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The study described in this thesis was designed to investigate the use of Immobilized Artificial Membrane (IAM) chromatography alone, or in combination with physiochemical descriptors, for predicting intestinal drug absorption in humans. / Thesis (MApSc(Pharmacy) )--University of South Australia, 2007.

The study described in this thesis was designed to investigate the use of Immobilized Artificial Membrane (IAM) chromatography alone, or in combination with physiochemical descriptors, for predicting intestinal drug absorption in humans.

The enantiomeric discrimination properties of a number of commercial chiral HPLC stationary phases have been examined using a series of 2-aminotetralin analogues. The mobile phase conditions for each column were varied in order to optimise the separation for the largest number of analytes. Factorial experimental design techniques were used to test the empirically derived mobile phase combinations. A primary limitation of factorial design optimisation strategies, the number of experiments required, was overcome by the development of a simultaneous factorial design method utilising the selectivity of mass spectrometric detection. This enabled the optimisation of the separation of the enantiomers of 12 compounds, using Chiral AGP, to be carried out concurrently. None of the columns tried was able to separate the enantiomers of more than half the analytes. The retention mechanism of the Chiral AGP column was shown to be via cation exchange and non-polar interactions. The ability to form a hydrogen bond between the carbon 8 substitution (acceptor) of the analyte and the stationary phase (donor) was found to be a prerequisite for the separation of enantiomers using Chiral AGP and Chiralcel OD. Neither a rule based nor a mathematical model constructed from calculation derived physicochemical and molecular data were sufficient to describe the enantioselectivity of the Chiral AGP stationary phase. A molecular graphics template model was used to demonstrate the importance of the spatial position of the hydrogen acceptor to the enantioselectivity of the column. Two 2-aminoteralins substituted at the carbon 8 position with a keto-pyrrol function were found to be enantioselectively cleared from in-vitro rat liver preparations dependent upon the substitutions attached to the carbon 2 nitrogen. Differences between these closely related structures were shown to be related to the hydroxylation of the parent molecule, probably, although not exclusively, via the cytochrome P450 isozyme CYP2D6. An in-vivo study using an intravenous dose route showed no sign of enantioselectivity or evidence of the major in-vitro metabolite.

We rely on oral drugs to treat several diseases and infections. Yet, the gut microbiome modifies oral drugs within the human gut by using enzymes, facilitating efficient chemical reactions. These drug modifications impact effective doses and outcomes for individuals. The gut microbiome can convert drugs destined for excretion back to active drugs, and the converse is also true, the microbiome can inactivate active drugs, and both may lead to toxic effects. There is no resource for cataloging bacterial drug-metabolizing genes within the human gut microbiome with analytical tools to annotate these genes in sequenced gut microbiomes. I created a resource called the Human Microbiome Drug Metabolism (HMDM) database. I analyzed 1,196 unpublished sequenced gut bacterial genomes from 8 healthy adult donors to predict genes that encode enzymes capable of metabolizing drugs using two in silico methods I developed, namely MAGIS and AutoPhylo. I reviewed the scientific literature and built an ontology-centric database, the HMDM, to catalog the bacterial drug-metabolizing genes and drugs they modify. I developed DME software to predict bacterial genes capable of metabolizing host-directed drugs using the HMDM data. We experimentally validated four novel AMR gene homologs predicted from the genomes. The HMDM is curated with 50 genes reported to metabolize drugs and 45 gene variants of the β-glucuronidase (uidA) gene. MAGIS was used to predict 246 putative bacterial drug-metabolizing genes. I predicted the three novel AMR gene homologs that resemble fosfomycin thiol transferase enzymes using AutoPhylo. The MIC experiment shows that fosD1, fosD2, and fosD3 have MIC of 8μg/mL, 8μg/mL, and >512μg/mL, respectively. The genes fosD1 and fosD2 are of unknown function, and FosD3 converts fosfomycin. The HMDM database is limited to bacterial genes. The in silico methods are critical for studying bacterial drug metabolism to predict drug fate and patient outcomes. / Thesis / Master of Science (MSc) / We use medications in our everyday life to treat infections and manage diseases. Yet, bacteria residing within the human gut can interact with these medications, which can cause undesirable outcomes. Many bacteria in the human gut produce biological catalysts known as enzymes that break down chemicals, including drugs. Medication is measured and given to an individual, called a dose, and the oral route is preferred. Enzymes break down oral and biliary system drugs, reducing the effective dose. As a result, medication becomes ineffective or toxic to the body. As such, we must study how each drug is affected by bacterial enzymes. I built a resource, the Human Microbiome Drug Metabolism (HMDM) database, to catalog all the bacterial genes that code for the enzymes reported in scientific papers to break down oral drugs. We can use the HMDM database to study bacterial enzymes that lead to poor drug efficacy.

