Università Degli Studi Del Piemonte Orientale “Amedeo Avogadro”

UNIVERSITÀ DEGLI STUDI DEL PIEMONTE ORIENTALE “AMEDEO AVOGADRO” DIPARTIMENTO DI SCIENZE DEL FARMACO Dottorato di Ricerca in “Scienza delle Sostanze Bioattive” CHEMICAL AND METABOLIC STABILITY STUDIES OF PROPARGYLAMINE-CONTAINING DRUGS Coordinatore Prof. Luigi PANZA Tutor Candidato Prof. Giorgio GROSA Dott.ssa Rossana CANAVESI XXVIII Ciclo TABLE OF CONTENTS List of abbreviations................................................................................................................................................ V CHAPTER 1. Introduction ....................................................................................................................................... 1 1.1 CHEMICAL STABILITY STUDIES ........................................................................................................... 1 1.1.1 Experimental approach ........................................................................................................................... 2 1.1.2 Degradation conditions .......................................................................................................................... 2 1.1.3 Drug product .......................................................................................................................................... 4 1.1.4 Stability-indicating method development .............................................................................................. 4 1.2 METABOLIC STABILITY STUDIES......................................................................................................... 5 1.2.1 Xenobiotics biotransformation ............................................................................................................... 6 1.2.2 Cytochrome P450 ................................................................................................................................... 7 1.2.3 In vitro models to study drug metabolism ............................................................................................ 10 1.3 PROPARGYLAMINE-CONTAINING DRUGS ....................................................................................... 14 1.3.1 Neurodegenerative disorders ................................................................................................................ 14 1.3.2 Alzheimer’s Disease (AD) ................................................................................................................... 15 1.3.3 Parkinson’s disease (PD) ...................................................................................................................... 16 1.3.4 Monoamine oxidase ............................................................................................................................. 18 1.3.5 Propargylamines ................................................................................................................................... 19 1.3.6 Multitarget-directed ligands (MTDLs) strategy ................................................................................... 21 1.3.7 Conclusions .......................................................................................................................................... 23 1.4 REFERENCES ............................................................................................................................................ 24 CHAPTER 2. Aim of the work .............................................................................................................................. 25 CHAPTER 3. New insights in oxybutynin chemical stability: identification in transdermal patches of a new impurity arising from oxybutynin N-oxide rearrangement .................................................................................... 27 3.1 INTRODUCTION ...................................................................................................................................... 27 3.2 EXPERIMENTAL ...................................................................................................................................... 28 3.2.1. Reagents and chemicals ...................................................................................................................... 28 3.2.2. Instrumentation and chromatographic conditions ............................................................................... 29 3.2.3 Forced degradation study of Oxy ......................................................................................................... 30 3.2.4. Isolation and purification of Oxy-EK .................................................................................................. 31 3.2.5. Synthesis of Oxy free base .................................................................................................................. 32 3.2.6 Synthesis of degradation products ........................................................................................................ 33 3.2.7. Evaluation of the mutagenic effects of Oxy-EK and Oxy ................................................................... 35 3.3 RESULTS AND DISCUSSION ................................................................................................................. 38 3.3.1 Forced degradation study of Oxy by LC-UV analysis ......................................................................... 38 3.3.2. Structural characterization of degradation products by LC-ESI-MS/MS analysis .............................. 41 3.3.3 Synthesis of Oxy-EK............................................................................................................................ 46 3.3.4 Analysis of transdermal patches ........................................................................................................... 47 3.3.5 Mutagenic activity of Oxy-EK ............................................................................................................. 49 I 3.4 CONCLUSIONS ......................................................................................................................................... 50 3.5 REFERENCES ............................................................................................................................................ 51 CHAPTER 4. New findings in the in vitro metabolism study of oxybutynin ........................................................ 53 4.1 INTRODUCTION ...................................................................................................................................... 53 4.2 EXPERIMENTAL ...................................................................................................................................... 54 4.2.1 Reagents and chemicals ....................................................................................................................... 54 4.2.2 Instrumentation and chromatographic conditions ................................................................................ 55 4.2.3 Synthesis of the reference compounds ................................................................................................. 56 4.2.4 Human and rat Liver Preparations........................................................................................................ 58 4.2.5 Phase I incubations with RLM, HLM .................................................................................................. 58 4.2.6 Phase II incubation with HLC fraction................................................................................................. 58 4.3 RESULTS ................................................................................................................................................... 59 4.3.1 Phase II incubation with human liver subcellular fractions ................................................................. 63 4.4 DISCUSSION ............................................................................................................................................. 65 4.5 REFERENCES ............................................................................................................................................ 67 CHAPTER 5. Forced degradation study of selegiline............................................................................................ 68 5.1 INTRODUCTION ...................................................................................................................................... 68 5.2 EXPERIMENTAL ...................................................................................................................................... 69 5.2.1 Reagents and chemicals ....................................................................................................................... 69 5.2.2 Instrumentation and chromatographic conditions ................................................................................ 70 5.2.3 Forced degradation conditions ............................................................................................................. 71 5.2.4 Isolation and purification of SG-EA .................................................................................................... 72 5.2.5 Synthesis of degradation product ......................................................................................................... 72 5.3 RESULTS AND DISCUSSION ................................................................................................................. 73 5.3.1 Forced degradation study and structural characterization of degradation products

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