61 |
Towards a test tube liver for drug metabolism studiesAchour, Brahim January 2013 (has links)
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.
|
62 |
Structure and biochemistry of the orphan cytochrome P450s CYP126A1 and CYP143A1 from the human pathogen Mycobacterium tuberculosisSwami, Shalini January 2015 (has links)
Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB) and poses a global threat to human health. A third of the world’s population is infected with Mtb. Multi-drug resistant and extensively drug resistant Mtb strains are widespread and development of new drugs is urgently needed to treat drug resistant TB. This thesis focuses on the Mtb cytochrome P450 (P450) enzymes CYP126A1 and CYP143A1. P450s are heme-binding enzymes that catalyse activation of molecular oxygen and the oxidation of substrates bound close to the heme. CYP126A1 and CYP143A1 are “orphans” in terms of their functional characterization, but potential drug targets in view of ability of azole-based P450 inhibitors to inhibit growth and viability of Mtb. The CYP126A1 and CYP143A1 genes were cloned and expressed in Escherichia coli. Expression conditions and strains were optimised to maximise soluble protein production and methods were developed to purify the P450s using affinity, ion exchange and size exclusion chromatography. Both P450s were shown to bind heme b, and heme was shown to be axially coordinated by a cysteine thiolate and a water molecule in both cases using UV-visible and electron paramagnetic resonance (EPR) spectroscopy. Both P450s bound carbon monoxide (CO) in their reduced forms to produce heme Fe2+-CO complexes with absorption maxima at ~450 nm – characteristic of P450s. CYP126A1 and CYP143A1 bound avidly to a range of inhibitors, including several azole drugs. As examples, binding constant (Kd) values of 13.8 µM and 21.9 µM were determined for clotrimazole and econazole with CYP143A1; while ketoconazole bound CYP126A1 with a Kd of 0.20 µM. Each of these drugs is very effective in inhibiting Mtb growth. EPR confirmed inhibitory coordination of both P450s by azole drug nitrogen atoms; though indirect coordination via a retained axial water ligand may also occur in some cases. Extinction coefficients were determined as έ420 = 125 mM-1 cm-1 (CYP126A1) and έ415 = 105 mM-1 cm-1 (CYP143A1). CYP126A1’s heme iron redox potential was shown to be unusually positive (E°’ = -80 mV). Light scattering studies showed CYP126A1 to be a monodisperse, monomeric protein. CYP143A1 is also mainly a monomer, but with a small proportion of an oligomeric form. Despite its polydispersity, CYP143A1 was crystallized and its structure solved by X-ray diffraction to a resolution of 1.9 Å, using molecular replacement with the Mtb P450 CYP142A1. A limited compound screen of typical P450 substrates failed to provide “hits” to identify CYP143A1 substrate selectivity, but the presence of polyethylene glycol in the CYP143A1 active site in crystals suggests fatty acids as potential substrates. CYP126A1 was crystallized for studies to identify binding modes of small molecules (“fragments”) identified to interact with CYP126A1 by NMR. Crystal structures of CYP126A1 in complex with two such fragments (NMR401 and NMR343) were determined to ~2.0 Å resolution in ongoing research to build Mtb P450 isoform-specific inhibitors. Compounds identified as CYP126A1 substrates/inhibitors identified by high-throughput screening were validated by UV-visible titrations with the P450, and binding modes and affinity established. In conclusion, this thesis provides novel insights into the biochemical, biophysical and structural properties of two novel Mtb P450s that are potential targets for new anti-TB drugs.
