Influence of CYP3A5*3 and ABCB1 C3435T on clinical outcomes and trough plasma concentrations of imatinib in Nigerians with chronic myeloid leukaemia.

WHAT IS KNOWN AND OBJECTIVE: Imatinib mesylate is the first-line drug for the treatment of Philadelphia/bcr-abl positive chronic myeloid leukaemia (CML). It is known to be metabolized mostly by CYP3A4 and CYP3A5 isoforms while its efflux is mediated by the transporters ABCB1 and ABCG2. Genetic polymorphism of some of these enzymes and transporters have been linked with inter-individual variations in the pharmacokinetics of the drug. This study, therefore, investigated the influence of CYP3A5*3, ABCG2 421C>A and ABCB1 3435 C>T genetic polymorphism on the clinical outcome and steady-state trough plasma concentration (TPC) of imatinib in Nigerians with CML. METHODS: A total of 110 Nigerians with CML each of whom had been receiving a 400 mg daily dose of imatinib for at least 1 month were genotyped for CYP3A5*3, ABCG2 421C>A and ABCB1 3435 C>T. The TPC of all the patients were determined by a validated HPLC method and possible relationships between genotypes, age, clinical outcome, sex, TPC and ethnicity were analysed. RESULTS AND DISCUSSION: Subjects of TT genotype of ABCB1 C3435T had higher frequencies of complete haematological response (CHR), complete cytogenetic response (CCR) and major molecular response (MMR) but these were not statistically significant (P < 0·05). No genetic polymorphism in ABCG2 421C>A was observed. However, significant associations were observed between TPC and various genotypes in both CYP3A5*3 (P < 0·001) and ABCB1 C3435T (P < 0·001). The GG and TT genotypes in CYP3A5*3 and ABCB1 C3435T, respectively, were linked with higher TPC. WHAT IS NEW AND CONCLUSION: This is the first pharmacogenetics study of CML patients in the Nigerian population with ethnic differences in the distribution of ABCB1 C3435T. Genetic polymorphisms in CYP3A5*3 and ABCB1 C3435T are associated with TPC in CML patients in this population.

Building Capability for Clinical Pharmacology Research in Sub-Saharan Africa.

A strong scientific rationale exists for conducting clinical pharmacology studies in target populations because local factors such as genetics, environment, comorbidities, and diet can affect variability in drug responses. However, clinical pharmacology studies are not widely conducted in sub-Saharan Africa, in part due to limitations in technical expertise and infrastructure. Since 2012, a novel public-private partnership model involving research institutions and a pharmaceutical company has been applied to developing increased capability for clinical pharmacology research in multiple African countries.

Clinical, Virologic, Immunologic Outcomes and Emerging HIV Drug Resistance Patterns in Children and Adolescents in Public ART Care in Zimbabwe.

OBJECTIVE: To determine immunologic, virologic outcomes and drug resistance among children and adolescents receiving care during routine programmatic implementation in a low-income country. METHODS: A cross-sectional evaluation with collection of clinical and laboratory data for children (0-<10 years) and adolescents (10-19 years) attending a public ART program in Harare providing care for pediatric patients since 2004, was conducted. Longitudinal data for each participant was obtained from the clinic based medical record. RESULTS: Data from 599 children and adolescents was evaluated. The participants presented to care with low CD4 cell count and CD4%, median baseline CD4% was lower in adolescents compared with children (11.0% vs. 15.0%, p<0.0001). The median age at ART initiation was 8.0 years (IQR 3.0, 12.0); median time on ART was 2.9 years (IQR 1.7, 4.5). On ART, median CD4% improved for all age groups but remained below 25%. Older age (≥ 5 years) at ART initiation was associated with severe stunting (HAZ <-2: 53.3% vs. 28.4%, p<0.0001). Virologic failure rate was 30.6% and associated with age at ART initiation. In children, nevirapine based ART regimen was associated with a 3-fold increased risk of failure (AOR: 3.5; 95% CI: 1.3, 9.1, p = 0.0180). Children (<10 y) on ART for ≥4 years had higher failure rates than those on ART for <4 years (39.6% vs. 23.9%, p = 0.0239). In those initiating ART as adolescents, each additional year in age above 10 years at the time of ART initiation (AOR 0.4 95%CI: 0.1, 0.9, p = 0.0324), and each additional year on ART (AOR 0.4, 95%CI 0.2, 0.9, p = 0.0379) were associated with decreased risk of virologic failure. Drug resistance was evident in 67.6% of sequenced virus isolates. CONCLUSIONS: During routine programmatic implementation of HIV care for children and adolescents, delayed age at ART initiation has long-term implications on immunologic recovery, growth and virologic outcomes.

