INTRODUCTION: Cytochrome P450 2B (CYP2B) is a drug-metabolizing enzyme subfamily found in both the brain and liver, which metabolizes clinical drugs, drugs of abuse (e.g. nicotine), toxicants and endogenous neurochemicals. Brain CYP2B’s role in the local metabolism of centrally acting substrates is important to investigate because of its ability to metabolize a variety of centrally active substrates. Additionally, CYP2B regulation by genetics, and exposure to xenobiotics, results in great inter-individual differences in the brain expression of this enzyme. METHODS: We investigated the time-course of rat brain CYP2B induction after chronic nicotine treatment. Using the rat model of brain CYP2B induction, combined with intracerebroventricular (ICV) inhibition of CYP2B, we assessed the effects of brain CYP2B in the response to the anaesthetic substrate, propofol. We also investigated the role of brain CYP2B-mediated activation of the pesticide chlorpyrifos on its neurotoxicity. RESULTS: Nicotine’s induction of rat brain CYP2B was long lasting, returning to basal levels by day 7, and was unaffected by nicotinic receptor blockade. Induction of CYP2B in rat brain, by chronic nicotine treatment, reduced the anaesthetic efficacy of propofol, through increased brain CYP2B-mediated metabolic inactivation. Inhibition of brain CYP2B, using mechanism based inhibitors of the enzyme, inhibited both basal and induced brain CYP2B activity, and prolonged propofol sleep time by reducing the local brain inactivation of the anaesthetic. Inhibition of rat brain, and not hepatic, CYP2B was able to effectively block local brain production of the toxic chlorpyrifos oxon, significantly attenuating the reductions in brain acetylcholinesterase activity and body temperature. Additionally, inhibition of brain CYP2B also significantly reduced the behavioural toxicity after chlorpyrifos exposure in a chlorpyrifos (CP) dose- and time-dependent manner. CONCLUSION: These studies indicate that rat brain CYP2B enzymes are active in vivo and play a meaningful role in the local metabolism of, and the response to, centrally acting substrates (i.e. propofol, chlorpyrifos). These data provide a first demonstration of the important role that brain CYP-mediated metabolism plays in the response to centrally acting substrates (i.e. clinical drugs, toxicants, endogenous neurochemicals), potentially contributing to the inter-individual variability seen in human responses to centrally active drugs and toxicants.

The process of in vitro-in vivo extrapolation (IVIVE) can be used to predict pharmacokinetics of drugs in patients using data from in vitro systems. This process relies on the use of experimentally obtained scaling factors, such as abundances of different drug-metabolising enzymes and microsomal protein content (MPPGL). The use of simulators is dependent on abundances and activities of pharmacokinetically relevant enzymes. The incorporation of inter-individual variability in abundances of enzymes, correlations between enzyme expression patterns, and relationships between genetic, physiological, and environmental factors and enzyme expression and activity can make predictions using IVIVE and simulations of pharmacokinetic experiments in virtual populations more accurate and realistic. Incorporation of variability and correlations can also assist in predicting extreme cases where drug therapy may be ineffective or may cause adverse effects. A meta-analysis of 52 studies was carried out to assess the reported abundances of cytochrome P450 and uridine glucuronosyltransferase (UGT) enzymes in adult Caucasian subjects. Some heterogeneity was found between studies and the weighted means and overall coefficients of variation were calculated. Some strong enzyme expression correlations were identified; CYP3A4/CYP3A5*1/*3 (rs = 0.66, p < 0.0001, n = 37), CYP3A4/CYP2C8 (rs = 0.79, p < 0.0001, n = 107), and CYP2C8/CYP2C9 (rs = 0.71, p < 0.0001, n = 72). A quantitative protocol based on targeted proteomics was used to quantify cytochrome P450 and UGT enzymes in adult liver samples (n = 24). The QconCAT standard used for quantification was successfully expressed in-house after optimisation of the expression protocol, and the utility of two strategies in expressing recalcitrant QconCAT proteins was highlighted; the use of a fusion partner and reshuffling the order of peptides in the sequence. The enzymes quantified in this study were CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2J2, 3A4, 3A5, 3A7, 3A43, and 4F2, and UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, and 2B15. Correlations of expression identified in the meta-analysis were confirmed and new correlations were demonstrated between UGT enzymes and between enzymes from the two families. Correlations between UGT enzymes were particularly strong and statistically significant. Relationships between enzyme expression levels and genotype, age, sex, smoking, and alcohol consumption were investigated. A significant effect of genotype on expression was seen for CYP3A5 (p < 0.0001). An overall moderate decline of expression with age was observed for all the enzymes under study; however, this relationship was not statistically significant in most cases. Gender did not have a considerable effect on expression, although some differences in expression were observed between male and female donors. Smoking seemed to induce the expression of all enzymes; however, statistically significant induction was demonstrated only in the cases of CYP2A6, CYP3A4, CYP3A7, and UGT1A1 (p < 0.05). Alcohol consumption was not shown to have a considerable effect on enzyme expression. Two pig livers were used to optimise some aspects of the experimental protocol including solubilisation and digestion of proteins. Pig MPPGL was measured and relative hepatic contents of drug-metabolising cytochrome P450 enzymes in pig liver were established using label-free quantification.