|
63 |
Análise de dados por imputação de sequenciamento de baixa cobertura Seleção de marcadores e genética populacional. /Alvarez, Marcus Vinicius Niz January 2020 (has links)
Orientador: Paulo Eduardo Martins Ribolla / Resumo: Introdução: O desenvolvimento de estratégias para redução no custo do sequenciamento de genoma completo (WGS) é importante para projetos que demandam por grandes quantidades de amostras. Uma estratégia de baixo custo é o sequenciamento de baixa cobertura aliado a técnicas de imputação para genotipagem eficiente e de confiabilidade adequada. A malária é uma das principais doenças transmitidas por artrópodes no mundo e o Brasil é considerado um país com alta incidência de malária, principalmente na região Amazônica, sendo principal vetor o mosquito Anopheles darlingi. Objetivo: O objetivo do presente estudo foi desenvolver estratégia para analisar dados de WGS de baixa cobertura de mosquitos Anopheles darlingi coletados no município de Mâncio Lima no Acre e verificar associação entre dados genéticos e dados de importância epidemiológica, tais como comportamento de picada, horário de atividade e distanciamento em escala microgeográfica. Materiais e métodos: Amostras de mosquitos Anopheles darlingi foram coletadas no município de Mâncio Lima - AC, entre 2016 e 2017. As bibliotecas foram preparadas com Nextera™ XT e sequenciadas no NextSeq500 da Illumina. Foi realizado genotipagem por sequenciamento e aplicado imputação. Estudos de associação ampla do genoma foram realizados com comportamento de picada e horário de atividade. Sinais de estratificação na população foram investigados por FST amplo no genoma e teste de permutação para significância. Resultados: Sinais fracos porém si... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Strategy development to reduce the cost of whole genome sequencing (WGS) is important for projects that demand large quantities of samples. A low-cost strategy is low-coverage sequencing combined with imputation techniques for efficient genotyping and sufficient confiability. Malaria is one of the main diseases transmitted by arthropods in the world and Brazil is considered a country with a high incidence of malaria, especially in the Amazon region with the main vector being the Anopheles darlingi mosquito. Objective: The objective of the present study was to develop a strategy to analyze low-coverage WGS data from Anopheles darlingi mosquitoes collected in the municipality of Mâncio Lima in Acre State and verify associations between genetic data and data of epidemiological importance, such as biting behavior, time of activity and distance on a microgeographic scale. Materials and methods: Samples of Anopheles darlingi mosquitoes were collected in the municipality of Mâncio Lima - AC, between 2016 and 2017. The libraries were prepared with Nextera ™ XT and sequenced on Illumina's NextSeq500. Genotyping by sequencing was performed and imputation was applied. Genome wide association studies were performed with biting behavior and time of activity. Population stratification signals were investigated by genome-wide FST and permutation test applied for significance. Results: Weak but significant stratification signals were identified considering distances of 2 to 3 k... (Complete abstract click electronic access below) / Mestre
|
64 |
Vliv cytochromů P450 na metabolismus protinádorových léčiv vázaných v apoferritinové nanočástici / Effect of cytochromes P450 on metabolism of anticancer drugs bound into apoferritin nanoparticleWilhelm, Marek January 2020 (has links)
Tumour-related diseases are the second most common cause of death in the Czech Republic, right after cardiovascular diseases. Nanomedicine - a novel scientific discipline - shows captivating potential in anticancer treatment with help of so called nanotranporters - nanoparticles capable of transporting other molecules. Encapsulation of a cytostatic drug into a nanoparticle improves its pharmacokinetical and pharmacodynamical properties which helps to reduce adverse side effects on non-tumour healthy tissue. In the scope of this diploma thesis apoferritin - apo-form of ferritin - was studied, since this nanotransporter shows promise for clinical use in anticancer treatment. Effect of hepatic microsomes from premedicated and control rats on biotransformation of doxorubicin cytostatic (Dox) in free and apoferritin nanoparticle-bound forms was investigated at pH 7,4. Over the course of biotransformation two types of metabolites - M1 and M2 - were observed. Regardless of the employed inductor all studied microsomes have exhibited similar metabolism of free doxorubicin and its apoferritin encapsulated form (ApoDox). Our results also imply that doxorubicin can be metabolically processed by rat hepatic microsomes in both free and ApoDox form with similar efficiency. We have also studied biotransformation...
|
65 |
Heterologní exprese a izolace lidských isoforem cytochromů P450 1A1/2 / Heterologous expression and isolation of human cytochromes P450 1A1/2Milichovský, Jan January 2011 (has links)
Cytochromes P450 form a large family of hemoproteins. Some of them are responsible for the metabolism of endogenous substrates, but their major role is in detoxification of exogenous substrates (xenobiotics), some of them are activated to reactive species forming covalent adducts with DNA and increasing intracellular oxidative stress. Cytochrome P450 are considered by very promiscuous in terms of their substrate specificity, thus one enzyme can typically oxidize many substrates. Cytochrome P450 1A1 prefers a planar aromatic compounds (e.g. polycyclic aromatic hydrocarbons, azo dyes, etc.). Cytochrome P450 1A2 elicits similar substrate specificity, but prefers slightly basic aromatic derivatives, for example caffeine. This work focuses on (i) the preparation of expression vectors containing genes encoding human cytochromes P450 1A1 and 1A2, (ii) their consequent expression in heterologous system followed by (iii) isolation of corresponding proteins. The genes coding both proteins were modified and transferred from older vectors to the more efficient to expression plasmids pET-22b. However, the new constructs did not produce stable native proteins. The modified genes were therefore transferred to the original expression plasmids pCW. The problem with the incorporation of native human form of...
