Throughput Lipophilicity Assays and Their Use for PET Tracer Optimization
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Artificial Tissue Binding Models: Development and Comparative Evaluation of High -Throughput Lipophilicity Assays and their Use for PET Tracer Optimization INAUGURALDISSERTATION zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Frauke Assmus aus Dessau, Deutschland Basel 2015 i Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Anna Seelig Prof. Dr. Beat Ernst Prof. Dr. Jörg Schibler, Dekan Basel, den 26.Februar 2013 ii Abstract Abstract The purpose of this thesis was to increase the efficiency of the Positron Emission Tomography (PET) tracer development process. Since many neuroimaging agents fail due to undesirably high non-specific binding (NSB) to brain tissue, we aimed at estimating the extent of NSB as early as possible, preferably before radioactive labeling and extensive animal testing. To this purpose we have developed, optimized and evaluated several in vitro assays with respect to their ability to predict brain tissue binding and, in particular, NSB in PET. A major goal of this thesis was the implementation of a miniaturized assay for the prediction of NSB in order to meet the demand for maximal efficiency, i.e. high throughput and minimal consumption of reagents, samples and animal tissue. Since octanol/water distribution coefficients ( log Doct ) are routinely measured in almost every research organization, we investigated whether is also useful for the prediction of brain tissue binding. In this context, we have developed a filter-based log Doct assay (Carrier Mediated Distribution System=CAMDIS) to overcome the drawbacks of the traditional shake flask technique, i.e. tedious phase separation and high consumption of reagents. Strategies have been developed to correct for drug adsorption to the assay construct in order to warrant both high throughput and high quality of the data. Even though the CAMDIS values were in excellent agreement with literature shake flask data, our results indicated that octanol is only a poor surrogate for tissue binding, as shown by the poor correlation between and the unbound fraction of drug in brain ( fu,brain ) available through equilibrium dialysis. The latter is the current industrial standard method for the measurement of tissue binding, however the technique is hampered by high consumption of animal tissue and low throughput. Apart from , another, more complex membrane surrogate system, namely the Parallel Artificial Membrane Permeation Assay (PAMPA), has found entry into many laboratories. We investigated whether the fraction of drug retained by the PAMPA barrier proves useful for the prediction of tissue binding. Since the default PAMPA setup at Roche was inappropriate in this respect, we optimized PAMPA towards better predictive power and compatibility with mass spectrometric analysis. Provided that PAMPA was conducted under optimized conditions (pH 7.4, brain polar lipids, without solubilizers), the membrane fraction was in much better agreement with tissue binding as compared to . Nevertheless, the predictive power was still unsatisfactory reflecting the fact that reverse micelles rather than iii Abstract lipid bilayers constitute the permeation barrier as revealed by NMR experiments. Since neither CAMDIS nor PAMPA yielded sufficiently reliable NSB - estimates, we developed a miniaturized label-free Lipid Membrane Binding Assay (LIMBA) allowing for the measurement of brain tissue/water distribution coefficients at minimal consumption of brain homogenate. LIMBA was highly predictive for the binding of drugs and molecular imaging probes to brain tissue and therefore provides a viable alternative to the equilibrium dialysis technique. LIMBA thus allows for more efficient optimization of potential PET tracers and should reduce the attrition rate in the late and particularly expensive stages in the PET tracer development process. Keywords: Tissue distribution, Non-specific binding, Equilibrium dialysis, PET tracer, Octanol/water distribution coefficient iv Table of Contents Table of Contents 1 Introduction ...................................................................................................................... 1 1.1 The Traditional Drug Discovery Process ................................................................................ 1 1.2 The FDA’s Critical Path Initiative .......................................................................................... 2 1.3 Molecular Imaging with PET .................................................................................................. 2 1.3.1 Principles of PET ............................................................................................................ 