Dichloroacetate (DCA) is an investigational drug for the treatment of genetic mitochondrial diseases. Its primary site of action is the pyruvate dehydrogenase (PDH) complex, which it stimulates by altering its phosphorylation state and stability. DCA is metabolized by and inhibits the bifunctional zeta-1 family isoform of glutathione transferase/maleylacetoacetate isomerase. Polymorphic variants of this enzyme differ in their kinetic properties toward DCA, thereby influencing its biotransformation and toxicity, both of which are also influenced by subject age. Results from open label studies and controlled clinical trials suggest chronic oral DCA is generally well-tolerated by young children and may be particularly effective in patients with PDH deficiency. Recent in vitro data indicate that a combined DCA and gene therapy approach may also hold promise for the treatment of this devastating condition.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24548号 / 薬科博第165号 / 新制||薬科||18(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 山下 富義, 教授 髙倉 喜信, 教授 寺田 智祐 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

No / Duocarmycin natural products are promising anti-cancer cytotoxins but too potent for systemic use. Re-engineering of the duocarmycin scaffold has enabled the discovery of prodrugs designed for bioactivation by tissue-specific cytochrome P450 enzymes. Lead prodrugs bioactivated by both P450 isoforms CYP1A1 and CYP2W1 have shown promising results in xenograft studies, however to fully understand the potential of these agents it is desirable to compare dual-targeting compounds with isoform-selective analogs. Such redesign requires insight into the molecular interactions with these P450 enzymes. Herein binding and metabolism of the individual stereoisomers of the indole-based duocarmycin prodrug ICT2700 and a nontoxic benzofuran analog ICT2726 were evaluated with CYP1A1 and CYP2W1, revealing differences exploitable for drug design. While enantiomers of both compounds bound to and were metabolized by CYP1A1, the stereochemistry of the chloromethyl fragment was critical for CYP2W1 interactions. CYP2W1 differentially binds the S enantiomer of ICT2726 and its metabolite profile could potentially be used as a biomarker to identify CYP2W1 functional activity. In contrast to benzofuran-based ICT2726, CYP2W1 differentially binds the R isomer of the indole-based ICT2700 over the S stereoisomer. Thus the ICT2700 R configuration warrants further investigation as a scaffold to favor CYP2W1-selective bioactivation. Furthermore, structures of both duocarmycin S enantiomers with CYP1A1 reveal orientations correlating with nontoxic metabolites and further drug design optimization could lead to a decrease of CYP1A1 bioactivation. Overall, distinctive structural features present in the two P450 active sites can be useful for improving P450-and thus tissue-selective-bioactivation. Significance Statement Prodrug versions of the natural product duocarmycin can be metabolized by human tissue-specific cytochrome P450 enzymes 1A1 and 2W1 to form an ultrapotent cytotoxin and/or high affinity 2W1 substrates to potentially probe functional activity in situ The current work defines the binding and metabolism by both P450 enzymes to support the design of duocarmycins selectively activated by only one human P450 enzyme. / National Institutes of Health and Yorkshire Cancer Research Program Grant (B381PA)

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction Earlier studies have shown the popularity of herbal products among people as traditional, complementary or alternative medication. One of the major clinical risks in the concomitant administration of herbal products and prescription medicine is pharmacokinetic herb-drug interaction (HDI). This is brought about by the ability of phytochemicals to inhibit or induce the activity of metabolic enzymes and transport proteins. The aim of this study was to investigate the potential of the crude extracts of popular medicinal herbs used in South Africa to