|
66 |
Investigating the Mechanisms Underlying Enhanced Bioavailability of Artemisinin Delivered Orally as Dried Leaves of Artemisia annuaDesrosiers, Matthew R. 05 May 2020 (has links)
Malaria, a disease caused by parasites of the Plasmodium genus, infects over 220 million people annually, resulting in over 400,000 deaths. Most of these deaths occur in Africa in children < 5 years of age. Artemisia annua L., an ancient Chinese medicinal herb, is known for its foremost phytochemical constituent, artemisinin (AN). Semisynthetic derivatives of AN form the primary component of artemisinin combination therapies (ACTs), the frontline treatment for malaria worldwide. However, ACTs have several drawbacks including cost and availability. Thus, cheaper, more readily available antimalarials are needed. Recent clinical data suggested dried leaves of A. annua (DLA) administered orally as a tea infusion may be as efficacious as ACTs despite a significantly lower AN dose delivered. In mice, AN plasma concentration was improved when administered as DLA compared to pure AN. I therefore hypothesized that phytochemicals within DLA enhanced the oral bioavailability of AN. To investigate this hypothesis, here I examined the effects of DLA on the underlying mechanisms that govern oral bioavailability. Using an in vitro human digestion model, I showed that AN solubility was greater when delivered as DLA, largely due to essential oil in the plant. Furthermore, AN intestinal permeability was enhanced in a Caco-2 cell model of the intestinal epithelium. Extracts, teas, and phytochemicals produced by Artemisia also inhibited the activity of CYP2B6 and CYP3A4, the enzymes responsible for first-pass AN metabolism in the liver. Additionally, AN tissue distribution was improved when delivered as DLA and AN accumulation in tissues was higher in female vs. male rats. Finally, I showed that DLA was a more efficacious anti-inflammatory than pure AN in rats, potentially due to enhanced AN bioavailability. Taken together, these results shed light on the mechanisms behind enhanced oral bioavailability afforded by DLA and demonstrate the potential for DLA to be used as a therapeutic for malaria and other diseases.
|
67 |
Analysis of the Cytochrome P450 and UDP-Glucuronosyltransferase Families and Vitamin D3- Supplementation in Anoxia Survival in Caenorhabditis elegansAgarwal, Sujata 12 1900 (has links)
Alteration in diet and knockdown of detoxification genes impacts the response of C. elegans to oxygen deprivation stress. I hypothesized that feeding worms a vitamin D3-supplementation diet would result in differential oxygen deprivation stress response. We used a combination of wet lab and transcriptomics approach to investigate the effect of a vitamin-D3 supplemented diet on the global gene expression changes and the anoxia response phenotype of C. elegans (Chapter 2). C. elegans genome consists of 143 detoxification genes (cyp and ugt). The presence of a significant number of genes in these detoxification families was a challenge with identifying and selecting specific cyp and ugt genes for detailed analysis. Our goal was to understand the evolution, phylogenetic, and expression of the detoxification enzymes CYPs and UGTs in C. elegans (Chapter 3). We undertook a phylogenetic and bioinformatics approach to analyze the C. elegans, detoxification family. Phylogenetic analysis provided insight into the association of the human and C. elegans xenobiotic/endobiotic detoxification system. Protein coding genes in C. elegans have been predicted to be human orthologs. The results of this work demonstrate the role of C. elegans in the identification and characterization of vitamin D3 induced alterations in gene expression profile and anoxia response phenotypes and the identification of human orthologs for the detoxification enzymes and provides insight into the gene expression pattern.
|
68 |
AROMATIC HYDROCARBON RECEPTOR-DEPENDENT MITOCHONDRIAL OXIDATIVE STRESSSENFT, ALBERT PAUL 22 May 2002 (has links)
No description available.
|
69 |
The Impact of CYP3A5 Genotype on the Interaction Between Tacrolimus and Intravenous Nicardipine in Kidney Transplant RecipientsHooper, David K. January 2010 (has links)
No description available.
|
70 |
Computational investigations of cytochrome P450 aromatase catalysis and biological evaluation of isoflavone aromatase inhibitorsHackett, John C. 22 December 2004 (has links)
No description available.
|
Page generated in 0.0779 seconds