No description available.

Vemurafenib is a potent serine/threonine kinase inhibitor and is registered as Zelboraf® for the treatment of metastatic melanomas harboring BRAFV600E mutations. There is a large individual variation in drug response and the side effects observed among patients treated with Zelboraf® has proven to be severe.  LC-MS/MS methods were developed to measure vemurafenib and its metabolites in human plasma for prediction of treatment outcome and side effects in order to individualize treatment with Zelboraf®.  A novel, rapid quantification method was developed for vemurafenib using a stable isotope labeled internal standard. The method was validated according to international guidelines with regard to calibration range, accuracy, precision, carry-over, dilution integrity, selectivity, matrix effects, recovery and stability. All parameters met the set acceptance criteria.  The first method suitable for quantifying vemurafenib metabolites in human plasma is presented. Lacking commercially available reference substances, human liver microsomes were used to produce the metabolites. In patient samples at steady-state five previously in vitro identified metabolites were quantified for the first time.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Thesis (Ph.D.)--Flinders University, School of Medicine, Dept. of Clinical Pharmacology. / Typescript bound. Includes bibliographical references: (leaves 172-189) Also available online.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

The impact of the intestine in determining the oral bioavailability of drugs has been extensively studied. Its large surface area, metabolic content and positioning at the first site of exposure for orally ingested xenobiotics means its contribution can be significant for certain drugs. However, prediction of the exact metabolic component of the intestine is limited, in part due to limitations in validation of in vitro tools as well as in vitro-in vivo extrapolation scaling factors. Microsomes are a well established in vitro tool for extrapolating hepatic metabolism, however standardised methodologies for preparation in the intestine are limited, in light of complexities in preparation (e.g. presence of multiple non-metabolic cells, proteases and mucus). Therefore, the aims of this study were to establish an optimised method of intestinal microsome preparation via elution in the proximal rat intestine, and to determine microsomal scaling factors by correcting for protein losses during preparation. In addition, to assess species in another preclinical species (dog) and human as well as assessing and regional differences in scaling factors and metabolism. Following optimisation of a reproducible intestinal microsome preparation method in the rat, the importance of heparin in limiting mucosal contamination was established. These microsomes were characterised for total cytochrome P450 (CYP) content, and CYP and uridine 5′-diphosphate glucuronosyltransferase (UGT) activities using maker probes of testosterone and 4-nitrophenol. Loss corrected microsomal scaling factors between two pools of n=9 rats was 9.6±3.5 (recovery 33%). A broad range of compounds (n=25) in terms of metabolic activity and physicochemical properties were screened in rat intestinal microsomes. The prediction accuracy relative to in house generated or literature in vivo estimates of the fraction escaping intestinal metabolism (FG) through in vitro-in vivo extrapolation of observed metabolism and the derived scaling factors and either Caco-2 permeability of physicochemical permeability estimates utilising the Qgut model. In the dog, regional differences in intestinal scaling factors and metabolic activities were explored, as well as relationships between the proximal intestine and liver in matched donors. Positive correlations in both hepatic activity and microsomal scalars were observed. Robust scaling factors were established using the 3 microsomal markers. A total of 24 compounds were screened for hepatic and intestinal metabolism in order to make in vivo estimates of FG, the fraction escaping hepatic metabolism (FH) and oral bioavailability (F). Estimates based on Caco-2 and physicochemical based scaling, as well as utilising a commercial PBPK software platform (ADAM model, Simcyp® v12) were broadly similar with generally reduced prediction accuracy in proximal physicochemical based Qgut scaling, and improved predictions using Caco-2 Qgut or PBPK approaches. Worse predictions were observed for compounds with high protein binding, transporter substrates and/or CYP3A inhibitors. Regional metabolism demonstrated peak metabolism in the proximal intestine, before declining distally. Human intestinal microsomes were prepared for jejunum and ileum tissue. Although samples were limited, regional differences in metabolic activities and scaling factors were also assessed, using correction markers and activity in 23 compounds. In all, 20 compounds overlapped between all three species. Comparison in Fa.FG between rat and human CYP3A substrates showed a modest relationship, however relationships between species and human were generally poor given the observed differing metabolic contributions of testosterone and 4-NP metabolite formation between species limited the observed relationships between species. However, within species, good estimates of oral bioavailability were observed. This is the largest know interspecies comparison of intestinal metabolism and scaling factors with microsomes prepared within the same lab.

