calibration Standards and Quality Control Samples ................................ ................................ ............................ 36 Method Validation ................................ ................................ ............................. 36 Extract Prepar ation and Quantification ................................ ............................. 37 Water extract ................................ ................................ .............................. 37 Ethanol extract ................................ ................................ ........................... 38 Visnagin Solubility ................................ ................................ ............................ 39 In water ................................ ................................ ................................ ...... 39 In Captisol ................................ ................................ ................................ 40 In standard Ammi visnaga water extract ................................ .................... 40 When added to standard Ammi visnaga water extract ............................... 41 Stability ................................ ................................ ................................ ............. 42 Visnagin in Captisol ................................ ................................ .................. 42 Visnagin and khellin in extracts ................................ ................................ .. 43 Results ................................ ................................ ................................ .................... 43

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6 Method Validation ................................ ................................ ............................. 43 Extract Quantification ................................ ................................ ....................... 44 Water extract ................................ ................................ .............................. 44 Ethanol extract ................................ ................................ ........................... 44 Visnagin Solubility ................................ ................................ ............................ 45 In water ................................ ................................ ................................ ...... 45 In Captisol ................................ ................................ ................................ 45 In standard Ammi visnaga water extract ................................ .................... 45 When added to standard Ammi visnaga water extract ............................... 46 Stability ................................ ................................ ................................ ............. 46 Visnagin in Captisol ................................ ................................ .................. 46 Visnagin a nd khellin in extracts ................................ ................................ .. 47 Discussion ................................ ................................ ................................ .............. 48 3 PHARMACOKINETIC EVALUATION OF VISNAGIN IN RATS AFTER INTRAVENOUS BOLUS ADMINISTRATION ................................ ......................... 62 Backgrou nd ................................ ................................ ................................ ............. 62 Specific Aims ................................ ................................ ................................ .......... 63 Material and Methods ................................ ................................ ............................. 63 Chemicals and Reagents ................................ ................................ ................. 63 Instrument ation and LC MS/MS Conditions ................................ ..................... 63 Chromatographic conditions ................................ ................................ ...... 63 Mass spectrometry conditions ................................ ................................ .... 64 Sample Preparation ................................ ................................ .......................... 64 Preparation of visnagin calibration standards and quality control samples ................................ ................................ ................................ .. 64 Plasma extraction ................................ ................................ ...................... 65 Method Validation ................................ ................................ ............................. 65 Pharmacokinetic Study ................................ ................................ ..................... 67 Animals ................................ ................................ ................................ ...... 67 Design of pharmacokineti c study in rats ................................ .................... 67 Preparation of visnagin solutions for administration to rats ........................ 68 Sample analysis ................................ ................................ ......................... 68 Pharmacokinetic Data Analysis ................................ ................................ ........ 69 No n compartmental analysis ................................ ................................ ...... 69 Compartmental analysis ................................ ................................ ............ 70 Results ................................ ................................ ................................ .................... 75 Method Validation ................................ ................................ ............................. 75 Pharmacokineti c Study and Sample Analysis ................................ .................. 76 Pharmacokinetic Data Analysis ................................ ................................ ........ 76 Non compartmental analysis ................................ ................................ ...... 76 Compartmental analysis ................................ ................................ ............ 77 Discussion ................................ ................................ ................................ .............. 78 4 PHARMACOKINETIC EVALUATION OF VISNAGIN AND AMMI VISNAGA AQUEOUS EXTRACT IN RATS AFTER ORAL ADMINISTRATION ...................... 97

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7 Background ................................ ................................ ................................ ............. 97 Specific Aims ................................ ................................ ................................ .......... 98 Material and Methods ................................ ................................ ............................. 98 Chemicals and Reagents ................................ ................................ ................. 98 Animals ................................ ................................ ................................ ............. 98 Design of Pharmacokineti c Study ................................ ................................ ..... 99 Preparation of Visnagin Solutions for Administration to Rats ........................... 99 Preparation of Ammi Visnaga Extract and Solutions for Administration to Rats ................................ ................................ ................................ ............. 100 Instrumentation and LC MS/MS Conditions ................................ ................... 101 Sample Preparation and Analysis ................................ ................................ .. 101 Pharmacokinetic Data Analysis ................................ ................................ ...... 102 Non compartmental analysis ................................ ................................ .... 102 Comp artmental analysis ................................ ................................ .......... 103 Results ................................ ................................ ................................ .................. 106 Pharmacokinetic Study and Sample Analysis ................................ ................ 106 Non Compartmental Analysis ................................ ................................ ......... 107 Compartmental Analysis ................................ ................................ ................. 108 Model building and selection ................................ ................................ .... 108 Bootstrapping and construction of non parametric 90% bootstrap percentile confidence intervals for param eter estimates ....................... 111 Visual predictive check ................................ ................................ ............ 111 Discussion ................................ ................................ ................................ ............ 111 5 PHARMACODYNAMIC EV ALUATION OF VISNAGIN AND AMMI VISNAGA AQUEOUS EXTRACT IN RATS ................................ ................................ ........... 134 Background ................................ ................................ ................................ ........... 134 Specific Aims ................................ ................................ ................................ ........ 136 Material and Metho ds ................................ ................................ ........................... 136 Chemicals and Reagents ................................ ................................ ............... 136 Pilot Studies ................................ ................................ ................................ ... 13 7 Animals ................................ ................................ ................................ ........... 138 Study Design ................................ ................................ ................................ .. 139 Preparation of Treatments for Administration to Rats ................................ ..... 140 Urine and Plasma Collection ................................ ................................ .......... 141 Tissue Collection and Quantification of Calcium Oxalate Crystal Deposition 142 Statistical Analysis ................................ ................................ .......................... 143 Results ................................ ................................ ................................ .................. 144 Discussion ................................ ................................ ................................ ............ 148 6 SUMMARY AND CONCLUSION ................................ ................................ .......... 175 APPENDIX NONMEM CONTROL STREAM ................................ .............................. 181 LIST OF REFERENCES ................................ ................................ ............................. 183

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8 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 194

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9 LIST OF TABLES Table page 2 1 Intra day accuracy and precision for HPLC UV analysis of khellin. .................... 54 2 2 Inter day accuracy and precision for HPLC UV analysis of khellin. .................... 54 2 3 Intra day accuracy and precision for HPLC UV analysis of visnagin. ................. 54 2 4 Inter day accura cy and precision for HPLC UV analysis of visnagin. ................. 54 2 5 Comparison of khellin and visnagin peak areas of ethanol extracts. .................. 55 2 6 Visnagin solubility in different concentrations of aqueous Captisol solution. .... 55 2 7 Stability of standard water Ammi visnaga extract with respect to visnagin and khellin after storage of the lyophilisate at 20 C. ................................ ................ 56 3 1 Intra day accuracy and precision for LC MS/MS analysis of visnagin in rat plasma ................................ ................................ ................................ .............. 86 3 2 Inter day accuracy and precision for LC MS/MS analysis of visnagin in rat plasma ................................ ................................ ................................ ............... 86 3 3 Results of non compartmental analysis ................................ ............................. 86 3 4 Summary statistics of standard two stage parameter estimation approach, one compartment body model ................................ ................................ ........... 87 3 5 Summary statistics of standard two stage parameter estimation approach, two compartment body model ................................ ................................ ........... 87 3 6 Non linear mixed effect modeling approach, objective function values and p value for likelihood ratio tests. ................................ ................................ ............ 87 3 7 Non linear mixed effect modeling approach, two compartment body model, final estimates, standard errors, parametric and non parametric confidence intervals for typical values ................................ ................................ ................. 88 3 8 Non linear mixed effect modeling approach, two compartment body model, final estimates and non parametric confidence intervals for inter subject and intra subject varia bility ................................ ................................ ....................... 88 4 1 Results of non compartmental analysis ................................ ........................... 119 4 2 Non linear mixed effect modeling approach, final estimates and non parametric confidence intervals for typical values of the model developed with all data and i.v. data only ................................ ................................ .......... 120

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10 5 1 Treatment groups of pharmacodynamic study. ................................ ................. 162 5 2 Summary statistics of the treatment groups and pair wise comparison results. 163

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11 LIST OF FIGURES Figure page 1 1 Image of Ammi visnaga L. ................................ ................................ .................. 31 1 2 Image of Ammi visnag a L. fruits (compound umbels) ................................ ........ 31 1 3 Chemical structures of khellin and visnagin ................................ ....................... 32 2 1 Percolator for Ammi visnaga ethanol extract preparation. ................................ .. 57 2 2 Exemplary HPLC UV chromatogram of standard water Ammi visnaga extract (tea preparation) ................................ ................................ ................................ 57 2 3 Peak area of khellin and visnagin vs. ethanol concentration of extraction solvent ................................ ................................ ................................ ............... 58 2 4 Exemplary HPLC UV chromatogram of alternative water Ammi visnaga extract (water bath) ................................ ................................ ............................ 58 2 5 Exemplary HPLC UV chromatogram of Ammi visnaga ethanol 50% (v/v) extract (water bath) ................................ ................................ ............................ 59 2 6 Visnagin solubility in different concentrations of aqueous Captisol solutions ... 59 2 7 Visnagin solubility in different concentrations of standard water Ammi visnaga extract ................................ ................................ ................................ .. 60 2 8 Stability of standard water Ammi visnaga extract with respect to visnagin and khellin after storage of the lyophilisate at 20 C. ................................ ................ 61 3 1 Exemplary LC MS/MS chromatogram of internal standard warfarin and visnagin ................................ ................................ ................................ ............. 89 3 2 Visnagin plasma concentration (untransformed and log transformed) vs. time, sorted by dosing group. ................................ ................................ ...................... 89 3 3 AUC last vs. dosing group and fitted simple linear regression of AUC last vs. dosing group ................................ ................................ ................................ ...... 90 3 4 C 0 vs. dosing group and fitted simple linear regression of C 0 vs. dosing group ................................ ................................ ................................ ................. 90 3 5 Lineweaver Burk plot of observed plasma concentrations and fitted simple linear regression of dc/dt vs. 1/C mid across 23 subjects. ................................ ..... 91 3 6 Non linear mixed effect modeling approach, two compartment body model, basic goodness of fit plots. ................................ ................................ ................. 92

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12 3 7 Non linear mixed effect modeling approach, two compartment body model, population and individual fits. ................................ ................................ .............. 93 3 8 Non linear mixed effect modeling approach, bootstrap distribution of parameter estimates ................................ ................................ .......................... 94 3 9 Leave one out cross validation method, left out dosing group: high dose. ......... 95 3 10 Leave one out cross validation method, left out dosing group: medium dose. ... 95 3 11 Leave one out cross validation method, left out dosing group: low dose. .......... 96 4 1 Visnagin plasma concentration vs. time profiles after administration of the pure compound and Ammi visnaga extract, sorted by dosing group .............. 121 4 2 AUC inf and AUC last of visnagin after administration of pure compound vs. Ammi visnaga extract, sorted by dosing group ................................ ................ 121 4 3 MRT inf and MRT last of visnagin after administration of pure compound vs. Ammi visnaga extract, sorted by dosing group ................................ ................ 122 4 4 Comparison of visnagin amount absorbed after an exemplary oral dose of visnagin or equivalent dose of Ammi visnaga extract using the final model. .... 122 4 5 Non linear mixed effect modeling approach, two compartment body model, basic goodness of fit plots. ................................ ................................ ............... 123 4 6 Non linear mixed effect modeling approach, two compartment body model, population and individual fits of i.v. visnagin data (log transformed), sorte d by dosing group. ................................ ................................ ................................ .... 124 4 7 Non linear mixed effect modeling approach, two compartment body model, population and individual fits of oral visnagin data (log transformed), sorted by dosing group. ................................ ................................ ............................... 125 4 8 Non linear mixed effect modeling approach, two compartment b ody model, population and individual fits of oral Ammi visnaga extract data (log transformed), sorted by dosing group. ................................ .............................. 126 4 9 Non linear mixed effect modeling approach, bootstrap distribution of mean parameter estimates ................................ ................................ ........................ 127 4 10 Non linear mixed effect modeling approach, bootstrap distribution of inter individual and residual variability parameter estimates ................................ .... 128 4 11 Visual predicti ve check for the final model. Visnagin low dose after i.v. bolus administration. ................................ ................................ ................................ .. 129

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13 4 12 Visual predictive check for the final mod el. Visnagin medium dose after i.v. bolus administration. ................................ ................................ ......................... 129 4 13 Visual predictive check for the final model. Visnagin high dose after i.v. bolus administration. ................................ ................................ ................................ .. 130 4 14 Visual predictive check for the final model. Visnagin low dose after oral administration. ................................ ................................ ................................ .. 130 4 15 Visual predictive check for the final model. Visnagin medium dose after oral administration. ................................ ................................ ................................ .. 131 4 16 Visual predictive check for the final model. Visnagin high dose after oral administration. ................................ ................................ ................................ .. 131 4 17 Visual predictive check for the final model. Ammi visnaga extract low dose after oral administration. ................................ ................................ ................... 132 4 18 Visual predictive check for the final model. Ammi visnaga extract medium dose after oral administration. ................................ ................................ .......... 132 4 19 Visual predictive check for the final model. Ammi visnaga extract high dose after oral administration. ................................ ................................ ................... 133 5 1 Light microscope images of an exemplary kidney section after Pizzolato staining and after transformation to a binary image following different threshold adjustments using ImageJ software ................................ ................. 164 5 2 Time course of pharmacodynamic study. ................................ ......................... 164 5 3 Bar graphs summarizing the results of the pilot studies. ................................ .. 165 5 4 Bar plot of average crystal area after four weeks, sorted by treatment group 166 5 5 Average body weight vs. time, sorted by treatment group ............................... 166 5 6 Average urinary volume vs. time, sorted by treatment group .......................... 167 5 7 Average urinary pH vs. time, sorted by treatment group ................................ 167 5 8 Average citrate in urine vs. time, sorted by treatment group ........................... 168 5 9 Average oxalate in urine vs. time, sorted by treatment group .......................... 168 5 10 Average calcium in urin e vs. time, sorted by treatment group ......................... 169 5 11 Average calcium in acidified urine vs. time, sorted by treatment group ........... 169 5 12 Average calcium in plasma vs. time, sorted by treatment group ...................... 170

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14 5 13 Average microalbumin vs. time, sorted by treatment group ............................. 170 5 14 Average creatinine in plasma vs. time, sorted by treatment group .................. 171 5 15 Average creatinine clearance vs. time, sorted by treatment group .................. 171 5 16 Average blood urea nitrogen vs. time, sorted by treatment group ................... 172 5 17 Average kidney injury molecule 1 vs. time, sorted by treatment group ............ 172 5 18 Average osteopontin vs. time, sorted by treatment group ............................... 173 5 19 Light microscope image of a urine sample showing calcium oxalate dihydrate crystals. ................................ ................................ ................................ ............ 173 5 20 Bar graph of weekly average visnagin plasma concentration 1 and 6 h after visnagin and Ammi visnaga extract administration. ................................ .......... 174

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15 LIST OF ABBREVIATIONS ANCOVA Analysis of covariance ANOVA Analysis of variance ARH(1) H eterogeneous autoregressive variance components AUC A rea under the plasma concentration time curve AVE Ammi visnaga L. extract BIC Bayesian i nformation c riterion BUN Blood urea nitrogen C aOx Calcium oxalate CaP Calcim phosphate CED C alcium enriched diet CI Confidence interval CL Clearance CrCL Creatinine clearance CT Control group CV Coefficient of variation EG Ethylene glycol ESWL E xtracorporeal shock wave lithotripsy EX Extract group FDA Food and drug administration FOCE F irst order conditional estimation GFR Glomerular filtration rate H&E Hematoxylin and eosin HPLC UV High performance liquid chromatography with ultraviolet light detection IIV Inter individual variability

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16 IS Internal standard i v Intravenous KIM 1 Kidney injury molecule 1 KUVA Khellin ultraviolet A light therapy LB Lineweaver Burk LC MS/MS Liquid chromatography tandem mass spectrometry LLOQ Low limit of quantification LSM L east squared mean MM Michaelis Menten MRT M ean residence ti me NaDC 1 S odium dicarboxylate co transporter NCA Non compartmental analysis NHANES National H ealth and Nutrition Examination Survey NLME N on linear mixed effect NT Nephrolithiasic group OFV O bjective function value OPN Osteopontin PC Positive control PCi Potassium citrate PD Pharmacodynamic PK Pharmacokinetic QC Quality control SD Standard deviation SEM Standard error of the mean STS Standard two stage

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17 TOEP T oeplitz variance components UN Unstructured variance components VG Pure compound (visnagin) group

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18 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EXPLORING THE EFFECTS OF AMMI VISNAGA L. ON NEPHROLITHIASIS PREVENTION : IN VIVO PHARMACOKINETIC AND PHARMACODYNAMIC EVALUATION OF AMMI VISNAGA L. EXTRACT AND VISNAGIN By Karin G Haug May 2013 Chair: Hartmut Derendorf Cochair: Veronika Butterweck Major: Pharmaceutical Sciences K idney stones are common in developed countri es. Causes are multifold including genetic predisposition, but dietary habits and lifestyle are most important contributors to nephrolithiasis formation. Over the last 30 years, the prevalence of stone disease in the USA has increased from 3.8 to 8.8%. After the first incidence the recurrence rate within five to ten years is higher than 50%. Usage of current drugs for kidney stone prevention is often limited by side effects and tolerance. Thus, the problem of recurrent stone formation remains. Ammi visnaga L. (syn. Khella, Apiaceae) fruit preparations have traditionally been used in the Middle East for the management of nephrolithiasis Visnagin, a furanoc hromone is one of the main compounds of Ammi visnaga with p otential effects on nephrolithi asis prevention. No information is available about the pharmacokinetic (PK) properties of visnagin Therefore, the PK of visnagin after intravenous (i.v.) bolus injection of visnagin as well as after oral administration of the pure compound and vi snagin in form of an aqueous Ammi visnaga extr a ct (AVE) was characterized in rats.

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19 Preceding the PK studies, a HPLC UV method for visnagin quantification in visnagin and AVE solutions and a LC MS/MS method for visnagin quantification in rat plasma w ere developed an d validated. The i.v. study revea l ed n on linear elimination kinetics for visnagin and a non linear mixed effect model that describes the observed data was developed and validated The o ral PK studies demonstrated that average visnagin plasma concentration exposure (AUC) and residence time w ere significantly increased when administered in form of AVE A developed PK model that describe s both i.v. and oral PK data s uggested that this effect is induced by the AVE compound khellin ac ting as competitive inhibit or of the saturable elimination process. A pharmacodynamic study was conducted to test if these observed characteristics result in a superior efficacy of AVE compared to an equivalent dose of visnagin Utilizing a developed method for consistent and reprod ucible calcium oxalate crystal induction, p reventive effects of visnagin and AVE that were demonstrated in previous studies could not be confirmed Conclusively, the need for research and new substances in the a rea of kidney stone prevention continues

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20 CHAPTER 1 INTRODUCTION Numbers and Facts about Kidney Stones Approximately 9% of Americans are affected by kidney stones [ 1 ] According to a study based on data from National Health and Nutrition Examination Survey (NHANES) II (1976 to 1 980) and III (1988 to 1994) the prevalence of urolithiasis has increased between the late 19 70s and early 19 90s from 3.8% to 5.2% among 20 to 74 year old r esidents of the United States The latest data from the 2007 2010 NHANES demonstrates that the rise in prevalence of stone disease is ongoing and has reached 8.8% [ 1 2 ] Many European countrie s and Southeast Asia noticed a similar trend [ 3 ] This trend is alarming, considering that the recurrence rate within five to ten years after the first occurrence of nephrolithiasis is 50% and higher [ 4 5 ] The range f or estimates of i ncidence rates lies between 0.1 to 0.3% per year for men and 0.06 to 0.1% per year for women [ 6 ] By the age of 70, 18.8 % of men and 9.4 % of women in the United States will have developed kidney stones [ 1 ] While originally men were more prone to form stones a gender specific shift has been observed towards women in the last years [ 7 ] Reasons for kidney stone formation are multifold, most importantly including dietary habits such as restricted fluid intake and diet high in protein and sodium, as well as obesity and family history [ 5 8 11 ] Enhanced rates of hypertension and obesity, which are linked to nephrolithiasis, also c ontribute to the increase in stone formation [ 12 ] Kidney stones have recently been identified as risk factor for chronic kidney disease [ 13 ] Treatment costs in the United States including inpatient, outpatient and emergency room services are approximately $2 billion. However, the actual economic burden including work loss is estimated to be around $5 billion [ 6 14 ]

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21 Pat hophysiology of Nephrolithiasis Kidney stones can be classified by their chemical composition. Calcium oxalate (CaOx) is the most common stone compound (~60%), followed by calcium phosphate (C aP) (~20%), uric acid (9%) and struvite (10%), leaving 1% for other stone types like cystine and drug stones [ 15 16 ] Mixed calcium stones containing both CaOx and CaP are not unusual. One fundamental condition for clinical stone formation is urinary supersaturation with stone forming salts. The supersaturated compound generally determines the stone type. Supersaturation is often a result of dietary factors and metabolic abnormalities such as hypercalciuria, hypocitraturia, hyperoxaluria and hyperuricosuria [ 5 16 ] It is widely accepted that stone development requires crystal formation, retention and accumulation in the kidney [ 15 ] Nucleation is the first step in crystal formation which is followed by crystal growth and aggregation. In order for stones to form, crystals need to be retained in the kidney [ 17 ] If this step is missing, crystals will not accumulate and can be excreted with urine without consequences [ 18 ] Currently, three pathways of stone formation and growth are discussed, namely free particle model, fixed parti [ 15 17 ] The free particle hypothesis suggests rapid crystal growth after initial nucleation, eventually getting stuck in the lumen of the nephron. In the fixed particle model formed nuclei in the lumen get attached to some underlying surface. Thereby the nuclei are fixed in a position which is exposed to supersaturated urine, thus promoting crystal growth. Renal cell injury is the most suspected condition facilitating crystal cell attachment. The third theory proposes that exposed crystalline deposits of interstitial calcium phosphate become the site of attachment for urinary crystals. The interstitial plaque can overgrow, usually with several layers of different

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22 stone salts, and form a fixed stone which may develop over many years. It is likely that the stone formation process varies based on the stone phenotype [ 17 ] In conclusion, the pathogenesis of kidney stone formation is a complex process. Although several theories exist to explain the pathogenesis of renal calculi, the exact cascade of events that lead s to kidney stone formation is still unclear. Management of S tone D is ease Treatment and Prevention Selection of treatment options depends on the stone size and location. It can be distinguished between ureteral and renal stones. For ureteral stones with diameter less than 10 mm and if symptoms like pain are well controlled, an option for init ial treatment is observation with periodic evaluation. Support is given by data demonstrating that the probability of spontaneous stone passage increases with decreasing stone diameter [ 19 ] If stones pass, the most will do so within four to six weeks [ 20 ] Medical expulsive therapy such as off label use of alpha blockers (tamsulosin) or the calcium channel blocker nifedipine may facilitate and reduce time for stone passage and can be offered to patients For stones with diameter greater than 10 mm active stone remo val is usually required. Both extracorporeal shock wave lithotripsy (ESWL) and ureteroscopy are considered acceptable first line treatments [ 21 ] Most renal stones require active stone removal for example by means of ESWL and percutaneous nephrolithotomy. Uric acid stones are exempted for which oral chemolysis is consid ered first line treatment. Unfortunately, the tend ency for stone recurrence is not altered by stone removal with ESWL [ 22 24 ] Additionally ESWL might show some significant side effects such as renal damage, ESWL induced hypertension or renal impairment [ 25 ] Effective kidney stone prevention is dependent on the stone type and the identification of risk factors for stone formation. Patients are

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23 advised to follow dietary recommendations which should be preceded by a metabolic evaluation [ 4 ] If dietary changes are not sufficie nt, initiation of additional medical treatment will be required. Depending on the chemical composition of the first stone current medical options for prevention include potassium citrate (PCi) off label use of thiazide diuretics and the anti gout drug all opurinol [ 26 ] For the prevention of CaOx stones, which represent the majority of stones, allopurinol is not an option, leaving thiazides and PCi However, in a great proportion of patients usage of these drugs is limited by side effects and tolerance resulting in loss of efficacy. Additionally, while PCi reduces the risk of CaOx stones the rise in urinary pH reduces the solubility of CaP, therefore increasing the risk of CaP stones [ 26 ] Despite major technical achievements for stone removal in the last three decades the problem of recurrent stone formation remains and a specific, satisfactory drug to use in clinical therapy for prevention of recurre nt stones is still lacking. Therefore, focus should be directed towards the development of new strategies for the prevention of kidney stone disease s Herbal medicines could play an important role in closing a gap Available literature on herbal medicines and their possible role in the management of urolithiasis has recently been reviewed [ 27 ] and revealed that phytotherapeutic age nts could be useful as e ither alternative or complementary therapy in the management of urolithiasis Ammi V isnaga Ammi visnaga L. belongs to the family of Apiaceae also known as carrot or parsley family [ 28 29 ] The plant is native to the Mediterranean region, Europe, Asia and North Africa. It is also cultivated in Argentina, Chile, Mexico and the USA [ 28 29 ] Ammi visnaga Daucus visnaga L.) and toothpick weed or herb, because the umbels have ligneous rays which

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24 are used as toothpicks [ 28 30 ] The annual or biennial herb grows up to one meter high. The leaves are pinnate with filiform tips and w hite flo wer s are grou ped in umbels which carry the fruits following pollination (Figure s 1 1 and 1 2) [ 29 31 32 ] The small fruits can be described as double achenes, grayish brown in color with five light colored ridges, ellipsoidal or broadly ovate and about 1.5 to 3 mm long and 1 mm wide with a faintly bitter taste (Figure 1 2) [ 29 32 ] Ammi visnaga fruit preparations, such as teas prepared from crushed or powdered seeds, have traditionally been used in the Middle East to ease urinary tract pain associated with kidney stones and to promote stone passage [ 28 30 33 ] V isnagin and khellin (Figure 1 3 ) are among the main compounds of Ammi visnaga fruits belonging to the category of furano chromones (2 4%) Other constituents include pyranocoumarins (0.2 0.5%) such as visnadin and saminidin, traces of flavon oids (kaempferol, quercetin, isorhamnetin), volatile oil, 12 18% fatty oils and 12 14% proteins [ 29 ] Many properties have been attributed to Ammi visnaga and its constituents such as antibacterial, antifungal, antiviral, antidiabetic, antiinflammatory and neuroprotective [ 34 39 ] The most prominent characteristic of Ammi visnaga extract (AVE) and compounds is peripheral and coronary vasodilator y and antispasmodic activity, which relaxes smooth muscles and explains the traditional use for cardiovascular diseases, such as angina pectoris, but also for bronchodilation and urolithiasis [ 33 40 4 5 ] Visnadin, khellin and visnagin are thought to be responsible for this effect due to calcium channel blocking activity [ 41 45 ] Vasodilatory and a ntispasmodic effects on the urinary tract could possibly explain the usage for urolithiais, acting as expulsive therapy

