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.

Estudos realizados em 1979 por Kelecom e Kanengiesser mostraram que extratos brutos obtidos a partir de amostras da esponja marinha Aplysina fulva não possuíam derivados bromados. Estes foram resultados inesperados, uma vez que esponjas pertencentes à ordem Verongida são conhecidos produtores de metabólitos derivados da dibromotirosina. O presente projeto teve como meta a investigação química de extratos obtidos de duas amostras de A. fulva, sendo uma coletada em São Sebastião (SP) e a outra em Angra dos Reis (RJ), objetivando verificar a ocorrência de derivados da bromotirosina e uma possível variabilidade química dependendo da distribuição geográfica das esponjas. Sete derivados da dibromotirosina foram isolados das duas amostras de Aplysina fulva: quatro compostos da amostra de Angra dos Reis e três da de São Sebastião. As estruturas dos compostos foram identificadas por análises espectroscópicas (como IV, UV, RMN 1H, RMN 13C, HMQC, COSY, HMBC) e de espectrometria de massas, e ainda por comparação com dados da literatura. Os resultados obtidos confirmaram que os derivados da dibromotirosina são bons marcadores quimiotaxonômicos para as esponjas da Ordem Verongida. Além disso, a variabilidade química observada para a A. fulva parece ser influência de fatores abióticos e bióticos como sazonalidade, disponibilidade de nutrientes, ou associação com diferentes micro-organismos. / Studies developed in 1979 by Kelecom and Kanengiesser showed that crude extracts obtained from samples of the marine sponge Aplysina fulva were devoid of bromotyrosine derivatives. These results were rather unexpected, since sponges belonging to the Order Verongida are well-known producers of bromotyrosine-derived metabolites. The present project aimed the chemical investigation of crude extracts obtainded from two samples of A. fulva, one collected at São Sebastião (SP) and the second from Angra dos Reis (RJ), in order to verify the occurrence of bromotyrosine derivatives and a possible chemical variability depending on the geographical distribution of the sponge. Seven bromoyrosine derivatives have been isolated from the two samples of A. fulva: four compounds from the Angra dos Reis sample and three from the São Sebastião sample. The structures of the compounds have been established by spectroscopic analysis, (including IV, UV, RMN 1H, RMN 13C, HMQC, COSY, HMBC) and mass spectrometry, as well as by comparison with literature data. The results obtained confirmed that bromotyrosine derivatives are good chemotaxonomic markers for sponges of the Order Verongida. Moreover, it appears that chemical variability of A. fulva may be influenced by abiotic and biotic factors, such as seasonality, nutrients availability, or association with distinct micro-organisms.

Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2009.

Selective antiretroviral! (ARV) drugs are primarily metabolized by cytochrome P450 (CYP) enzymes, characteristically predisposed to variation, and are therefore primarily responsible for ARV pharmacokinetic variability and associated drug interactions. For the majority of ARV drugs, the therapeutic window is narrow and imminent toxicities due to CYP inhibition or sub-therapeutic drug levels as a result of CYP induction is inevitable. Animals provide a metabolism replica to conduct detailed investigations. We endeavored to establish a rat model to screen for variability in metabolism of selective ARV drugs responsible for treatment failure and drug interactions, over time in the liver and serum. Male Sprague-Dawley rats (n = 24) were divided into 6 groups: methylcellulose, 160mg/kg/day (n = 24) (control); efavirenz, 160mg/kg/day (n = 18); ritonavir, 20 mg/kg/day (n = 18); ritonavir, 20 mg/kg/day and verapamil 5 mg/kg/day (n = 18); Kaletra® (ritonavir/lopinavir), 20 mg/kg/day, (n = 18); Kaletra® (ritonavir/lopinavir), 20 mg/kg/day and verapamil 5 mg/kg/day (n = 18). Treatment duration varied from one day (single dose), 7 or 21 days. Blood samples were collected after decapitation on days 1, 7 and 21. A sensitive and rapid liquid chromatograph (LC) interfaced to a quadrupoie mass spectrometer (MS) and coupled with electrospray ionization (ESI) method was employed for the blood sample determinations. One single injection was required to simultaneously quantify efavirenz, lopinavir and ritonavir within the linear concentration range of 78 - 5000 ng/ml. Efavirenz blood levels increased statistically significantly (p < 0.05) from day 1 to day 21 with distinct steady state achievement prior to day 7. The levels of ritonavir increased statistically significantly (p < 0.05) from day 7 to 21 when administered alone and statistically significantly (p < 0.01) from day 1 to 21 when administered as the ritonavir/lopinavir combination. The levels of lopinavir also increased statistically significantly (p<0.01) from day 1 and 21 in the ritonavir/lopinavir combination. However, the inclusion of a P-glycoprotein inhibitor, verapamil, increased both the ritonavir (administered alone) and lopinavir blood levels significantly (p < 0.05) at day 1. The ritonavir levels were also significantly increased on day 21 (p < 0.05). When verapamil was added to the ritonavir/lopinavir combination the levels of ritonavir increased statistically significantly (p < 0.01) from day 1 to 21. A rat model can be used to detect changes in metabolism over time as measured by blood levels. The influence of drug interactions, such as verapamil, on ARV drug metabolism can be investigated by this model. These results will be substantiated by PCR liver results in the future. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2009.

