Collen Masimirembwa

Identification of human cytochrome P 450 s that metabolise anti-parasitic drugs and predictions of in vivo drug hepatic clearance from in vitro data

ObjectiveKnowledge about the metabolism of anti-parasitic drugs (APDs) will be helpful in ongoing efforts to optimise dosage recommendations in clinical practise. This study was performed to further identify the cytochrome P450 (CYP) enzymes that metabolise major APDs and evaluate the possibility of predicting in vivo drug clearances from in vitro data.MethodsIn vitro systems, rat and human liver microsomes (RLM, HLM) and recombinant cytochrome P450 (rCYP), were used to determine the intrinsic clearance (CLint) and identify responsible CYPs and their relative contribution in the metabolism of 15 commonly used APDs.Results and discussionCLint determined in RLM and HLM showed low (r2=0.50) but significant (P<0.01) correlation. The CLint values were scaled to predict in vivo hepatic clearance (CLH) using the venous equilibrium model. The number of compounds with in vivo human CL data after intravenous administration was low (n=8), and the range of CL values covered by these compounds was not appropriate for a reasonable quantitative in vitro–in vivo correlation analysis. Using the CLH predicted from the in vitro data, the compounds could be classified into three different categories: high-clearance drugs (>70% liver blood flow; amodiaquine, praziquantel, albendazole, thiabendazole), low-clearance drugs (<30% liver blood flow; chloroquine, dapsone, diethylcarbamazine, pentamidine, primaquine, pyrantel, pyrimethamine, tinidazole) and intermediate clearance drugs (artemisinin, artesunate, quinine). With the exception of artemisinin, which is a high clearance drug in vivo, all other compounds were classified using in vitro data in agreement with in vivo observations. We identified hepatic CYP enzymes responsible for metabolism of some compounds (praziquantel—1A2, 2C19, 3A4; primaquine—1A2, 3A4; chloroquine—2C8, 2D6, 3A4; artesunate—2A6; pyrantel—2D6). For the other compounds, we confirmed the role of previously reported CYPs for their metabolism and identified other CYPs involved which had not been reported before.ConclusionOur results show that it is possible to make in vitro–in vivo predictions of high, intermediate and low CLint drug categories. The identified CYPs for some of the drugs provide a basis for how these drugs are expected to behave pharmacokinetically and help in predicting drug–drug interactions in vivo.

High prevalence of the CYP2B6 516G→T(*6) variant and effect on the population pharmacokinetics of efavirenz in HIV/AIDS outpatients in Zimbabwe

ObjectiveThe study sought to investigate the relationship between efavirenz exposure and the CYP2B6 516G→T(*6) genotype in HIV/AIDS outpatients, using pharmacokinetic modelling and simulation.MethodsBlood samples where obtained from 74 outpatients treated with a combination regimen including 600xa0mg efavirenz daily for a duration of at least 3 weeks at clinics in Harare, Zimbabwe. The subjects were genotyped for the major CYP2B6 variant, CYP2B6*6, associated with reduced enzyme activity, using a PCR-RFLP method. Efavirenz plasma concentrations were determined by HPLC-UV. Population pharmacokinetic modelling and simulation of the data were performed in NONMEM VI.ResultsA high allele frequency of the CYP2B6*6 allele of 49% was observed. Efavirenz plasma concentrations were above 4xa0mg/L in 50% of the patients. Genotype and sex were identified as predictive covariates of efavirenz disposition. Pharmacokinetic parameter estimates indicate that a dose reduction to 400xa0mg efavirenz per day is possible in patients homozygous for the CYP2B6*6 genotype without compromising therapeutic efficacy.ConclusionThe CYP2B6*6 allele occurs at a high frequency in people of African origin and is associated with high efavirenz concentrations. Simulations indicate that an a priori 35% dose reduction in homozygous CYP2B6*6 patients would maintain drug exposure within the therapeutic range in this group of patients. Our preliminary results suggest the conduct of a prospective clinical dose optimization study to evaluate the utility of genotype-driven dose adjustment in this population.

Metabolic stability for drug discovery and development: pharmacokinetic and biochemical challenges.