inhibit major cytochrome P450 (CYP) enzymes and transport proteins through in vitro assessment. Methods Medicinal herbs were obtained from traditional medical practitioners and 15 were selected for this study. The selected herbal products were extracted and incubated with human liver microsomes to monitor the following reactions as markers for the metabolic activities of the respective CYP: phenacetin O-deethylation (CYP1A2), diclofenac 4‟-hydroxylation (CYP2C9), S-mephenytoin 4‟- hydroxylation (CYP2C19) and testosterone 6β-hydroxylation (CYP3A4). In addition, the influence of Lessertia frutescens (formerly Sutherlandia frutescens) and Hypoxis hemerocallidea was investigated on more isozymes: coumarin 7-hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), paclitaxel 6α-hydroxylation (CYP2C8), bufuralol 1‟-hydroxylation (CYP2D6), chlorzoxazone 6- hydroxylation (CYP2E1) and midazolam 1‟-hydroxylation (CYP3A4/5). The generation of the CYPspecific substrates/metabolites were monitored and quantified with the aid of LC-MS/MS. The metabolic clearance of midazolam using cryopreserved hepatocytes was monitored in the presence of Lessertia frutescens and Hypoxis hemerocallidea. The potential of both to inhibit human ATP-binding cassette (ABC) transporter activity was assessed using recombinant MDCKII and LLC-PK1 cells overexpressing human breast cancer resistant protein (BCRP) and human P-glycoprotein (P-gp), respectively. Similarly, the potential for interactions with human organic anion transporting polypeptide (OATP1B1 and OATP1B3) was assessed using recombinant HEK293 cells over-expressing OATP1B1 and OATP1B3, respectively. Results Bowiea volubilis, Kedrostis Africana, Chenopodium album, Lessertia frutescens (methanolic extract), Hypoxis hemerocallidea, Spirostachys africana and Lessertia frutescens (aqueous extract), in ascending order of potency demonstrated strong inhibition of CYP1A2 activity (IC50 = 1-100 g/mL). Similarly, Emex australis, Alepidea amatymbica, Pachycarpus concolor, Lessertia frutescens, Capparis sepiaria, Kedrostis africana and Pentanisia prunelloides inhibited CYP2C9 with IC50 less than 100 g/mL. The following demonstrated strong inhibition of CYP2C19 with IC50 values less than 100 g/mL: Acacia karroo, Capparis sepiaria, Chenopodium album, Pachycarpus concolor, Ranunculus multifidus, Lessertia frutescens and Zantedeschia aethiopica. CYP3A4 was inhibited by Lessertia frutescens, Hypoxis hemerocallidea, Spirostachys Africana, Bowiea volubilis, Zantedeschia aethiopica, Chenopodium album, Kedrostis Africana, Acacia karroo, Emex australis, Pachycarpus concolor, Ranunculus multifidus, Capparis sepiaria and Pentanisia prunelloides. Time-dependent (irreversible) inhibition of CYP3A4/5 (KI = 296 μg/mL, kinact = 0.063 min-1) and delay in the production of midazolam metabolites in the human hepatocytes, leading to a 40% decreased midazolam upscaled in vivo clearance, was observed with Lessertia frutescens. Further, Lessertia frutescence inhibited the activity of P-gp (IC50 = 324.8 μg/mL), OATP1B1 (IC50 = 10.4 μg/mL) and OATP1B3 (IC50 = 6.6 μg/mL). Hypoxis hemerocallidea inhibited the activity of OATP1B1 (IC50 = 118.7 μg/mL) and OATP1B3 (IC50 = 290.1 μg/mL) with no potent inhibitory effects on P-gp. None of the two inhibited the activity of BCRP within the tested concentrations. Conclusion The result indicates the potential for HDI between the selected medicinal herbs and the substrates of the enzymes investigated in this study, if sufficient in vivo concentrations are achieved. / AFRIKAANSE OPSOMMING: Inleiding Vroeëre studies het aangedui dat die gebruik van plantaardige produkte as tradisionele, aanvullende en alternatiewe medikasie baie gewild is. Een van die grootste kliniese risiko‟s geassosieer met die gelyktydige gebruik van plantaardige produkte met voorskrifmedikasie is farmakokinetiese kruiegeneesmiddel interaksies (HDI). Hierdie interaksies word veroorsaak deur die vermoë van plantchemikalieë om die aktiwiteit van metaboliese ensieme en transportproteïene te inhibeer of