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25 and thereby facilitating and reducing time for passage of smaller stones. Sensitization of skin is a known side effect of khellin [ 46 ] due to its photoactivity but this property has also been used in combination with ultraviolet A light ( K UVA khellin ultraviolet A therapy ) for the treatment of vitiligo a pigmentation disorder of the skin [ 47 49 ] However there is insufficient evidence to recommend KUVA in the official guidelines for vitiligo treatment [ 49 ] According to the German Commission E monograph of Ammi visnagae fructus from 198 6 updated 199 4 [ 46 ] claimed indications for use are as fol lows : Khella preparations are used in the treatment of angina pectoris, coronary insufficiency, paroxysmal tachycardia, extrasystoles, presbycardia with hypertension, asthma, whooping cough as well as cramp like discomforts in the lower abdomen.Preparatio ns containing khella fruit are also used as adjunctive therapy in the prevention of premature aging of the heart, circulat ory and vascular systems, after cardiac infarction, nervous heart disorders, hypertension, bronchitis, bronchial asthma and coughs, sp asms of the gastrointestinal biliary and urinary tracts, disorders of the hepatobiliary system, urolithiasis (renal calculus), tendency to form stones after surgery, kidney insufficiency, for the reduction of hormone based ureter dilation in the 2 nd and 3 rd trimesters of pregnancy or due to contraceptive use, as supportive antibacterial therapy in acute and chronic pyelonephritis in therapy resistant cases, for menopausal complaints, depressions as well as arteriosclerosis and associated symptoms. Neverth eless the efficacy of Ammi visnaga preparations for the listed indications has not been adequately substantiated [ 46 ] In later research, p revention of nephrolithiasis by an AVE was demonstrated by Khan et al. who reported a reduction of oxalate and calcium con tent in rat kidney s after administration of khella tea preparation [ 50 ] Also, our working group showed that an aqueous AVE as well as the pure compounds visnagin and khellin reduced cell injury after CaOx exposure in vitro and decreased CaOx crystal deposition in vivo in a commonly used nephrolithiasis model in rats [ 51 52 ] B ased on the results visnagin

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26 seems to be slightly more promising in the prevention of kidney stones than khellin. Interestingly while the extract demonstrated significant changes on the urinary chemistry of treated rats the pure compounds did not significantly affect those parameters [ 52 ] Thus, the question was raised if AVE possesses a superior efficacy over an equivalent d ose of the pure compound visnagin due to the fact that an extract is a multi component mixture In fact, there are examples where an extract shows a superior efficacy compared to the isolated individual compounds such as Hypericum perforatum L. [ 53 54 ] Possible reasons for additive or even synergistic effects of plant extracts can be categorized as pharmacodynamic (PD) or pharmacokinetic (PK) effects. An extract can contain several active components which may affect one or even multiple targets. These effects can sum up or be synergistic and this phenomenon can be classified as PD effects. A PK effect would be present if b yproducts of an extract trigger a change in a PK parameter of an active component, such as extent of absorption or clear ance, ultimately resulting in an altered plasma exposure (area under the curve (AUC)) of the active compound. A superior eff icacy of an extract compared to an isolated compound would justify the usage of an extract over pure compounds T h erefore visnagin and aqueous plant extract were selected for thorough PK and further PD evaluation for this dissertation project Pharmacokin etics and Pharmacodynamics PK quantifies the time course of drug and metabolite concentrations in the body and aids finding and optimizing dose, dosage regimen and dosage form. The fate of a drug after administration can be divided into liberation (in case drug needs to be released from a formulation) absorption, distribu tion, metabolism and excretion. PD describes the intensity of desired or side effects that is achieved by certain drug

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27 concentrations at the effect site, without accounting for drug concen tration changes over time. The aim of a pharmacological drug therapy is to achieve drug concentrations in the body (particularly at the effect site, which might be unknown) that produce a desired PD effect by utilization of a suitable dosing regimen includ ing administration site and do sage form Therefore, it is anticipated to find a correla tion between drug concentration and PD effect and to link PK and PD so that the time course of effects in response to a dosing regimen can be described [ 55 ] Quantification of var iability within and between individuals in PK parameters and PD effects is important for drug application in a broad population F actors explaining part of the variability, so called covariates like age, weight, disease status etc., are identified and impl emented, if available Ideally, a ll knowledge ca n be integrated in PK, PD and combined PK / PD models Among others, such models can be used for simulation of clinically relevant scenarios (e.g. variation of dose or dosing interval in case of multiple dosing ) and help to understand dose response relationships and to identify correct doses and dosing regimens. The development and utilization of models is used throughout the different stages of drug development and reaches back to stages as early as preclinical de velopment, where data is obtained from different animal species. N umerous pharmacological characteristics are attributed to m ost herbal medicines. However, in vitro studies, poorly designed in vivo experiments and/or historical tradition are often the source of claimed properties and indications. In in vivo studies i t is typically not tested if suspected active ingredients actually reach the systemic circulation and to which extent. PK characterization is a crucial step in drug

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28 development. I n orde r to gain wider credibility herbal medicines must undergo similar scrutiny to which synthetic drugs are subjected, including PK evaluation. Hypothesis and Specific Aims The main hypothesis claims that Ammi visnaga extract prevents kidney stone formation a nd is superior over an equivalent dose of visnagin. One possible cause for superior efficacy of AVE would be the change of PK parameters of visnagin, which has previously been demonstrated to be an active principle, resulting in enhanced plasma levels of v isnagin after AVE administration. Since n o information is available about the PK properties of visnagin and PK evaluation is required in the development of a new drug, the PK of visnagin in rats was thoroughly investigated in this dissertation project prio r to PD analysis Although intravenous (i.v.) administration would not be a preferred route for a drug used in preventive treatmen t, it is important to initially evaluate the PK of a new substance in an i.v. study This approach provides sound estimates of PK parameters and avoids for example confusion of absorption and elimination rate constant in a so called flip flop case. Thus, an i.v. PK experiment with three different doses of visnagin was performed. Evaluation of dose proportionality and description of the data by a suitable PK model including model parameter estimation were specific aims. Following an i.v. study, PK evaluation after oral administration was the next logical step. T he PK properties of visnagin after oral administration of the pure comp ound visnagin and visnagin in form of an aqueous AVE were characterized Enhanced plasma levels of key compounds after administration of plant extracts could lead to superior efficacy of an extract compared to an individual substance and might justify the usage of an extract over pure compounds.

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29 Thus, the comparison of visnagin plasma exposure after administration of the two preparations was of particular interest in th e oral PK studies In the previously conducted in vivo study AVE and the pure compounds visnagin and khellin were tested in separate experiments and doses of the pure compounds were not in accordance with their concentrations in the extract This makes it difficult to assess if AVE shows a superior efficacy. In order to confirm preventive effects on nephrolithiasis and test the hypothesis of superior PD efficacy of AVE over an equivalent dose of visnagin, a PD study in rats was conducted to test visnagin and AVE in one experiment using equivalent doses. Certain changes and improvements were made to the study design. An objective and efficient method for quantif icati on of CaOx crystal deposits in histological sections of rat kidneys was developed and used for analysis. A protocol for consistent and reproducible generation of CaOx crystal deposits in rat kidneys was determined, based on pilot studies using ethylene gly col in varying concentrations with or without dietary modification Preceding the PK and PD experiments it was inevi table to develop and validate analytical method s for quantification of vis nagin in visnagin solutions, AVE s and rat plasma The dissertati on was partitioned in to four parts with respective specific aims: P art 1: Characterization of AVEs and visnagin solutions Specific Aims: To develop and validate a high performance liquid chromatography with ultraviolet light detection (HPLC UV) method to quantify the key compou nds visnagin and khellin in AVEs To compare water and ethanol extracts of Ammi visnaga with respect to their visnagin and khellin concentrations.

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30 To determine visnagin solubility in water and standard aqueous AVE and explore options to enhance visnagin solubility. To determine the stability of visnagin solutions upon storage and AVEs with respect to visnagin and khellin upon reconstitution and storage. Part 2: PK evaluation of visnagin in rats after i.v. bolus administration Specific Aims: To develop and validate a liquid chromatography tandem mass spectrometry (LC MS/MS) method for quantification of visnagin in rat plasma. To evaluate the PK characteristics of visnagin in rats after i.v. bolus administration with respect to l inear or non linear PK. To develop an adequate PK model for the description of the observed data, including parameter estimation. Part 3: PK evaluation of visnagin and aqueous AVE in rats after oral administration Specific Aims: To evaluate the PK charact eristics of visnagin after oral administration of the pure compound visnagin and visnagin in form of standard aqueous AVE in rats. To compare visnagin plasma exposure after administration of the two preparations To develop an extenstion of the PK model th at was based on i.v. data for the description of the observed data (oral and i.v.), including parameter estimation. Part 4: PD evaluation of visnagin and aqueous AVE in rats Specific Aims: To develop a method for objective quantification of CaOx crystal d eposits in histological sections of rat kidneys. To determine a method for consistent and reproducible induction of CaOx crystal deposits in rat kidneys to serve as nephrolithiasis model in rats. To evaluate the effects of AVE and visnagin on kidney stone prevention and to investigate if AVE is superior over visnagin.

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31 Figure 1 1. Image of Ammi visnaga L. [ 31 ] Reprinted under creative commons license ( http://creativecommons.org/licenses/by/3.0/ ). Figure 1 2. Image of Ammi visnaga L. fruits (compound umbels) [ 32 ] Reprinted under creative commons license ( http://creativecommons.org/licenses/by/3.0/ ).

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32 Figure 1 3 Chemical structures of khellin (left) and visnagin (right)

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33 CHAPTER 2 CHARACTERIZATION OF AMMI VISNAGA EXTRACTS AND VISNAGIN SOLUTIONS Background Several pharmacokinetic ( PK ) studies and a pharmacodynamic ( PD ) study were performed for this dissertation pr oject, which required dosing of Ammi visnaga extract (AVE) and visnagin to rats. Throughout th ose experiments it was crucial to confirm concentrations of prepared visnagin so lutions and to quantify extracts of Ammi visnaga with respect to the marker compounds visnagin and khellin. Only if the typical concentrations of visnagin and khellin in an extract are known it can be assured that administered extract preparations contain equivalent amounts of pure compounds and thus, results after extract and isolated agent administration can be compared A standard quantification method is high performance liquid chromatography with ultraviolet light detection ( HPLC UV ) The aim for this project was to develop and validate a method that can distinguish and quantify visnagin and khellin within a short run time and with a simple mobile phase composition using isocratic elution. Ammi visnaga fruit preparations, such as teas prepared from crus hed or powdered seeds, have traditionally been used in the Middle East to ease urinary tract pain associated with kidney stones and to promote stone passage [ 28 30 33 ] A water extract was prepared based on the traditional tea preparation Utilization of less polar ethanol water mixtures as extraction solvent might facilitate higher yields of visnagin and khellin E thanol and ethanol water mixtures are solvent s commonly used for extract preparation European Pharmacopoea ( Ph.Eur. 7, Band 1 ) [ 56 ] The ideal concentration of the ethanol water mi xture that yield s maximum extraction of visnagin and khellin w as investigated in this project. Variations

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34 of extract preparation methods, such as percolation and water bath extraction, were tested and compared to each other. Solutions of extract and visnag in were used f or administration by oral gavage and for intravenous administration (visnagin only) Therefore, it was necessary to determine the solubility of visnagin and to explore possible options to enhance its solubility. Supportive byproducts in extra cts can enhance the solubility of other components of the extract which might lead to an increased resorption rate ultimately resulting in elevated plasma exposure of key compounds [ 57 58 ] An increase in plasma expo sure after extract administration could result in a superior efficacy of such an extract compared to an equivalent dose of a pure compound, if that compound is a major active ingredient. Against this background, the solubility of visnagin in w ater and in a solution of the standard aqueous AVE was compared. A la st point to address was the stability of freeze dried standard water extract after reconstitution and of prepared visnagin solutions Since most experiments last for days or even weeks the stability of freeze dried extract after storage needed to be investigated as well Specific Aims To develop and validate a HPLC UV method to quantify the key compounds visnagin and khellin in AVEs T o compare water and ethanol extracts of Ammi visnaga with respect to their visnagin and khellin concentrations. To determine visnagin solubility in water and standard aqueous AVE and explore options to enhance visnagin solubility.

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35 To determine the stability of visnagin solutions upon storage and AVEs with respect to visnagin and khellin upon reconstitution and storage Material and Methods Chemicals and Reagents Visnagin (>97%) and khellin were obtained from Sigma Aldrich (St. Loui s, MO, USA). Methanol was purchased from Fisher Scientific (Pittsburgh, PA, USA). Ethanol 190 proof USP was obtained from Decon Laboratories Inc. (King of Prussia, PA, USA). All chemicals used were analytical grade. Ammi visnaga L. fruits (DAB 10 quality, invoice 12119085) were kindly provided by Martin Ba uer Group Finzelberg GmbH & Co. KG (Andernach, Germany). Captisol cyclodextrin sulfobutyl ethers, sodium salts) was purchased from Cydex Pharmaceut icals (Lenexa, KS, USA) (meanwhile acq uired by Ligand Pharmaceuticals Inc. (La Jolla, Ca, USA) HPLC grade deionized water was prepared using a Barnstead Nanopure Diamond UV ultra pure water system (Dubuque, IA, USA). Instrumentation and HPLC UV Conditions Chromatographic separation was performed on a Symmetr y C 18 4.6 mm x 50 mobile phase was comprised of deionized water and methanol (55:45, v/v) and was degased prior to use utilizing helium gas. Mobile phase was delivered at a flow rate of 0. 5 mL /min and the i njection volume of samples was 5 L Auto sampler rinse solution consisted of 50% methanol. The wavelength for UV detection was set to 250 nm. An integrated LC 2010 HPLC system from Shimadzu Scientific Instruments Inc. (Columbia, MD, USA), comprised of auto sampler, pump, column oven and UV/VIS

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36 SP1 software from Shimadzu. Preparation of V isnagin and K hellin C alibration S tandards and Q uali ty C ontrol S amples Separate s tock solution s (1 mg/m L ) of visnagin and khellin w ere prepared in methanol Calibration standards for a six point calibration curve ranging from 2 to 1 2 g/mL were prepared by adequately diluting the respective stock solution in mobile phase after filtration of the stock solution through Millex sterile syringe filter units (Millipore, Cork, Ireland) to remove any floating contamination Independent quality control (QC) samples were prepared at concentrations of 2 6, and 12 g/mL in the same manner. S tock solution s of both compounds were demonstrated to be stable for at least four weeks at 4C This was confirmed by repeated preparation of calibration curves from the same stock solution over four weeks and compari son of slope and intercept values to those from calibration curves prepared with fresh standards Method Validation Method validation included determination of calibration curve, accuracy and precision. Validations for visnagin and khellin were performed s eparately, but in the same fashion. For evaluation of the relationship between instrument response and nominal compound concentrations, one se t of calibration standards was prepared per run. The calibration curve was fitted by simple linear regression of t he peak area on the nominal standard concentration in g/ mL The criterion for acceptance of the calibration curve was a coefficient of determination (R 2 ) of 0.99 9 or greater. Accuracy and precision were determined utilizing three concentrations of independent QC samples (n=3 ): one low QC ( 2 g/m L ), one middle QC ( 6 g/m L ), and

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37 one high QC ( 12 g/m L ). Calculation of QC sample concentrations was based on the fitted regression equation. Validation was carried out on three independent days. Intra and inter day accuracy was determined as the deviation of the calculated average to the nominal concentration I ntra and inter day precision was expressed as coefficient of determination (CV) at each concentration. Extract Preparation and Qu antification Water extract The standard aqueous AVE was prepared according to the method described previously [ 51 52 ] Briefly, one part (weight, e.g. 40 g) Ammi visnaga fruits were grinded Inc., M iramar, FL, USA). Ten parts (volume, e.g. 400 mL ) boiling water (100C) w ere poured over the grinded fruits and the mixture was steeped for 5 min at room temperature with occasional swirling. Subsequently, the aqueous mixture was filtered through No. 2 Wha tman filter paper circles (pore size 8 UK) and freeze dried (FreeZone 6, Labc onco, Kansas City, MO, USA). Three samples of lyophilisate w ere dissolved in methanol (2 mg/mL) filtered through Millex non sterile syr inge filter units (Millipore, Cork, Ireland) diluted 1:10 in mobile phase and quantified with respect to the key compound s visnagin and khellin by the validated HPLC UV method The lyophilisate was kept in an amber colored bottle at 20C An alternative water extract was prepared in a similar manner. Instead of boiling water, ambient temperature water was added to the grinded fruits and the mixture was steeped for 30 min in a water bath at 40 C with occasional swirling. Subsequently, the aqueous mixture w as filtered through No. 2 Whatman filter paper circles (pore size 8 For direct comparison of the two water extract preparations, freshly prepared

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38 extracts (without freeze drying step) were quantified in triplicate with respect to the key compound s visnagin and khellin by the validated HPLC UV method after being filtered through Millex on sterile syringe filter units and dilut ion 1:100 in mobile phase Ethanol extract A series of ethanol extracts was prepared in duplicate using ethanol c oncentrations in creasing stepwise from 0 to 100% (v / v ) ethanol in steps of 10% For each extract preparation 3 g Ammi visnaga fruits were grinded for 30 sec in a regular coffee grinder. 30 mL of the respective extraction solvent was poured over the grinde d fruits and the mixture was steeped for 30 min in a water bath at 40 C with occasional swirling Subsequently, each mixture was filtered through No. 2 Whatman filter paper circles (pore size 8 ). After cooling down to room temperature, f reshly prepared extracts were filtered through Millex sterile syringe filter units and diluted 1:100 in mobile phase The validated HPLC UV method was used to quantify the area under the curve of the key compound s visnagin and khellin in order to determine the extraction solvent yielding maximum compound concentrations. A different method was tested for extract preparation, namely percolation, European Pharmacopoea (Ph.Eur. 7, Band 1) [ 56 ] Therefore, one part A mmi visnaga fruits were grinded for 30 sec in a regular coffee grinder Approximately 1/3 part of 50% ( v/v ) ethanol was poured over the grinded fruits and the mixture was covered and steeped for 2 h. Afterwards, the slurry was transferred into a self buil (Figure 2 1). A glass liquid chromatography column with a valve was used for this purpose. Cotton was placed at the bottom of the column to serve as filter. The drug solvent slurry was poured on top of the cotton layer

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39 and compressed under m ild pressure. A final cotton layer on top prevented swirling of the drug upon addition of extraction solvent. The valve was opened and solvent was added until extract started dropping. Subsequently, the valve was closed and solvent was added to cover the t op layer of cotton. The percolator was sealed with aluminum foil and allowed to stand for 24 h. Afterwards, the valve was opened to achieve a dropping frequency of three drops/minute. Sufficient solvent needed to be added to cover all drug throughout the percolation process, which was discontinued after approximately two parts of solvent were added and recovered. Leftover liquid in the drug slurry was squeezed out and add ed to the ex t ract. Ethanol was removed from the extract using vacuum evaporation to concentrate the extract prior to freeze drying. Three samples of lyophilisate w ere dissolved in methanol (2 mg/mL), filtered through Millex sterile syringe f ilter units diluted 1:10 in mobile phase and quantified with respect to the key compound s visnagin and khellin by the validated HPLC UV method The lyophilisate was kept in an amber colored bottle at 20C For direct comparison of the percolation method with the method using the same extraction solvent (50% (v/v) ethanol) but steeping the mixture for 30 min in a water bath at 40 C the extract using the water bath method was freeze dried and three samples were processed the same way as the percolation ext ract and were analyzed with respect to the key compound s visnagin and khellin by the validated HPLC UV method. Visnagin Solubility In water In order to evaluate its water solubility, visnagin was added in excess (2.5 to 3 mg) to six 1.5 mL Eppendorf centrifuge tubes containing 1 mL water each. Three of the

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40 samples were shaken for 1 h and the other three samples for 24 h at maximum speed (25 rpm) on a Lab Line Maxi Rotator (Lab Line Instruments Inc., Melrose Park, IL, USA ) at room temperature. Subsequently, samples were centrifuged at 10. 000 rpm for 15 min. The clear supernatant was diluted in mobile phase and analyzed with respect to visnagin by the validated HPLC UV method. In Captisol For the PK studies, visnagin solut ions with a concentration of 2.5 mg/mL were targeted due to the anticipated doses and dosing volume limitations. Captisol a uniquely modified and Food and Drug Administration ( FDA ) accepted cyclodextrin, was tested for its adequacy to enhance the aqueous solubility of visnagin to concentrations that were sufficient to achieve the required dosing concentrations Therefore, v isnagin was added in excess to each of three aqueous solutions containing 40, 30, 20, 15, 10, 5, and 2.5 % Capti sol The mixtures were shaken for 72 h at maximum speed (25 rpm) on a Lab Line Maxi Rotator at room temperature. Subsequently, samples were centrifuged twice at 10.000 rpm for 1 0 min. The clear supernatant was diluted in mobile phase and analyzed with re spect to visnagin by the validated HPLC UV method. Based on the results, 25% aqueous Captisol solution was tested for its adequacy to produce visnagin solutions with a concentration of 2.5 mg/mL. For this experiment, the shaking time was reduced to 30 min T he solution was filtered through Millex non sterile syringe filter units prior to analysis In standard Ammi visnaga water extract Visnagin solubility in standard aqueous AVE was initially assessed by analysis of three samples of the freshly prepared standard water extract (without freeze drying step) after filtration through Millex sterile syringe filter units and 1:100

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41 dilution in mobile phase. Additionally, concentrations of 100, 250, 500, and 1000 mg/mL AVE in water were prepared in triplicate in 1.5 mL Eppendorf centrifuge tubes The mixtures were shaken for 1h at maximum speed (25 rpm) on a Lab Line Maxi Rotator at room temperature and afterwards centrifuged at 10.000 rpm for 15 min. The supernatant of the two low conce ntrations were filtered through Millex sterile syringe filter units and adequately diluted in mobile phase (1:200 for 100 mg/mL and 1:500 for 250 mg/mL). The supernatant of the two high concentrations was too viscous to filter and thus, filt ration through Millex sterile syringe filter units was carried out after an initial 1:10 dilution in mobile phase (final dilution for 500 and 1000 mg/mL: 1:1000). Dilutions were quantified with respect to visnagin by the validated HPLC UV me thod When added to standard Ammi visnaga water extract The solubility of visnagin upon addition to standard aqueous AVE reconstituted in water was determined to test if visnagin solubility increases when AVE is used as solvent instead of water. Therefore, visnagin was added in excess (approximately 5 mg) to three 1.5 mL Eppendorf centrifuge tubes containing 50 mg AVE in 1 mL water each, while three other tubes contained only 50 mg AVE in 1 mL water each (no external visnagin addition). The mixtures were sh aken for 1h at maximum speed (25 rpm) on a Lab Line Maxi Rotator at room temperature and filtered through Millex non sterile syringe filter units Filtrates were diluted in mobile phase (1:200 if external visnagin was added, 1:100 without exter nal visnagin) and quantified with respect to visnagin by the validated HPLC UV method Based on the results of the visnagin content in the samples containing AVE only it c ould be calculated how much visnagin

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42 dissolved from the external visnagin excess and contributed to the overall visnagin concentration of the filtrate. Stability V isnagin in C aptisol Visnagin stability in 25% aqueous Captisol and most suitable storage conditions were evaluated. Therefore, 10 mg visnagin were added to 4 mL of 25% Captisol solution and shaken for 30 min at maximum speed (25 rpm) on a Lab Line Maxi Rotator at room temperature. One part of the solution was stored at room temperature while the other part was stored at 4C. After one day, the solution t hat was stored at 4C c ontained needle type precipitation s Both solutions were filtered through Millex HV sterile syringe filter units diluted in mobile phase and quantified with respect to visnagin by the validated HPLC UV method T he solutions were reanalyzed one week after preparation Since the soluti on that was stored at 4C demonstrated again needle type precipitation s it was filtered through a Millex sterile syringe filter unit An aliquot of the solution stored at room temperature was not fil tered again, while another aliquot was filtered through a sterile Millex filter unit (Millipore, Cork, Ireland) in order to assess any loss due to sterile filtration which will become necessary for parenteral administration during intrav enous PK studies. Two weeks after preparation, the solution stored at room temperature was reanalyzed without further filtration. Long term stability of visnagin in 25% Captisol at room temperature was also investigated. Visnagin solution with an anticipa ted concentration of 1.5 mg/mL was quantified after preparation as well as after ten months of storage at room temperature.

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43 V isnagin and khellin in extracts The standard water extract was analyzed directly after freeze drying and after one two and three weeks of storage in an amber colored bottle at 20C After three weeks, the extract was freeze dried again to remove any adsorbed water and was afterwards analyzed as well as after five and nine weeks of storage under addition of a SORB IT package Addi tionally, a sample of the extract after freeze drying again at week three was analyzed 2 h after being reconstituted in water to assess its stability in water upon reconstitution The ethanol extract obtained by percolation was reassessed after four weeks For analysis of freeze dried extracts t hree samples of lyophilisate w ere dissolved in methanol (2 mg/mL) filtered through Millex sterile syringe filter units diluted 1:10 in mobile phase and quantified with respect to the key compound s visnagin and khellin by the validated HPLC UV method The only exception was when the stability upon reconstitution in water was tested, in which case water instead of methanol was used for reconstitution. Results Method Validation Utilizing the described column, settings and mobile phase comprised of deionized water and methanol (55:45, v/v), khellin and visnagin could be separated with good resolution within a run time of 15 min. The retention times were approximately 9.7 min and 12.6 min for khellin and visnagin respectively (Figure 2 2) All calibration curves met the acceptance criteria. For both khellin and visnagin, i ntra and inter day accuracy was less than 4% deviation and precision was less than 2% CV for all three levels of QC s The results of the validation are listed in Tables 2 1 to

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44 2 4 The limit of quantification was defined as the concentration of the low QC at 2 g/mL Extract Quantification Water extract The quantitative amount s of khellin and visnagin in the standard water extract directly after freeze drying were 32 2 mg (3.1 % CV) and 15 2 mg (2.8% CV) per g lyophilisate, respectively A representative HPLC UV chroma togram is depicted in Figure 2 2 The drug:extract ratio was 5 6:1, meaning that five to six parts plant material yield one part lyophilisate. The quantitative amount s of khellin and visnagin in the standard water extract without freeze drying were 0.73 mg/mL (2. 9 % CV) and 0.34 mg/mL (2.4 % CV) respectively When preparing the alternative fresh water extract by steeping the drug water mixture for 30 min in a 40C water bath, slightly more khellin and visnagin could be extracted, namely 0.76 mg/mL (2.3 % CV) khellin and 0.35 mg/mL (1.7 % CV) visnagin Ethanol extract The results of the comparison of khellin and visnagin peak areas following extraction using differe nt concentrations of ethanol (0 to 100% (v/v)) are listed in Table 2 5 and graphically summarized in Figure 2 3 The extraction power with respect to khellin and visnagin increase d with increasing ethanol concentration to reach a maximum at 50% (v/v) before decreasing when the water content of the solvent was further reduced. The concentrations of khellin and visnagin in the 50% (v/v) ethanol extract w ere elevated by about 60 to 7 0% compared to the extract when only water was

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45 used as solvent. Exemplary HPLC UV chromatograms of the extracts utilizing water and 50% (v/v) ethanol as extraction liquid are s hown in Figures 2 4 and 2 5 respectively The quan titative amounts of khellin and visnagin in the extract using the percolation method with 50% (v/v) ethanol were 54 0 mg (3.6 % CV) and 26 6 mg (3.2% CV) per g lyophilisate, respectively. The quantitative amounts of khellin and visnagin in the extract using the water bath method with 50% (v/v) ethanol were slightly increased with 55 5 mg (3.0 % CV) and 27 6 mg (2.9 % CV) per g lyophilisate, respectively. Visnagin Solubility In water The aver age concentration of visnagin dissolved in water after shaking for 1 h and 24 h was 0.16 mg/mL (3.4 % CV) and 0.16 mg/mL (7.2 % CV), respectively. Thus, 1 h was sufficient to dissolve the maximum amount of visnagin in water. In Captisol Visnagin solubility in different concentrations of aqueous Captisol solution is listed in Table 2 6 and depicted in Figure 2 6 Visnagin solubility increase d approximately linear with increasing concentrations of the cyclodextrin. In order to achieve visnagin concentrations of 2.5 mg/mL, more than 20%, but possibly less than 30% Captisol are required. When a solution of 2.5 mg/mL visnagin in 25% aqueous Captisol was prepared followed by shaking for 30 min and filtering through a Millex non sterile syringe filter unit, 2.19 mg/mL (1.14 % CV) visnagin could be recovered in the solution. In standard Ammi visnaga water extract The average concentration of visnagin in the freshly prepared standard aqueous AVE (without freeze drying) was 0.34 mg/mL (2.4 % CV).