Metabolic stability refers to the susceptibility of compounds to biotransformation in the context of selecting and/or designing drugs with favourable pharmacokinetic properties. Metabolic stability results are usually reported as measures of intrinsic clearance, from which secondary pharmacokinetic parameters such as bioavailability and half-life can be calculated when other data on volume of distribution and fraction absorbed are available. Since these parameters are very important in defining the pharmacological and toxicological profile of drugs as well as patient compliance, the pharmaceutical industry has a particular interest in optimising for metabolic stability during the drug discovery and development process. In the early phases of drug discovery, new chemical entities cannot be administered to humans; hence, predictions of these properties have to be made from in vivo animal, in vitro cellular/subcellular and computational systems. The utility of these systems to define the metabolic stability of compounds that is predictive of the human situation will be reviewed here. The timing of performing the studies in the discovery process and the impact of recent advances in research on drug absorption, distribution, metabolism and excretion (ADME) will be evaluated with respect to the scope and depth of metabolic stability issues.Quantitative prediction of in vivo clearance from in vitro metabolism data has, for many compounds, been shown to be poor in retrospective studies. One explanation for this may be that there are components used in the equations for scaling that are missing or uncertain and should be an area of more research. For example, as a result of increased biochemical understanding of drug metabolism, old assumptions (e.g. that the liver is the principal site of first-pass metabolism) need revision and new knowledge (e.g. the relationship between transporters and drug metabolising enzymes) needs to be incorporated into in vitro-in vivo correlation models. With ADME parameters increasingly being determined on automated platforms, instead of using results from high throughput screening (HTS) campaigns as simple go/no-go filters, the time saved and the many compounds analysed using the robots should be invested in careful processing of the data. A logical step would be to investigate the potential to construct computational models to understand the factors governing metabolic stability. A rational approach to the use of HTS assays should aim to screen for many properties (e.g. physicochemical parameters, absorption, metabolism, protein binding, pharmacokinetics in animals and pharmacology) in an integrated manner rather than screen against one property on many compounds, since it is likely that the final drug will represent a global average of these properties.

In Vitro High Throughput Screening of Compounds for Favorable Metabolic Properties in Drug Discovery.

Drug metabolism can have profound effects on the pharmacological and toxicological profile of therapeutic agents. In the pharmaceutical industry, many in vitro techniques are in place or under development to screen and optimize compounds for favorable metabolic properties in the drug discovery phase. These in vitro technologies are meant to address important issues such as: (1) is the compound a potent inhibitor of drug metabolising enzymes (DMEs)? (2) does the compound induce the expression of DMEs? (3) how labile is the compound to metabolic degradation? (4) which specific enzyme(s) is responsible for the compounds biotransformation? and (5) to which metabolites is the compound metabolized? Answers to these questions provide a basis for judging whether a compound is likely to have acceptable pharmacokinetic properties in vivo. To address these issues on the increasing number of compounds inundating the drug discovery programs, high throughput assays are essential. A combination of biochemical advances in the understanding of the function and regulation of DMEs (in particular, cytochromes P450, CYPs) and automated analytical technologies are revolutionizing drug metabolism research. Automated LC-MS based metabolic stability, fluorescence, radiometric and LC-MS based CYP inhibition assays are now in routine use. Automatible models for studying CYP induction based on enzyme activity, quantitative RT-PCR and reporter gene systems are being developed. We will review the utility and limitations of these HTS approaches and highlight on-going developments and emerging technologies to answer metabolism questions at the different stages of the drug discovery process.

Novel variants of major drug-metabolising enzyme genes in diverse African populations and their predicted functional effects

Pharmacogenetics enables personalised therapy based on genetic profiling and is increasingly applied in drug discovery. Medicines are developed and used together with pharmacodiagnostic tools to achieve desired drug efficacy and safety margins. Genetic polymorphism of drug-metabolising enzymes such as cytochrome P450s (CYPs) and N-acetyltransferases (NATs) has been widely studied in Caucasian and Asian populations, yet studies on African variants have been less extensive. The aim of the present study was to search for novel variants of CYP2C9, CYP2C19, CYP2D6 and NAT2 genes in Africans, with a particular focus on their prevalence in different populations, their relevance to enzyme functionality and their potential for personalised therapy. Blood samples from various ethnic groups were obtained from the AiBST Biobank of African Populations. The nine exons and exon-intron junctions of the CYP genes and exon 2 of NAT2 were analysed by direct DNA sequencing. Computational tools were used for the identification, haplotype analysis and prediction of functional effects of novel single nucleotide polymorphisms (SNPs). Novel SNPs were discovered in all four genes, grouped to existing haplotypes or assigned new allele names, if possible. The functional effects of non-synonymous SNPs were predicted and known African-specific variants were confirmed, but no significant differences were found in the frequencies of SNPs between African ethnicities. The low prevalence of our novel variants and most known functional alleles is consistent with the generally high level of diversity in gene loci of African populations. This indicates that profiles of rare variants reflecting interindividual variability might become the most relevant pharmacodiagnostic tools explaining Africans diversity in drug response.