te induseer. Die doel van hierdie studie is om ondersoek in te stel na die moontlikheid van onsuiwer ekstrakte van gewilde Suid-Afrikaanse medisinale kruie om die belangrikste sitochroom P450 (CYP)- ensieme en transportproteïene te inhibeer. Hierdie ondersoek sal plaasvind deur middel van in vitrostudies. Metodes Medisinale kruie is verkry vanaf tradisionele genesers, waaruit ʼn totaal van 15 kruie geselekteer is vir gebruik tydens hierdie studie. Die geselekteerde kruie is geëkstraheer en met menslike lewermikrosome geïnkubeer om die volgende reaksies as merkers vir die metaboliese aktiwiteit van die onderskeie CYP-ensieme te moniteer: fenasetien-O-deëtilasie (CYP1A2), diklofenak-4‟- hidroksilasie (CYP2C9), S-mefenitoïen-4‟-hidroksilasie (CYP2C19) en testosteroon-6β-hidroksilasie (CYP3A4). Afgesien van die voorafgaande, is ook die invloed van Lessertia frutescens en Hypoxis hemerocallidea op verskeie ander iso-ensieme ondersoek. Hierdie iso-ensieme is soos volg: koumarien-7-hidroksilasie (CYP2A6), bupropioonhidroksilasie (CYP2B6), paklitaksiel-6α-hidroksilasie (CYP2C8), bufuralol-1‟-hidroksilasie (CYP2D6), chloorsoksasoon-6-hidroksilasie (CYP2E1) en midasolaam-1‟- hidroksilasie (CYP3A4/5). Die produksie van CYP-spesifieke substrate/metaboliete is gemoniteer en deur middel van LC-MS/MS-analises gekwantifiseer. Die metaboliese opruiming van midasolaam deur middel van krio-gepreserveerde hepatosiete is gemoniteer in die teenwoordigheid van Lessertia frutescens en Hypoxis hemerocallidea. Die moontlikheid van beide om menslike ATPbindingskasset (ABC)-transporteerderaktiwiteit te inhibeer is bepaal deur die gebruik van rekombinante MDCKII- en LLC-PK1-selle wat onderskeidelik menslike borskanker-weerstandige proteïen (BCRP) en menslike P-glikoproteïen (P-gp) potensieel. Op ʼn soortgelyke wyse is die moontlikheid vir interaksies met menslike organiese anion-transportpolipeptiede (OATP1B1 en OATP1B3) bepaal deur rekombinante HEK293-selle te gebruik wat onderskeidelik OATP1B1 en OATP1B3 potensieel. Resultate Bowiea volubilis, Kedrostis Africana, Chenopodium album, Lessertia frutescens (metanol-ekstrak), Hypoxis hemerocallidea, Spirostachys africana en Lessertia frutescens (water-ekstrak), in toenemende potensie, het sterk inhibisie van CYP1A2-aktiwiteit (IC50 = 1-100 g/mL) getoon. In ooreenstemming met die voorafgaande resultate het Emex australis, Alepidea amatymbica, Pachycarpus concolor, Lessertia frutescens, Capparis sepiaria, Kedrostis africana en Pentanisia prunelloides CYP2C9 met IC50–waardes van minder as 100 g/mL geïnhibeer. Die volgende het sterk inhibisie van CYP2C19 met IC50-waardes van minder as 100 g/mL getoon: Acacia karroo, Capparis sepiaria, Chenopodium album, Pachycarpus concolor, Ranunculus multifidus, Lessertia frutescens en Zantedeschia aethiopica. CYP3A4 is deur Lessertia frutescens, Hypoxis hemerocallidea, Spirostachys Africana, Bowiea volubilis, Zantedeschia aethiopica, Chenopodium album, Kedrostis Africana, Acacia karroo, Emex australis, Pachycarpus concolor, Ranunculus multifidus, Capparis sepiaria en Pentanisia prunelloides geïnhibeer. Tydafhanklike (onomkeerbare) inhibisie van CYP3A4/5 (KI = 296 μg/mL, kinact = 0.063 min-1) en vertraging in die produksie van midasolaammetaboliete in menslike hepatosiete wat aanleiding gee tot ʼn 40% afname in midasolaam bepaal in vivo opruiming, is waargeneem met Lessertia frutescens. Lessertia frutescens het ook die aktiwiteit van P-gp (IC50 = 324.8 μg/mL), OATP1B1 (IC50 = 10.4 μg/mL) en OATP1B3 (IC50 = 6.6 μg/mL) geïnhibeer. Hypoxis hemerocallidea het die aktiwiteit van OATP1B1 (IC50 = 118.7 μg/mL) en OATP1B3 (IC50 = 290.1 μg/mL) geïnhibeer met geen betekenisvolle effekte op P-gp nie. Geen een van die twee het die aktiwiteit van BCRP geïnhibeer binne die konsentrasies waarin getoets is nie. Gevolgtrekking Die resultate van hierdie studie dui aan dat wanneer voldoende in vivo-konsentrasies bereik word, die moontlikheid vir kruie-geneesmiddel interaksies tussen die geselekteerde medisinale kruie en ensiemsubstrate ʼn werklikheid word.