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46 The concent rations of visnagin in solutions containing 100, 250, 500, and 10 0 0 mg/mL AVE in water were analyzed to be 1.6 (1.5% CV), 3.6 (2.0% CV), 6.2 (3.2% CV), and 7.3 (13.1 % CV) mg/mL, respectively ( Figure 2 7 ) When added to standard Ammi visnaga water extract The average concentration of visnagin in the solution containing 50 mg standard aqueous AVE in 1 mL water was 0.80 mg/mL or back calculated 15.8 mg (3.2 % CV) per g lyophilisate. The average concentration in the solution containing 50 mg standard aqueous AV E in 1 mL water plus externally added visnagin was 1.97 mg/mL. The estimate of 15.8 mg visnagin per g lyophilisate was utilized to calculate how much of the dissolved visnagin originated from the added AVE. An average of 0.78 mg/mL visnagin originated from the extract, while 1.19 mg/mL visnagin dissolved from the externally supplied visnagin powder, which represents an increase of more than 600% in the solubility of visnagin. Stability Visnagin in Captisol After one day of storage at room temperature and 4C, the measured concentrations of visnagin in the anticipated 2.5 mg/mL visnagin in 25% aqueous Captisol solution follo wing filtration were 2.38 (0.58 % CV) and 2.06 (6.11 % CV) mg/mL, respectively. After one week of storage at 4C followed by filtration ( non sterile) to remove needle like precipitations the measured concentration of visnagin further decreased to 1.29 mg/mL (1.33 % CV). Thus, storage at 4C is not suitable. The solution stored at room temperature s till contained 2.39 mg/mL (1.01 % C V) if not filtered again

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47 and 2.41 mg/mL (1.49 % CV) after 0.22 sterile filtration. Hence, filtration through 0.22 sterile syringe filter units does not affect the recovered visnagin concentration. After two weeks of storage at room temperature, there was not enough solution left to analyze three replicates. However, one aliquot could be quantified and the visnagin concentration in this solution was determined to be 2.53 mg/mL. The concentration of visnagin i n the anticipated 1.5 mg/mL visnagin in 25% aqueous Captisol solution after preparation and after ten months of storage at room temperature was 1.23 mg/mL and 1.36 mg/mL, respectively. Visnagin and khellin in extracts The results of the stability tests of AVE are presented in Table 2 7 and visualized in Figure 2 8 After initial freeze drying, t he quantitative amount s of khellin and visnagin in the standard water extract were 32.2 mg (3.1 % CV) and 15.2 mg (2.8 % CV) per g lyophilisate, respectively The amounts decreased slightly over the first weeks reaching 29.1 mg (0.8 % CV) and 13.4 ( 0.8% CV) after three weeks. This decline was accompanied by a change in the appearance of the lyophilisate towards becoming somewhat sticky. Assuming that some water adsor ption might have occurred, the lyophilisate was freeze dried again. The quantitative amounts of khellin and visnagin increased after this process to 31.1 mg (2.1% CV) and 14.4 mg (2.6% CV) per g lyophilisate, respectively. After further storage with a SORB IT package, the amounts of khellin and visnagin in the lyophilisate stayed relatively stable. At week three after freeze drying the lyophilisate again, it was tested if the extract is stable upon recons titution in water. 30.4 mg (1.3 % CV) khellin and 14.0 mg (1.5% CV) visnagin per g lyophilisate could be found in the extract 2 h after reconstitution in

PAGE 48

48 water when back calculated to the lyophilisate. The values are slightly lower compared to the ones when dissolved in methanol and immediately analyzed. The ethanol extract after percolation was reassessed after four weeks of storage at 20C. The quantitative amounts of khellin and visnagin were negligi ble decreased with 53.0 mg (1.6 % CV) and 25.0 mg (2.0% CV) per g lyophilisate, respectively. Discussion The first aim of this study was t o develop and vali date a HPLC UV method to quantify visnagin and khellin which wa s required to pursue all further objectives of this project Emphasis was placed on validating a practical, not necessarily new method, compr ising a short run time with a simple mobile phase composition using isocratic elution knowing that methods for determination of khellin and visnagin in AVEs exist [ 59 61 ] Also, it was not t he purpose to develop a method that gives detailed information about the extract composition with respect to other compounds. The presented method fulfills its expectations and can be used to quantify khellin and visnagin in AVEs at sufficiently low concentrations within a run time of 15 min utilizing isocratic elution and a mobile phase consisting solely of the inexpensive solvents methanol and water. Stability of stock solutions over four weeks was confirmed and standard solutions of khellin and visnagin were previously found to be stable on an auto sampler at 20C [ 60 ] and thus, this was not tested again in this validation. The quantitati ve amount s of khellin and visnagin in the standard water AVE directly after freeze drying were 32.2 mg and 15.2 mg per g lyophilisate, respectively These results are in agreement with the amounts reported in previous experiments, when 28.1 mg khellin and 17.2 mg visnagin per g lyophilisate were found [ 60 ] Slight

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49 discrepancies might be explained by natural variation of the different plant material used for extract preparation. When the standard water extract based on classic tea preparation wa s compared to an alternative method using a water bath at 40C extracted amounts of khellin and visnagin were slightly elevated using the water bath method. Due to the minor differences in extraction power, the shorter preparation time and the analogy to the traditional tea preparation, it was decided to adhere to the standard water extract preparation. The usage of 50% (v/v) ethanol as solvent provided a significant increase of about 60 70% in extraction power with respect to khellin and visnagin. The rec overy of khellin and visnagin increase d with increasing ethanol concentrations to reach a maximum at 50% (v/v) before decreasing when the water proportion of the solvent was further reduced. Both khellin and visnagin are rather lipophilic compounds (partit ion coefficient 1.72 and 1.87) explain ed why ethanol as low polar solvent compared to water enhances the extraction power of those compounds. A certain amount of water is usually necessary for moisture expansion of the plant material This could contribute to the observation of decreasing amounts of khellin and visnagin in extracts using ethanol concentrations higher than 50% (v/v). Extracted a mounts of khellin and visnagin were slightly elevated when the water bath method at 40C was compared to the percolation method both using 50% (v/v) ethanol This difference might be explained by an improved extraction power due to the elevated temperatur e during extraction using the water bath method. Given the higher instrumental and time effort there seems to be no reason to perform percolation

PAGE 50

50 extraction when recovery of khellin and visnagin is the main purpose. Percolation extraction is conducted at room temperature and thus, can be beneficial for compounds sensitive to elevated temperature, which is not the case for khellin and visnagin. Although higher amounts of khellin and visnagin could be extracted using 50% (v/v) ethanol as solvent, the standar d water AVE will be used in future PK and PD studies due to its similarity to the traditional tea preparation. The water solubility of visnagin was determined to be 0.16 mg/mL, which is by far too low for the anticipated concentration of dosing solutions o f 2.5 mg/mL for the PK studies. Therefore, Captisol a uniquely modified and FDA accepted cyclodextrin, was tested for its adequacy to enhance the aqueous solubility of visnagin to concentrations that were sufficient to achieve the required dosing concent rations. As previously mentioned, concentrations of 2.5 mg/mL visnagin were targeted while keeping the concentration of the cyclodextrin as low as possible. The experiment testing a seri es of aqueous Captisol solutions revealed an approximately linear inc rease of visnagin solubility with increasing Captisol concentrations. The solubility in 20% aqueous Captisol (1.43 mg/mL) was lower than expected, given the linear trend for the different Captisol concentrations. The added amount of visnagin in this case was not high enough since all visnagin was dissolved. Thus, the actual visnagin solubility is supposedly higher than the presented value Nevertheless more than 20% but less than 30% aqueous Capti sol solution should be suitable to dissolve enough visnagin to reach a concentration of 2.5 mg/mL When a solution of 2.5 mg/mL visnagin in 25% aqueous Captisol was prepared followed by shaking for 30 min and filtering through a Millex HV non ste rile syringe filter unit, 2.19 mg/mL visnagin could be recovered in the

PAGE 51

51 solution. The lower than expected solubility wa s most likely due to weighing inaccuracies or compound loss during preparation, rather than limited solubility. Furthermore, it is more i mportant to know the actual visnagin concentration in the solution than to achieve exactly the anticipated concentration, since the dosing volume can be slightly adjusted after determination of the concentration to ensure administration of the targeted dos e. Thus, 25% aqueous Captisol was selected as solvent for visnagin for future PK and PD studies. The average concentration of visnagin in freshly prepared standard water AVE (without freeze drying) was 0.34 mg/mL, which is more than double as high as the visnagin solubility in water. This indicates that AVE contains certain byproducts that enhance the solubility of visnagin. When increasing concentrations of AVE we re reconstituted in water, recovered visnagin increase d as well initially linear but levelin g off around concentrations of 7.3 mg/mL in 1000 mg/mL AVE. The CV also increased for higher AVE concentrations, which can be explained by higher viscosity of those solutions, making accurately pipetting more challenging. Furthermore, visnagin solubility o f the 500 and 1000 mg/mL AVE solutions should be evaluated with caution. These solutions were too viscous to directly filter them prior to dilution in mobile phase and analyzing visnagin by the validated HPLC UV method Hence, filtration was performed afte r the first dilution step in mobile phase. Therefore, it is possible that visnagin that was only suspended in the viscous AVE mixture was transferred and dissolved in the first dilution step and thus, measured visnagin solubility might represent a false increased value However, there is no doubt that visnagin solubility is increased in AVE compared to water. When visnagin was added to a solution containing 50 mg/mL AVE,

PAGE 52

52 1.19 mg/mL of the externally added visnagin could be dissolved in addition to 0.78 mg/mL visnagin already contained in the AVE. Given a water solubility of 0.16 mg/mL, this represents a tremendous increase in solubility. Visnagin stability in 25% aqueous Captisol is only ensured if stored at room temperature, but not at 4C. It is not clear why needle type precipitates appear ed upon storage at 4C, but it is assumed that the precipitate represents Captisol which would explain why visnagin concentrations in the solution decrease d after precipitate removal. The visnagin solution of targeted 2.5 mg/mL in 25% aqueous Captisol d id not deteriorate within two weeks of storage at room temperature. The concentration even increased slightly from 2.38 mg/mL one day after preparation to 2.53 mg/mL two weeks after preparation. This increase ca nnot be explained, because the solution was filtered one day after preparation through a Millex non sterile syringe filter unit. Thus, even if not all visnagin was dissolved after one day, no undissolved visnagin could have passed through the fi lter to get in solution at a later time. However, a similar increase in visnagin concentration was observed after ten months of storage at room temperature. Fluctuations in the calibration standards of the HPLC UV method could possibly give explanation to this phenomenon. Nevertheless visnagin in 25% aqueous Captisol is sufficiently stable and does not need to be prepared freshly throughout planned PK and PD studies lasting maximal three and five weeks, respectively. The amounts of khellin and visnagin in standard water AVE decreased slightly within three weeks of storage accompanied by a change in the appearance of the lyophilisate towards becoming somewhat gooey. It was assumed that water adsorption had occurred, either during storage or during weekly op ening of the container for

PAGE 53

53 stability testing. After freeze drying the extract again after three weeks of storage at 20C, the quantitative amounts of khellin and visnagin increased almost back to the original values after initial determination. After furt her storage with a SORB IT package for water adsorption the amounts of khellin and visnagin in the lyophilisate stayed relatively stable. Thus, it was concluded that water adsorption caused the decreased measurements of khellin and visnagin, because a sa mple of AVE for analysis contained comparatively less compounds due to its higher water content. Consequently, it was decided to add SORB IT packages to the storage container of freshly prepared AVE lyophilisate f or future experiments AVE was sufficientl y stable 2 h after reconstitution in water. For both PK and PD studies, AVE solutions will be prepared just before administration and most likely will be dosed much earlier than 2 h after reconstitution. The stability of AVEs seems to be independent of the preparation method, since the amounts of khellin and visnagin in the ethanol extract after percolation decreased only negligible compared to the fresh lyophilisate. In conclusion, a HPLC UV method for quantification of khellin and visnagin was developed and validated. The method was applied to analyze AVEs and visnagin solutions with respect to compound concentrations and to answer questions regarding solubility and stability. 25% aqueous Captisol was determined as adequate solven t for preparation of visnagin solutions for PK and PD studies.

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54 Table 2 1. Intra day (at three separate days, n=3) accuracy and precision for HPLC UV analysis of khellin Quality Control Samples ( mL ) Day 2 6 12 1 0.86 1.49 0.97 Accuracy Deviation ( % ) 2 3.64 1.22 0.51 3 3.78 0.99 0.01 1 0.41 0.18 0.12 Precision CV ( % ) 2 0.52 0.47 0.07 3 0.70 0.10 0.28 Table 2 2. Inter day (averaged over three separate days, n=3) accuracy and precision for HPLC UV analysis of khellin Quality Control Samples ( mL ) 2 6 12 Accuracy Deviation ( % ) 2.76 0.24 0.15 Precision CV (%) 1.69 1.50 0.75 Table 2 3. Intra day (at three separate days, n=3) accuracy and precision for HPLC UV analysis of visnagin Quality Control Samples ( mL ) Day 2 6 12 1 1.8 0.61 0.13 Accuracy Deviation ( % ) 2 3.15 2.45 2.06 3 1.88 0.32 0.42 1 0.15 0.12 0.1 Precision CV ( % ) 2 0.09 0.09 0.11 3 0.55 1.75 0.16 Table 2 4. Inter day (averaged over three separate days, n=3) accuracy and precision for HPLC UV analysis of visnagin Quality Control Samples ( mL ) 2 6 12 Accuracy Deviation ( % ) 2.27 0.72 0.78 Precision CV ( % ) 0.78 1.58 1.15

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55 Table 2 5. Comparison of khellin and visnagin peak areas of ethanol extracts Khellin Visnagin Ethanol ( % (v/v) ) Peak A rea 0 7535574 3715940 10 8269544 4150124 20 9096457 4715564 30 9822153 5276633 40 10222434 5619820 50 10805379 5993292 60 10260819 5727682 70 10255759 5714376 80 9819805 5472332 90 8780438 4854082 Table 2 6. Visnagin solubility (mean ( SD ) n=3) in different concentrations of aqueous Captisol solution Captisol ( % ) Visnagin ( mg/ mL ) 2.5 0.305 (0.032) 5 0.526 (0.034) 10 0.860 (0.070) 15 1.227 (0.049) 20 1.430 (0.063) 30 3.050 (0.026) 40 4.346 (0.073)

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56 Table 2 7 Stability of standard water Ammi visnaga extract with respect to visnagin and khellin after storage of the lyophilisate at 20 C. Compound per g lyophilisate (mean ( SD ) n=3) after freeze drying, repeated freeze drying after three weeks and after reconstitution in water for 2 h. W eek Khellin (mg/g L yophilisate) Visnagin (mg/g L yophilisate) 1. F reeze d rying 0 32.3 (1.0) 15.2 (0.4) 1 31.1 (0.2) 14.3 (0.1) 2 30.6 (0.3) 13.7 (0.1) 3 29.1 (0.2) 13.2 (0.1) 2. F reeze drying 3 31.1 (0.6) 14.4 (0.4) 5 29.9 (0.5) 13.9 (0.2) 9 31.6 (0.1) 13.8 (0.01) 2 h in W ater 3 30.4 (0.4) 14 .0 (0.2)

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57 Figure 2 1. Percolator for Ammi visnaga ethanol extract preparation Figure 2 2 Exemplary HPLC UV chromatogram of standard water Ammi visnaga extract (tea preparation) reconstituted in methanol after filtration and dilut ion 1:10 in mobile phase

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58 Figure 2 3 P eak area (mean, n=2) of khellin and visnagin vs. ethanol concentration of extraction solvent (% (v/v)) Figure 2 4 Exemplary HPLC UV chromatogram of alternative water Ammi visnaga extract (water bath) after filt r ation and dilut ion 1:100 in mobile phase

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59 Figure 2 5 Exemplary HPLC UV chromatogram of Ammi visnaga ethanol 50% (v/v) extract (water bath) after filt ration and dilut ion 1:100 in mobile phase Figure 2 6 Visnagin solubility in different concentrations of aqueous Captisol solutions (mean SD, n=3)

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60 Figure 2 7 Visnagin solubility in different concentrations of standard water Ammi visnaga extract (mean SD, n=3)

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61 Figure 2 8 Stability of standard water Ammi visnaga extract with respect to visnagin and khellin after storage of the lyophilisate at 20 C. Compound per g lyophilisate (mean SD, n=3) vs. time after freeze drying, repeated freeze drying after three weeks and after being reconst ituted in water for 2 h.

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62 CHAPTER 3 PHARMACOKINETIC EVALUATION OF VISNAGIN IN RATS AFTER INTRAVENOUS BOLUS ADMINISTRATION 1 Background Herbal medicines could be useful as an alternative or complementary therapy for urolithiasis management as revealed by data from in vitro and in vivo experiments as well as clinical trials [ 27 ] The furanoc hromones visnagin and khellin (Figure 1 3 ) are the main compounds of Ammi visnaga L. (syn. Khella, Apiaceae) fruits with potential effects o n kidney stone prevention. As previously demonstrated by our working group, the aqueous plant extract as well as the pure compounds visnagin and khellin decreased calcium oxalate (CaOx) deposition in vivo and reduced cell in jury after CaOx exposure in vitro [ 51 52 ] Based on these results visnagin seems to be slightly more promising in the prevention of kidney stones than khellin. Kha n et al. reported a reduction of oxalate and calcium content in rat kidney s after administration of khella tea preparation [ 50 ] Pharmacokinetic (PK) characterization is a crucial step in drug development. Moreover, in order to gain w ider credibility herbal medicines must undergo similar scrutiny to which synthetic drugs are subjected, including PK evaluation. Said [ 62 ] investigated the PK properties o f khellin in rats. Further studies about the PK characteristics of khellin were performed in rabbits [ 63 ] Even though the therapeutic use of visnagin has been studied for years [ 41 43 64 65 ] no information is ava ilable about its PK properties. 1 This chapter was originally published in the European Journal of Pharmaceutical Sciences. Haug KG, Weber B, Hochhaus G, Butterweck V Nonlinear pharmacokinetics of visnagin in rats after intravenous bolus administration. Eur J Pharm Sci 2012; 45: 79 89

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63 Specific Aims To develo p and validate a liquid chromatography tandem mass spectrometry ( LC MS/MS ) method for quantification of visnagin in rat plasma. To evaluate the PK characteristics of visnagin in rats after intravenous ( i.v. ) bolus administration with respect to linear or n on linear PK To develop an adequate PK model for the description of the observed data, including parameter estimation. Material and Methods Chemicals and Reagents Visnagin (>97%), warfarin (98%), heparin sodium salt (202 U/mg), D (+) g lucose (>99.5%) and ammonium acetate (>99%) were obtained from Sigma Aldrich (St. Louis, MO, USA). Captisol cyclodextrin sulfobutyl ethers, sodium salts) was purchased from Cydex Pharmaceut icals (Lenexa, KS, USA) (meanwhile acquired by Ligand Pharmaceuticals Inc. (La Joll a, Ca, USA) Methanol, ethyl acetate and formic acid (>88%) were obtained from Fisher Scientific (Pittsburgh, PA, USA). All chemicals used were analytical grade. HPLC grade deionized water was prepared using a Barnstead Nanopure Diamond UV ultra pure water system (Dubuque, IA, USA). Instrumentation and LC MS/MS C onditions Chromatographic conditions Chromatographic separation was performed on a Symmetry C 18 4.6 mm x 50 mobile phase was comprised of 0.1% formic acid, 5 mM ammonium acetate in deionized water and methanol (15:85, v/v). Prior to use, mobile phase was filtered through an Express Plus lipore, Cork, Ireland)

PAGE 64

64 and dega sed utili zing helium gas. Mobile phase was delivered at a flow rate of 0.6 mL /min and the injection volume of samples was 10 Auto sampler rinse solution consisted of 50% methanol. Mass spectrometry conditions The LC MS/MS system was composed of a PerkinElmer Se ries 200 LC pump and auto sampler, coupled with a Micromass Quattro LC triple quadrupole mass spectrometer. The Micromass Quattro LC was equipped with an electrospray ionization source and operated in the positive ion mode. Data was acquired and processe d using MassLynx V3.5 software (Micromass Limited, Manchester, UK). Following instrument parameters were applied: capillary voltage of 3.7 kV, source block temperature of 120C, desolvation temperature of 400C, desolvation gas flow o f 706 L /h, cone gas f low of 65 L /h and collision energy of 22 eV. Quantification was carried out using multi reaction mode of the transitions of m/z 230.8 216.1 for visnagin and 309.2 162.9 for warfarin, with a scan time of 0.4 s ec per transition for visnagin and 0.1 s ec p er transition for warfarin. Sample P reparation Preparation of visnagin calibration standards and quality control samples Visnagin stock solution (1 mg/ mL ) was prepared in methanol. Visnagin stock solution was demonstrated to be stable for at least four weeks at 4C. Working solutions for a seven point calibration curve ranging from 1 to 100 ng/ mL were prepared by adequately diluting the stock solution in 50% methanol. Calibration standards were prepared by spiking nine parts of blank rat plasma with one part of respective working solutions. Independent quality control (QC) samples were prepared at concentrations of 1, 5, 50, and 100 ng/ mL in the same manner.

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65 Plasma extraction Calibration standards and QC samples were extracted prior to LC MS/MS analysis. Warfarin solution (100 200 ng/ mL prepared in 50% methanol) was added as internal standard (IS) to each sample (100 ). Subsequently, 1 mL ethyl acetate was added to extract visnagin and warfarin from the plasma to the organic solvent. Therefore, the samples were mixed for 5 min on a vortex shaker and centrifuged at 10.000 rpm for 15 min at room temperature. Clear supernatant (950 ) was transferred to borosilicate glass vials and ethyl acetate was completely evaporated at room temperature utilizing a vacuum centrifuge. Residues were stored at 4C overnight until reconstitution in 60 of mobile phase ( co mposition as described under chromatographic conditions ). The reconstituted solutions were shaken in the glass vials for 90 s ec on a multi vortexer and afterwards centrifuged at 3000 rpm for 5 min at 10C. Reconstituted samples (50 ) were transferred to auto sampler vials and analyzed within 4 h after reconstitution. Method Validation Method validation included determination of calibration curve, a ccuracy, precision and recovery according to the criteria of the United States Food and Drug Administration (FDA) Bioanalytical Method Validation Guidance [ 66 ] For evaluation of the relationship between instrument response and nominal visnagin concen trations, two sets of calibration standards were prepared per run. The calibration curve was fitted by simple linear regression of the average peak area ratio of visnagin to warfarin on the nominal standard visnagin concentration in ng/ mL A we ighting fact or of 1/y was used. Accuracy and precision were determined on three independent days utilizing four concentrations of independent QC samples (n=5): two low QCs (1 and 5 ng/ mL ), one

PAGE 66

66 middle QC (50 ng/ mL ), and one high QC (100 ng/ mL ). Calculation of QC sample concentrations was based on the fitted regression equation. Intra and inter day accuracy criteria were met when the mean value did not exceed 15% of the nominal value at each concentration, except for the LLOQ, where 20% was accepted. Determined intra a nd inter day precision should not exceed 15% of the coefficient of variation (CV) at each concentration, except for the LLOQ, where 20% was accepted. Recovery and matrix effects were determined in triplicate for three different concentrations. For evaluati on of extraction recovery, peak area ratios of visnagin to warfarin of spiked plasma samples were compared to those of blank plasma samples spiked with visnagin after extraction. Matrix effects were determined by comparison of peak area ratios of visnagin to warfarin of blank plasma samples spiked with visnagin after extraction with those of compound spiked injection solutions. Blank samples (blank plasma processed without IS) and zero samples (blank plasma processed with IS) were positioned in between the calibration samples and analyzed to assess possible carry over. Both short term temperature stability and post preparative stability tests were performed. For short term temperature stability, visnagin plasma samples at concentrations 10 and 100 ng/ mL were prepared in triplicate and frozen at 20C. Samples were thaw ed 4 h prior to extraction. For post preparative stability, visnagin in plasma (50 ng/ mL ) was extracted and analyzed immediately and, additionally, at four different time points up to 4 h after being reconstituted in mobile phase.

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67 Pharmacokinetic S tudy Animals Eight male Sprague Dawley rats with a catheter inserted in the jugular vein (approximately 300 g body weight) were purchased from Harlan Laboratories (Indianapolis, IN, USA). Animals were individually housed in plastic cages and received water ad libitum and standard chow ( during experiments. Rats were maintained on a 12 h/12 h light/dark cycle and allowed to adapt to their environment for one week before b eing used in experiments. All animal experiments were performed according to the policies and guidelines of Institutional Animal Care and Use Committee of the University of Florida, Gainesville, FL, USA (IACUC approval #201003686) Design of pharmacokineti c study in rats Each rat received three different doses of visnagin (1.25, 2.5, and 5 mg/kg) by i.v. bolus administration in a randomly assigned order, separated by one week of washout period. Visnagin solution was injected through the i.v. catheter at a v olume of 2 mL /kg. Small blood samples (300 ) were collected from the sublingual vein into heparinized tubes [ 67 ] the day before dosing (baseline values) and at time points 5, 10, 20, 30 min and 1, 2, 3, 4, 6 and 8 h post dosing. The baseline values were taken to confirm the sufficiency of the washout period. In order to maintain their functionality, catheters were f lushed twice a week by withdrawing a small volume of blood followed by flushing with sterile isotonic saline. Afterwards, catheters were filled with sterile heparinized 25% dextrose solution.