Genetic polymorphism of cytochrome P450 CYP2D6 in Zimbabwean population.

The objective of this study was to determine the CYP2D6 genotype of a black Zimbabwean population. Genotyping was carried out using Eco RI and Xba I RFLP, and allele-specific PCR amplification. Of 114 Zimbabwean samples analysed, no individual homozygous for any of the defect allelic forms CYP2D6A, CYP2D6B or CYP2D6D or combinations thereof was found. The allele frequencies of the three defect genes were 0, 1.8 and 3.9%, respectively. No subject carrying the Xba I 44 kb haplotype, indicative for poor metabolizers among Caucasians, was identified, whereas five individuals being heterozygous with a 29/42 kb haplotype were seen. Three out of the four CYP2D6B alleles found were associated with the 29/42 kb haplotype. Our findings are in agreement with the 0-2% prevalence of poor metabolizers (PMs) in the black populations previously phenotyped. The very low frequency of the CYP2D6B allele in the Zimbabwean population is different from very recent data from black Americans (allele frequency = 8.5%) and might indicate the Caucasian ancestry of this allele. Taken together, our data indicate important interethnic differences in the CYP2D locus between Caucasian, Asian and different black populations.

Low CYP1A2 activity in rural Shona children of Zimbabwe.

Caffeine is increasingly used as a biochemical probe for liver function, in cancer epidemiology, and in pharmacogenetics, with its recognized ability to assess the activities of CYP1A2, xanthine oxidase, and N-acetyltransferase-2. The activity of these hepatic enzymes was tested in 45 Shona children from a rural area of Zimbabwe with use of caffeine as a probe. Many of these rural black children had lower indexes of CYP1A2 activity than otherwise on our extensive records; the average value (3.78 +/- 2.9) was significantly (p < 0.001) lower than that of healthy white urban children from Zimbabwe (8.86 +/- 3.36) or from Canada (7.92 +/- 1.88), or that of healthy Canadian adults (5.96 +/- 2.4). A higher CYP1A2 activity in children than in adults is usual. The low CYP1A2 activity of the children from rural Zimbabwe calls for medical studies and suggests a widespread and perhaps serious impairment of certain liver functions. Causes could be parasitic infections with Schistosoma mansoni, causing schistosomiasis, which are endemic, in addition to generally poor nutrition and frequent iodine deficiency. By contrast, the xanthine oxidase activity in rural Shona children was slightly higher than that reported for a healthy Canadian adult population. The N-acetyltransferase activities were comparable in both the rural and urban children and were also similar to those reported in a population study of healthy adult Canadians.

Phenotyping and genotyping of S-mephenytoin hydroxylase (cytochrome P450 2C19) in a Shona population of Zimbabwe.

