No description available.

No description available.

No description available.

Pharmacogenetics involves research into the hereditary basis for the different responses of individuals to drugs or other environmental pollutants. Several functional genetic polymorphisms of drug metabolising enzymes, transporters, receptors and other drug targets have been identified and characterised and these polymorphisms may be responsible for interethnic differences in drug disposition and disease risk. Few studies have focussed on ethnic African populations. Several genes that have known genetic polymorphism and have clinical implications for disease risk and/or treatment of patients were evaluated in a sample of the Ghanaian (West African) population. Catechol-O-methyltransferase (COMT) catalyses the 0-methylation of neurotransmitters, catechol hormones and drugs such as levodopa and methyldopa. Ethnic differences in COMT activity have been observed in several populations. Previous studies suggest that the homozygous low activity allele (COMT*L) is less common in individuals of African origin than Caucasians. COMT genotyping was performed using a mini-sequencing method in 195 healthy Ghanaians. The frequency of the homozygous low activity allele was 6%. In Caucasians it is 31%. This study provides confirmation that the low activity COMT allele is less common in individuals of African origin. This finding may be important clinically with regards to the treatment of many neuropsychiatric disorders and in the pathophysiology of various human disorders including oestrogen-induced cancers, Parkinson's disease, depression and hypertension. This thesis aimed to determine the allele frequency of therapeutically important genetic polymorphisms in an African (Ghanaian) population. The data was then compared to other ethnic populations. The marked racial and ethnic differences in the frequency of functional polymorphisms in these drug- and xenobiotic-metabolising enzymes, transporters, receptors and other drug targets shows that ethnic origin needs to be considered in studies aimed at discovering whether specific genotypes or phenotypes are associated with disease risk or drug toxicity. Genotyping prior to treatment may be essential, as 95% of the Ghanaian subjects genotyped had between one and four mutations in the therapeutically important genes analysed. Genotyping assays specific for predominant mutant alleles should be used in different ethnic groups.

Repaglinide is currently recommended as an in vivo CYP2C8 probe by the U.S. Food and Drug Administration (FDA), but the kinetic characterisation and enzymes involved in the elimination of this drug have not been fully delineated. In addition to its complex metabolism, polymorphism in the SLCO1B1 gene encoding for the hepatic uptake transporter organic anion transporting polypeptide 1B1 (OATP1B1) has been shown to impact repaglinide pharmacokinetics, further complicating the prediction of repaglinide clearance and drug-drug interactions (DDIs). The aim of this thesis was to firstly perform a systematic analysis of repaglinide metabolic pathways and thereby assess the contribution of specific enzymes to its clearance, and to secondly increase the understanding of repaglinide as a victim drug by implementing obtained in vitro metabolism data together with reported hepatic uptake parameters into a physiologically-based pharmacokinetic (PBPK) model. Furthermore, reported repaglinide DDIs, repaglinide AUC in different SLCO1B1 genotype groups and repaglinide P450 metabolite ratios were collated and critically analysed. The metabolism of repaglinide was characterised using a range of in vitro systems, namely pooled cryopreserved human hepatocytes, human liver microsomes (HLMs), human S9 fractions and recombinant P450 enzymes. The impact of in vitro systems on the analysis of repaglinide metabolic pathway was investigated and the importance of individual metabolic pathways studied. Definite differences in formation clearance ratios were found between CYP3A4 and CYP2C8 for the formation of M1 and M4 metabolites, resulting in a 60- and 0.05-fold M1:M4 ratio in recombinant CYP3A4 and CYP2C8, respectively. A major system difference was seen in clearances for the formation of M2, which is suggested to be a main metabolite of repaglinide in vivo. An approximately 7-fold higher unbound intrinsic clearance was observed in hepatocytes and S9 fractions in comparison to microsomes; the involvement of aldehyde dehydrogenase in M2 formation was shown for the first time. This systematic analysis revealed a comparable in vitro contribution from CYP2C8 and CYP3A4 to the metabolism of repaglinide.