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68 Preparation of visnagin solutions for administration to rats Bas ed on the selected doses and administration volume, the visnagin solutions for administration of the three dosing groups were required to have concentrations of 0.625 1.25, and 2.5 mg/ mL respectively. Due to its limited water solubility (0.16 mg/ mL Chap ter 2 ) visnagin was dissolved in 25% aqueous Captisol solution. 25% Captisol was previously identified as adequate to enhance the aqueous solubility of visnagin to concentrations that were sufficient to achieve the required dosing concentrations (Chapter 2) Visnagin solutions were prepared on the day before the study was initiated. The day preceding the weekly experiments, solutions were sterilized by filtering through sterile Millex and visnagin concentrations were analyzed in triplicate by a validated high performance liquid chromatography with ultraviolet light detection ( HPLC UV ) method (Chapter 2) The dosing volume of 2 mL /kg was adjusted if the actual concentration of the dosing solutions d eviated from their anticipated concentrations in order to ensure administration of the defined doses throughout the study. Sample analysis Blood samples were stored on ice for a maximum of 4 h until centrifugation at 4000 rpm for 15 min at 4C. All plasma samples were stored at 20C until extraction Reconstituted samples were injected into the LC MS/MS system along with two sets of calibration standards and three replicates of three concentrations of QC samples ( 5, 50 and 100 ng/ mL ) per run. A calibration curve was fitted by linear regression as described in method validation Sample calculation was based on the fitted regression equation. For each run, accuracy and precision were monitored with the QC samples A run was accepted if at least 67% of the Q C samples were within 15% (20% for LLOQ) of the

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69 nomina l value. 33% of the QC samples were allowed to be outside the 15% limit, as long as not all replicates of one concentration exceeded the 15% limit [ 66 ] Pharmacokinetic D ata A nalysis Both non compart mental and compartmental PK analysis methods were applied to the plasma concentration time data to assess the PK behavior of visnagin after i.v. bolus administration. Plasma concentration time data was treated as being obtained from 23 individual subjects ( ) throughout the analysis. Data points below the determined LLOQ were excluded from the PK analysis. Non compartmental analysis Plasma concentration time profiles were visually examined for a fi rst assessment of the PK characteristics of visnagin. Non compartmental analysis (NCA) of the (Pharsight orm weighting for selection of the data points that were included in the terminal slope determination of the area under the plasma concentration time curves (AUC). By means of the inbuilt descriptive statistics tool, mean and standard deviation (SD) of important PK parameters were obtained, namely AUC over the time interval from zero to last quantifiable time point ( AUC last ), AUC over the time interval from zero extrapolated to infinity ( AUC inf z ), terminal half life (t 1/2 ), clearance (CL), volume of z (V z ), concentration at time point zero ( C 0 ) and mean residence time (MRT ). Plots of average AUC last vs. dosing group and average C 0 vs. dosing group were created in R

PAGE 70

70 version 2.12.2 (The R Foundation for Statistical Computing) and utilized for determination of linear or non linear PK of visnagin within the tested dosing range. Lack of fit tests were performed to formally test a linear relationship of average AUC last over dosing group and average C 0 over dosing group (alpha = 0.05) [ 68 ] Compartmen tal analysis Model building and selection A stepwise model building approach was applied to obtain a compartmental model that describes the fate of visnagin after i.v. bolus administration. The compartment model should adequately reflect the capacity limi ted elimination process of visnagin (as suggested by the NCA results ). The kinetics of capacity limited processes are well described by the Michaelis Menten (MM) equation ( Equation 3 1 ) [ 69 70 ] (3 1) where dC/dt is the rate of decline of drug concentration C, V max ( unit: concentration/time ) represents the theoretical maximum elimination rate of the saturable process, and K M is the drug concentration at which the elimination rate is equal to one half of V max Thus, the rate of drug elimination from the central compartment was represented by MM kinetics in the compartment model. One two and three compartmen t models were considered throughout the model building process. All distribution processes were assumed to follow first order kinetics. Firstly, initial estimates for V max and K M were obtained by means of the Lineweaver Burk (LB) linearization method of th e MM equation ( Equation 3 2 ) [ 71 ]

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71 (3 2) where C mid represents the plasma concentration at the midpoint of a sampling interval. For each subject and sampling interval, dC/dt and C mid were calculated and simple linear regression of dt/dC as a function of 1/C mid across subjects was performed in R version 2.12.2. LB estimates for V max ( Equation 3 3 ) and K M ( Equation 3 4 ) were calculated as functions of the estimated inter cept ( ) and slope ( ) parameters from the simple linear regression equation. (3 3) (3 4) Secondly, a standard two stage (STS) parameter estimation approach was performed in SAS 9.2 (PROC Model) (SAS Institute Inc., Cary, NC, USA) to obtain estimates for distribution rate constants (multi compartment model) and updated estimates for volume of distribution of the central compartment (V C ), V max and K M Each individual plasma concentration time profile was fitted to a one and two compartment body model. At this model building stage, implementation of a third compartment was not possible because the number of data points was exceeded by the number of pa rameters (seven) in a three compartment model for most subjects. A system of differential equations was used to parameterize the models. Equation 3 5 and Equation s 3 6/3 7 were used to parameterize the one and two compar tment body model, respectively.

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72 (3 5) (3 6) (3 7) where X is the amount of visnagin in the central body compartment at time t, P is the amount of visnag in in the peripheral body compartment at time t, V C is the volume of the central compartment, and k 12 and k 21 are the distribution rate constants. Initial estimates for V max and K M V C and the distribution rate constants were based on the LB estimates, NCA estimates, and educated guesses, respectively. Residual plots and goodness of fit plots (ODS graphics option in SAS 9.2) were visually examined for evaluation of the appropriateness of the estimated parameters. For both the one and two compartment bod y model, STS estimates for V max ( ), K M ( ), V c ( ), k 12 ( ), and k 21 ( ) were obtained as the median of the distribution of the individual model fitting parameters. The median was selected to reduce the impact of outliers on the STS est imates. Only subjects for which the model fitting algorithm converged successfully were included in the calculation of the STS estimates. In addition, other summary statistics (average, standard deviation, 10 th and 90 th percentiles) were calculated across subjects for which the model fitting algorithm converged successfully. However, these statistics were not further included in the model building process. Thirdly, a non linear mixed effect (NLME) modeling approach was applied to obtain final estimates for the typical values of V max K M V C and the distribution rate

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73 constants as well as estimates for inter subject and residual variability. All plasma concentration time profiles were simultaneously fitted to a one two or three compartment body model in NONMEM VI (ICON, Dublin, Ireland). Subroutine ADVAN6 TRANS1 was used to parameterize the models as a system of differential equations. Differential equations for the one and two compartment model are given above. Equations 3 8 through 3 10 were used to pa rameterize the three compartment body model. (3 8) (3 9) (3 10) where P 1 is the amount of visnagin in the shallow peripheral body compartment at time t, P 2 is the amount of visnagin in the deep peripheral body compartment at time t, and k 13 and k 31 are distribution rate constants. For all compartment models, V C V max and K M w ere assumed to follow a log 1 2 3 and 1 2 2 2 3 2 1 2 3 represented the typical values for V C V max and K M respectively. The distribution rate constants (k 12 k 21 k 13 and k 31 ) were assumed to be fixed across subjects. The typical values of the distribution rate constants k 12 k 21 k 13 and k 31 4 5 6, 7 respectively. The intra subject variability was assumed to follow a constant CV error model. Initial estimates for the model parameters were based upon the STS estimates. The first order

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74 conditional estimation (FOCE) with interactions method was used to obtain parameter estimates for the one two and three compartment body model. If the model e stimation procedure did not converge successfully, initial estimates were updated until minimization was successful. Census (version 1.1.1 http://census.sourceforge.net/ ) was used to process the NONMEM output f iles. Selection of a final model was based upon formal statistical testing (likelihood ratio tests on objective function value (OFV), alpha = 0.01) and visual assessment of typical goodness of fit plots and distribution of the individual parameter estimate s. Bootstrapping and construction of non parametric 90% bootstrap percentile confidence intervals for parameter estimates 1000 independent bootstrap samples were obtained from the original 23 subjects and individually fitted to the final model (Wings for NONMEM http://wfn.sourceforge.net/ ). The percentage of bootstrap runs that resulted in reasonable estimates for the typical value parameters was calculated and used as a measure for the stability of the final mod el. The bootstrapping distribution of each parameter was visually compared to the respective estimate of the original data set. For each model parameter, a 90% bootstrap percentile confidence interval (CI) was obtained by choosing the 5 th and 95 th percenti le of the distribution of bootstrap estimates as lower and upper bound, respectively (R version 2.12.2). Final model validation and visual predictive check The predictive power of the final model was evaluated by a leave one out cross validation method. Therefore, model parameter estimates were obtained by fitting the plasma concentration data of only two dosing groups to the final model (NONMEM VI). Subsequently, obtained model

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75 parameters were used as input to generate plasma concentration time profiles of 1000 hypothetical subjects of the left out dosing group (NONMEM VI). The 5 th 50 th and 95 th percentile of the distribution of the simulated plasma concentration time data were obtained at each time point and visually compared to actual data of that dosing group (R version 2.12.2). The median (50 th percentile) was used as a measure for central te ndency. The 5 th and 95 th percentile were used to construct a prediction interval that served as a measure for variability. The procedure was performed three times, leaving out each dosing group once. Results Method V alidation The retention times were approximately 1.23 min and 1.37 min for visnagin and warfarin, respectively (Figure 3 1 ) All calibration curves met the acceptance criteria. Intra and inter day accuracy and precision were within the tolerated limits for 5, 50 and 100 ng/ mL but did not meet the acceptance criteria for 1 ng/ mL (Tables 3 1 and 3 2 ). Therefore, 5 ng/ mL was determined as LLOQ. The average matrix effect for the three concentrations was 126.2% with a CV of 0.94%. The mean recovery for the three concentrations was determined t o be 74.5% with a CV of 1.87%. Visnagin could not be detected in blank and zero samples. Thus, no detectable carry over was interfering with sample analysis. Short term temperature stability over 4 h could be demonstrated for visnagin in plasma. Bias and CV for 10 ng/ mL samples were 0.97% and 2.61%, respectively. Determination of bias and CV for 100 ng/ mL samples yielded 7.06% and 1.59%, respectively.

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76 Post preparative stability of visnagin after extraction and reconstitution in mobile phase could be confi rmed for at least 4h. The predicted value at each of the five tested time points after reconstitution was within 15% of the nominal concentration of 50 ng/ mL and the CV of the samples was 8.4%. Thus, LC MS/MS analysis of plasma samples was performed within 4 h after reconstitution Pharmacokinetic S tudy and S ample A nalysis Seven rats received visnagin in all three doses. One rat did not receive the low dose of 1.25 mg/kg because of congestion of the drug administration catheter in the third study week. For all runs, calibration curves and QC samples met the required acceptance criteria. Pharmacokinetic D ata A nalysis Non compartmental analysis The average plasma concentration time profiles (untransformed and log transformed) are de picted in Figure 3 2 Visnagin plasma concentrations fell below the LLOQ no later than 8 h after i.v. bolus injection Results of NCA are presented in Table 3 3. Mean AUC last after doses of 1.25, 2.5, and 5 mg/kg were 1.03, 3.61, and 12.6 mg*h/L respectively. A plot of average AUC last vs. dosing group ( Figure 3 3 ) demonstrated visual evidence for a disproportionate increase in average AUC last with increasing dose. A lack of fit test confirmed a non linear relationship of average AUC last and admi nistered dose (p value = 0.0007). Mean C 0 after doses of 1.25, 2.5, and 5 mg/kg were determined to be 2.57, 5.22, and 9.44 mg/ L respectively. A plot of average C 0 vs. dosing group ( Figure 3 4 ) did not provide any evidence for a disproportionate increase i n average C 0 with increasing

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77 dose. A lack of fit test confirmed the linear relationship of mean C 0 and administered dose (p value = 0.2688). The extrapolated AUC was on average below 5% with one outlier (21.5%) in the high dosing group which explains the l arge SD compared to the other two groups ( Table 3 1 ). Compartmental analysis Model building and selection Lineweaver Burk method Simple linear regression of dt/dC over 1/C mid across subjects yielded estimates for and of 0.212 and 0.464, respectively ( Figure 3 5 ). The coefficient of determination (R 2 ) was 0.689. and were determined according to Equations 3 3 and 3 4 as 4.71 mg/( L *h) and 2.18 mg/ L respectively. Model building and selection Standard two stage parameter estimation The model fitting algorithm converged successfully for twelve and eight subjects for the one and two compartment model, respectively. and were determined to be 10.1 mg/( L *h), 3.5 mg/ L, and 0.2 L for the on e compartment model, respectively. and were determined to be 2.16 mg/(L*h), 0.13 mg/L 0.18 L 1.01 1/h and 1.72 1/h for the two compartment model, respectively. Summary statistics for the one and two compartment model ar e displayed in Table s 3 4 and 3 5, respective ly. Model building and selection Non linear mixed effect modeling approach Model fitt ing to the plasma concentration time data was successful for the one and two compartment model. Convergence criteria were not met for the three compartment model despite attempting several sets of initial estimates. OFVs and associated p values for the likelihood ratio tests are present ed in Table 3 6 Basic goodness of fit plots are shown in Figure 3 6 Population and individual fits for the 23 subjects are

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78 displayed in Figure 3 7 The two compartment body model was selected as final model based upon the result of the formal likelihood ratio test (p value < 0.0001) and visual inspection of the goodness of fit plots and distribution of the individual parameter estimates (not shown). The typical values of the final parameter estimates we re 2.09 mg/ ( L h ) (V max ), 0.08 mg/L (K M ), 0.175 L (V C ) 1.0 0 1/h (k 12 ), and 1.22 1/h (k 21 ). Associated inter s ubject variability estimates (% CV) for V max K M and V C were 21.8, 70.9, and 9.2, respectively. Intra subject variability (constant CV error model) was estimated to be 7.0%. Bootstrapping and construct ion of non parametric 90% bootstrap percentile confidence intervals for parameter estimates 99.8% of 1000 independent bootstrap samples that were fitted to the two compartment body model resulted in reasonable estimates for the typical value parameters. T he bootstrapping distribution of each parameter is displayed in Figure 3 8 90% bootstrap percentile CIs are given in Table s 3 7 and 3 8 for each of the model parameters. Final model validation and visual predictive check The results of the leave one out cross validation method are shown in F igures 3 9 to 3 11 for each of the three cases. High agreement between simulated and observed concentrations was achieved for both central tendency (median) and variability (prediction interval) in all three cases. Discussion The administered doses of 1.25, 2.5, and 5 mg/kg were selected based on PK pilot studies. The concentrations of the visnagin solutions for administration were determined based upon two limiting factors. Firstly, th e dosing volume should not exceed 5 mL /kg for i.v. bolus administration. This limitation to the dosing volume is considered good practice [ 72 ] Secondly, the aqueous solubility of visnagin is low

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79 (determined to be 0.16 mg/ mL Chapter 2 ) and limits the preparatio n of concentrated visnagin solutions in water. A dosing volume of 2 mL /kg was selected for practical reasons. Thus, an aqueous solubility of visnagin of 2.5 mg/ mL had to be achieved in order to deliver a dose of 5 mg/kg 25% aqueous Captisol a uniquely m odified and FDA accepted cyclodextrin, was tested as adequate to enhance the aqueous solubility of visnagin to concentrations that were sufficient to achieve the required dosing concentrations. Supported by safety data, Captisol can be used to develop for mulations for parenteral, oral, ophthalmic, nasal topical and inhalative administration routes [ 73 74 ] Several formulations containing Captisol have been approved by the FDA and are in clinical use ( i.e. Abilify Injection IM, Geodon IM, Cerenia SC and Vfe nd IV [ 75 ] ). It has been extensively discussed and reviewed if drug complexation by cyclodextrins alters the PK of drugs [ 76 78 ] It can be concluded that in general the intrinsic PK of drugs is not or only little affected by cyclodextrins [ 78 ] Drug release from the complex is usually fast and complete, especially after parenteral administ ration. Most drugs exhibit drug:cyclodextrin binding constants ( K1:1) in the range of 100 to 10. 000 1/ M. For these drugs, dilution is the major driving force for complex dissociation and it is sufficient for quantitative release of the drug from the comple x [ 74 78 ] It is reasonable to assume only a weak complexation of visnagin with the cyclodextrin. Therefore, it can be assumed that the effect of dilution after parenteral adm inistration is sufficient for a rapid and complete dissociation of the visnagin cyclodextrin complex and that Captisol does not influence the PK of visnagin. It also needs to be kept in mind that i f a drug is poorly water soluble, parenteral drug formulat ions often include co

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80 solvents, surfactants, pH modifiers, etc. to achieve sufficient drug solubility. Since it is not always clear if these additives affect the PK of the drug, the question about which formulation should actually serve as control remains unanswered [ 78 ] The semi logarithmic plot of the plasma concentration time profiles ( Figure 3 2 ) exhibits steeper slopes at later time points and non parallelism of the profiles at the three dose levels. These characteristics point to non linear PK of visnagin. Sources of non linearity are typically saturable components in the system and can be classified into three categories, namely non linear absorption, non linear distribution and no n linear elimination (including metabolism) [ 79 ] Since visnagin was administered int ravenously, non linear absorption can be excluded. Saturable protein binding and/or tissue distribution are common causes of non linearity in distribution. Reasons for non linear elimination include, but are not limited to, capacity limited metabolic enzym es, saturable transporters involved in renal and/or biliary elimination and autoinduction [ 79 ] Average AUC last after doses of 1.25, 2.5, and 5 mg/kg were 1.03, 3.61, and 12.6 mg*h/ L respectively. Figure 3 3 illustrates a disproportionate increase in AUC last with increasing dose. Moreover, the fitted regression line which is shown in Figu re 3 3 deviates significantly from a line connecting the observed values (lack of fit). The non linear relationship of AUC last and dosing group was further confirmed by a formal statistical test for a linear relationship (lack of fit test, p value = 0.0007 ). Considering the relationship between AUC and CL, namely AUC = d ose/ CL, it becomes apparent that CL decreases for higher doses ( Table 3 3 ). Dose dependence of CL (non linearity in CL) is a typical characteristic of non linear elimination kinetics. Therefore, it was concluded that visnagin exhibits non linearity in an elimination process However,

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81 detailed information about the physiological structures or mechanisms that are involved in the capacity limited elimination process cannot be determined wit hout further experiments. Average C 0 valu es we re 2.57, 5.22, and 9.44 mg/L after doses of 1.25, 2.5, and 5 mg/kg, respectively. Fi gure 3 4 depicts a linear increase in C 0 with increasing dose. In addition, the fitted regression line is almost congruent wi th a line connecting the observed values ( Figure 3 4 ). A lack of fit test further verified the linear relationship of C 0 and administered dose (p value = 0.2688). Given the equation C 0 = d ose/V z it is evident that V z must be dose independent for the linear relationship to hold. Hence, visnagin possesses linearity in V z at least up to the tested dose of 5 mg/kg and no saturable mechanism in a distribution process is expected. The goals of the compartmental PK analysis were to confirm the capacity limited elimination of visnagin that was suggested by the NCA and to describe a compartmental model that adequately characterizes the PK of visnagin after i.v. bolus administration. The step wise model building approach comprising LB linearization and STS model fi tting was necessary to obtain good initial estimates for the data fitting in NONMEM. While NCA provided evidence for non linear PK in CL, it did not give any information about V max and K M First ballpark estimates for V max and K M were obtained by applicati on of the LB linearization method of the MM equation to plasma concentration time data. However, knowledge of a possible multi compartment behavior of visnagin after i.v. bolus administration could not be acquired by the LB method. Moreover, in addition to well known disadvantages of linearization methods ( e.g. susceptibility to outliers and influential points, highly biased weighting of points ), the LB estimates for

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82 V max and K M are biased if a one compartment model is incorrectly assumed [ 69 80 81 ] Thus, a STS approach was performed to refine initial parameter estimates for the NLME modeling approach ( Table s 3 4 and 3 5 ) and to gain information about the necessity of a multi compartment PK model for visnagin. For the STS approach, convergence criteria were achieved in less than 50% of the subjects for both the one and two compartment body model. Interestingly, convergen ce rates for the one and two compartment model were increased for subjects with lower doses and higher doses, respectively (data not shown). Hence, the STS approach demonstrated a certain tendency that a one compartment model is more appropriate for low d oses whereas a two compartment model is more appropriate for high doses. Similar behavior has been widely observed in PK analysis of other compounds [ 82 ] In summary, LB and STS estimates of the model parameters should be interpreted carefully. Their primary use was to obtain good initial estimates for the NLME modeling approach rather than to base inference about PK charac teristics of visnagin on them. In fact, NONMEM model fitting was not successful based on arbitrary selection of initial estimates for V max and K M confirming the importance of the first two steps of the model building approach. The principal obje ctives of the NLME modeling approach were to select a final model and to obtain reliable estimates for the typical values of the model parameters. Estimation of inter subject and intra subject variability was secondary. The two compartment body model with capacity limited elimination process was selected as final model. Strong agreement between individual and population fits and the observed plasma concentration time profiles ( Figure 3 7 ) and the narrow 90% parametric CIs for the typical values of the param eter estimates (Table 3 7 ) confirmed the adequateness

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83 of the final model. Hence, the outcome of the NLME modeling approach provided further evidence for the capacity limited elimination of visnagin. Final estimates for inter subject and intra subject vari ability were reasonable (Table 3 8). These estimates should be interpreted carefully since the 23 plasma concentration time profiles were sampled from eight subjects but treated as if sampled from 23 individual subjects. However, considering the 23 profile s independently of each other did not affect the validity of the typical value estimates because of the sufficient wash out period between dosing events and the random design of the experiment. The high percentage of successful non parametric bootstrappin g runs and the unimodal distributions of the bootstrap estimates (typical values) demonstrated the robustness of the final model ( Figure 3 8) Moreover, non parametric 90% bootstrap CIs were in close agreement with their parametric equivalents. The validi ty of the final model was confirmed impressively by the leave one out cross validation method. For each of the three doses, the average plasma concentration time profile (median) and the associated variability (prediction interval) could be predicted accur ately using data from the other two dosing groups ( Figure s 3 9 to 3 11 ). Thus, the final model seems to possess predictive power even outside the doses that were included in this study. The final model could be used for simulation of single dose i.v. exper iments with different doses, multiple dose i.v. experiments, and single or multiple dose oral experiments. Information from these simulations could be further utilized to improve the design of prospective visnagin experiments ( e.g more effective sampling schemes) and to reduce the number of animals and resources. Thus, this PK model for visnagin could tremendously affect prospective visnagin research,

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84 help to understand the effect of visnagin on kidney stone prevention, and accelerate the development of a commercially available visnagin preparation. To recapitulate, a pplication of a NLME modeling approach to visnagin i.v. bolus data confirmed the capacity limi ted elimination process that was suggested by the NCA and demonstrated that a two compartment body model is necessary to adequately characterize the PK of visnagin. Furthermore, this project demonstrated nicely the advantages of the NLME modeling approach over the LB estimation method and STS approach for non linear PK data. However, it also showed the importance of NLME model fitting. No n linearity in PK could be a drawba ck for a potential drug in the drug development process since it is usually an undesirable characteristic in clinical settings. Non linearity often implies a lack of dose proportionality that can affect safety and efficacy [ 83 ] In case of visnagin, which exhibits saturable elimination, higher doses result in disproportional higher plasma concentrations. The risk of adverse effects increases and can be of major concern if a drug has a narrow therapeutic window. In theory, if the daily dose exceeded the maximum elimination rate, the drug would accumulate endlessly in the body. In reality, at least a small portion of th e drug is eliminated by a parallel first order elimination [ 79 ] Even if a drug is el iminated by multiple pathways of which only one is capacity limited, non linearity will still be detectable. Requirements are that more than 20 30% of the drug are cleared by the saturable pathway and plasma concentrations are high enough to exceed the K M of that enzyme or transporter [ 84 ]

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85 While non linea rity is in general an unwanted drug property, it might only be a problem if it occurs at therapeutic concentrations. Therefore, it needs to be explored if the tested dosing range is reasonable for therapeutic purposes. Previous PD experiments by our workin g group were based on oral dosing of 5 and 10 mg/kg visnagin suspensions and resulted in a decrease of CaOx deposition in rats [ 52 ] Thus, the visnagin plasma concentrations achieved by the selected oral doses can be con sidered therapeutic in this species. An oral PK study of visnagin in rats becomes inevitable in order to determine the absolute bioavailability of visnagin. Knowledge of the absolute bioavailability allows the comparison of visnagin plasma concentrations a fter administration of oral and i.v. bolus doses. Moreover, it will be possible to evaluate if the selected i.v. bolus doses were therapeutic.