The S-mephenytoin hydroxylase has recently been identified as cytochrome P450 2C19 (CYP2C19). This enzyme metabolizes mephenytoin, diazepam, omeprazole, and citalopram and has been shown to be polymorphically distributed. One clinical implication of CYP2C19-dependent drug metabolism for persons who reside in tropical regions is in the use of the antimalarial drug chloroguanide hydrochloride, which is apparently biotransformed to its active metabolite by this isozyme. In this investigation we studied mephenytoin metabolism in 103 black Zimbabwean Shona subjects. Four were identified as poor metabolizers (4%). This prevalence is comparable to that in white subjects (2% to 5%) but lower than the 15% to 20% incidence of poor metabolizers among Oriental subjects. Of the subjects phenotyped, 84 were genotyped for the G-->A mutation in exon 5 of CYP2C19, which creates a cryptic splice site, causing the production of a nonfunctional protein. Three of the four poor metabolizers were homozygous for this mutation, whereas the fourth one was heterozygous. The G-->A mutation has been shown to predict the incidence more than 60% of poor metabolizers among white subjects and Japanese subjects, and in the current investigation we also obtained a similar relationship in the black population.

Characterisation of praziquantel metabolism by rat liver microsomes using cytochrome P450 inhibitors.

The metabolism of praziquantel (PZQ) was studied in microsomes isolated from livers of differently pretreated rats and in the presence of various inhibitors of cytochrome P450 (P450) isoforms. Microsomes from phenobarbitone (PB)-pretreated rats metabolised PZQ to its major metabolite 4OH-praziquantel (4OH-PZQ) at a greater rate than those from 20-methylcholanthrene (MC) and saline (SA) pretreated rats. The Vmax for the PB microsomes was 600 nmol 4OH-PZQ formed/mg/min x 10(-3) compared to 91.4 nmol/mg/min x 10(-3) for MC and 238 nmol/mg/min x 10(-3) for SA microsomes. These results indicate that PZQ is metabolised by PB-inducible isoforms of P450. Inhibitor studies were conducted with microsomes from SA-pretreated animals. In these studies, caffeine, disulfuram, and tolbutamide were poor inhibitors of the metabolism of PZQ to 4OH-PZQ, with I50 values not determinable. Quinidine and quinine inhibited the hydroxylation of PZQ but with high Ki values. 17 alpha-Ethynylestradiol, cimetidine and diphenylhydramine were effective inhibitors of the formation of 4OH-PZQ, with 17 alpha-ethynylestradiol being the most potent with a Ki of 0.5 +/- 0.05 microM. From the known specificities of these P450 inhibitors, it is therefore concluded that cytochromes P450 1A2, 2E1, 2C9-10, and 2D6 probably do not contribute significantly to the metabolism of PZQ to its major metabolite in rats. It is likely that cytochromes P450 2B1 and 3A, both inducible by PB, are predominantly responsible for the formation of 4OH-PZQ.

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 compound's 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.

Molecular epidemiology of Plasmodium falciparum antifolate resistance in Vietnam: genotyping for resistance variants of dihydropteroate synthase and dihydrofolate reductase.

Using PCR techniques, we analysed the dihydropteroate synthase (DHPS) mutations associated with sulphonamide resistance and the dihydrofolate reductase (DHFR) mutations associated with resistance to pyrimethamine and cycloguanil in samples from Plasmodium falciparum-infected Vietnamese patients. Of the 40 samples analysed, 39 had DHFR mutations associated with high level resistance to pyrimethamine, whereas only three had mutations at position 164, which is linked to cross resistance to both DHFR inhibitors. The DHPS, 437Gly variant associated with very mild resistance to sulphadoxine was found in 38 out of the 40 samples. Of seven samples resistant to Fansidar in vivo, only two were fully explained by the currently documented DHPS mutations. The treatment failure could be due to a high level of pyrimethamine resistance caused by the detected mutations. Most patients, however, were cured with a single dose of Fansidar in spite of the high number of resistance mutations found.

Genetic polymorphism of drug metabolising enzymes in African populations: implications for the use of neuroleptics and antidepressants.