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3-Methylcholanthrene (MC) is a model polycyclic aromatic hydrocarbon that induces cytochrome P450 1A1 (CYP1A1) expression. This laboratory has shown previously that aromatic hydrocarbons, which are important environmental toxicants, down-regulate the expression of rat liver CYP2C11. Recent observations also suggested that CYP2C8, a human enzyme that metabolizes antineoplastic and antidiabetic drugs, among others, is down-regulated in response to aromatic hydrocarbon exposure in primary human hepatocytes. I examined the regulation of CYP2C8 at the mRNA level by MC in two human hepatocellular carcinoma cell lines, HepG2 and HepaRG. MC down-regulated CYP2C8 mRNA levels in HepG2 cells at 24 hours and in HepaRG cells at 48 hours. CYP1A1 mRNA was induced by MC in both cell lines and HepaRG cells appeared to be more sensitive than HepG2 cells to MC-induced cytotoxicity. Further studies are warranted to define the mechanisms and functional impacts of the modulation of this important human CYP by environmental toxicants.

3-Methylcholanthrene (MC) is a model polycyclic aromatic hydrocarbon that induces cytochrome P450 1A1 (CYP1A1) expression. This laboratory has shown previously that aromatic hydrocarbons, which are important environmental toxicants, down-regulate the expression of rat liver CYP2C11. Recent observations also suggested that CYP2C8, a human enzyme that metabolizes antineoplastic and antidiabetic drugs, among others, is down-regulated in response to aromatic hydrocarbon exposure in primary human hepatocytes. I examined the regulation of CYP2C8 at the mRNA level by MC in two human hepatocellular carcinoma cell lines, HepG2 and HepaRG. MC down-regulated CYP2C8 mRNA levels in HepG2 cells at 24 hours and in HepaRG cells at 48 hours. CYP1A1 mRNA was induced by MC in both cell lines and HepaRG cells appeared to be more sensitive than HepG2 cells to MC-induced cytotoxicity. Further studies are warranted to define the mechanisms and functional impacts of the modulation of this important human CYP by environmental toxicants.

No description available.

The UDP-glucuronosyltransferases (GT) represent a major family of drug-metabolising enzymes, but little is known about their multiplicity in man. The aims of this project were to purify and characterise a GT isozyme from human liver, and to investigate glucuronidation in rat and human hepatocytes, with the aim of using human hepatocytes in primary culture as an <i>in vitro</i> model for the study of human drug metabolism. Chromatofocusing of human liver microsomes produced separation of GT isozymes, providing evidence for heterogeneity. However, purification in an active form was not achieved, due to lability in the presence of detergent. Rat liver 17β-hydroxysteroid-GT was purified, and antibodies raised against this protein recognised a single protein in human liver microsomes. Rates of glucuronidation of 1-naphthol and phenolphthalein were significantly higher in rat hepatocyte homogenates than in hepatocytes, the magnitude of the difference being greater for 1-naphthol. This was attributed to the presence of excess UDPGA in homogenate assays and the limitation imposed by lipophilicity on substrate uptake into cells by passive diffusion. In contrast, the rate of bilirubin glucuronidation was greater in hepatocytes, possibly as a result of intact carrier-mediated uptake mechanisms, combined with a suitable environment for efficient delivery of bilirubin to the endoplasmic reticulum by membrane-membrane transfer. The same three substrates were glucuronidated at a reduced rate in human compared with rat hepatocytes, apparently due to a lower intracellular UDPGA level and isozyme-specific differences in intrinsic activity and latency. As a result, the rate of glucuronidation of all these substrates was greater in human hepatocyte homogenates than in hepatocytes. Isozyme-specific changes were observed in GT activities in human hepatocytes in primary culture, indicating the need to develop culture systems allowing stable expression of these enzymes before such a model could be used for predictive human drug metabolism.