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86 Table 3 1 Intra day (at three separate days, n=5) accuracy and precision for LC MS/MS analysis of visnagin i n rat plasma Quality Control Samples ( ng/ mL ) Day 1 5 50 100 1 42.04 10.8 5.68 13.91 Accuracy Deviation ( % ) 2 61.95 1.58 4.82 2.06 3 54.17 0.92 0.54 6.51 1 14.63 3.99 5.7 3.54 Precision CV (%) 2 26.06 7.71 4.12 4.66 3 9.84 6.38 3.21 9.11 Table 3 2 Inter day (averaged over three days, n=5) accuracy and precision for LC MS/MS analysis of visnagin in rat plasma Quality Control Samples ( ng/ mL ) 1 5 50 100 Accuracy Deviation ( % ) 51.99 6.19 0.43 7.99 Precision CV ( % ) 29.32 6.95 7.46 9.11 Table 3 3 Results of non compartmental analysis (mean (SD)) Dosing Group ( mg/kg ) 1.25 2.5 5 n 7 8 8 AUC last ( mg*h/ L) 1.03 (0.13) 3.61 (0.80) 12.60 (1.94) AUC inf ( mg*h/ L) 1.08 (0.13) 3.68 (0.82) 13.35 (3.10) AUC extrapolated ( % ) 4.93 (3.44) 1.80 (2.81) 4.30 (7.68) z (1/h) 3.01 (0.60) 2.18 (0.97) 1.06 (0.44) t 1/2 (h) 0.23 (0.04) 0.37 (0.13) 0.83 (0.52) CL (L/h) 0.38 (0.05) 0.24 (0.06) 0.13 (0.03) V z (L) 0.13 (0.03) 0.12 (0.03) 0.14 (0.06) C 0 ( mg/ L) 2.57 (0.58) 5.22 (0.43) 9.44 (1.58) MRT (h) 0.31 (0.06) 0.63 (0.14) 1.38 (0.57) n : number of subjects, AUC last : AUC over the time interval from zero to last quantifiable time point, AUC inf : z : terminal elimination rate constant, t 1/2 : terminal half life, CL: clearance, V z z C 0 : concentration at time point zero, MRT: mean residence time

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87 Table 3 4 Summary statistics of standard two stage p arameter estimation approach, one compartment body model, n = 12, only subjects for which model fittin g was successful were included SD: standard deviation, P10: 10 th percentile, P90: 90 th percentile V C (L) V max ( mg /(L *h) ) K M (mg/L) Mean 0.2 25 13.9 Median 0.2 10.1 3.5 SD 0.02 49.4 26.1 P10 0.18 5.38 1.42 P90 0.22 24.1 24.1 Table 3 5 Summary statistics of standard two stage parameter estimation approach, two compartment body model, n = 8, only subjects for which model fitting was successful were included, SD: standard deviation, P10: 10 th percentile, P90: 90 th percentile V C (L) V max ( mg /(L *h) ) K M (mg/L) k 12 (1/h) k 21 (1/h) Mean 0.19 6.79 8.36 1.33 3.49 Median 0.18 2.16 0.13 1.01 1.72 SD 0.02 12 22 0.83 4.42 P10 0.17 1.41 0.02 0.64 0.87 P90 0.21 14.7 20.4 2.43 7.38 Table 3 6 Non linear mixed effect modeling approach, objective function values (OFV) and p value for likelihood ratio tests # Compartments # Parameters OFV P value 1 3 142.62 2 5 288.484 0.000 3 7 288.484 1

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88 Table 3 7 Non linear mixed effect modeling approach, two compartment body model, final estimates, standard errors (SE M ), parametric and non parametric confidence intervals (CI) for typical values, non parametric CI s are bootstrap percentile intervals 90% CI ( P arametric) 90% CI ( N on parametric) Parameter Estimate SE M Lower B ound Upper B ound Lower B ound Upper B ound V C (L) 0.175 0.005 0.166 0.184 0.168 0.182 V max (mg/(L *h) ) 2.09 0.154 1.837 2.343 1.81 2.204 K M ( mg/ L) 0.08 0.021 0.045 0.116 0.037 0.141 k 12 (1/h) 1 .00 0.071 0.883 1.117 0.853 1.23 k 21 (1/h) 1.22 0.097 1.06 1.38 0.944 1.68 Table 3 8 Non linear mixed effect modeling approach, two compartment body model, final estimates and non parametric confidence intervals (CI) for inter subject and intra subject variability (expressed as %CV), non parametric CI s are bootstrap percentile intervals 90% CI (N on parametric) Parameter Estimate ( %CV ) L ower B ound U pper B ound 1 2 9.2 5.8 11.7 2 2 21.8 16.1 25.7 3 2 70.9 11.6 89.2 2 7 .0 6.1 8.2

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89 Figure 3 1. Exemplary L C MS/MS chromatogram of internal standard warfarin (top) and visnagin (bottom) Figure 3 2 Visnagin plasma concentration (untransformed (left) and log transformed (right) ) (mean SD) vs. time, sorted by dosing group

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90 Figure 3 3. AUC last (mean SD) vs. dosing group (solid line) and fitted simple linear regression of AUC last vs. dosing group (dashed line, for visual comparison) Figure 3 4. C 0 (mean SD) vs. dosing group (solid line) and fitted simple linear regression of C 0 vs. dosing group (dashed line, for visual comparison)

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91 Figure 3 5. Lineweaver Burk plot ( Equation 3 2 ) of observed plasma concentrations and fitted simple linear regression of dc/dt vs. 1/ C mid across 23 subjects

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92 Figure 3 6. Non linear mixed effe ct modeling approach, two compartment body model, basic goodness of fit plots

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93 Figure 3 7. Non linear mixed effect modeling approach, two compartment body model, population and individual fits

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94 Figure 3 8 Non linear mixed effect modeling approach, bootstrap distribution of parameter estimates (n = 998), vertical black bar represents the parameter estimate obtained by fitting the original data

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95 Figure 3 9. Leave one out cross validation method left out dosing group: high dose Figure 3 10. Leave one out cross validation method, left out dosing group: medium dose

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96 Figure 3 11. Leave one out cross validation method, left out dosing group: low dose

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97 CHAPTER 4 PHARMACOKINETIC EVALUATION OF VISNAGIN AND AMMI VISNAGA AQUEOUS EXTRACT IN RATS AFTER ORAL ADMINISTRATION 1 Background A pharmacokinetic ( PK ) study of visnagin after intravenous (i.v.) bolus injection in rats was performed to characterize the PK properties of visnagin and identified non linear elimination kinetics for visnagin [ 85 ] Furthermore, a non linear mixed effect model that describes the observed data was developed and validated C haracterization of the PK properties of a new substance after i.v. administration is crucial as initial step in the PK evaluation of that substance. However, i.v. bolus administration is not a typical route of a dministration for a potential drug for kidney stone prevention. Therefore, PK evaluation after oral administration is the next logical step. T his study was performed to characterize the PK properties of visnagin after oral administration of the pure compou nd visnagin and visnagin in form of an aqueous Ammi visnaga extract ( AVE ) which is similar to a traditional tea preparation, in rats. Enhanced plasma levels of key compounds after administration of plant extracts compared to isolated compounds of those extracts have been observed in certain examples [ 53 54 86 ] Such differences in plasma exposure could lead to superior efficacy of an extract compared to an individual substance and might justify the usage of an extract over pure compounds. Thus, the comparison of visnagin plasma exposure after administration of the two preparations was of particular interest in this study. 1 Parts of this chapter were originally published in Planta Medica. Haug KG, Weber B, Hochhaus G, Butterweck V Pharmacokinetic Evaluation of Visnagin and Ammi visnaga Aqueous Extract after Oral Administration in Rats. Planta Med 2012; 78: 1831 1836.

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98 Specific Aims To evaluate the PK characteristics of visnagin after oral administration of the pure comp ound visnagin and visnagin in form of standard aqueous AVE in rats To compare visnagin plasma exposure after administration of the two preparations To develop an extenstion of the PK model that was based on i.v. data for the description of the observed data (oral and i.v.) including parameter estimation. Material and Methods Chemicals and R eagents Visnagin (>97%), warfarin (98%), and ammonium acetate (>99%) were obtained from Sigma Aldrich (St. Louis, MO, USA). Captisol cyclodextrin sulfobutyl ether s, sodium salts) was purchased from Cydex Pharmaceuticals (Lenexa, KS, USA) (meanwhile acquired by Ligand Pharmac euticals Inc. (La Jolla, Ca, USA) Methanol, ethyl acetate and formic acid (>88%) were obtained from Fisher Scientific (Pittsburgh, PA, USA). A ll chemicals used were analytical grade. Ammi visnaga L. fruits (DAB 10 quality, invoice 12119085) were kindly provided by Martin Bauer Group Finzelberg GmbH & Co. KG (Andernach, Germany). HPLC grade deionized water was prepared using a Barnstead Nanopure Diamond UV ultra pure water system (Dubuque, IA, USA). Animals The study was conducted in two parts. In the first and second part, visnagin and standard aqueous AVE were orally administered, respectively. For each part, twelve male Sprague Dawley rats (app roximately 300 g body weight) were purchased from Harlan Laboratories (Indianapolis, IN, USA). Animals were housed in pairs in plastic cages and received water ad libitum and standard chow ( 8604 Teklad Rodent Diet,

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99 during experiments. Rats were ma intained on a 12 h/12 h light/dark cycle and allowed to adapt to their environment for one week before being used in experiments. All animal experiments were performed according to the policies and guidelines of Institutional Animal Care and Use Committee of the University of Florida, Gainesville, FL, USA (IACUC approval #201003686). Design of Pharmacokinetic S tudy Three different doses of visnagin (2.5, 5, and 10 mg/kg) and three different doses of AVE (extract standardized on visnagin, containing equivalent doses of 2.5, 5, and 10 mg/kg visnagin) were orally administered to a total number of 24 rats. Each rat recei ved two of the three doses separated by a sufficient (one week) washout period. Hence, a total number of 48 plasma concentration time profiles (eight per dosing group for both treatments) could be obtained. Both preparations were administered as solutions by oral gavage at a volume of 4 mL/kg utilizing a feeding needle. Small blood samples (300 ) were collected from the sublingual vein into heparinized tubes [ 67 ] the day before dosing (baseline values) and at time points 2, 5, 10, 20, 30 min and 1, 2, 3, 4, 6, 8 h post dosing for visnagin and the day before dosing (baseline values) and at time points 5, 10, 20, 30 min and 1, 2, 4, 6, 9, 12, 24 h post dosing for AV E, respectively. Details on the different sampling schedules are given in the discussion. Preparation of Visnagin S olutions for A dministration to R ats Based on the selected doses and administration volume, the visnagin solutions for administration of the t hree dosing groups were required to have concentrations of 0.625, 1.25, and 2.5 mg/mL, respectively. Due to its limited water solubility (0.16 mg/mL Chapter 2 ) visnagin was dissolved in 25% aqueous Captisol solution. 25% Captisol was previously identifi ed as adequate to enhance the aqueous solubility of

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100 visnagin to concentrations that were sufficient to achieve the required dosing concentrations. Prepared visnagin solutions were filtered through Millex non sterile syringe filter units (Millipo re, Cork, Ireland). The day preceding the weekly experiments visnagin concentrations were analyzed in triplicate by a validated high performance liquid chromatography with ultraviolet light detection ( HPLC UV ) method (Chapter 2) No significant change in v isnagin concentrations was observed throughout this study or the previous i.v. study lasting three weeks [ 85 ] confirming stability of visnagi n in Captisol solution during prolonged storage (see also Chapter 2) Preparation of Ammi V isnaga E xtract and S olutions for A dministration to R ats The extract was prepared according to t he method described previously [ 51 52 ] (Chapter 2) Briefly, 40 g Ammi visnaga fruits were grinded for 30 sec in a regular coffee mL boiling water (100C) was poured over the grinded fruits and the mi xture was steeped for 5 min at room temperature with occasional swirling. Subsequently, the aqueous mixture was filtered through No. 2 Whatman filter paper circles (pore size 8 dried (FreeZone 6, Labconco, Kan sas City, MO, USA). The lyophilisate was quantified in triplicate with respect to the key compound visnagin by the validated HPLC UV method and kept in an amber colored bottle at 20C. It has previously been demonstrated that the freeze dried extract is s table with regard to visnagin for at least two months under the named conditions. Furthermore, stability of visnagin in the extract 2 h after reconstitution in water has been confirmed ( Chapter 2 ). The administered doses of AVE should contain equivalent am ounts of visnagin as after pure compound administration to achieve comparable results. Given the

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101 selected doses and administration volume, the AVE solutions for administration of the three dosing groups were required to contain 0.625 1.25 and 2.5 mg/mL v isnagin, respectively. Therefore, based on the result of the quantification of AVE with respect to visnagin three differently concentrated extract solutions were prepared freshly every day prior to dosing. AVE solutions were prepared by dissolving appropri ate amounts of lyophilisate in deionized water and were administered within 2 h after preparation. Instrumentation and LC MS/MS C onditions Liquid chromatography tandem mass spectrometry ( LC MS/MS ) analysis was performed as previously described [ 85 ] Briefly, chromatographic separation was performed on a Symmetry C 18 Milford, MA, US A) at ambient temperature. The mobile phase was comprised of 0.1% formic acid, 5 mM ammonium acetate in deionized water and methanol (15:85, v/v). Mobile phase was delivered at a flow rate of 0.6 mL/min and the injection volume of r details about the mass spectrometry conditions can be found elsewhere [ 85 ] Sample Preparation and A nalysis A seven point calibration curve ranging from 1 to 100 ng/mL as well as independent quality control (QC) samples (5, 50, and 100 ng/mL) were prepared as previously described [ 85 ] Blood samples were stored on ice for a maximum of 4 h until centrifugation at 4000 rpm for 15 min at 4C. All plasma samples were stored at 20C until extraction as described in detail elsewhere [ 85 ] prior to LC MS/MS analysis. Extracted samples were analyzed by a validated LC MS/MS method [ 85 ] (Chapter 3) After reconstitution, samples were injected into the LC MS/MS system

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102 along with two sets of calibration standards and three replicates of three concentrations of QC samples ( 5, 50 and 100 ng/mL) per run. T he calibration curve was fitted by simple linear regression of the average peak area ratio of visnagin to warfarin (internal standard) on the nominal standard visnagin concentration in ng/mL. A weighting factor of 1/y was used. Sample calculation was based on the fitted regression equation. For each run, accuracy and precision were monitored with the QC samples 20% deviation for the coefficient of variation (CV) and mean bias of each concentration of QC samples was tolerated. Pharmacokinetic Data Analysis Both non compartmental and compartmental PK analysis methods were applied to the plasma concentration time data to assess the PK behavior of and explain possible differences between visnagin after oral administration of the pure compound and visnagin in fo rm of standard aqueous AVE Plasma concentration time data w as treated as being obtained from individual subjects throughout the analysis. Non c ompartmental a nalysis Non compartmental analysis (NCA) of the individual plasma concentration time profiles was (Pharsight selection of the data points that were included in the terminal slope calculation. If no satisfying fit was obtained, points for terminal slope calculation would be selected determination of the area under the plasma concentration time curves (AUC). The latest version of R (Th e R Foundation for Statistical Computing) was utilized to calculate the median and standard deviation (SD) of important PK parameters, namely AUC over the

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103 time interval from zero to last quantifiable time point ( AUC last ), AUC over the time interval from ze ro extrapolated to infinity ( AUC inf ), AUC extrapolated, maximum observed concentration ( C max ), time at C max (t max z ), terminal half life (t 1/2 ), area under the first moment curve over the time interval from zero to la st quantifiable time point ( AUMC last ), AUMC over the time interval from zero extrapolated to infinity ( AUMC inf ), AUMC extrapolated, mean residence time over the time interval from zero to last quantifiable time point (MRT last ), MRT over the time interval from zero extrapolated to infinity (MRT inf ), clearance uncorrected for F (CL/F), and volume of distribution during the terminal phase uncorrected for F (V z /F). F is the fraction of dose absorbed (oral bioavailability), which cannot be estimated from extrav ascular data only. Statistical analysis was performed in R. Non parametric Wilcoxon Mann Whitney Rank Sum tests [ 87 ] were used for comparison of AUC last AUC inf MRT last and MRT inf after pure visnagin and AVE administration at a given dose level. P values were le testing due to the three dosing levels. An alpha level of 0.05 was considered statistically significant. Compartmental analysis Modeling was performed using non linear mixed effect modeling software NONMEM version 7.2 ( ICON, Dublin, Ireland ), which allo ws estimation of population means for model paramete rs and quantification of inter individual variability (IIV) and residual (unexplained intra individual ) variability. The FOCE algorithm in NONMEM with interaction option was used for estimation Subrouti ne ADVAN6 TRANS1 was utilitzed to parameterize the models as system of differential equations. IIV was modeled using exponential random effects models. Model selection was based on visual assessment

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104 of goodness of fit plots, precision of parameter estimates ( % relative standard error (RSE) and 90% non parametric confidence interval (CI) ) distribution of the individual parameter estimates and the objective function value (OFV) provided by NONMEM. One nested model was considered superior to another when the OFV was reduced by 3.84 points ( chi squared p value < 0.05, 1 degree of freedom). Model building and selection. A stepwise model building approach was applied to develop a compartmental model that describes the fate of visnagin after i.v. bolus administration as we ll as after oral administration of the pure compound and visnagin in form of AVE. Overall data from three PK studies w as available and used : 23 profiles after i.v. bolus administration o f three different doses of visnagin (i.v. visnagin data), 24 profiles after oral administration of three different doses of visnagin (oral visnagin data) and 18 profiles after oral administration of three different doses of AVE (oral AVE data) (see results In th e first step, only i.v. and oral visnagin data were utilized and fitted simultaneously to the structural model that was previously de veloped for the data after i.v. bolus administration Therefore, a n absorption process needed to be integrated in to the existing two compartment model with non linear elimination in order to adequately describe oral visnagin data Different types of absorption models were explored, i.e. first order rate (with fixed or dose dependent oral bioavailability) zero order rate, Michaelis Menten kinetics and Weibull type absorption. In the second step, oral AVE data was fitted simultaneously with the previously used i.v. and oral visnagin data. In order to acc ount for differences in visnagin plasma exposure after oral administration of pure compound and AVE, relevant model parameters (K M V max F and Weibull type absorption parameters and k d ) were

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105 estimated separately for pure compound and AVE data Therefore an additional term (theta) for the difference in the parameter mean estimate between pure compound and AVE data was introduced but the same IIV was applied In case of the oral bioavailability term F, two independent mean parameter estimates were obtain ed for visnagin and AVE data to avoid estimates larger than the natural boundary of 1. The control stream of the final model is given as example in the Appendix. Common parameter estimates of the model s using data from the i.v. PK study and all three PK studies were compared Bootstrapping and construction of non parametric 90% bootstrap percentile confidence intervals for parameter estimates. 1000 independent bootstrap samples were obtained from the o riginal 65 subjects and individually fitted to the fin al model ( Perl speaks NONMEM (PsN) version 3.5.3 http://psn.sourceforge.net/ ). Oral bioavailability was fixed to the final model parameter estimate, since estimates near the natural boundary of 1 were observed rep eatedly during bootstrap test runs. The percentage of successful bootstrap runs was calculated and used as a measure for the stability of the final model. The bootstrapping distribution of each parameter was visually compared to the respective estimate of the original data set. For each model parameter, a 90% bootstrap percentile CI (non parametric CI) was obtained by choosing the 5 th and 95 th percentile of the distribution of bootstrap estimates as lower and upper bound, respectively (R version 2.1 4.1 ). V i sual predictive check. The selected final PK model was evaluated by a visual predictive check. Therefore, model parameter estimates of the final model were used as input to generate plasma concentration time profiles of at least 1000 subjects (number

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106 varie d depending on the subjects in the original data sets) per treatment and dosing group (NONMEM 7.2 ). The 5 th 50 th and 95 th percentile of the distribution of the simulated plasma concentration time data were obtained at each time point and visually compared to the observed raw data of the respective treatment and dosing group ( SAS 9.2, SAS Institute Inc., Cary, NC, USA ). The median (50 th percentile) was used as a measure for central tendency. The 5 th and 95 th percentile were used to con struct a prediction interval that served as a measur e for variability. Results Pharmacokinetic S tudy and S ample A nalysis The quanti tative amount of visnagin was 1 6 .6 mg per g of freeze dried extract. Thus, solutions containing 37.7, 75.5, and 150.9 mg/mL lyophilisate were prepared for AVE administration. Eight plasma concentration time profiles were obtained for each dose of pure visnagin. Only seven, six and five profiles f or the low, medium and high dose of AVE, respectively, could be used for analysis due to administration difficulties in some rats leading to incomplete dosing of those animals. For all runs, calibration curves and QC samples met the required acceptance cr iteria. For all profiles, maximal one data point fell below the low limit of quantification of 5 ng/mL. Those data points were included in the plasma concentration time plots and non compartmental data analysis for better estimation of the terminal slope but were eliminated from compartmental analysis. Average visnagin plasma concentration time profiles (geometric mean standard error of the mean (SEM)) after pure compound and AVE administration are depicted in Figure 4 1 It demonstrates that the average visnagin plasma concentration is higher

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107 when it was administered in form of AVE compared to the administration of the pure compound for all three doses. After 8 h, the median visnagin plasma concentration was below 0.08 mg/L for all three doses when it wa s given in form of the pure compound. On the other hand, after administration of AVE, the median visnagin plasma concentration was larger than 0.07 mg/L for all three doses after 9 h. Non Compartmental Analysis Results of NCA are presented in Table 4 1 T he duration of plasma sample collection was in most cases sufficient to capture the complete plasma concentration time profiles. Profiles with an extrapolated AUC and extrapolated AUMC greater than 50% were excluded from summary statistic calculation (medi z AUC extrapolated, AUC inf t 1/2 CL/F, V z /F and MRT inf AUMC extrapolated, AUMC inf respectively ( see iscussion and Table 4 1 ). After removal of those profiles, the extrapolated AUC and AUMC were close to zero for all groups except for the 10 mg/kg dosing group of visnagin (AUC and AUMC) and AVE (AUMC) ( Table 4 1 ). Visnagin plasma exposure (AUC inf and AUC last ) was statistically significantly increased when administered as AVE compared to an equivalent dose of the pure compound for al l three dosing levels except for the 10 mg/kg dosing group (AUC inf p value: 0.052) ( Table 4 1 and Figure 4 2 ). For the 2.5 mg/kg dosing group, the AUC inf of the AVE group (2.72 mg*h/L) was even increased by more than 10 fold compared to that of the pure c ompound group (0.15 mg*h/L) For the high dosing group, AUC inf of the AVE group (35.67 mg*h/L) was increased by 3.5 fold compared to that of the pure compound group (10.14 mg*h/L). For both the AVE and the pure compound, AUC inf and AUC last increased dispro portionately with an increase in dose ( Table 4 1 and Figure 4 2 ).

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108 The MRT inf and MRT last of visnagin were statistically significantly prolonged when given as AVE compared to an equivalent dose of the pure compound for all three dosing groups ( Table 4 1 and Figure 4 3 ). C max values after AVE administration were elevated (AVE vs. pure compound: 0.52 vs. 0.17 (2.5 mg/kg), 1.38 vs. 0.84 (5 mg/kg), and 3.61 vs. 2.03 mg/L (10 mg/kg)) and occurred at later time points (t max : AVE vs. pure compound: 1 vs. 0.42 ( 2.5 mg/kg), 2 vs. 1 (5 mg/kg), and 6 vs. 1 h (10 mg/kg)) compared to equivalent doses of the pure compound. The terminal half life increased with dose for both AVE and pure compound reaching a maximum value of 1.94 h for the 10 mg/kg pure compound treatmen t group ( Table 4 1 ). Compartmental Analysis Model building and selection When fitting i.v. and oral visnagin data simultaneously the model using Weibull type absorption provided the best fit. A Weibull function [ 88 ] was implemen ted in the differential equation s that were used to parameterize the two compartment body model with capacity limited elimination, represented by Michaelis Menten kinetics (Equations 4 1 and 4 2): (4 1) (4 2) where X is the amount of visnagin in the central body compartment at time t, F represents the fraction of dose absorbed (oral bioavailability), k d corresponds to the scale parameter and shape parameter of the Weibull function V max ( unit:

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109 amount /time ) represents the theoretical maximum elimination ra te of the saturable process, K M is the drug concentration at which the elimination rate is e qual to one half of V ma x P is the amount of visnagin in the peripheral body compartment at time t, V C is the volume of the central compartment, and k 12 and k 21 are the distribution rate constants. The accumulated amount of visnagin absorbed into the centr al compartment is represented by the integrated form of the Weibull function (Equation 4 3): (4 3) Simultaneous fitting of i.v. and oral visnagin and oral AVE data to the interim model resulted in a mediocre description of the observed data. In particular, population profiles after AVE administration which demonstrate increased and extended visnagin plasma exposure were underp redicted when the same structural model with out separate estimates of model mean parameters w as used for pure compound and extract data Introducing a n additional term (theta) for K M for oral AVE data to account for the observed difference s in the profiles resulted in the strongest improvement in data description ( delta OFV 60.7 points, p value < 0.0001 ) The IIV term o n the K M expression corresponding to AVE data was demonstrated to be redundant and was therefore removed from the model. Distinguishing V max only and V max and K M at once for AVE and pure compound data provided inferior fits and no further improvement respectively, compared to two different K M estimates. Likewise, s eparate estimates of oral bioavailability F in addition to different K M estimat es did not improve the model Visual inspection of distribution of IIV estimates sorted by PK study reveale d a trend towards elevated shape parameter ( ) estimates for oral visnagin data as against oral

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110 AVE data. Thus, it was tested if separate parameter estimates could further advance the model. This hypothesis was confirmed given a significant drop in OFV ( delta OFV 9.1 points, p value =0.0026) Visually, the effect on the absorption profiles caused by the differences in estimates was marginal as can be seen by the comparison of two simulated absorption profil es after an exemplary oral dose of 1.5 mg visnagin, equivalent to 5 mg/kg visnagin or 302 mg/kg AVE administered to a rat weighing 300 g (Figure 4 4) In contrast to the shape par ameter no further improvement was achieved by allowing separate scale parameter s (k d ) for AVE and pure compound data. The residual variability was described using a proportional error model for i.v. visnagin data and a combined (proportional plus additive ) error model for oral visnagin and AVE data. Despite the very small effect, the additional residu al variability portion of the oral data was required in the residual error model based on the OFV In summary, t he final model was comprised of a two comp art ment body model with Weibull type absorption, non linear elimination and separate estimates for K M and if visnagin was orally administered in form of the extract. The typical values of the final parameter estimates are 0.176 L (V C ), 0.339 mg/h (V max ), 0. 112 mg/L (K M pure compound), 1.255 mg/L (K M AVE), 1.00 1/h (k 12 ), 1.14 1/h (k 21 ), 0.879 (F), 0.285 1/h (k d ), 0.904 ( oral visnagin) and 0.752 ( AVE) Associated IIV estimates (%CV) for V C V max K M k d and are 9.9, 22.6, 93.9, 39.8 and 15.8, respectively. Residual variability of the i.v. and oral data was estimated to be 6.9% (proportional error i.v.) and 23.6% (proportional error oral) and 0.001 mg/L (additional error oral), respectively. Table 4 2 lists all fi nal model parameter estimates along with parameter estimates of the previously developed model using solely i.v. visnagin data for the purpose of comparison. Common parameter estimates of

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111 the two models do not differ significantly. Goodness of fit plots an d population and individual fits for the 65 subjects are displayed in Figures 4 5 and Figures 4 6 to 4 8 respectively. The observed data was well described by the final model for all doses and treatments. Bootstrapping and construction of non parametric 9 0% bootstrap percentile confidence intervals for parameter estimates 90.1 % of bootstrap runs were successful The main reason for termination (95% of the cases ) was due to rounding errors. The bootstrapping distribution of each parameter is depicted in Figure s 4 9 and 4 10. 90% bootstrap percentile CIs (non parametric CIs) for each parameter are given in Table 4 2. Visu a l predictive check The results of the visual predictive check are shown in Figure s 4 1 1 to 4 1 9 High agreement between simulated and ob served concentrations was achieved for both central tendency (median) and variability (prediction interval) for most of the nine treatment dosing group combinations. Discussion The main purpose of this study was to compare the visnagin plasma exposure after administration of an aqueous AVE and visnagin in form of the pure compound. The visnagin plasma exposure was significantly increased and extended for all three doses when visnagin was given in form of its extract and not as a sole agent. A statistica l significance could be concluded for all comparisons but the AUC inf for the 10 mg/kg dosing group. Removal of plasma concentration time profiles with an extrapolated AUC and z AUC

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112 extrapolated, AUC inf t 1/2 CL/F, V z /F and MRT inf AUMC extrapolated, AUMC inf respectively, was necessary because those estimates did not seem to be reliable or z in those cases because of the previously demonstrated non linear elimination kinetics of visnagin [ 85 ] (Chapter 3) appears to be very likely. However, the similarity of the results for AUC last and AUC inf AUMC last and AUMC inf as well as MRT last and MRT inf suggests that removal of those plasma concentration time profiles does not limit the conclusions with respect to extent and duration of visnagin plasma exposure aft er administrat ion of the pure compound and AVE. The shorter duration of plasma sampling for the pure visnagin treatment groups (8 h) is possibly a limiting factor for comparing the visnagin plasma exposure after administration of AVE and the pure compound. 8 h sampling for the pure compound was selected based upon the previously conducted i.v. study [ 85 ] (Chapter 3) and an oral pilot study. How ever, the median plasma concentrations after 8 h for all three pure compound dosing groups were low (<0.08 mg/L) and therefore, measuring any significant visnagin plasma concentration at later time points is very unlikely. Hence, the difference in the plas ma sampling schedules between the pure compound and AVE treatment groups should not affect the conclusions with respect to the visnagin plasma exposure. The disproportionate i ncrease of both AUC last and AUC inf with an increasing dose that was observed for both pure compound and AVE confirmed the non linear PK of visnagin that was shown in the previous i.v. study [ 85 ] Wit hout further studies, the exact source of non linear elimination as observed in the i.v. study cannot be determined.