Evaluation of Human Liver Slices and Reporter Gene Assays as Systems for Predicting the Cytochrome P450 Induction Potential of Drugs in Vivo in Humans

PurposeThe aim of the study was to investigate the feasibility of predicting human in vivo cytochrome P450 (CYP) induction properties of drugs using in vitro methods.MethodsThe CYP induction potential of compounds was tested in human liver slices and in reporter gene assays for the aryl hydrocarbon receptor (AhR) and the pregnane X receptor (PXR).ResultsIn human liver slices, CYP activities decreased dramatically over the experimental period, whereas mRNA levels could reliably be used to investigate CYP1A, 2C9, and 3A4 induction. However, the interindividual variations and demanding experimentation limit the use of liver slices in screening programs. Reporter gene assays are robust and reliable assays, amenable to high throughput screening. Several compounds activated AhR. The relevance of this activation, however, needs to be further investigated since there are no clear reports on drugs inducing CYP1A in vivo. The results from the PXR assay could be used to correctly classify compounds with known CYP3A induction properties when relating in vivo AUCtot to PXR EC50 values.ConclusionsLiver slices are a valuable model to study the regulation of a larger number of enzymes by single compounds. The PXR reporter gene assay could be used as a reliable screening method to predict CYP3A induction in vivo.

Phenotype and genotype analysis of debrisoquine hydroxylase (CYP2D6) in a black Zimbabwean population Reduced enzyme activity and evaluation of metabolic correlation of CYP2D6 probe drugs

Objective: Debrisoquine hydroxylase (CYP2D6) is responsible for the oxidative metabolism of many clinically used drugs. Since this enzyme has been poorly studied in the southern part of Africa, we examined the CYP2D6 phenotypes and genotypes in 103 unrelated black Zimbabweans.Methods:Phenotyping for CYP2D6 activity was done using debrisoquine and metoprolol as probe drugs by measuring the urinary metabolic ratio (MR) of parent drug to metabolite concentration ratios. Genotyping was done using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP) and sequencing analyses with respect to CYP2D6 variants of interest.Results and conclusion:Phenotyping with debrisoquine revealed two poor metabolisers (PMs), whereas 5 subjects out of 94 were PMs using metoprolol as probe drug. Genotypes predictive of the poor metaboliser status were observed for the two subjects who were PMs with both probe drugs, whereas no mutations could explain the PM phenotype for metoprolol among the three remaining subjects, a fact possibly explained by lack of compliance in metoprolol intake. There was a moderate correlation of 0.67 between the debrisoquine and metoprolol metabolic ratios in the 89 subjects who were extensive metabolisers for both probe drugs. The median values for the metabolic ratios for debrisoquine and metoprolol as probe drugs were 1.00 and 1.35, respectively, which are higher than those observed in Caucasian populations. This is indicative of a decreased capacity for metabolism of CYP2D6 substrates by Zimbabweans compared to Caucasians. Evaluation of the DNA samples for the known allelic variants CYP2D6A, CYP2D6B, CYP2D6C,CYP2D6D or CYP2D6Ch1 yielded no explanation for these results.

Novel Metabolites of Amodiaquine Formed by CYP1A1 and CYP1B1: Structure Elucidation Using Electrochemistry, Mass Spectrometry, and NMR