Fish may be exposed to an array of pharmaceuticals that are discharged into the aquatic environment, paralleling advances in medical knowledge, research and technology. Pharmaceuticals by their nature are designed to target specific receptors, transporters, or enzymes. Nuclear receptors (NRs) are often a key component of the therapeutic mechanism at play, and many of these are conserved among vertebrates. Consequently, fish may be affected by environmental pharmaceutical exposure, however there has been relatively little characterisation of NRs in fish compared with in mammals. In this thesis common carp (C. carpio) were exposed to selected pharmaceuticals in vitro and in vivo to investigate effects centred on the pregnane X receptor (PXR) and peroxisome proliferator-activated receptor alpha (PPARα), two key NRs involved in organism responses to pharmaceutical exposure. The PXR acts as a xenosensor, modulating expression of a number of xenobiotic metabolising enzymes (XMEs) in mammals. In a primary carp hepatocyte model it was shown that expression of a number of XMEs was altered on exposure to rifampicin (RIF), as occurs in mammals. This response was repressed by addition of ketoconaozle (KET; PXR-antagonist), indicating possible PXR involvement. The genes analysed showed up-regulation on exposure to ibuprofen (IBU) and clofibric acid (CFA), but not clotrimazole (CTZ) or propranolol (PRP). The lack of response to mammalian PXR-agonist CTZ was unexpected. In contrast, the same XME genes were found to be up-regulated in vivo after 10 days of exposure of carp to CTZ, although this response occurred only for a relatively high exposure concentration. CTZ was found to concentrate in the plasma (with levels up to 40 times higher than the water). Development and application of a reporter gene assay to measure PXR activation in carp (cPXR) and human PXR showed CTZ activation of cPXR, supporting data from the in vivo studies. Furthermore, activation was seen at concentrations as low as 0.01 μM. Interestingly RIF did not induce a response in the cPXR reporter gene assay, contrasting with the hepatocyte culture work. Taken together, the data presented here suggests divergence in the PXR pathway between mammals and fish in terms of ligand activation and downstream gene targets. PPARα was investigated in carp in vivo using CFA as a mammalian PPARα-agonist. Overall the resulting data suggested a broadly similar role for this NR in lipid homeostasis in fish as for mammals, with a number of PPARα-associated genes and acyl-coA oxidase (ACOX1) activity up-regulated in response to CFA exposure. A number of XMEs were also up-regulated by CFA (in vivo and in vitro), potentially extending the role of PPARα in fish (carp) to regulation of xenobiotic metabolism. The work presented has provided further characterisation of PXR and PPARα in fish. Elucidation of these pathways is vital to provide meaningful data in terms of establishing toxicity and mechanism-of-action data for pharmaceuticals and other compounds in fish, to allow validation of read-across approaches and ultimately aid in their environmental risk assessment. In vitro approaches are attractive ethically, financially and can provide useful mechanistic characterisation of compounds and the primary hepatocyte model and reporter gene assays used here show potential for the screening of pharmaceutical compounds in fish. However, further understanding of the metabolism of drugs and chemicals in fish is required to establish the true value of these methods for informing on possible effects in fish, in vivo.

Variations in drug metabolism may alter drug efficacy and cause toxicity; better understanding of the mechanisms and risks shall help to practice precision medicine. At the 21st International Symposium on Microsomes and Drug Oxidations held in Davis, California, USA, in October 2-6, 2016, a number of speakers reported some new findings and ongoing studies on the regulation mechanisms behind variable drug metabolism and toxicity, and discussed potential implications to personalized medications. A considerably insightful overview was provided on genetic and epigenetic regulation of gene expression involved in drug absorption, distribution, metabolism, and excretion (ADME) and drug response. Altered drug metabolism and disposition as well as molecular mechanisms among diseased and special populations were presented. In addition, the roles of gut microbiota in drug metabolism and toxicology as well as long non-coding RNAs in liver functions and diseases were discussed. These findings may offer new insights into improved understanding of ADME regulatory mechanisms and advance drug metabolism research. (C) 2017 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.