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113 Since after an oral dose of 10 mg/kg pure visnagin less than 1/1000 of the dose could be recovered in a 24 h urine specimen (data not shown ) renal elimination of unchanged drug as the main route of elimination can be excluded Supported by the drug properties of visnagin (low molecular weight (M r 230.2) lipophilic (partition coefficient 1.87) ) this points towards elimination mainly via capa city limited liver metabolism. Performing i n vitro studies involving hepatocytes, liver derived cell lines or microsomes could be helpful to confirm this hypothesis [ 89 ] The observed differences i n AUC s after administration of visnagin as extract and pure compound can be explained by differences in clearance and/or differences in oral bioavailability such as differences in absorption from the gut, differences in gut metabolism and/or differences in hepatic first pass metabolism The standard approach for estimating oral bioavailability, which is the comparison of dose normalized AUCs after oral and i.v. administration, is only valid if clearance is constant (linear PK). Since visnagin exhibits non linearity in clearance this approach cannot be used for oral bioavailability determination. Additionally, it cannot be clarified if it is the same saturable process underlying the elimination of visnagin after pure compound and AVE administration without performing another study. One option would be an i.v. bolus study with equivalent doses of AVE. If the mean parameters for the theoretical maximum elimination rate of the saturable process ( V max ) and K M the drug concentration at which the elimination rate is equal to one half of V max are estimated identical or at least similar, it will be very likely that the saturable process eliminating visnagin is the same and is unchanged for both preparations. However, even after sterile filtration it is not advisabl e to inject a relatively unknown multi component mixture

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114 into the systemic circulation of rats Another option that avoids i.v. injection of AVE would be the simultaneous administration of AVE orally and visnagin intravenously. If a byproduct in AVE affect s the elimination process of visnagin, the shape of the terminal phase of the plasma concentration time profiles and associated estimates of V max and K M will change compared to after i.v. bolus administration of pure visnagin. The non linear elimination ki netics of visnagin complicates this approach. In order to avoid further studies a different, model based approach was applied to obtain estimates of oral bioavailability and clearance parameters for pure compound and AVE data. Therefore, i.v. and oral visn agin data were first fitted simultaneously to an appropriate model and subsequently all available data was used to fit a combined model Due to the lack o f i.v. AVE data certain assumptions were required for parameter estimation following oral AVE adminis tration. When fitting i.v. and oral visnagin data s imultaneously, best description was achieved with the model using Weibull type absorption All other absorption models failed to describe the terminal phase particularly of the profiles following the high visnagin oral dose A n initial fast absorption with maximum plasma concentrations being reached after approximately one hour was followed by a slower absorption limit ing the rate of elimination The Weibull function is characterized by two parameters (shape parameter and scale parameter k d ) [ 88 ] providing a more flexible shape than for example a zero or first order rate absorption, and thus, was able to appropriat ely describe the observed absorption behavior In order to eliminate possible differences in dissolution as source of differences in AUC it was anticipated to administer visnagin as solution, since AVE lyophilisate

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115 appeared to be completely dissolved in water Captisol was used as vehicle for visnagin to enhance the aqueous solubility of visnagin and enable its administration as solution. I t has been extensively discussed and reviewed if drug complexation by cyclodextrins alters the PK of drugs [ 76 78 ] It can be concluded that in general the intrinsic PK of drugs is not or only little affected by cyclodextrins especially if drug release from the complex is fast and complete [ 78 ] However, it cannot be completel y ruled out that after oral administration of visnagin in Captisol the complex dissociates upon dilution in the gastrointestinal tract and as consequence visnagin precipitates. The portion being dissolved based on the intrinsic solubility would be quickly absorbed, while the rest of the dose would be dissolved slowly, thus limiting the supply for further absorption over hours. Hence, although visnagin was administered as solution a dissolution step might have become necessary if visnagin actually precipita ted upon dilutio n in the gastrointestinal tract, which could explain the observed absorption behavior. Since visnagin is a small and lipophilic drug membranes would not represent a barrier and a fast absorption would have be en expected if visnagin was act ually dissolved. Another explanation how Captisol could potentially affect the intrinsic PK of visnagin after oral administration is possible. For most drugs, dilution after administration is the major driving force for dissociation of the drug :cyclodextrin complex and is sufficient for rapid and quantitative release of the drug from the complex [ 74 77 90 ] This holds particularly true for parenteral administration of small volumes and in this case the int rinsic PK of a drug is not affected by cyclodextrins [ 78 ] However, if large volumes of cyclodextrin solution are administered to rodents, this volume can

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116 dominate the gastrointestinal tra ct of the rodent and the dilution effect for complex dissociation might be insufficient [ 90 ] After initial partial dilution and dissociation f urther complex dissociation might only occur slowly after free drug has been absorbed. Visnagin absorption after oral administration of AVE was also well described using a Weibull function. It is questionable if a simpler absorption model would be sufficient. Visnagin solubility is increased in AVE, likely due to certain byproduct s (Chapter 2) It cannot be forseen if this solubility enhancing effect continues upon dilution and pH change in the gastrointestinal tract or if visnagin precipitates requiring a dissolution step prior to absorption. It seems reasonable to assume that th e increased AUC of visnagin after extract administration is due to the presence of khellin in the extract (khellin to visnagin ratio of AVE is approximately 2:1, based on HPLC UV analysis of AVE (Chapter 2) ), a furanochromone possessing an additional methoxy group compared to visnagin (Figure 1 3 ). If khellin is a substrate of the same saturable process as vis nagin, saturation occurs at comparatively lower levels of visnagin and thus, plasma levels of visnagin are higher. This can be expressed by a change in V max or K M after extract administration, reflecting a non competitive and competitive inhibition by khel lin, respectively. Given the structural similarities of khellin and visnagin, the latter appears more likely. The final model with two separate estimates of K M for pure c ompound and AVE data confirms the hypothesis of khellin acting as competitive inhibito r. In the presence of khellin the affinity of visnagin to the protein involved in the saturable process is decreased and higher visnagin concentrations are required to achieve half maximum velocity of the process. Thus, K M appears to be increased when visn agin is administered in form of the extract.

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117 It is important to emphasize that the underlying assumption of the developed model is that only K M is affected by khellin or other extract compounds with regard to the clearance process Visnagin is almost compl etely absorbed as suggested by the oral bioavailability estimate of 0.879 and bioavailability is assumed to be unchanged after AVE administration. Solely the absorption profiles after pure compound and AVE administration depart slightly (Figure 4 4) as exp ressed by different Weibull function shape parameter estimates. A two compartment body model with Weibull type absorption, non linear elimination and separate estimates for K M and if visnagin was orally administered in form of the extract was selected a s final model Strong agreement between individual and population fits, in general narrow non parametric 90% bootstrap CI s reflecting precise parameter estimates, and high agreement between simulated and observed concentrations for both central tendency (median) and variability (prediction interval) for most of the nine treatment dosing group combinations in the visual predictive check confirmed the adequacy of the final model. The high percentage of successful bootstrapping runs and the unimodal distributions of the bootstrap estimates demonstrated the robustness of the selected model. In conclusion, a PK model that describes the data obtained from all performed P K studies could be developed and validated. It captures characteristics like non linear elimination and atypical absorption. The model also supports the hypothesis that visnagin plasma exposure is elevated after extract administration because khellin acts as competitive inhibitor of the saturable elimination process that was observed after i.v. and oral administration of pure compound and AVE. Captisol might have affected the

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118 PK of visnagin after oral administration. However, if visnagin was given in form of a suspension, differences in exposure compared to AVE administration would likely be even more pronounced. I ncreased exposure of visnagin after extract administration could result in a superior pharmacodynamic efficacy of AVE compared to an equivalent d ose of visnagin, assuming that visnagin is in deed a major active ingredient. Thus, the efficacy of visnagin and AVE in kidney stone prevention should be evaluated in a properly designed study.

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119 Table 4 1. Results of non co mpartmental analysis (me dian (SD )) Dosing Group (mg/kg) 2.5 5 10 Compound VG AVE VG AVE VG AVE n 8 7 8 6 8 5 AUC last (mg*h/L) 0.21 (0.29) 2.72 (0.65) 2.45 (2.40) 10.11 (2.66) 9.85 (4.09) 35.42 (11.59) AUC inf (mg*h/L) + 0.15 (0.33) 2.72 (0.67) 2.45 (2.49) 10.22 (2.49) 10.14 (7.86) 35.67 (11.42) AUC extrap. (%) + 0.10 (2.37) 0.08 (3.99) 0.05 (4.45) 0.05 (5.22) 6.06 (16.84) 0.71 (1.31) C max (mg/L) 0.17 (0.15) 0.52 (0.15) 0.84 (0.63) 1.38 (0.45) 2.03 (0.85) 3.61 (0.47) t max (h) 0.42 (0.36) 1 (1.67) 1 (0.52) 2 (1.86) 1 (0.53) 6 (3.27) z (1/h) + 2.46 (0.88) 0.81 (0.21) 1.39 (0.72) 0.67 (0.75) 0.36 (0.94) 0.39 (0.13) t 1/2 (h) + 0.28 (0.45) 0.86 (0.41) 0.50 (0.57) 1.14 (2.88) 1.94 (2.19) 1.77 (0.83) AUMC last (mg*h 2 /L) 0.23 (0.56) 10.56 (2.82) 4.89 (5.74) 50.0 0 (6.43) 29.24 (16.6 2 ) 227.8 (110.7) AUMC inf (mg*h 2 /L) ++ 0.1 0 (0.77) 10.59 (3.55) 4.9 0 (6.6 0 ) 52.0 0 (19.4 0 ) 21.14 (12.17 ) 235.0 (107.7) AUMC extrap. (%) ++ 0.63 (8.19) 0.28 (9.14) 0.16 (12.15) 0.17 (18.75) 7.5 0 (11.24) 3.06 (3.51) MRT last (h) 1.05 (0.55) 3.58 (0.49) 2.37 (0.49) 4.62 (0.77) 3.37 (0.66) 6.43 (1.26) MRT inf (h) ++ 1.04 (0.62) 3.58 (0.71) 2.45 (0.59) 4.69 (2.19) 2.84 (0.73) 6.59 (1.16) CL/F (L/h) + 5.24 (5.47) 0.30 (0.08) 0.65 (0.41) 0.17 (0.04) 0.31 (0.26) 0.10 (0.04) V z /F (L) + 1.86 (2.38) 0.41 (0.22) 0.62 (0.33) 0.26 (0.77) 0.86 (0.87) 0.36 (0.25) VG: visnagin, AVE: Ammi visnaga extract, n : number of subjects, AUC last : AUC over the time interval from zero to last quantifiable time point, AUC inf : AUC over the time interval from zero extrapolated to infinity, C max : maximum observed concentration, t max : time at C max z : terminal elimination rate constant, t 1/2 : terminal half life, AUMC last : AUMC over the time interval from zero to last quantifiable time point, AUMC inf : AUMC over the time interval from zero extrapolated to infinity, MRT last : mean residence time over the time interval from zero to last quantifiable time point, MRT inf : mean residence time over the time interval from zero extrapolated to infinity, CL/F: clearance uncorrected for oral b ioavailability (F), V z /F: z uncorrected for F + Plasma concentration time profiles with an extrapolated AUC greater than 50% were excluded from calculation for those parameters, n = 7, 7, 8, 6, 6, 5 ++ Plasma concentration time profiles with an extrapolated AUMC greater than 50% were excluded from calculation for those parameters, n = 7, 7, 8, 6, 4, 5

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120 Table 4 2. Non linear mixed effect modeling approach, final estimates and non parametric confidence in tervals (CI) for typical values of the model developed with all data (left) and i.v. data only (right). N on parametric CI s are bootstrap percentile intervals ). All Data i.v. Data 90% CI 90% CI (Non parametric) (Non parametric) Lower Bound Upper Bound Lower Bound Upper Bound Parameter Estimate Estimate PK Model V C (L) 0.176 0.170 0.183 0.175 0.168 0.182 V max (mg/h) 0.339 0.292 0.391 0.367 0.321 0.418 K M (mg/L) 0.112 0.056 0.185 0.080 0.037 0.141 A dd. K M (AVE) (mg/L) 1.143 0.853 1.513 ---k 12 (1/h) 1.00 0.858 1.166 1.00 0.853 1.230 k 21 (1/h) 1.14 0.918 1.401 1.22 0.944 1.680 F 0.879 -----k d (1/h) 0.285 0.241 0.334 ---0.752 0.689 0.815 ---A 0.152 0.0071 0.231 ---Inter individual variability (IIV) IIV V C (%CV) 9.9 6.4 12.9 9.2 5.8 11.7 IIV V max (%CV) 22.6 18.5 25.7 21.8 16.1 25.7 IIV K M (%CV) 93.9 58.2 118.6 70.9 11.6 89.2 IIV k d (%CV) 39.8 23.7 51.9 ---15.8 10.3 20.0 ---Residual Variability Prop. Error i.v. (%CV) 6.9 6.0 7.8 7.0 6.1 8.2 Prop. Error Oral (%CV) 23.6 19.6 27.0 ---Add. Error Oral (mg/L) 0.001 0.0006 0.003 ---

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121 Figure 4 1 Visnagin plasma concentration (geometric mean SEM) vs. time profiles after administration of the pure compound (a) and Ammi visnaga extract (b), sorted by dosing group (Low: 2.5 mg/kg, Medium: 5 mg/kg, High: 10 mg/kg) n is given in Table 4 1 Figure 4 2 AUC inf (a) and AUC last (b) (median + SD) of visnagin after administration of pure compound vs. Ammi visnaga extract sorted by dosing group (Low: 2.5 mg/kg, Medium: 5 mg/kg, High: 10 mg/kg), numbers represent Bonferroni adjusted p values, n is given in Table 4 1 AUC inf values that are based upon extrapolated AUC > 50% were excluded

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122 Figure 4 3 MRT inf (a) and MRT last (b) (median + SD) of visnagin after administration of pure compound vs. Ammi visnaga extract sorted by dosing group (Low: 2.5 mg/kg, Medium: 5 mg/kg, High: 10 mg/kg), numbers represent Bonferroni adjusted p values n is given in Table 4 1 MRT inf values that are based upon extrapolated AUMC > 50% were excluded Figure 4 4. Comparison of visnagin amount absorbed (mg) afte r an exemplary oral dose of 1.5 mg visnagin or equivalent dose of Ammi visnaga extract using the final model.

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123 Figure 4 5 Non linear mixed effect modeling approach, two compartment body model with Weibull type absorption and non linear elimination basi c goodness of fit plots

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124 Figure 4 6 Non linear mixed effect modeling approach, two compartment body model with Weibull type absorption and non linear elimination population and individual fits of i.v. visnagin data (log transformed), sorted by dosing group.

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125 Figure 4 7 Non linear mixed effect modeling approach, two compartment body model with Weibull type absorption and non linear elimination population and individual fits of oral visnagin data (log transformed), sorted by dosing group.

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126 Figure 4 8 Non linear mixed effect modeling approach, two compartment body model with Weibull type absorption and non linear elimination population and individual fits of oral Ammi visnaga extract data (log transformed), sorted by dosing group.

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127 Figure 4 9. Non linear mixed effect modeling approach, bootstrap distribution of mean parameter estimates vertical black bar represents the parameter estimate obtained by fitting the original data

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128 Figure 4 10. Non linear mixed effect modeling appr oach, bootstrap distribution of inter individual and residual variability parameter estimates vertical black bar represents the parameter estimate obtained by fitting the original data

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129 Figure 4 11 Visual predictive check for the final model Visnagin low dose (1.25 mg/kg) after i.v. bolus administration Figure 4 12 Visual predictive check for the final model. Visnagin m edium dose (2.5 mg/kg) after i.v. bolus administration.

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130 Figure 4 13 Vis ual predictive check for the final model. Visnagin high dose (5 mg/kg) after i.v. bolus administration. Figure 4 14 Visual predictive check for the final model. Visnagin low dose (2.5 mg/kg) after oral administration.

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131 Figure 4 15 Visual predictive check for the final model. Visnagin medium dose (5 mg/kg) after oral administration. Figure 4 16 Visual predictive check for the final model. Visnagin high dose (10 mg/kg) after oral administration.

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132 Figure 4 17 Visual predictive check for the fin al model. Ammi visnaga extract low dose ( 150.8 mg/kg equivalent to 2.5 mg/kg visnagin ) after oral administration. Figure 4 18 Visual predictive check for the final model. Ammi visnaga extract medium dose (302 mg/kg, equivalent to 5 mg/kg visnagin) af ter oral administration.

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133 Figure 4 19 Visual predictive check for the final model. Ammi visnaga extract high dose ( 603.6 mg/kg, equivalent to 10 mg/kg visnagin) after oral administration.

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134 CHAPTER 5 PHARMACODYNAMIC EVALUATION OF VISNAGIN AND AMMI VISNAGA AQUEOUS EXTRACT IN RATS Background A thorough evaluation of the pharmacokinetic ( PK ) properties of visnagin after i .v. and oral administration of visnagin and after oral administration of standard water Ammi visnaga L. (syn. Khel la, Apiaceae) extract ( AVE ) was performed [ 85 91 ] N on l inear elimination kinetics was identified for visnagin and a non linear mixed effect model that describes the observed data after i.v. bolus administration was developed and validated It was demonstrated that average visnagin plasma concentration was signi ficantly higher when it was orally administered in form of AVE compared to the oral administration of the pure compound for all three tested doses Furthermore v isnagin plasma exposure ( area under the plasma concentration time curve (AUC) over the time in terval from zero extrapolated to infinity ( AUC inf ) and AUC over the time interval from zero to last quantifiable time point ( AUC last ) ) was significantly increased and visnagin resided significantly longer in the body ( mean residence time (MRT) over the time interval from zero extrapolated to infinity ( MRT inf ) and MRT over the time interval from zero to last quantifiable time point ( MRT last ) ) when it was given as AVE compared to an equivalent dose of the pure compound for all three tested doses. A non lin ear mixed effect model that describes all observed data after i.v. and oral administration supports the hypothesis that visnagin plasma exposure is elevated after extract administration because, due to structural similarities, the extract compound khellin acts as competitive inhibitor of the saturable elimination process that was observed after i.v. and oral administration of pure compound and AVE Assuming that visnagin is a major active ingredient, t hese observed characteristics could result in a superior pharmacodynamic

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135 ( PD ) efficacy of AVE compared to an equivalent dose of visnagin Prevention of nephrolithiasis by an AVE was previously demonstrated by Khan et al. who reported a reduction of oxalate and calcium content in rat kidney s after administration of khella tea preparation [ 50 ] Our working group showed that AVE as well as the pure compounds visnagin and khellin reduced cell injury after calcium oxalate (CaOx) exposure in vitro and decreased CaOx deposition in vivo in rats [ 51 52 ] However, in the in vivo study AVE and the pure compounds visnagin and khellin were tested in separate experiments and doses of the pure compounds were not in accordance with their concentrations in the ex tract Additionally, analysis of crystal deposits was performed by visual examin ation of hematoxylin and eosin ( H&E ) stained kidney sections using a light microscope and manual counting of visible deposits with a semi quantitative scoring system. In orde r to confirm preventive effects on nephrolithiasis and test the hypothesis of superior PD efficacy of AVE over an equivalent dose of visnagin a PD study in rats was conducted to test visnagin and AVE in one experiment using equivalent doses. Visnagin plas ma levels after the two treatments were measured weekly at two different time points to confirm presence and quantify visnagin concentration in the systemic circulation. Furthermore, a less subjective and more efficient method to count CaOx crystal deposit s in histological sections of rat kidneys was developed and used for analysis. A protocol for consistent and reproducible generation of CaOx crystal deposits in rat kidneys was de termined, using only ethylene glycol ( EG ) in varying concentrations with or w ithout dietary modification Besides visnagin and AVE, the standard treatment for kidney stone prevention, namely potassium citrate (PCi) was also evaluated as positive control.

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136 Specific Aims To develop a method for objective quantification of CaOx crysta l deposits in histological sections of rat kidneys. To determine a method for consistent and reproducible induction of CaOx crystal deposits in rat kidneys to serve as nephrolithiasis model in rats. To evaluate the effects of AVE and visnagin on kidney sto ne prevention and to investigate if AVE is superior over visnagin Material and Methods Chemicals and Reagents Visnagin (Aldrich CPR ), ethylene glycol anhydrous (99.8%) and potassium citrate tribasic monohydrate were purchased from Sigma Aldrich (St. Louis, MO, USA). Captisol cyclodextrin sulfobutyl ethers, sodium salts) was obtained from Cydex Pharmaceuticals (Lenexa, KS, USA) (meanwhile acq uired by Ligand Pharmaceuticals Inc. (La Jolla, Ca, USA) All chemica ls used were of analytical grade except visnagin, for which Aldrich CPR did not provide analytical data Therefore, v isnagin (Aldrich CPR ) was compared to previously purchased visnagin (>97%, Sigma Aldrich) by a validated HPLC UV method. Ammi visnaga L. fru its (DAB 10 quality, invoice 12119085) were kindly provided by Martin Bauer Group Finzelberg GmbH & Co. KG (Andernach, Germany). Commercial kits were used to determine urinary concentration s of citrate (R Biopharm AG, Darmstadt, Germany) and oxalate (Trini ty Biotech, Jamestown, N Y USA). For the quantification of calcium, creatinine, microalbumin and blood urea nitrogen (BUN) reagents as well as calibrator and control solutions for the BioLis 24i Chemistry Analyzer were purchased from Carolina Liquid Chemi stries (Winston Salem, NC, USA) Immunoassays from R&DSystems ( Minneapolis, MN, USA ) were utilized to

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137 measure urinary concentration s of kidney injury molecule 1 (KIM 1) and osteopontin (OPN). Protocol 10% buffered formalin was obtained from Fisher Scientif ic (Pittsburgh, PA, USA). RNAlater RNA Stabilization Reagent was obtained from Qiagen (Valencia, CA, USA). HPLC grade deionized water was prepared using a Barnstead Nanopure Diamond UV ultra pure water system (Dubuque, IA, USA). Pilot Studies Preceding the main study for PD evaluation of standard aqueous AVE and visnagin, a series of pilot st udies was performed to determine a method for consistent and reproducible induction of CaOx crystal deposits in rat kidneys Finding such a nephrolithiasis model in rats was necessary in order to allow testing for significant effects of preventive treatm ent. C riteria for considered methods for crystal induction included administration of EG with the drinking water in varying concentrations for at least four but no longer than eight weeks with or without dietary modification. In particular, 0.75, 1.0, and 1.25% EG added to drinking water for four weeks was tested without dietary modification ( standard chow : (1.36% calcium) ) re spectively Additionally, 1.25% EG was assessed for six and eight weeks, respectively, without dietary modification and for four weeks with a modified diet containing 20% lactose, 2% calcium and 1.25% phosphorus (96348 Teklad Rodent ). Finally 0.75 and 1.25% EG was evaluated for four weeks in combination with 8604 diet enriched in calcium (2% calcium )) respectively. Four male Sp rague Dawley rats were used per method. Basis for co mparing various induction protocols was an objective quantification of CaOx crystal deposits in histological sections of rat kidneys. Therefore, at the end of

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138 each pilot study rats were sacrificed by an overdose of isoflurane (5%) followed by decapitation Afterwards, k idney s were harvested and briefly washed in ice cold saline prior to vertical slicing and fixation in 10% buffered formalin for approximately 21 h. Subsequently, fixation liquid was replaced by 70% ethanol and sent to McKnight Brain Institut e Cell and Tissue Analysis Core Histology Resource Lab at the University of Florida for paraffin embedding, sectioning and histochemical staining. Two 5 cuts stained utilizing the Pizzolato method which turns CaOx containing structures black [ 92 ] (Figure 5 1 A ) Four l ight microscope images of the cuts (depicting papilla, medulla and left and right cortex area) were randomly taken at 5x magnifications and analyzed using the latest version of ImageJ software Briefly, images were first converted to 8 bit grayscale format before only kidney tissue area was manually selected if necessary and threshold was adjust ed followed by conversion to a binary image. As a result, only kidney tissue shows up as black ar ea (Figure 5 1, B) and pixel counts of kidney area can be determined Additionally, by setting a different threshold, only CaOx containing structures will remain black (Figure 5 1, C) and thus, pixel counts of the crystal area can be quantified. The relation of those two pixel counts yield s an objective measurement of percent kidney area covered by crystals. Results were averaged for each rat (16 images per rat: four images per kidney section, two sections per kidney, two kidneys per rat). Animals Fifty male Sprague Dawley rats (weighing 130 150 g) were purchased from C harles River Laboratories (Wilmington, MA, USA). Animals were housed in pairs in plastic cages and were maintained on a 12 h/12 h light/dark cycle. Rats were allowed to adapt to their environment for one week while receiving water ad libitum and standard

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1 39 c (1.36% c a lcium )) During the actual experiment animals received depending on the treatment group ad libitum water or water co ntaining 1.25% EG and standard chow or CED (89166 Teklad Rodent Diet, H ched in calcium (2% calcium )). All animal experiments were performed according to the policies and guidelines of Institutional Animal Care and Use Committee of the University of Florida, Gainesville, FL, USA (IACUC approval #201003686 and #201207258 (after change of princip al investigator) ). Study D esign The animals were divided into five groups ( n = 10 per group) (Table 5 1) : (1) no EG, standard diet, vehicle (healthy animals, control (C T ) group); (2) EG 1.25%, CED, vehicle (nephrolithiasic (NT) group); (3) EG 1.25 %, CED, PCi 2.5 g/kg (positive cont rol (PC) group ); (4) EG 1.25%, CED, AVE 463 mg/kg (extract (EX) group); (5) EG 1.25%, CED, visnagin 7.5 mg/kg (pure compound (VG) group). Treatments were randomly assigned to the rats. Based on the results of the conducted pilot studies, 1.25% EG and CED were utilized for crystal induction ( C T group exempted). Since the C T group received normal water and standard chow in contrast to the other groups, control rats could not be combined in a cage with rats from othe r groups. Thus, a control rat always shared the cage with another control rat, while rats from the other groups were never combined with rats from the same treatment group. All treatments were administered once daily as solutions by oral gavage at a volume of 5 mL/kg utilizing a feeding needle, which is considered good practice in the oral administration of volumes to rats [ 72 ] The animals of the C T and NT group received water (vehicle) instead of treatment.