An aldehyde metabolite of amodiaquine and desethylamodiaquine has been identified. The aldehyde was the major metabolite formed in incubations with two recombinantly expressed human cytochromes P450 (rP450s), namely, CYP1A1 and CYP1B1. The aldehyde metabolite was also formed, to a lesser extent, in both human and rat liver microsomes. When comparing results from incubations with liver microsomes from 3-methylcholanthrene-treated rats (inducing CYP1A1 and CYP1B1) with those from noninduced rats, a 6-fold increase of the aldehyde metabolite was observed in the rat liver microsomes after 3-methylcholanthrene treatment. The metabolic oxidation was mimicked by the electrochemical system, and the electrochemical oxidation product was matched with the metabolite from the in vitro incubations. The electrochemical generation of the aldehyde metabolite was repeated on a preparative scale, and the proposed structure was confirmed by NMR. Trapping of the aldehyde metabolite was done with methoxyl amine. Trapping experiments with N-acetyl cysteine revealed that the aldehyde was further oxidized to an aldehyde quinoneimine species, both in the rP450 incubations and in the electrochemical system. Three additional new metabolites of amodiaquine and desethylamodiaquine were formed via rCYP1A1 and rCYP1B1. Trace amounts of these metabolites were also observed in incubations with liver microsomes from 3-methylcholanthrene-treated rats. Tentative structures of the metabolites and adducts were assigned based on liquid chromatography/tandem mass spectrometry in combination with accurate mass measurements.

Electrochemical generation of electrophilic drug metabolites: characterization of amodiaquine quinoneimine and cysteinyl conjugates by MS, IR, and NMR.

The chemical reactivity of electrophilic metabolites usually prevents their detection in vivo since, by definition, they are relatively short-lived and are likely to undergo one or more structural modifications to form more stable final products. Electrochemical oxidation provides a means to generate reactive metabolites in an environment without the presence of such nucleophiles. This paper describes the results of our MS, MS/MS, NMR, IR, and computational studies on oxidation products (and conjugates) that have been generated electrochemically from the antimalarial agent amodiaquine. The electrophilic quinoneimine metabolite of amodiaquine was the major oxidation product following electrochemical oxidation at +600 mV. The absence of biological nucleophiles in the electrochemical experiment facilitated (i) the acquisition of a clean IR spectrum of the amodiaquine quinoneimine and (ii) the addition of biologically relevant nucleophiles under controlled conditions. The addition of cysteine gave four cysteinyl conjugates, while the addition of glutathione gave four glutathionyl conjugates. The product ion spectra of the conjugates formed in the electrochemical experiment were used to identify suitable fragments for selected reaction monitoring (SRM) to selectively search for these conjugates in human liver microsomal (HLM) incubations. The four cysteinyl conjugates, as well as the four glutathionyl conjugates, were also detected as metabolites in HLM. The experiment with cysteine was repeated on a preparative scale that allowed characterization of the major conjugates by (1)H NMR. Desethylamodiaquine, the major metabolite formed in human liver microsomes, was also generated electrochemically by oxidation of amodiaquine at +1200 mV followed by reduction at -800 mV. In conclusion, the EC-ESI/MS technique provides the unique opportunity to generate reactive metabolites in the absence of biological nucleophiles, which enables studies that can give insight into the nature of these reactive intermediates. Such knowledge is valuable for risk assessment of new compound classes and can be complementary to computer-based structure-activity relationships of carcinogenicity, mutagenicity, and teratogenicity.

Genetic polymorphism of cytochrome P450 1A1 (Cyp1A1) and glutathione transferases (M1, T1 and P1) among Africans.

Abstract The co-ordinate expression and regulation of the drug metabolising enzymes, cytochrome P4501A1 (CYP1A1) and glutathione transferases (GSTM1, GSTT1 and GSTP1), and their metabolic balance in the cells of target organs may determine whether exposure to carcinogens results in cancer. Besides showing variability in activity due to induction and inhibition, these enzymes also exhibit genetic polymorphism that alter enzyme levels and activity. We determined frequencies of common allelic variants of CYP1A1 and glutathione (M1, T1 and P1) among Tanzanians, South African Venda and Zimbabweans using PCR/restriction fragment length polymorphism techniques. The CYP1A1 Val462 mutant variant was found at a frequency of 1.3% among 114 subjects. The GSTM1*0 genotype was found at a frequency of 29% and 33% among Tanzanian psychiatric patients and healthy volunteers, respectively. Similarly, the GSTT1*0 polymorphism was present with a frequency of 25% in both the psychiatric patients and healthy controls. The frequency of GSTP1 Val105 variant was 16%, 12% and 21% among Tanzanians, South African Venda and Zimbabweans, respectively. We conclude here that CYP1A1 Val462 polymorphism is very rare among Africans. This is the first report of the GSTP1 Val105 variant frequency in African populations. We show here that there are no differences in frequencies of the variant alleles for CYP1A1, GSTM1, GSTT1 and GSTP1 in the three African populations.