Selective antiretroviral! (ARV) drugs are primarily metabolized by cytochrome P450 (CYP) enzymes, characteristically predisposed to variation, and are therefore primarily responsible for ARV pharmacokinetic variability and associated drug interactions. For the majority of ARV drugs, the therapeutic window is narrow and imminent toxicities due to CYP inhibition or sub-therapeutic drug levels as a result of CYP induction is inevitable. Animals provide a metabolism replica to conduct detailed investigations. We endeavored to establish a rat model to screen for variability in metabolism of selective ARV drugs responsible for treatment failure and drug interactions, over time in the liver and serum. Male Sprague-Dawley rats (n = 24) were divided into 6 groups: methylcellulose, 160mg/kg/day (n = 24) (control); efavirenz, 160mg/kg/day (n = 18); ritonavir, 20 mg/kg/day (n = 18); ritonavir, 20 mg/kg/day and verapamil 5 mg/kg/day (n = 18); Kaletra® (ritonavir/lopinavir), 20 mg/kg/day, (n = 18); Kaletra® (ritonavir/lopinavir), 20 mg/kg/day and verapamil 5 mg/kg/day (n = 18). Treatment duration varied from one day (single dose), 7 or 21 days. Blood samples were collected after decapitation on days 1, 7 and 21. A sensitive and rapid liquid chromatograph (LC) interfaced to a quadrupoie mass spectrometer (MS) and coupled with electrospray ionization (ESI) method was employed for the blood sample determinations. One single injection was required to simultaneously quantify efavirenz, lopinavir and ritonavir within the linear concentration range of 78 - 5000 ng/ml. Efavirenz blood levels increased statistically significantly (p < 0.05) from day 1 to day 21 with distinct steady state achievement prior to day 7. The levels of ritonavir increased statistically significantly (p < 0.05) from day 7 to 21 when administered alone and statistically significantly (p < 0.01) from day 1 to 21 when administered as the ritonavir/lopinavir combination. The levels of lopinavir also increased statistically significantly (p<0.01) from day 1 and 21 in the ritonavir/lopinavir combination. However, the inclusion of a P-glycoprotein inhibitor, verapamil, increased both the ritonavir (administered alone) and lopinavir blood levels significantly (p < 0.05) at day 1. The ritonavir levels were also significantly increased on day 21 (p < 0.05). When verapamil was added to the ritonavir/lopinavir combination the levels of ritonavir increased statistically significantly (p < 0.01) from day 1 to 21. A rat model can be used to detect changes in metabolism over time as measured by blood levels. The influence of drug interactions, such as verapamil, on ARV drug metabolism can be investigated by this model. These results will be substantiated by PCR liver results in the future. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2009.

Estudos realizados em 1979 por Kelecom e Kanengiesser mostraram que extratos brutos obtidos a partir de amostras da esponja marinha Aplysina fulva não possuíam derivados bromados. Estes foram resultados inesperados, uma vez que esponjas pertencentes à ordem Verongida são conhecidos produtores de metabólitos derivados da dibromotirosina. O presente projeto teve como meta a investigação química de extratos obtidos de duas amostras de A. fulva, sendo uma coletada em São Sebastião (SP) e a outra em Angra dos Reis (RJ), objetivando verificar a ocorrência de derivados da bromotirosina e uma possível variabilidade química dependendo da distribuição geográfica das esponjas. Sete derivados da dibromotirosina foram isolados das duas amostras de Aplysina fulva: quatro compostos da amostra de Angra dos Reis e três da de São Sebastião. As estruturas dos compostos foram identificadas por análises espectroscópicas (como IV, UV, RMN 1H, RMN 13C, HMQC, COSY, HMBC) e de espectrometria de massas, e ainda por comparação com dados da literatura. Os resultados obtidos confirmaram que os derivados da dibromotirosina são bons marcadores quimiotaxonômicos para as esponjas da Ordem Verongida. Além disso, a variabilidade química observada para a A. fulva parece ser influência de fatores abióticos e bióticos como sazonalidade, disponibilidade de nutrientes, ou associação com diferentes micro-organismos. / Studies developed in 1979 by Kelecom and Kanengiesser showed that crude extracts obtained from samples of the marine sponge Aplysina fulva were devoid of bromotyrosine derivatives. These results were rather unexpected, since sponges belonging to the Order Verongida are well-known producers of bromotyrosine-derived metabolites. The present project aimed the chemical investigation of crude extracts obtainded from two samples of A. fulva, one collected at São Sebastião (SP) and the second from Angra dos Reis (RJ), in order to verify the occurrence of bromotyrosine derivatives and a possible chemical variability depending on the geographical distribution of the sponge. Seven bromoyrosine derivatives have been isolated from the two samples of A. fulva: four compounds from the Angra dos Reis sample and three from the São Sebastião sample. The structures of the compounds have been established by spectroscopic analysis, (including IV, UV, RMN 1H, RMN 13C, HMQC, COSY, HMBC) and mass spectrometry, as well as by comparison with literature data. The results obtained confirmed that bromotyrosine derivatives are good chemotaxonomic markers for sponges of the Order Verongida. Moreover, it appears that chemical variability of A. fulva may be influenced by abiotic and biotic factors, such as seasonality, nutrients availability, or association with distinct micro-organisms.