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140 Preparation of Treatments for Administration to R ats P Ci: PCi solution was prepared freshly every day by dissolving PCi in water to achieve a concentration of 500 mg/mL. AVE: The extract was prepared according to the method described previously [ 51 52 ] (Chapter 2) Briefly, 40 g Ammi visnaga fruits were grinded for 30 sec in a FL, USA). 400 mL boiling water (100C) was poured over the grinded fruits and the mixture was steeped for 5 min at room temperature with occasional swirling. Subsequently, the aqueous mixture was filtered through No. 2 Whatman filter paper circles (pore size 8 Labconco, Kansas City, MO, USA). The procedure was repeated until the required amount of lyophilisate for the entire experiment was produced. All batches of lyophilisate were combined and kept in an am ber colored bottle containing a SORB IT package at 20C. The week before the experiment, l yophilisate was quantified in triplicate with respect to the key compound visnagin by a validated high performance liquid chromatography with ultraviolet light detection ( HPLC UV ) method (C hapter 2 ) Aliquots of lyophilisate were prepared based on the extr act quantification and stored at 20C. AVE solution was prepared freshly every day by addition of water to achieve an AVE solution containing 1.5 mg/mL visnagin. It has previously been demonstrated that the freeze dried extract is stable with regard to vi snagin for at least two months under the named conditions. Furthermore, stability of visnagin in the extract for at least 2 h after reconstitution in water has been confirmed ( C hapter 2 ). Visnagin: Based on the selected dose and administration volume, the visnagin solution for administration was required to have a concentration of 1.5 mg/mL. Due to its

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141 limited water solubility (0.16 mg/mL Chapter 2 ) visnagin was dissolved in 25% aqueous Captisol solution. 25% Captisol was previously identified as adequa te to enhance the aqueous solubility of visnagin to concentrations that were sufficient to achieve the required dosing concentration (Chapter 2) The week before the experiment, the visnagin solution was analyzed in triplicate by the validated HPLC UV meth od to verify the anticipated concentration. It has previously been confirmed that visnagin in 25% Captisol is stable for the duration of the experiment ( C hapter 2) Urine and Plasma C ollection A scheme of the time course of the PD study is depicted in Fi gure 5 2 At day 0, 7, 14, 21 and 28 animals were placed in metabolic cages for 24 h for urine collection. Prior to collection, sodium azide w as added to the urine collection tubes to achieve a concentration of 0.02% for bacterial growth prevention. After 24 h, urine volume and pH (EMD ColorPhast pH strips) were determined. A 5 mL aliquot was transferred to a separate tube and pH was adjusted to pH 1.5 with 6N hydrochloric acid ( HCl ) Both acidified and non acidified urine were centrifuge d at 2800 rpm for 10 min at 20C to remove any particles. The supernatant of the acidified urine was used for the determination of oxalate and total calcium whereas citrate, free calcium creatinine, microalbumin, KIM 1 and OPN were measured in the non ac idified supernatant. For the determinatio n of KIM 1 and OPN aliquots of non acidified urine were stored at 80C until analysis of the samples collected at day 0, 7, and 28. All other biomarkers were analyzed within 4 h after collection at all time points. All assays were carried out of the urine collection (non acidified) at day 28 was examined under the light microscope (20x magnification s ) for CaOx crystals.

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142 In additio vein into heparinized tubes [ 67 ] the day before the first treatment (baseline values) and 1 and 6 h after dosing at day 7, 14 and 21. On the last day of the experiment (day 28) blood collection was performed 1 h post dosing only. All blood samples were stored on ice for a maximum of 2 h until centrifugation at 4000 rpm for 15 min at 4C. Plasma was recovered and 1 h samples were used freshly for the determi nation of BUN, creatinine and calcium Unused 1 h and 6 h plasma samples were stored at 20C until quantification of visnagin concentrations in both 1 an d 6 h plasma samples of animals treated with AVE and visnagin. Plasma extraction and analysis by the validated liquid chromatography tandem mass spectrometry ( LC MS/MS ) method were performed as previously described [ 85 ] (Chapter 3) Calculation of creatinine clearance (Cr CL) was performed according to Eq uation 4 1. (4 1) Tissue C ollection and Q uantification of C alcium O xalate C rystal D eposition After 28 days rats were sacrificed by an overdose of isoflurane (5%) fo llowed by decapitation. The right kidney was immediately sliced vertically submerged in RNAlater RNAStabilization Reagent and stored at 80C. The left kidney was harvested for histopathological investigation of CaOx deposition and processed as described for the pilot studies In order to reduce time consuming image analysis for the main study, one image was taken per kidney section at 1.25x magnifications instead of four images at 5x magnifications yielding two values per rat (one per kidney section, two sections per kidney, one kidney per rat), which were averaged.

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143 Statistical A nalysis Statistical analysis was carried out in SAS (version 9.2 SAS Institute Inc., Cary, NC, USA ). Percent k idney area covered by crystals was analyzed by one way analysis of variance ( ANOVA ) using a mixed model approach (proc mixed) assuming cage effect as random and treatment group as fixed effect. If a rat died prior to study completion, the value for kidney area covered by crystals would not be included in the analysis. Aft er concluding a significant treatment effect, pair wise comparisons were conduct ed for following combinations: C T vs. NT, PC vs. NT, EX vs. NT, VG vs. NT, and EX vs. VG. P to t he listed comparisons of interest. An alpha level of 0.05 was considered statistically significant. Following parameters were measured at multiple time points: body weight (prior to placing rat in metabolic cage); urinary volume, urinary pH calcium (in b oth acidified and non acidified urine), microalbumi n, citrate, oxalate, KIM 1, OPN, CrCL BUN and plasma calcium If a rat died throughout the study, values collected until that time would be included in the analysis. Time series data was analyzed by one w ay analysis of covariance ( ANCOVA ) using a mixed model approach (proc mixed) assuming cage effect as random and treatment group and time including their interaction as fixed effects. Baseline values at time point zero were incorporated as covariate to acco unt for pre treatment differen ces of the respective variable For each repeated measures variable, three different covariance structures that are reasonable for the selected study design were compared, namely heterogeneous autoregressive variance component s (ARH(1)), toeplitz variance components (TOEP) and unstructured variance components (UN). The most suitable

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144 covariance structure for each variable was chosen based on the Bayesian Information Criterion (BIC, smaller is better). In case of significant inte raction (alpha level of 0.2), pair wise comparisons of interest (see kidney area covered by crystals) were carried out utilizing least squared mean (LSM) values of the last measured time point (week four). Additionally it was tested for treatment differen ces at each time level by partition of interaction LSM effects, concluding statistical significance at an alpha level of 0.05. If interaction was not significant, named comparisons were performed based on treatment main effects (averaged over time). P valu account for multiple testing due to the five comparisons of interest. An alpha level of 0.05 was considered statistically significant. Generation of f igures was performed using R software version 2.14.1 (The R Fou ndation for Statistical Computing). A bar graph of average percent kidney area covered by crystals was generated using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) utilizing LSM values. In figures depicting time series data, the baseline v alues at week zero represent mean values ( + standard error of the mean ( SEM ) ), while the values at week one through four are model based LSMs ( + model based SE M ). Results Two sets of five point calibration curves (2 visnagin (AldrichC PR) and the other using visnagin (>97%, Sigma Aldrich) resulted in superimposable lines after simple linear regression of the peak area of visnagin on the nominal standard visnagin concentration in Thus, it was concluded that visnagin (AldrichCPR) and visnagin (>97%, Sigma Aldrich) were of the same analytical grade.

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145 The results of the pilot studies are presented in F igure 5 3 Only the addition of 1.25% E G to the drinking water in combination with CED could meet the requirements of reproducible and consistent CaOx deposit generation in rat kidneys and thus, was determined as induction method for the main study The pilot study in which the lactose diet was fed had to be t erminated after only five days due to neuro toxic effects of EG Thus, results ar e not depicted in F igure 5 3 but c rystals were present in the kidneys of all four animals. However, this fast induction of crystals hardly simulates human conditions and was therefore not selected as suitable. The quantit ative amount of visnagin was 16. 2 mg per g of freeze dried extract. Therefore, solutions containing 92. 6 mg/mL lyophilisate (equivalent to 1.5 mg/mL visnagin) were prepare d daily for AVE administration. Two rats each of the C T and PC group died during the first week due to treatment unrela ted reasons and one rat of the NT group was sacrificed because of neuro toxic effects. In week three, one rat of the PC group was found dead in the metabolic cage. Hence, plasma and urine samples for all time points and values for crystal area in the kidney were available for 8, 9, 7, 10 and 10 rats of the C T NT, PC, EX and VG group, respectively. All animals receiving no treatment during CaOx crystal induction and none of the control rats generated CaOx deposits in the kidney. Crystals were present throug hout all areas of the kidney, but cumulated particularly in the renal tubules in the cortex area. Widening of the tubular lumen was a typical morphological finding in all kidneys of animals receiving EG and CED. Crystal aggregates at the renal papillary ti p were not uncommon. An exemplary image of a Pizzolato stained kidney section, as well as after

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146 ImageJ proc essing is depicted in Figure 5 1 (A C) A bar graph comparing the average percent kidney area covered by crystals ( + SE M ) for the individual groups is presented in Figure 5 4 LSMs of crystal area of the treatment groups and pair wise comparison results can be found in Table 5 2 The average area of crystals in the kidney of nephrolithiasic rats was significantly higher com pared to healthy animals. While the extract did not reduce the crystal area in contrast to nephrolithiasic rats, visnagin significantly increased the mean area of crystals in the kidney in comparison to both NT and EX groups. Although the difference to nep hrolithiasic animals was not statistically significant, PCi treated rats showed a strong trend towards reduced CaOx deposits. Ten out of 1 4 repeatedly measured parameters showed significant interaction between treatment and time, namely body weight (prior to placing rat in metabolic cage); urinary pH, citrate calcium (in both acidified and non acidified urine) plasma creatinine, CrCL microalbumin, BUN and OPN. At each time level, differences between treatment groups were statistically significant (p valu e <0.05, results not shown). The variables urinary volume, oxalate, plasma calcium and KIM 1 did not demonstrate significant interaction between treatment and time. For each parameter, LSMs (based on week four in case of interaction; based on treatment mai n effects averaged over time in case of no significant interaction) for the five groups, as well as the selected covariance structure are presented in Table 5 2 Time trends of the individual variables are visualized in Figure s 5 5 to 5 1 8 The body weigh t of control rats after four weeks was significantly higher than that of nephrolithiasic animals (C T vs. NT: 373.15 vs. 280.43 g). A strong tendency towards elevated weight of PCi treated rats could be observed (PC: 312.93 g), whereas the

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147 average weight of the EX and VG group was similar to nephrolithiasic animals (EX: 287.95 g; VG: 268.65 g). In comparison to the NT group, urinary volume was significantly increased for PCi treated animals (PC vs. NT: 47.70 vs. 26.44 mL/ 24h ) and less pronounced for rats of the EX group (EX: 35.44 mL/ 24h ). A significantly higher pH could be observed in the urine of the C T and PC group, as against to nephrolithiasic rats (C T NT and PC: 6.58, 5.45 and 7.43). The PC, EX and VG group, but not the healthy animals demonstrated sig nificantly increased urinary citrate levels with respect to the NT group. A significant difference in urinary oxalate output was only observed for the comparison of control to nephrolithiasic animals, with less oxalate in the C T group (C T vs. NT: 0.76 vs. 3.75 mg/24h). Although not significant, the oxalate level of PCi treated rats tended to be higher than that of NT rats (PC: 5.04 mg/24h). In general, calcium levels were higher in acidified urine than in unadjusted urine. Both acidified a nd non acidified urine of the PC and EX group demonstrated significantly elevated calcium values. The same pattern was observed for plasma calcium, in which case AVE treated animals additionally showed significantly higher levels compared to the VG group. CrCL w as significantly increased, whereas microalbumin plasma creatinine BUN and KIM 1 were significantly decreased in healthy animals when compared to nephrolithiasic rats. No significance was demonstrated for any of the comparisons involving OPN. Urine of control rats was clear of CaOx crystals. Most nephrolithiasic animals demonstrated some CaOx crystals of rather small size in their urine. However, in some samples almost no crystals could be spotted. Urine samples of the PC group were either free of crystals or contained some isolated crystals small to medium in size. All rats that received AVE as treatment had urinary crystals present. Most samples

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148 contained plenty of small to medium sized crystals, except for two cases, where also large crystals were present. Only two of the samples showed rather few crystals. About a third of the urine samples of the VG group contained either few small or almost no crystals. In another third some small to medium sized crystals were spotted, while the last third showed plenty of crystals also small to medium in size. Only two samples contained additionally large crystals. Overall, o bserved crystals exhibited an envelope shape (Figure 5 1 9 ) For all LC MS/MS runs for plasma analysis, calibration curves and QC sampl es met the required acceptance criteria. Weekly average visnagin plasma concentrations ( SEM ) for the EX and VG group 1 and 6 h post dosing are depicted in Figure 5 20 1 h after the first dose, average visnagin plasma levels were similar for the two grou ps. The following weeks, 1 h post dosing levels after visnagin administration were approximately double as high as after a dose of AVE. However, 6 h after pure visnagin administration, the compound was already eliminated, whereas visnagin was still present in the p lasma of AVE treated animals. Discussion The induction of hyperoxaluria and CaOx crystals by administration of 1.25% EG in the drinking water and CED for four weeks was based on the outcome of a series of pilot studies. The purpose of the conducte d pilot studies was to find a method for consistent and reproducible CaOx crystal induction to serve as nephrolithiasis model in rats in order to allow testing for significant effects of preventive treatments. Typically, acute or chronic hyperoxaluria is i nduced by certain agents which is essential for solid phase formation in the urinary passages [ 93 ] Direct administration of sodium or ammonium oxalate via intraperitoneal injection leads to acute hyperoxaluria, whereas

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149 oxalate precursors like EG, hydroxyl l proline or glycolic acid have been used to induce chronic hyperoxaluria [ 94 100 ] Dietary modifications or pH reducing substances like ammonium acetate are often dispensed concomitantly [ 94 97 99 ] For our study we intended to induce chronic hyperoxaluria to simulate conditions for crysta l formation similar to humans [ 97 ] Most experience exists for EG as inducing agent [ 101 ] Thus, criteria for considered methods included administration of EG in varying concentrations for at le ast four but no longer than eight weeks with or without dietary modification. The selected protocol of 1.25% EG and CED was the only one resulting in CaOx crystal generation in the kidneys of all tested animals. The fact that all animals receiving no treat ment during CaOx crystal induction and that the average area of crystals in the kidney of nephrolithiasic rats was significantly higher compared to healthy animals, confirmed that the selected method was reproducible and adequate for CaOx crystal induction For crystal quantification of the main study it was decided to take images of kidney sections with a lower magnification (1.25x) compared to the pilot studies, which captures the major portion of a kidney section. Thus, one instead of four images per sec tion, as performed during the pilot studies, was taken and analyzed. Prior to this decision, images of 1.25x magnifications of kidney sections from one of the pilot studies were taken and the results were compared to the values when images of 5x magnificat ions were analyzed. Since no significant difference could be detected in the results, the methods were determined to be comparable, but using a lower magnification translates to a tremendous saving in time required for analysis. PCi treated rats showed a s trong tendency towards reduced CaOx deposits in contrast to the NT group, although the difference was not statistically significant. This

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150 might be explained by the varia bility (SE M ) between the rats in each group. In opposition to our expectations, neither AVE nor visnagin administration prevented CaOx crystal deposition in the kidney. Visnagin treatment even deteriorated the condition by significantly raising the crystal area in comparison to the NT and EX group. An explanation for this worsening following visnagin administration is still lacking Captisol has an excellent safety profile and does not exhibit nephrotoxicity associated with beta cyclodextrin [ 73 74 ] Also, after oral administration Captisol absorption is negligible [ 77 ] Thus, worsening of the condition due to the cyclodextrin is unlikely The findings about AVE and visnagin are in contrast to the outcome of a previously conducted similar study with AVE, visnagin and khellin [ 52 ] where PCi, AVE, visnagin and khellin demonstrated a significant reduction in CaOx crystal deposition score compared to nephrolithiasic animals. While the experiments showed similarities to a certain extent, several discrepancies with respect to the design and methods were present, which might account for differences in the results. First, CaOx depositions were generated by addition of 0.75% EG and 1% ammonium chloride to the drinking water for a peri od of two weeks. Second, analysis of crystal deposits was performed by unblinded visual examin ation of H&E stained kidney sections using a light microscope and manual counting of visible deposits with a semi quantitative scoring system. Finally, AVE and th e pure compounds visnagin and khellin were tested in separate experiments and doses of the pure compounds were not in accordance with their concentrations in the extract, thus comparability might be questioned. Our design and methods represent an improveme nt since crystal quantification was performed in an objective semi automated manner. Additionally, the visnagin dose was in accordance with its amount in

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151 the extract and both treatments were administered in one study, which makes their results comparable. Our method for crystal induction was intended to simulate conditions for crystal formation similar to humans Therefore, we decided to use no additional intervention for crystal formation besides EG and calcium, whic h naturally occurs in the diet, and to allow more time for crystals to form, being retained and grow. For the body weight a similar pattern could be observed as for crystal area. Healthy control rats were on average the heaviest group, followed by PCi treated animals. The weight of the NT and EX group was similar and visnagin treated rats exhibited the lowest weight which correlates with the highest crystal deposition in this group. This suggests that animals with less CaOx deposits tend to have a higher body weight and that body weight can be used as indicator of general condition. The significantly higher urinary output following PCi treatment was also seen in the previously conducted study [ 52 ] and this diuretic effect may contribute to the preventive effects of PCi. Increase d urinary output after AVE administration was not only o bserved by Vanachayangkul et al. [ 52 ] and Khan et al. [ 50 ] but also confirmed in the present study, suggesting that AVE exhibit s diuretic effects to a certain extent The elevated urinary pH of the PC group was mainly caused by the alkali load that PCi provides after rapid metabolism of o rally dosed citrate to bicarbonate [ 102 103 ] The selected dose of 2.5 g/kg PCi was based on the previous study [ 52 ] The increase in s ystemic pH causes a raise in intracellular pH which diminishes citrate metabolism in proximal tubular cells. Consequently, more citrate is available in the cells shifting the lumen to cell citrate gradient so that less citrate is reabsorbed [ 102 103 ] Additionally, following an alkali load the luminal pH raises reducing the divalent form of the citrate

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152 cation (citrate 2 ) in the lumen. Since citrate 2 is the substrate for the apical sodiu m dicarboxylate co transporter (NaDC 1) luminal apical uptake into proximal tubular cells is decreased [ 103 104 ] Thus, less freely filtered citrate is re absorbed in the proximal tubules, which is the main site of citrate reabsorption [ 105 106 ] and more citrate is excreted with the urine [ 102 104 ] These effect s are reflected in the tremendously increased urinary citrate levels following PCi treatment. Animals treated with AVE and visnagin exhibited elevated urinar y citrate values without a change in pH in comparison to the NT group. A potential explanation could be a pH independent direct inhibition of the NaDC 1 transporter or citrate metabolism [ 60 ] Hypocitraturia is a well known risk factor for n ephrolithiasis [ 5 16 26 102 107 111 ] Increasing urinary citrate levels can prevent future crystal development by formation of soluble complexes wi th calcium, thereby directly reducing freely available calcium [ 109 112 113 ] The observed diminished values of citrate as well as the reduction in urinary pH in nephrolithiasic animals are typical and could possibly be the result of a metabolic acidosis induced by EG administration. It is well known that acidosis decreases citrate excretion [ 114 ] Administration of EG concentrations higher than 0.75% may result in metabolic acidosis [ 96 101 ] Induction of hyperoxaluria by EG has therefore been criticized, because effects of hyperoxaluria might not be clearly distinguishable from effects due to metabolic acidosis [ 96 101 ] Nevertheless, this model is still widely used because most experience exists for EG as inducing agent and also because of its simplicity [ 101 115 116 ] As mentioned before, one of our requests for a suitable method was a consistent and reproducible induction of crystals in all rats in order to evaluate preventive treatment effects. Although some studies found the administration of 0.75%

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153 EG wit h drinking water for four weeks to be sufficient for crystal induction [ 97 115 117 ] this could not be consistently reproduced in our pilot studies. Even the addition of calcium in form of C ED generated crystal s in just one out of four tested rats. Thus, we selected the only method that produced crystals in all animals namely 1.25% EG and CED knowing that there is a chance of al so inducing metabolic acidosis. Hyperoxaluria was present in all rats receiving EG, but most pronounced wa s the effect of elevated urinary oxalate after treatment with PCi. This result is in accordance to the previously conducted study [ 52 ] An explanation for increased oxalate output of the PC group can be given by the concomitant high citrate excretion. Cit rate is known to build soluble complexes with calcium, thereby directly reducing freely available calcium [ 109 112 113 ] Thus, less insoluble CaOx complexes can be formed resulting in excessive elimination of free oxalate with the uri ne. This is one mechanism how PCi prevents CaOx nephrolithiasis. Furthermore, the calcium citrate complex directly inhibits all aspects of crystallization including crystal agglomeration and growth, which is another mechanism how PCi prevents stone formati on [ 109 112 ] Urinary calcium excretion was elevated in all ani mals receiving CED. A comparison of urinary calcium of the C T and NT group is not meaningful since control rats did not receive CED. Statistical significant elevation could be observed for PCi and AVE treated rats and less pronounced for the VG group, comp ared to nephrolithiasic animals. Part of this effect might be explained by higher calcium availability in the urine in form of soluble calcium citrate since all three groups also demonstrated significantly increased citrate levels. In case of the PC group where citrate and calcium levels we re elevated the most, the chelation of calcium with citrate probably caused fewer CaOx

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154 crystals being retained in the kidney. The kidney section image results support this explanation, since PCi treated rats present ed l ess CaOx deposits as against the NT group. However, e levated urinary citrate levels after AVE and visnagin administration did not positively affect crystal deposition, maybe because the increase in citrate was not sufficient. The elevated microalbumin leve ls in the urine especially of PCi and AVE treated animals might also contribute to the observed hyper calciuria. About 5 0% of circulating calcium is not bound to proteins and approximately 5 10% is complexed with small diffusible anions such as citrate [ 104 118 121 ] Only those two fractions are freely filterable [ 104 118 119 ] Almost all filtered calcium (~99%) is reabsorbed throughout the kidney, the proximal tubule being the main absorption site with 65% of filtered load [ 120 122 123 ] Due to the observed microalbuminuria after PCi AVE and less pronounce d after visnagin treatment it can be assumed that plasma albumin levels are decreased and thus, more unbound calcium might be available for glomerular filtration and renal excretion [ 119 ] Additionally, PCi and AVE treated rats show ed the highest urinary output. The major site of calcium elimination is the kidney and an inc rease in calcium excretion could also be due to an increase in the glomerular filtration rate [ 124 ] Plasma calcium concentrations are closely regulated within a nar row range varying by less than 6% throughout the day [ 125 126 ] We observed an increase of about 10% for the PC and EX groups along with hypercalciuria and elevated microalbu min levels in the urine. These laboratory findings are in accordance with a rare cause of hypercalcemia, namely milk alkali syndrome [ 124 127 128 ] This syndrome can be observed after ingestion of lar ge amounts of calcium and absorbable alkali [ 128 ] and was seen as common toxicity following a calcium laden milk and antacid regimen

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155 developed by Sippy [ 129 ] for the treatment of peptic ulcer disease. The excessive intake of calcium and alkali can generate hypercalcemia in the first place with concomitant hypercalciuria elevated blood creatini ne albuminuria, diuresis and metabolic alkalosis, particularly but not exclusively, if renal impairment preexist [ 124 128 130 131 ] On the one hand, hypercalcemia raises renal calcium ex cretion, but on the other hand several mechanisms also limit calcium excretion, which aids maintenance of hypercalcemia. For example, diuresis as initial consequence of hypercalcemia leads to volume depletion, which reduces the glomerular filtration rate and thus, the excretion of calcium and bicarbonate [ 124 128 ] Metabolic alkalosis enhances calcium reabsorption in the distal tubule, which also maintains hypercalcemia [ 124 ] Chronic usage over years may cause abnormal calcifications including nephrocalcinosis but after acute o r intermediate usage, symptoms are usually relieved quickly and renal functi on return s to normal status after withdrawal of milk and alkali [ 124 ] It can be speculated that r ats receiving the combination of CED and PCi might have developed a milk alkali syndrome in addition to hyperoxaluria and CaOx crystals which could explain elevated plasma calcium levels and to some extent the hypercalciuria, diuresis, microalbuminuria and reduced creatinine clearance. However, diminished creatinine clea rance and microalbuminuria may also be effects of impaired renal function as a consequence of C aOx crystal depositions Also, the EX group demonstrates similar changes of laboratory parameters as the PC group, but it remains unclear what the alkali source in AVE could be. Several prognostic markers of kidney function and injury were selected to assess disease progression and treatment effects. Microalbuminuria is an indication for

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156 impaired glomerular filtration barrier function, since the glomer o lus is rela tively impermeable for proteins like albumin under normal conditions [ 132 133 ] Large amounts of albumin passing the glomerolus both reflects and induces glomerular damage, eventually resulting in glomerular dysfunction [ 133 ] It is often used as predictor for the development of diabetic nephropathy [ 134 135 ] Also, a lbumin has recently been accepted by regulators as renal drug safety biomarker in preclinical testing [ 136 ] The glomerular filtration rate (GFR) is the usual expression for overall kidney function and is commonly estimated by the CrCL [ 137 140 ] In order to calculate CrCL, 24 h measurements of creatinine serum (or plasma) and urine concentrations as well as urine flow are required An increase in serum creatinine reflects a decrease in its glomerular filtration and, consequently, in the GFR. The u sage of CrCL as estimat or of GFR has been critiqued because part of the creatinine excretion results from proximal tubular secretion, which is even more pronounced if severe renal insufficiency is present [ 140 141 ] Thus, GFR might be overest imated. Nevertheless, it is still widely used and in our study i t wa s only one of several parameter s to assess kidney function. The measurements of serum creatinine (usually measured in plasma) [ 142 ] and BUN are standard clinical tests for detection of kidney injury [ 136 141 143 145 ] Disadvantages of t hese traditional biomarkers include no s pecificity with respect to the damaged tissue and insensitivity to mild or moderate injuries because they are rather biomarkers of overall kidney function and not necessarily kidney injury [ 136 143 146 ] Therefore, we also evaluated the novel biomarkers KIM 1 and OPN to compare if differences can be detected earlier than with the traditional biomarkers. In several studies KIM 1 and OPN have been suggested and confirmed as sensitive endp oints for acute kidney injury and