Polychlorinated biphenyls (PCBs) continue to pose a health concern because of their predominance in the diet and air as well as in environmental samples and humans. PCB congeners with 3 or 4 chlorine substituents in ortho position have been associated with neurodevelopmental disorders. Hydroxylated metabolites (OH-PCBs) of these PCBs are also potentially toxic to the developing brain. Metabolism studies have mainly focused on animal models. However, preliminary data from this dissertation work have revealed PCB metabolism differences between laboratory animal models and humans in terms of metabolite profiles, chiral signatures. More concerning, biotransformation of chiral PCBs is poorly investigated in humans. The objective of this dissertation research was to study the biotransformation of chiral and prochiral PCBs to chiral hydroxylated metabolites in humans and rats and to identify individual human P450 enzymes involved in the metabolism of these PCBs. I chose chiral PCB congeners 2,2',3,4',6-pentachlorobiphenyl (PCB 91); 2,2',3,5',6-pentachlorobiphenyl (PCB 95), 2,2',3,3',4,6'-hexachlorobiphenyl (PCB 132) and 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) for this investigation because they are environmentally relevant and their metabolism has been studied in rodents and other laboratory animal species (Kania-Korwel et al., 2016a). Prochiral PCB congeners 2,2′,4,6′-tetrachlorobiphenyl (PCB 51) and 2,2′,4,5,6′-pentachlorobiphenyl (PCB 102) were selected because their considerable presence in technical PCB mixtures. To test the hypothesis that P450 enzyme and species differences mediate the congener-specific enantioselective metabolism of chiral PCBs to hydroxylated metabolites, I sought to establish structure-metabolism relationships by studying the enantioselective metabolism of structurally diverse chiral PCBs by human liver microsomes (HLMs). Racemic PCB 91, PCB 95 and PCB 132 were incubated in vitro with pooled or individual donor HLMs at 37 °C, and levels and chiral signatures of the parent PCB and its hydroxylated metabolites were determined by high-resolution gas chromatography equipped with time-of-flight mass spectrometry (GC/TOF-MS) or electron capture detection (GC-ECD). Hydroxylated metabolites formed were identified and metabolic schemes for these PCBs proposed. I found inter-individual differences in the formation of OH-PCBs by individual donor HLMs. Comparison of the metabolite profiles of PCB 91, PCB 95, PCB 132 and PCB 136 (PCB 136 metabolism by HLMs was investigated by other researchers) revealed congener-specific differences in the oxidation of PCBs by human cytochrome P450 enzymes. PCB 91 and PCB 132 were mainly hydroxylated in meta position, with the 1,2-shift metabolites being the major metabolites formed from both PCB congeners by HLMs. In contrast, PCB 95 and PCB 136 were primarily hydroxylated in the para position. Moreover, we determined human P450 isoforms involved in the metabolism of neurotoxic PCBs using in silico and in vitro approaches. In silico predictions suggested that chiral PCBs are metabolized by CYP1A2, CYP2A6, CYP2B6, CYP2E1, and CYP3A4. Experimentally we found that CYP2A6, CYP2B6 and to a minor extent CYP2E1 were the enzymes involved in the metabolism of these chiral PCBS. We also investigated nonchiral sources of chiral OH-PCBs by studying the P450- and species-dependent biotransformation of prochiral PCB 51 and PCB 102 to chiral OH-PCB metabolites. Prochiral PCB 51 and PCB 102 were incubated with liver microsomes prepared from male Sprague-Dawley rats pretreated with various inducers of P450 enzymes including phenobarbital (PB), dexamethasone (DEX), isoniazid (INH), β-naphthoflavone (BNF), clofibric acid (CFA) or corn oil (CO); and untreated male cynomolgus monkeys, Hartley albino guinea pigs, New Zealand rabbits, golden Syrian hamsters; and untreated female Beagle dogs. PCB 51 and PCB 102 were metabolized to 2,2',4,6'-tetrachlorobiphenyl-3'-ol (OH-PCB 51) and 2,2',4,5,6'-pentachlorobiphenyl-3'-ol (OH-PCB 102), respectively. The formation of both metabolites was P450 isoforms- and species-dependent. Moreover, OH-PCB 51 and OH-PCB 102 were chiral and were formed enantioselectively in all microsomes investigated. Taken together, my findings demonstrate (1) considerable inter-individual variability in the congener-specific metabolism of PCBs to OH-PCBs; (2) the enantioselective formation of OH-PCBs by human CYP2A6, CYP2B6, and CYP2E1; and (3) that chiral PCB metabolites are formed enantioselectively from prochiral PCB congeners. Interestingly, the metabolism of PCBs by CYP2A6 appears to involve arene oxide intermediates, as suggested by the formation of 1,2-shift products as major metabolites of PCB 91 and PCB 132. In contrast, 1,2-shift products are minor PCB metabolites formed in rodents. Therefore extrapolation of hepatic metabolism across species may not be consistent and these differences should be considered in future toxicity and risk assessment studies.