Metabolism of most drugs influences their pharmacological and toxicological effects. Drugs particularly affected are those with a narrow therapeutic window and that are subjected to considerable first-pass metabolism. Much of the interindividual and interethnic differences in effects of drugs is now attributable to genetic differences in their metabolism. Genetic polymorphisms have been described for many drug-metabolising enzymes in Caucasian and Oriental populations, the most well-characterised being those for cytochrome P450 2D6, cytochrome P450 2C19, glutathione S-transferases, and N-acetyl transferase 2. African populations have been studied to a lesser extent, but it is apparent that populations within Africa are heterogeneous with respect to these polymorphisms. In addition, although some allelic variants are common to all populations throughout the world (e.g., CYP2D6*5), some allelic variants are specific for an African population (e.g., CYP2D6*17). The polymorphisms give rise to enzymes with changed or no activity towards drug substrates. Two of the most important enzymes for metabolism of neuroleptics and other psychoactive drugs are CYP2D6 and CYP2C19. This article compares the current information on polymorphisms of these two enzymes in African and other populations and discusses the implications of these polymorphisms for neuropharmacotherapy.

Phenotyping of the glutathione S-transferase M1 polymorphism in Zimbabweans and the effects of chloroquine on blood glutathione S-transferases M1 and A.

The frequency of the null allele phenotype of glutathione S-transferase (GST) M1 was investigated in 114 Zimbabweans and results for a subset of 63 subjects were compared with genotyping by PCR. In addition, the effect of the antimalarial chloroquine on blood levels of GSTM1 and GSTA in 19 subjects was studied. Quantification of GSTs was by enzyme linked immunosorbent assays (ELISA). Thirty percent of the subjects were of the GSTM1 null phenotype. Comparison of results of phenotyping by ELISA and genotyping by PCR showed that 16% of samples were in discordance; unknown mutations in the GSTM1 gene in the Zimbabwean population may explain this observation. Chloroquine decreased levels of blood GSTM1 and GSTA by 50% or more. In populations treated with chloroquine, these decreases in GST activities might lead to compromised ability to detoxify xenobiotics, could confound GSTM1 phenotyping and might invalidate use of GSTA as an indicator of liver damage.

Monolithic silica rod liquid chromatography with ultraviolet or fluorescence detection for metabolite analysis of cytochrome P450 marker reactions.

In vitro cytochrome P450 assays are used in metabolism studies in support of early phases of drug discovery to investigate, e.g., metabolic stability, enzyme inhibition and induction by new chemical entities. LC-UV and LC-fluorescence are traditional analytical tools in support of such studies. However, these tools typically comprise different methods of relatively low throughput for the various metabolites of probe reactions. In recent years, LC-MS methods have been developed to increase throughput. Increased throughput can also be achieved by means of modern chromatographic tools in combination with UV and fluorescence detection. This approach is especially suitable when cytochrome P450 isoforms are investigated by means of single probe incubations. Here, an LC-UV/fluorescence system based on a monolithic porous silica column is described for the analysis of metabolites of nine cytochrome P450 marker reactions [phenacetin to paracetamol (CYP1A2), coumarin to 7-hydroxycoumarin (CYP2A6), paclitaxel to 6alpha-hydroxypaclitaxel (CYP2C8), diclofenac to 4-hydroxydiclofenac (CYP2C9), mephenytoin to 4-hydroxymephenytoin (CYP2C19), bufuralol to 1-hydroxybufuralol (CYP2D6), chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1), midazolam to 1-hydroxymidazolam (CYP3A4), and testosteron to 6beta-hydroxytestosteron (CYP3A4)]. While offering sensitivities and linear ranges comparable to previously reported methods, the set-up described here provides ease of use and increased throughput with maximum cycle times of 4.5 min.

Effects of phenobarbital and 3-methylcholanthrene pretreatment on the pharmacokinetics of praziquantel in rats.