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As a critical parameter in pharmacokinetics, prediction of clearance is an integral aspect of drug discovery programmes. Since the liver is the major site of xenobiotic biotransformation, accurate prediction of hepatic clearance (CLh) is vital. The use of cellular and subcellular in vitro systems for this purpose is common practice; however, prediction accuracy tends to be poor. The aim of this thesis was to explore potential contributing factors to the underprediction of in vivo clearance, specifically with relation to the in vitro methodology of hepatocyte clearance assays, which is largely unstandardised. Literature data analyses highlighted an overall clearance-dependent trend of underprediction in both human and rat hepatocytes, indicating a fundamental in vitro system bias which is independent of species. During initial investigation of incubation conditions, the format of hepatocyte assays (suspension in microcentrifuge tubes, 96-well plates, 24-well plates and short-term monolayer culture) was demonstrated to influence determined intrinsic clearance (CLint). Differences in midazolam CLint were observed not only between suspended and short-term cultured hepatocytes, but also between suspended hepatocytes in different vessels/plate formats. The applicability of 1 µM as a generic substrate incubation concentration for determination of CLint by substrate depletion was evaluated in rat hepatocytes using nine well-characterised drugs. For seven of the nine drugs, a statistically significantly (p < 0.05) higher CLint was observed in determinations of 0.1 µM substrate as opposed to 1 µM, highlighting the potential for false determinations using current practices. Cofactor depletion in isolated hepatocytes was investigated based on previous speculation as the cause of clearance-dependent underprediction of in vivo clearance. Although moderate increases in CLint were observed with the addition of NADPH to hepatocyte incubations, this was subsequently attributed to the replenishment of NADPH in membrane-damaged hepatocytes. Retained functionality of metabolic enzymes in cells which would generally be considered non-viable by trypan blue exclusion was indicated in comparisons of unpurified and Percoll-purified cryopreserved hepatocytes. This phenomenon was conclusively demonstrated in incubations of permeabilised hepatocytes supplemented with NADPH, revealing a need for re-evaluation of the use of plasma membrane integrity (trypan blue exclusion) as a measure of viability in metabolic studies. ATP content was considered as a potential alternative measure; however no significant correlations were found between ATP content, trypan blue exclusion and the CLint of nine drugs in associated preparations. The effect of shaking on CLint in rat hepatocytes was also examined. For 10 out of 12 drugs, CLint determined at 900 rpm was significantly (p < 0.05) higher than in static incubations. Three potential mechanisms were hypothesised: plasma membrane damage, increased substrate distribution throughout the bulk medium and reduction in the depth of the unstirred water layer (UWL) surrounding cells. Shaking of saponin-permeabilised hepatocytes (supplemented with NADPH to maintain metabolism) also increased the determined CLint of saquinavir, indicating a rate-limitation other than membrane permeation. However, shaking of ultra-sonicated hepatocytes in which the plasma membrane was entirely destroyed (also supplemented with NADPH) did not change the determined CLint of saquinavir, revealing the rate-limitation of UWL permeation in both intact and permeabilised cells. The depth of such an UWL in vitro is likely to be artificially greater than in vivo; therefore reduction of UWL depth through incubation shaking is proposed as a physiologically sound approach by which to increase in vitro CLint. In addition, a framework of experiments and related equations is presented by which intact and permeabilised hepatocytes in static and shaken conditions may be utilised to identify the rate-determining process and contribution of individual processes to the in vitro CLint of a drug. The effects of substrate concentration and shaking were also evaluated in human hepatocytes. Significant increases in determined CLint of drugs with use of 0.1 µM substrate (as opposed to 1 µM) and shaking at 900 rpm were demonstrated, confirming equivalent potential in vitro sources of underprediction between rat and human. These highly significant findings reveal the existing limitations of in vitro assays and potential flaws in current practice in in vitro determinations of CLint. Appropriate consideration of the properties of in vitro systems, including the presence of the UWL, should lead to improved predictions of in vivo clearance.

The aim of this study was to investigate the induction of CYP1A by DMSO, to determine whether DMSO induced other P450 isozymes (CYP2B) and to compare the effects of DMSO and another differentiating agent, sodium butyrate. Induction of CYP1A-dependent ethoxyresorufin-O-deethylase (EROD) was observed in the presence of increasing concentrations of DMSO. All concentration investigated (1%, 1.5% and 2%) caused induction (2-3 fold), and enhanced BA-induction of EROD. Enhancement of BA-induction was greater with 1% and 1.5% DMSO (2.5-3 fold over BA alone) than with 2% (1.8-fold). DMSO alone did not increase CYP1A1 RNA levels. Hepatocytes treated with BA and DMSO together exhibited a 1.3-fold greater increase in RNA levels than with BA alone. Western blotting indicated that CYP1A1 protein was increased by inducers (BA, DMSO and isosafrole), but that CYP1A2 was not. This indicates that the CYP1A1 isozyme is responsible for EROD activity in these cultures, and that the CYP1A2-induction mechanism is lost in rat hepatocytes cultured under the conditions of these experiments. This observation was confirmed by the lack of CYP1A2-dependent phenacetin-O-deethylase (POD) activity in culture. The substituted benzimidazole omeprazole has been shown to induce CYP1A isozymes in human hepatocyte cultures. In this study omeprazole was not effective in inducing EROD activity in rat hepatocytes or <I>in vivo</I> in the rat. This confirms that rat hepatocytes are not a good model for CYP1A induction in man. DMSO appears to be isozyme specific, since CYP2B-dependent pentoxyresorufin-O-dealkylase (PROD) activity was not increased by DMSO, and phenobarbitone (PB) induction of PROD was enhanced only slightly by DMSO on day 3 of culture (4-fold over control; 1.5-fold over PB alone). Sodium butyrate and DMSO were both shown to induce differentiation of rat hepatocyte, with maintenance of low level of γ-glutamyl transferase activity, and maintenance of a more rounded morphology.