3 1.3.2 The Value of PET in the Drug Discovery and Development Process............................. 6 1.3.3 Shortcomings and Challenges of Developing New PET Tracers ................................. 11 2 Rationale and Aims of Research ................................................................................... 17 3 Capsules ........................................................................................................................... 28 4 Manuscripts ..................................................................................................................... 35 4.1 Carrier Mediated Distribution System (CAMDIS): A New Approach for the Measurement of Octanol/Water Distribution Coefficients ...................................................................................... 35 4.2 The Impact of Drug Adsorption to Microtiter Plates on the Accuracy of LogDoct ............... 84 4.3 Drug Adsorption to Teflon and Excipient-Aided Drug Recovery ...................................... 128 4.4 Evaluation and Management of Excipient-Related Matrix Effects in HPLC-ESI/MS Analysis of PAMPA-Permeability .................................................................................................. 169 4.5 31P and 1H-NMR Studies on the Molecular Organization of Lipids in the PAMPA Permeation Barrier .......................................................................................................................... 205 4.6 Evaluation of a Novel Lipid Membrane Binding Assay (LIMBA) for the Assessment of Brain Tissue Binding ...................................................................................................................... 248 4.7 Label-Free Assay for the Assessment of Non-specific Binding of Positron Emission Tomography Tracer Candidates ...................................................................................................... 293 5 Summary ....................................................................................................................... 331 6 Acknowledgements ....................................................................................................... 348 7 Curriculum Vitae .......................................................................................................... 350 v List of Abbreviations List of Abbreviations AD Alzheimer's Disease ACN Acetonitrile AD Cross sectional area ADME Absorption, Distributions, Metabolism, Elimination Bavail Receptor density which is available for drug binding BBB Blood-brain barrier Bmax Total receptor density BLM Black lipid membrane BP Binding potential BPL Brain polar lipids BTL Brain total lipids C Concentration CND Concentration of free plus non-specifically bound tracer CS Concentration of tracer bound specifically to receptors C p Concentration of unmetabolized tracer in plasma CAMDIS Carrier Mediated Distribution System CE Capillary electrophoresis CHAPS Na· 3-[(3-Cholamidopropyl) dimethylammonio]-1- propanesulfonate CPI Critical Path Initiative CT Computer tomography CTAB Cetyltrimethylammonium bromide D2 receptor Dopamine D2 receptor DMPK Drug Metabolism and Pharmacokinetics DMSO Dimethyl sulfoxide ESI Electrospray ionization source FaSSIF Simulated gastrointestinal fluid in the fasted state FDA Food and Drug Administration FDG 2-(F-18)Fluoro-2-Deoxy-D-Glucose FeSSIF Simulated gastrointestinal fluid in the fed state vi List of Abbreviations fND Free fraction of drug in brain (PET nomenclature) f p Free fraction of drug in plasma (PET nomenclature) fu,brain Unbound fraction of drug in brain GCA Glycocholic acid HP - β - CD Hydroxypropyl-β-cyclodextrine HPLC High performance liquid chromatography IND Investigational new drug K1,k2 Rate constants governing the transfer of a PET tracer from plasma into tissue and vice versa k3,k4 Rate constants governing receptor association and receptor dissociation of a PET tracer Kpu Tissue/plasma water partition coefficient LIMBA Lipid Membrane Binding Assay LogBB Brain/blood distribution coefficient LogDbrain Brain tissue/water distribution coefficient LogDoct Octanol/water distribution coefficient LogDPAMPA PAMPA-lipid/water distribution coefficient MRI Magnetic resonance imaging ms Manuscript MS Mass spectrometry NCE New chemical entity NMP N-methyl-2-pyrrolidone NMR Nuclear magnetic resonance NSB Non-specific binding PAMPA Parallel Artificial Membrane Permeability Assay PBPK modeling Physiologically based pharmacokinetic modeling Pe Effective permeability PE Phosphatidylethanolamine PET Positron Emission Tomography PFGNMR Pulsed field gradient NMR PG Propylene glycol P-gp P-glycoprotein vii List of Abbreviations PK/PD Pharmacodynamic/pharmacokinetic pKa Negative logarithm of the acid dissociation constant POPC 1-Palmitoyl-2-oleoyl-phosphatidylcholine