Vitamin K1 pharmacokinetics. in a clinical study and VKORC1 enzyme kinetics. using HPLC methodology

Transcription

1 Vitamin K1 pharmacokinetics in a clinical study and VKORC1 enzyme kinetics using HPLC methodology Inaugural-Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn Vorgelegt von Milka Marinova aus Levski, Bulgarien Bonn, Juni 2012

2 TABLE OF CONTENTS 5 LIST OF ABBREVIATIONS 10 LIST OF FIGURES 13 LIST OFTABLES INTRODUCTION Biological role of vitamin K Discovery, structure Vitamin K dependent proteins Vitamin K cycle Biochemical measures Determinants of vitamin K serum levels Nongenetic determinants Dietary intake of vitamin K Absorption, transport and cellular uptake of vitamin K Tissue stores of vitamin K and distribution Metabolism and excretion of vitamin K Age and gender Xenobiotics Lifestyle Genetic Determinants VKOR, gamma glutamyl carboxylase CYP4F2, ABCC Apolipoprotein E Pharmacokinetics Objectives MATERIALS AND METHODS Materials Equipment Reagents, disposables and Standards List of chemicals List of consumable material HPLC columns, lamps, GC columns Commercial kits Plasmids Buffers Primer sequences Biological material and enzymes Animals Standards and vitamin K-spiked serum preparation Vitamin K epoxides Solutions and dilutions Vitamin K-spiked human serum 44

3 Bile acid calibration curves Cholesterol and 7a-hydroxycholesterol calibration curves Molecular biological methods PCR Performance Creation of oligo-nucleotides for the PCR Polymerase chain reaction Agarose gel electrophoresis DNA isolation from agarose gels Preparative plasmid isolation DNA digestion by endonuclease restriction enzymes Plasmid transformation in E.coli DH5a DNA sequencing and ethanol-acetate-precipitation Cell biological and enzymological methods Cultivation of mammalian cell lines Subcultivation of cell cultures Long-term storage of cells Cell transfection Harvesting of full-cell homogenate Lowry protein assay VKORC1 enzymatic activity assay Determination of kinetic constants Bioanalytical methods High-Performance Liquid Chromatography HPLC Instrumentation Sample preparation and hexane extraction of vitamin K from serum Isolation of vitamin K fraction from lipid extracts using Sep-Pak silica cartridges Final analytical stage of the vitamin K determination assay Separation on RP-18 column Post-column zinc reduction Fluorescence detection Calibration and calibration curve of vitamin K Gas-Chromatography Mass-Spectrometry Gas-Chromatography Mass-Spectrometry GC-MS and GC-FID analyses Determination of vitamin K in human serum by GC-MS GC-MS bile acid analysis GC-FID analysis of cholesterol and GC-MS analysis of 7ahydroxycholesterol Bioanalytical Method Validation Selectivity 62

4 Range of determination; Limit of detection (LOD); Limit of quaritification (LOQ) Calibration/ Standard curve Lower limit of quantification (LLOQ) Concentration-response Accuracy, precision Recovery Quality Assurance Stability Freeze and thaw stability Short-term stability Long-term stability Stock Solution stability Post-preparative stability Vitamin K in mouse tissue "Influence of VKORC1 promoter polymorphism C G>A on the vitamin K1 pharmacokinetics"- phase I clinical study Study period and subject definition Trialdesign Investigational product Indication Trial procedure Trial schedule Primary aim, primary endpoint Secondary aims Inclusion and exclusion criteria, criteria for stopping trial Inclusion criteria Exclusion criteria Criteria for stopping trial First approaches in human volunteers Statistical and pharmacokinetic analysis RESULTS Enzymological characterization of the VKORC Separation and quantification of K Vitamins VKORC1 enzyme kinetics: determination of kinetic constants Michaelis-Menten curves Lineweaver-Burk graphs K m and V ma* determination Quinone generation during K10 and K20 substrate competition Validation of the quantitative analysis of vitamin K1 (phylloquinone) and vitamin K2 (menaquinone-4) in human serum by HPLC with fluorescence detection after solid-phase extraction Specificity, selectivity 82

5 Range of determination Limit of detection (LOD) Limit of quantification (LOQ), lower limit of quantification (LLOQ) Calibration/ Standard curve Accuracy - robin round tests, KEQAS participation Precision Recovery Quality assurance Stability Freeze and thaw stability Short-term stability Long-term stability Stock Solution stability Post-preparative stability Vitamin K levels in mouse tissue Vitamin K1 determination assay and pharmacokinetic analysis - preliminary studies in human volunteers Raw concentration - time data after/ without administration of Konakion MM 2 mg Pharmacokinetic analysis Graphic presentation of the data Serum concentration vs time profiles of vitamin K1 following p.o. administration of Konakion MM 2 mg Serum concentration vs time profiles of vitamin K1 following i.v. administration of Konakion MM 2 mg Pharmacokinetic Parameters after p.o./ i.v. administration of Konakion MM 2 mg Pharmacokinetic Parameters of volunteer A Pharmakocinetic parameters of volunteer B Pharmakocinetic parameters of volunteer C Pharmakocinetic parameters of volunteer D Bile acid, cholesterol and 7D-hydroxycholesterol concentrations Bile acid concentrations A2,p.o A2,i.v A B2,p.o B2,i.v., B Cholesterol and 7n-hydroxycholesterol concentrations A2,p.o., A2,i.v A B2,p.o B2,i.v., B Phase I clinical study "Influence of VKORC1 promoter polymorphism C G>A on the vitamin K1 pharmacokinetics" DISCUSSION Enzymological characterization of the VKORC1 150

6 Bioanalytical method Validation of vitamin K determination Vitamin K levels in mouse tissue Pharmacokinetic analysis - preliminary studies in human volunteers Pharmacokinetic analysis - phase I clinical study SUMMARY APPENDIX LITERATURE 177