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157 also for chronic nephrotoxicity [ 143 144 147 ] While KIM 1 is predomin antly expressed in the proximal tubule OPN c an be found from the glomerolus through the distal tubule [ 143 ] Except for OPN, all other kidney function and injury biomarkers detected a statistically significant worsening in nephrolithiasic rats as against healthy control rats. For the parameters with interaction, significant differences were already apparent after the first week. No distinction of the treatment effects could be made based on the comparisons of each biomarker. Certain trends could be detected, but also not consis tently. For example, after PCi treatement plasma creatinine and BUN were less increased, whereas CrCL was less decreased compared to nephrolithiasic animals. However, out of all five groups microalbuminuria was most pronounced in the PC group, although th is might be due to the hypothesized milk alkali syndrome. KIM 1 and OPN were not very effective biomarkers in our study. Despite markedly increased values in all animals receiving 1.25% EG and CED, the large variability made it almost impossible to conclud e any significant differences. It is not clear if this variability is solely between rat variability, or if it was also due to technical imperfections, like large within and/or between plate variability pipetting inaccuracies or fluctuations of the plate reader. A contribution of both factors is also possible. Overall, it can only be concluded that k idney injury was already substantial after one week of induction, given the significant differences in kidney function biomarkers between the C T and NT group already after the first week The method of crystal induction might have been too severe for treatments to be preventive and resulted in irreversible damage. However, other protocols did not provide consistent crystal generation in all rats and it is a cha llenge to

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158 simulate human conditions with only mild changes, but also trying to evaluat e treatment effects if just some animals develop few crystals. The typical envelope shape indicate s that observed crystals in the urine were mostly CaOx dihydrate, which is the most common form in the urine of patients with idiopathic calcium urolithiasis [ 148 ] It is difficult to say if there is a correlation between number and size of crystals found in urine samples and the area in the kidney covered by crystals, because in the urine of both nephrolithiasic and PCi treated animals onl y few or no crystals were present. However, crystal area in the kidney was considerably smaller in PCi treated rats, as against the NT group. There are different approaches to explain the results. For nephrolithiasic animals it seems likely, that almost no crystals could be found in the urine because most CaOx was retained in the kidney in for m of crystals. The urine of the PC group demonstrated only few crystals not because CaOx was heavily retained in the kidney but because urinary citr ate levels we re v ery high and thus, ca lcium was chelated with citrate and could not form crystals. In urine of all animals of the EX group crystals were spotted whereas only a smaller portion of VG treated rats demonstrated urinary crystals. However the crystal area of t he VG group was significantly higher as against the EX group. Both treatment groups exhibited elevated citrate levels in the urine, but by far less than the PC group. It can be hypothesized that after VG treatment less crystals could be found in the urine, because more crystals were retained i n the kidney, compared to the EX group. In both cases there was still enough CaOx reaching the urine to show up as crystals, because citrate levels wer e not high enough to chelate most calcium. A remaining question is why nephrolithiasic animals demonstrated basically no urinary crystals while having similar

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159 or even less area in the kidney covered by crystals, compared to the EX and VG group, respectively. A possible explanation could be that the urine of the EX and VG group contain ed more calcium, but not enough citrate for complete chelation, leaving enough calcium to form CaOx crystals in the urine. Nevertheless, in humans it is evident that crystalluria cannot be equated with the formation of stones, since crysta lluria is seen in both n ormal subjects as well as stone formers [ 148 ] Visnagin plasma concentratio ns 1 and 6 h after dosing were measured weekly in order to explain possible differences in treatment effects between the EX and VG group. Two time points are certainly not enough to achieve a detailed description of the plasma concentration time profiles, but can give at least a certain insight in how much drug had reached the circulation. Time points were selected b ased on the data of the PK studies after oral administration of visnagin and AVE in rats [ 91 ] In these studies three doses of pure visnagin (2.5, 5, and 10 mg/kg) and equiva lent doses of AVE were utilized The current PD study investigated 7.5 mg/kg visnagin and an equivalent dose of AVE. Thus, values lying between the midd le and high dose of the oral PK study may be taken a s reference. Maximal visnagin plasma concentrations (C max ) were expected to be reached 1 h after administration of pure compound. Time to reach maximal visnagin plasma concentrations after AVE administrat ion has been demonstrated to take rather l onge r, presumably around 3 to 4 h post dosing. Thus, for the EX group 1 h values are unlikely to present maximal concentrations that were reached. This also explains why plasma concentrations 1 h after pure compound administration were generally around double as high compared to the EX group, although it ha d previously been demonstrated that higher C max values we re reached after equivalent doses of AVE. Plasma concentrations

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160 1 h after visnagin administration were approximately in the range expected for a dose of 7.5 mg/kg, but between week fluctuations were relatively high. Pl asma levels 1 h after AVE dosing were more stable, but somewhat lower as deflected from the oral PK study results. After pure compound a dministration, v isnagin plasma levels drop quickly and therefore, 6 h post dosing was selected as trough level time point, despite knowing that mean residence time after AVE administration is usually extended. For all three weeks, no visnagin could be dete cted anymore 6 h after pure compound dosing, whereas visnagin still resided in plasma of the EX group, even though somewhat less than deflec ted from the oral PK study. These differences could explain why visnagin treated rats were generally in a worse cond ition compared to AVE treated animals. However, it is still not clear why v isnagin treatment even deteriorated the condition by significantly raising the crystal area in comparison to the NT and EX group Neither after pure compound, nor after AVE administ ration a steady state was reached. This was not surprising given a half life between 1 and 2 h and once daily dosing, where plasma levels basically drop to zero before the next dose was given. A more frequent dosing could have lead to a steady state at hig her plasma levels and possibly to a prevention of nephrolithiasis However, dosing twice or three times a day would not have been feasible and the non linear elimination complicate s steady state predictions. In conclusion, preventive effects of visnagin an d AVE for kidney stones could not be confirmed, although demonstrated in previous studies. Discrepancies in the outcomes might be due to different study designs and analysis. Our method for consistent crystal induction might have been too severe for AVE an d visnagin to show effective prevention. The lack of reaching steady state visnagin plasma concentrations

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161 possibly contributed to this outcome. Nevertheless, our protocol could consistently produce CaOx crystals in rats and preventive treatment with PCi sh owed a reduction in crystal area, although not statistically significant. This protocol should be further investigated and used for assessment of preventive treatments. Overall, it is evident that the need for research and new substances in the area of kid ney stone prevention continues.

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162 Table 5 1. Treatment groups of pharmacodynamic study EG: ethylene glycol, PCi: Potassium citrate, AVE: Ammi visnaga extract, CED: calcium enriched diet Group Treatment Diet n 1 Control (CT) No EG, Vehicle Standard Diet 10 2 Nephrolithiasic (NT) EG 1.25%, Vehicle CED 10 3 Positive Control (PC) EG 1.25%, PCi 2.5 g/kg CED 10 4 Extract (EX) EG 1.25%, AVE 463 mg/kg CED 10 5 Pure Compound (VG) EG 1.25%, Visnagin 7.5 mg/kg CED 10

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163 Table 5 2 Summary statistics ( LSM (SD)) of the treatment groups and pair wise comparison results Group Covariance structure C T NT PC EX VG Crystal area (%) 0.05 (0.39)** 1.42 (0.27) 0.57 (0.30) 1.45 (0.35) # 2.44 (0.25)* Body weight (g) + 373.15 (11.00)*** 280.43 (10.37) 312.93 (11.12) 287.95 (9.84) 268.65 (9.84) UN Volume (urine) (mL/24h) 32.12 ( 3.04 ) 26.44 ( 2.39 ) 47.70 ( 2.42 )*** 35.44 ( 2.33 ) 30.14 ( 2.15 ) TOEP pH (urine) + 6.58 (0.12)*** 5.45 (0.13) 7.43 (0.13)*** 5.50 (0.12) 5.38 (0.11) TOEP Citrate (mg/24h) + 38.15 (5.74) 30.48 (5.80) 153.94 (6.13)*** 68.60 (5.49)*** 62.46 (5.15)*** TOEP Oxalate (mg/24h) 0.76 (0.41)*** 3.75 (0.37) 5.04 (0.38) 3.18 (0.35) 3.73 (0.33) ARH(1) Calcium (urine) (mg/24h) + 0.69 (0.47) 1.28 (0.49) 4.26 (0.49)*** 3.65 (0.45)** 2.63 (0.42) TOEP Calcium (acidif. urine) (mg/24h) + 0.79 (0.49) 1.74 (0.51) 4.17 (0.51)** 4.12 (0.47)** 2.78 (0.44) TOEP Calcium (plasma) (mg/dL) 10.21 (0.18) 10.61 (0.14) 11.47 (0.15)*** 11.32 (0.13)** ## 10.65 (0.13) TOEP Microalbumin (mg/24h) + 0.06 (0.19)*** 1.23 (0.19) 1.90 (0.20) 1.84 (0.18) 1.54 (0.17) TOEP Creatinine (plasma) (mg/dL) + 0.51 (0.08)*** 0.94 (0.08) 0.72 (0.09) 1.08 (0.08) 0.98 (0.07) UN CrCL (mL/min) + 1.60 (0.09)*** 0.64 (0.09) 0.88 (0.09) 0.47 (0.09) 0.54 (0.08) ARH(1) Blood urea nitrogen (mg/dL) + 16.03 (4.65)*** 54.82 (4.45) 41.14 (4.96) 57.02 (4.19) 57.34 (4.16) ARH(1) KIM 1 (ng/24h) 6.41 (12.68)** 75.12 (12.67) 73.87 (14.27) 87.12 (11.81) 108.56 (11.56) TOEP Osteopontin (ng/24h) + 76.83 (533.88) 1679.66 (535.31) 2287.66 (572.16) 3408.89 (506.81) 3183.70 (477.56) UN + Repeatedly measured parameters with significant interaction between treatment and time LSMs are based on week 4 measurement. In case of no significant interaction, LSMs are based on treatment main effects averaged over time. p value < 0.05 vs. NT, ** p value < 0.01 vs. NT, *** p value < 0.001 vs. NT # p value < 0.05 vs. VG, ## p value < 0.05 vs. VG

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164 A B C Figure 5 1 Light microscope images (1.25x magnifications) of an exemplary kidney section after Pizzolato staining (A) and after transformation to a binary image following different threshold adjustments using ImageJ software (B and C) Figure 5 2. Time cours e of pharmacodynamic study.

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165 Figure 5 3. Bar graphs summarizing the results of the pilot studies. Average crystal area per rat for different methods of crystal induction. Methods: A: 0.75% EG, standard diet, four weeks; B: 1.0% EG, standard diet, four we eks; C: 1.25% EG, standard diet, four weeks; D: 1.25% EG, standard diet, six weeks; E: 1.25% EG, standard diet, eight weeks; F: 0.75% EG, CED, four weeks; G: 1.25% EG, CED, four weeks.

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166 Figure 5 4 Bar plot of average crystal ar ea (+ SE M ) after four weeks, sorted by treatment group (PCi 2.5 g/kg, AVE 463 mg/kg, visnagin 7.5 mg/kg) p value < 0.05 vs. NT, ** p value < 0.01 vs. NT, # p value < 0.05 vs. VG Figure 5 5 Average body weight (LSM + SEM) vs. time, sorted by treatment group (PCi 2.5 g/kg, AVE 463 mg/kg, visnagin 7.5 mg/kg).

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167 Figure 5 6 Average urinary volume (LSM + SEM) vs. time, sorted by treatment group (PCi 2.5 g/kg, AVE 463 mg/kg, visnagin 7.5 m g/kg). Figure 5 7 Average urinary pH (LSM + SEM) vs. time, sorted by treatment group (PCi 2.5 g/kg, AVE 463 mg/kg, visnagin 7.5 mg/kg).

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168 Figure 5 8 Average citrate in urine (LSM + SEM) vs. time, sorted by treatment group (PCi 2.5 g/kg, AVE 463 mg/kg, visnagin 7.5 mg/kg). Figure 5 9 Average oxalate in urine (LSM + SEM) vs. time, sorted by treatment group (PCi 2.5 g/kg, AVE 463 mg/kg, visnagin 7.5 mg/kg).

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169 Figure 5 10 Average calcium in urine (LSM + SEM) vs. time, sorted by treatment group (PCi 2.5 g/kg, AVE 463 mg/kg, visnagin 7.5 mg/kg). Figu re 5 1 1 Average calcium in acidified urine (LSM + SEM) vs. time, sorted by treatment group (PCi 2.5 g/kg, AVE 463 mg/kg, visnagin 7.5 mg/kg).

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170 Figure 5 1 2 Average calcium in plasma (LSM + SEM) vs. time, sorted by treatment group (PCi 2.5 g/kg, AVE 46 3 mg/kg, visnagin 7.5 mg/kg). Figure 5 1 3 Average microalbumin (LSM + SEM) vs. time, sorted by treatment group (PCi 2.5 g/kg, AVE 463 mg/kg, visnagin 7.5 mg/kg).

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171 Figure 5 1 4 Average creatinine in plasma (LSM + SEM) vs. time, sorted by treatment g roup (PCi 2.5 g/kg, AVE 463 mg/kg, visnagin 7.5 mg/kg). Figure 5 1 5 Average creatinine clearance (LSM + SEM) vs. time, sorted by treatment group (PCi 2.5 g/kg, AVE 463 mg/kg, visnagin 7.5 mg/kg).

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172 Figure 5 1 6 Average blood urea nitrogen (LSM + SEM) vs. time, sorted by treatment group (PCi 2.5 g/kg, AVE 463 mg/kg, visnagin 7.5 mg/kg). Figure 5 1 7 Average kidney injury molecule 1 (LSM + SEM) vs. time, sorted by treatment group (PCi 2.5 g/kg, AVE 463 mg/kg, visnagin 7.5 mg/kg).

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173 Figure 5 1 8 Average osteopontin (LSM + SEM) vs. time, sorted by treatment group (PCi 2.5 g/kg, AVE 463 mg/kg, visnagin 7.5 mg/kg). Figure 5 1 9 Light microscope image (20x magnifications) of a urine sample showing calcium oxalate dihydrate crystals

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174 Figure 5 20 Bar graph of w eekly average v isnagin plasma concentration (mean SEM) 1 and 6 h after visnagin (7.5 mg/kg) and Ammi visnaga extract (493 mg/kg, equivalent to 7.5 mg/kg visnagin) administration

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175 CHAPTER 6 SUMMARY AND CONCLUSION Suffering from kidney stones is a common phenomenon in developed countries and the prevalence has increased over the last dec a des. Causes are multifold including genetic predisposition, but dietary habits and lifestyle are most important co ntributors to nephrolithiasis formation. Calcium oxalate (CaOx) is the most common stone compound (~60%), followed by calcium phosphate (~20 %) and m ixed calcium stones containing both CaOx and calcium phosphate are not unusual. It can be distinguished betw een ureteral and renal stones and stone size and location determine the s election of treatment options which range from observation with periodic evaluation f or small ureteral stones to extracorporeal shock wave lithotripsy (ESWL) and percutaneous nephrolithotomy for most renal stones. Unfortunately, the propensity for stone recurrence is about 50% and generally not altered by stone removal but significant side effects might occur e.g. with ESWL. Effective kidney stone prevention is dependent on the stone type and the identification of risk factors for stone formation. Patients are advised to follow dietary recommendations which can be accompanied by additional medical treatment such as potassium citrate (PCi) and off label use of thiazide diuretics However, in a great proportion of patients usage of these drugs is limited by side effects and tolerance, resulting in loss of efficacy. Despite the major technical achievements for stone re moval in the last three decades the problem of recurrent stone formation remains and a specific, satisfactory drug to use in clinical therapy for prevention of recurrent stones is still lacking. Herbal medicines could play an important role in closing this gap as revealed by a vailable literature on herbal medicines and their possible role in the management of urolithiasis Ammi visnaga L. (syn. Khella,

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176 Apiaceae) fruit preparations, such as teas prepared from crushed or powdered seeds, have traditionally bee n used in the Middle East to ease urinary tract pain associated with kidney stones and to promote stone passage Based on this traditional use and more recent promising findings in in vitro and in vivo studies about Ammi visnaga and the isolated compound v isnagin from Ammi visnaga extracts (AVE) in the prevention of kidney stones this herb was thor o u ghly investigated in the presented dissertation thesis. The development and validation of a high performance liquid chromatography with ultraviolet light detection (HPLC UV) method for visnagin and khellin quanitification was fundamental to determine their concentrations in AVE and visnagin solutions which was the basis for all performed pharmac okinetic (PK) and pharmacodynamic (PD) studies. With the successfully developed and validated method, different AVEs could be characterized with respect to thei r khellin and visnagin content and visnagin solubility experiments and stability experiments could be conducted. It was demonstrated that visnagin solubility in water is poor, but can be enhanced by addition of the Food and Drug Administration (FDA) accepted cyclodextrin Captisol and that AVE signi ficantly increases visnagin solubility compared to water. Stability of visnagin in 25% Captisol solution and freeze dried AVE for the duration of the studies as well as in reconstituted AVE for a sufficient amount of time could be confirmed. PK characteri zation is a crucial step in drug development and in order to gain wider credibility herbal medicines must undergo similar scrutiny to which synthetic drugs are subjected, including PK evaluation. So far, no information of the PK of visnagin was available. Therefore, the PK of visnagin in rats after intravenous (i.v.) bolus injection

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177 was characterized utilizing three doses of visnagin solubilized in 25% Captisol Prior to this i.v. study, a liquid chromatography tandem mass spectrometry (LC MS/MS) method fo r visnagin quantification in rat plasma was developed and validated. The experiments revealed n on linear elimination kinetics for visnagin and a non linear mixed effect model incorporating a saturable elimination that describes the observed data after i.v. administration was developed and validated A leave one out cross validation suggested that t he final model possess es predictive power even outside the doses that were included in th e study. Thus, t he final model could be used for simulation of single dos e i.v. experiments with different doses, multiple dose i.v. experiments, and single or multiple dose oral experiments assuming a certain absorption model Information from these simulations could be further utilized to improve the design of prospective vis nagin experiments ( e.g more effective sampling schemes) and to reduce the number of animals a nd resources. C haracterization of the PK properties of a new substance after i.v. administration is essential as initial step in the PK evaluation of that substance. However, since i.v. bolus administration is not a typical route of administration for a potential drug for kidney stone prevention PK evaluation after oral administration was the next logical step. Therefore, PK studies were performed in rats to characterize the PK properties of visnagin in rats after oral administration of the pure compound visnagin and visnagin in form of an aqueous AVE which is similar to a traditional tea preparation. It was dem onstrated that average visnagin plasma concentration was significantly higher when it was administered in form of AVE compared to the administration of the pure compound for all three tested doses Furthermore v isnagin plasma exposure (area

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178 under the plas ma concentration time curves ( AUC )) was significantly increased and visnagin resided s ignificantly longer in the body when it was given as AVE compared to an equivalent dose of the pure compound for all three tested doses. The disproportionate i ncrease of AUC with an increasing dose that was observed for both pure compound and AVE confirmed the non linear PK in particular saturable elimination, of visnagin that was shown in the previous i.v. study Supported by the drug properties of visnagin (low molecula r weight lipophilic) it is suggested that elimination mainly occurs via capacity limited liver metabolism. Performing i n vitro studies involving hepatocytes, liver derived cell lines or microsomes could be helpful to confirm this hypothesis. The observed differences in the AUC s after administr ation of visnagin as extract compared to the pure compound can be explained by differences in clearance and/or differences in oral bioavailability. A model based approach was applied to obtain estimates o f oral bioavailability and clearance parameters for pure compound and AVE data. Therefore, i.v. and oral visnagin data were first fitted simultaneously to an appropriate model and subsequently all available data was used to fit a combined model. Due to the lack of i.v. AVE data, certain assumptions were required for parameter estimation following oral AVE administration. A PK model that describes the data obtained from all performed PK studies could be developed and validated. Characteristics like atypical absorption and non linear elimination were implemented utilizing flexible Weibull type absorption and Michaelis Menten kinetics, respectively. The model supports the hypothesis that visnagin plasma exposure is elevated after extract administration because, due to structural similarities, the extract compound

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179 khellin acts as competitive inhibitor of the saturable elimination process that was observed after i.v. and oral administration of pure compound and AVE. This was incorporated by separate estimates of t he Michaelis Menten constant ( K M ) for pure compound and AVE data. The underlying assumption of the developed model was that only K M is affected by khellin or other extract compounds with regard to the clearance process. Captisol might have affected the PK of visnagin after oral administration. However, if visnagin was given in form of a suspension, differences in exposure compared to AVE administration would likely be even more pronounced. I ncreased exposure of visnagin after extract administration could r esult in a superior PD efficacy of AVE compared to an equivalent dose of visnagin, assuming that visnagin is in deed a major active ingredient. In order to confirm preventive effects on nephrolithiasis and test the hypothesis of superior PD efficacy of AVE over an equivalent dose of visnagin, a PD study in rats was conducted to test visnagin and AVE in one experiment using equivalent doses. Visnagin plasma levels after the two treatments were measured weekly at two different time points to confirm presence a nd quantify visnagin concentration in the systemic circulation. An objective and efficient method to count CaOx crystal deposits in histological sections of rat kidneys was developed and used for analysis. A protocol for consistent and reproducible generat ion of CaOx crystal deposits in rat kidneys was determined, using 1.25% ethylene glycol added to drinking water together with a diet containing an increased calcium content compared to the standard chow. Besides visnagin and AVE, the standard treatment for kidney stone prevention, namely P Ci was also evaluated as positive control.

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180 In conclusion, preventive effects of visnagin and AVE for kidney stones could not be confirmed, although demonstrated in previous studies. Discrepancies in the outcomes might be due to different study designs and analysis. There is not much known about pos sible mechanisms of AVE and visnagin in nephrolithiasis prevention. The interaction with sodium dicarboxylate co transporter ( NaDC 1 ) and calcium channels has been suggested. Our method for consistent crystal induction might have been too severe for AVE an d visnagin to show effective prevention. The lack of reaching steady state visnagin plasma concentrations possibly contributed to this outcome. Nevertheless, our protocol could consistently produce CaOx crystals in rats and preventive treatment with PCi sh owed a reduction in crystal area, although not statistically significant. This protocol should be further investigated and used for assessment of preventive treatments. Overall, it is evident that there is still need for research and new substances in the area of kidney stone prevention.

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181 APPENDIX NONMEM CONTROL STREAM $PROBLEM Nonlinear Visnagin oral $INPUT ID TIME DV MDV AMT GRP CMT EVID BW TRT ODOS $DATA .. \ DATAFILES \ visnagin_oral_iv_extract_no_BLQ.csv IGNORE=@ WID E $SUBROUTINE ADVAN6 TRANS1 TOL=5 $MODEL COMP=(CENTRAL) COMP=(PERIPH) $PK VMAX=THETA(1)*EXP(ETA(1)) IF (TRT.EQ.3) THEN KM=(THETA(2)+THETA(9)) ELSE KM=THETA(2)*EXP(ETA(2)) ENDIF V1=THETA(3)*EXP(ETA(3)) K12=THETA(4) K21=THETA(5) BA =THETA(6) KD=THETA( 7)*EXP(ETA(4)) IF (TRT.EQ.3) THEN BETA=THETA(8)*EXP(ETA(5)) ELSE BETA=(THETA(8)+THETA(10))*EXP(ETA(5)) ENDIF S1=V1 $ERROR IPRED=F Y=F*(1+ERR(1))+ERR(2) IF (TRT.EQ.2) Y=F*(1+ERR(3)) $DES DADT(1)=(BA*ODOS*KD**BETA*BETA*(T+0.001)**(BETA 1)*EXP( 1*(KD*(T+0.0 01))**BETA) ) VMAX*A(1)/V1/(KM+A(1)/V1) K12*A(1)+K21*A(2) DADT(2)=K12*A(1) K21*A(2) $THETA (0,0.366,1) ;VMAX (0,0.08,1) ;KM (0,0.175,1) ;V1 (0,1) ;K12 (0,1.22) ;K21

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182 0.87862 FIX ;BA (0,0.4,1) ;KD (0,0.8,1) ;BETA (0,0.8,5) ;KMEX (0,0.5,1) ;BETAEX $OMEGA 0.0475 ;VMAX 0.502 ;KM 0.00853 ;V1 (0.09) ;KD (0.09) ;BETA $SIGMA (0.025);Err1 (0.025);Err2 (0.025);Err3 $ESTIMATION METHOD =1 INT SIGDIGITS=3 PRINT=10 NOABORT MSFO=MSFO.OUTPUTFILE $COV $TABLE ID TIME IPRED GRP TRT CWRES CWRESI NOPRINT ONEHEADER FILE=AL LRECORDS.TXT $TABLE ID TRT ETA(1) ETA(2) ETA(3) ETA(4) ETA(5) GRP VMAX KM V1 BETA BA KD NOPRINT ONEHEADER NOAPPEND FILE=FIRSTRECORDS.TXT

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194 BIOGRAPHICAL SKETCH Karin G Haug was bor n and raised in Germany. She received her Pharmacy state examination degree in summer 2006 after completing Pharmacy School at University of Tuebingen Germany. Karin interned at Curtin University in Perth, Western Australia investigating anti inflammator y properties of Euphorbia hirta a plant traditionally used by Australian aborigines. After an internship in a public pharmacy in Tuebingen, Germany, she r eceived her Pharmacy license. In spring 2009 Karin joined graduate school at University of Florida in order to pursue her Ph.D. in pharmaceutical sciences under the supervision of Dr. Veronika Butterweck During this time she worked on her dissertation thesis xploring the effects of Ammi visnaga L. on nephrolithiasis prevention : i n vivo pharmacokinetic and pharmacodynamic evaluation of Ammi visnaga L. As teaching assistant Karin was responsible to support various courses in Pharmacy School at University of Florida. Besides the stu dies for her dissertation project, Karin was enrolled into several courses, particularly in the Department of Statistics, in order to graduate with an additional Ph.D. m inor in s tatistics. She did summer internships in 2011 and 2012 in the Pharmacometrics group at Boehringer Ingelheim, Germany. Karin presented her studies in form of publications and oral and poster presentations at national and international conferences.