The effects of phenobarbital (PB) and 3-methylcholanthrene (MC) pretreatment on the pharmacokinetics of praziquantel (PZQ), a schistosomicide were studied in Sprague-Dawley rats. Blood samples at different time intervals were obtained by severing the tail vein and were analyzed for unchanged PZQ by HPLC. The PB-pretreated rats showed a 6-fold decrease in AUC, a 5-fold decrease in Cmax and an 8-fold increase in CLtot compared to the saline treated controls. The MC-pretreated rats and their olive-oil treated controls did not show any statistically significant differences in the above parameters. These results suggest that PZQ is extensively metabolised by PB-inducible cytochrome P-450 isoforms and not by MC-inducible isoforms. These findings also suggest that the bioavailability of praziquantel could be altered to a significant extent in humans taking drugs that are phenobarbital type inducers.

Inhibitory effects of antiparasitic drugs on cytochrome P450 2D6.

The interaction of antiparasitic drugs with the polymorphic cytochrome P450 2D6 was studied in human liver microsomes. Of ten different drugs tested, three quinolines, oxamniquine, primaquine and chloroquine inhibited microsomal CYP2D6-catalysed formation of 1'hydroxybufuralol at concentrations that might have clinical consequences in drug use. These drugs inhibited competitively bufuralol metabolism with Ki values of 22, 23 and 15 microM, respectively, indicative of high affinity for the CYP2D6-active site. The results imply that oxamniquine, primaquine and chloroquine could be substrates of cytochrome P4502 D6 or that they are potent non-substrate inhibitors of the enzyme similar to quinidine. In either case, the inhibition of CYP2D6 by these agents could lead to interference with in vivo population-phenotyping procedures in the tropical regions where treatment with the drugs is common.

The effect of chloroquine on the pharmacokinetics and metabolism of praziquantel in rats and in humans.

It is likely that a proportion of people treated with the anti-schistosomicidal drug praziquantel (PZQ) is also taking other drugs such as chloroquine (CHQ), a widely used anti-malarial. The effect of CHQ on the pharmacokinetics and metabolism of PZQ in rats and in humans was therefore studied. CHQ decreased the bioavailability of PZQ and reduced its maximum serum concentrations to a significant extent in rats and in humans. The clearance was increased to a statistically significant extent in rats but not in humans because of the wide interindividual variation. The effect of CHQ on PZQ pharmacokinetics was unexpected since drugs that inhibit hepatic drug metabolism usually increase the bioavailability of PZQ. We found that CHQ inhibits non-competitively the metabolism of PZQ to its major metabolite, 4-hydroxy-praziquantel, with a Ki of 1.65 mM in rat hepatic microsomes. Maximum concentrations attained by CHQ in serum, however, are low compared to the Ki value and significant inhibition is therefore unlikely in vivo. The explanation for CHQ's effect on the pharmacokinetics of PZQ may be due to other effects of CHQ rather than to a direct effect on drug-metabolizing enzymes.

Application of higher throughput screening (HTS) inhibition assays to evaluate the interaction of antiparasitic drugs with cytochrome P450s.

In this study we have evaluated the application and reliability of using fluorescence (FLUO)-based high throughput screening assays with recombinant CYPs (rCYP). This was accomplished by screening 29 clinically important antiparasitic drugs for inhibition of the five major drug-metabolizing CYPs (-1A2, -2C9, -2C19, -2D6, and -3A4). Data from FLUO/rCYP assays were compared with that obtained by conventional HPLC assays using human liver microsomes (HLM) and rCYPs. The K(i) values showed good correlations: FLUO/rCYP and HPLC/rCYP (r(2) = 0.81), HPLC/rCYP and HPLC/HLM (r(2) = 0.82), and FLUO/rCYP and HPLC/HLM (r(2) = 0.72). Niclosamide had substrate-dependent contrasting effects on CYP2C9 activity with an apparent activation (400%) of 7-methoxy-4-trifluoromethylcoumarin demethylase activity and potent inhibition (K(i) = 6.00 microM) of diclofenac 4-hydroxylase activity. Potent inhibitors of CYP1A2 were artemisinin, dihydroartemisinin, thiabendazole, primaquine, and niclosamide (K(i) = 0.43, 3.67, 1.54, 0.22, and 2.70 microM, respectively). Proguanil, cycloguanil, amodiaquine, and desethylamodiaquine inhibited CYP2D6 (K(i) = 6.76, 5.97, 2.1, and 4.13 microM, respectively). Considering the C(max) of these drugs, artemisinin, thiabendazole, primaquine, amodiaquine, and desethylamodiaquine may cause clinically important interactions because they are predicted to inhibit 67 to 99% of the activities of the CYPs they interact with. In addition, our results suggest CYP1A2 inhibition as the mechanism behind the observed thiabendazole/theophylline and primaquine/antipyrine interactions in vivo.