Matrix metalloproteinases (MMPs) play a significant role in degrading the extracellular matrix in cancer development and metastasis. Overexpression of matrix metalloproteinases in tumour tissues relative to normal tissues has been exploited as a target for peptide-based therapeutics, to improve therapeutic index of currently used agents. The stability of MMP-activated prodrugs in normal tissue or organs is a significant challenge for their success in the clinic. In an in vitro study, the stability of twenty six prodrugs was studied in mouse liver, kidney, lung and tumour homogenates using HPLC and LC/MS. Selected agents were studied in vivo. Each prodrug has a characteristic amino acid sequence with dominant FITC N-terminal end cap. All prodrugs were conjugated to a colchicine derivative (ICT 2552) which is a vascular disrupting agent causing tumour vasculature shutdown and consequently, tumour necrosis. ICT 3146, ICT 3019, ICT 3120 and ICT 3115 prodrugs showed significant stability in normal tissues and considerable activation in certain tumour tissues compared to the lead compound ICT 2588. Also, the selectivity of promising prodrugs to the MMP family was confirmed by using leupeptin (serine, cysteine and threonine protease inhibitor), pepstatin A (aspartate protease inhibitor), phosphoramidon (nepralysin inhibitor), ilomastat (metalloproteinase inhibitor) and BML-P115 (matrix metalloproteinase inhibitor). Moreover, members of the MMP family responsible for cleaving the selected prodrugs were identified using recombinant MMP enzymes. Furthermore, a LC/MS-MS method was developed to specifically detect and quantify MMP-16 protein expression in H460 tumour. MMP- 16 was responsible for the cleavage of ICT 3146 and ICT 3115. Therefore, MMPactivated prodrugs could be a useful therapeutic approach to avoid off-site toxicities of currently used anti-tumour agents.

The influence of hyperlipidemia on the pharmacodynamic and pharmacokinetic aspects of lipophilic drugs was explored. The antiarrhythmic, amiodarone, and the antifungal, ()-ketoconazole, were used as model drugs. Experimental hyperlipidemia was induced in rat using poloxamer 407 and two sensitive novel HPLC assays were developed. In a multiple dosing study, hyperlipidemia increased amiodarone plasma concentrations, heart concentrations and electrocardiographic changes. The amiodarone heart uptake could not be totally attributed to its unbound fraction, where the cardiac very low density lipoprotein receptors seemed to play a role in the uptake of bound drug. Amiodarone liver metabolism in presence and absence of hyperlipidemia was studied using isolated primary rat liver hepatocytes. The metabolism of amiodarone was lower in hepatocytes isolated from hyperlipidemic than those from normolipidemic rats. Hyperlipidemic serum resulted in a decrease in amiodarone metabolism and when coincubated, the expected decrease in unbound fraction seemed to resulted in greater inhibition of metabolism. ()-Ketoconazole showed stereoselectivity in its pharmacokinetics in rat with (+)-ketoconazole showing higher plasma concentrations than its antipode. This was attributed to its higher protein binding. There was no difference in the total bioavailability of the two enantiomers. Ketoconazole enantiomers exhibited nonlinear pharmacokinetics. In normolipidemic rat plasma ketoconazole enantiomers were more than 95% bound to lipoprotein deficient fraction. Hyperlipidemia resulted in shifting both enantiomers 20% to very low density and low density lipoprotein fractions. In a pharmacokinetic assessment, hyperlipidemia was found to increase ketoconazole enantiomer volume of distribution. Moreover, the stereoselectivity ratios of most pharmacokinetic parameters were changed. After oral dosing, the uptake of (-)-ketoconazole was significantly decreased. Since ketoconazole is used as a potent CYP3A inhibitor, alteration in liver concentrations of (-)-ketoconazole, the more potent inhibitory enantiomer, could decrease its CYP inhibitory potential. Hyperlipidemia potentiated the CYP-mediated interaction between ketoconazole and midazolam with significantly higher midazolam AUC and lower clearance. This was attributed to the inhibitory action of ketoconazole and the effect of hyperlipidemia on the binding of midazolam. Hyperlipidemia was found to unexpectedly decrease midazolam unbound fraction in plasma. In conclusion, the findings could explain some unexpected dose versus effect outcomes in hyperlipidemic patients receiving amiodarone or ketoconazole. / Pharmaceutical Sciences