A novel mutant variant of the CYP2D6 gene (CYP2D6*17) common in a black African population: association with diminished debrisoquine hydroxylase activity.

1. The debrisoquine hydroxylase (CYP2D6) is polymorphically distributed. Not only are there differences in the proportions of extensive metabolisers to poor metabolisers in various ethnic groups, but there are also pronounced variations in the metabolic capacity among those classified as extensive metabolisers. 2. The mean debrisoquine metabolic ratio of Caucasian extensive metabolisers is lower than that for a number of African populations. In the present study, we have searched for novel CYP2D6 mutations to explain the diminished enzyme activity in African populations. 3. Three Zimbabwean Shona subjects with EM phenotypes (metabolic ratios for debrisoquine of 0.4, 1.5 and 10.5 respectively) were selected and the open reading frame of the CYP2D6 gene of each was sequenced. 4. The subject with metabolic ratio of 10.5 was found to be homozygous for an allele with a nucleotide exchange in exon 2, 1111C-->T causing a 107Thr-->Ile amino acid exchange in a conserved region of the enzyme. In addition, he was homozygous for the 2938C-->T and 4268G-->C mutations causing 296Arg-->Ser and 486Ser-->Thr amino acid substitution found in the CYP2D6*2 allele. 5. Seventy-six Zimbabwean Shona subjects were subsequently genotyped for the 1111C-->T mutation and for the intron 1 gene conversion present in the CYP2D6*2 gene. The 1111C-->T mutation was found at an allele frequency of 34% and was only present in alleles carrying the gene conversion in intron 1 indicative for the CYP2D6*2 gene. 6. This allele (CYP2D6*17), containing the 1111C-->T, 2938C-->T and 4268G-->C mutations, was found to be strongly associated with lower capacity for debrisoquine hydroxylation. We therefore postulate that the CYP2D6*17 allele might contribute to the molecular basis of the previously established diminished debrisoquine hydroxylase activity in African Bantu populations.

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 responsble 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.

Arginines 97 and 108 in CYP2C9 are important determinants of the catalytic function.

Human cytochrome P450 2C9 (CYP2C9) is one of the major drug metabolising enzymes which exhibits a broad substrate specificity. The B-C loop is located in the active-site but has been difficult to model, owing to its diverse and flexible structure. To elucidate the function of the B-C loop we used homology modelling based on the Cyp102 structure in combination with functional studies of mutants using diclofenac as a model substrate for CYP2C9. The study shows the importance of the conserved arginine in position 97 and the arginine in position 108 for the catalytic function. The R97A mutant had a 13-fold higher K(m) value while the V(max) was in the same order as the wild type. The R108 mutant had a 100-fold lower activity with diclofenac compared to the wild-type enzyme. The other six mutants (S95A, F100A, L102A, E104A, R105A, and N107A) had kinetic parameters similar to the CYP2C9 wild-type. Our homology model based on the CYP102 structure as template indicates that R97, L102, and R105 are directed into the active site, whereas R108 is not. The change in catalytic function when arginine 97 was replaced with alanine and the orientation of this amino acid in our homology model indicates its importance for substrate interaction.