Comparing coordination uranyl(vi) complexes with 2-(1H-imidazo[4,5-b]phenazin-2-yl)phenol and derivatives.

Four derivatives of 2-(1H-imidazo[4,5-b]phenazin-2-yl)phenol have been synthesized and characterized structurally using X-ray crystallography. Coordination complexes with uranyl (UO22+) and copper (Cu2+) were prepared and absorption/emission spectra detailed. We observed increased fluorescence upon uranyl binding, in stark contrast to rapid quenching observed with the addition of copper. These phenomena have been further examined by DFT computational methods.

Disposition of debrisoquine and nortriptyline in Korean subjects in relation to CYP2D6 genotypes, and comparison with Caucasians.

AIMS: To study the influence of the CYP2D6*10 allele on the disposition of debrisoquine and nortriptyline. METHODS: The pharmacokinetics of debrisoquine and nortriptyline and their main metabolites were determined in ten Koreans with the CYP2D6*1/*1 (n = 5) and CYP2D6*1/*10 (n = 5) genotypes after single oral doses of 20 mg debrisoquine and 25 mg nortriptyline, respectively. The data were compared with previously published findings from 21 Caucasians with 0, one, two, three, four or 13 functional CYP2D6 genes. RESULTS: The AUC0-8 of 4-hydroxydebrisoquine was significantly lower in Koreans with CYP2D6*1/*10 genotype compared with CYP2D6*1/*1[95% confidence interval (CI) for the ratio between means 1.17, 1.85]. No other genotype-related differences were found in the plasma kinetics of nortriptyline and debrisoquine, or their hydroxy metabolites. The AUCnortriptyline/AUC10-hydroxynortriptyline ratio did not differ between the *1/*1 and *1/*10 genotype groups (95% CI for the ratio of means 0.60, 1.26). Similarly, there was no difference between these genotypes with respect to the AUCdebrisoquine/AUC4-hydroxydebrisoquine ratio (95% CI for the ratio of mean values 0.38, 1.46). Both Korean genotype groups had similar AUCs and parent compound/metabolite AUC ratios of debrisoquine and nortriptyline to Caucasians with two functional CYP2D6 genes. CONCLUSIONS: Heterozygosity for CYP2D6*10 decreases the CYP2D6-dependent elimination of nortriptyline and debrisoquine to only a limited degree. Further studies in subjects homozygous for CYP2D6*10 are required to elucidate fully the pharmacokinetic consequences of this CYP2D6 genotype in Orientals.

In-vivo phenotyping for CYP3A by a single-point determination of midazolam plasma concentration.

We investigated whether a single plasma midazolam concentration could serve as an accurate predictor of total midazolam clearance, an established in-vivo probe measure of cytochrome P450 3A (CYP3A) activity. In a retrospective analysis of data from 224 healthy volunteers, non-compartmental pharmacokinetic parameters were estimated from plasma concentration-time curves following intravenous (IV) and/or oral administration. Based on statistical moment theory, the concentration at the mean residence time (MRT) should be the best predictor of the total area under the curve (AUC). Following IV or oral midazolam administration, the average MRT was found to be approximately 3.5 h, suggesting that the optimal single sampling time to predict AUC was between 3 and 4 h. Since a 4-h data point was common to all studies incorporated into this analysis, we selected this time point for further investigation. The concentrations of midazolam measured 4 h after an IV or oral dose explained 80 and 91% of the constitutive interindividual variability in midazolam AUC, respectively. The 4-h midazolam measurement was also an excellent predictor of drug-drug interactions involving CYP3A induction and inhibition. Compared with baseline values, the direction and magnitude of change in midazolam AUC and the 4-h concentration were completely concordant for all study subjects. We conclude that a single 4-h midazolam concentration following IV or oral administration represents an accurate marker of CYP3A phenotype under constitutive and modified states. Moreover, the single-point approach offers an efficient means to phenotype and identify individuals with important genetic polymorphisms that affect CYP3A activity.

Identification of functionally variant MDR1 alleles among European Americans and African Americans.

MDR1 (P-glycoprotein) is an important factor in the disposition of many drugs, and the involved processes often exhibit considerable interindividual variability that may be genetically determined. Single-strand conformational polymorphism analysis and direct sequencing of exonic MDR1 deoxyribonucleic acid from 37 healthy European American and 23 healthy African American subjects identified 10 single nucleotide polymorphisms (SNPs), including 6 nonsynonymous variants, occurring in various allelic combinations. Population frequencies of the 15 identified alleles varied according to racial background. Two synonymous SNPs (C1236T in exon 12 and C3435T in exon 26) and a nonsynonymous SNP (G2677T, Ala893Ser) in exon 21 were found to be linked (MDR1*2 ) and occurred in 62% of European Americans and 13% of African Americans. In vitro expression of MDR1 encoding Ala893 (MDR1*1 ) or a site-directed Ser893 mutation (MDR1*2 ) indicated enhanced efflux of digoxin by cells expressing the MDR1-Ser893 variant. In vivo functional relevance of this SNP was assessed with the known P-glycoprotein drug substrate fexofenadine as a probe of the transporter's activity. In humans, MDR1*1 and MDR1*2 variants were associated with differences in fexofenadine levels, consistent with the in vitro data, with the area under the plasma level-time curve being almost 40% greater in the *1/*1 genotype compared with the *2/*2 and the *1/*2 heterozygotes having an intermediate value, suggesting enhanced in vivo P-glycoprotein activity among subjects with the MDR1*2 allele. Thus allelic variation in MDR1 is more common than previously recognized and involves multiple SNPs whose allelic frequencies vary between populations, and some of these SNPs are associated with altered P-glycoprotein function.

In vivo inhibition of human CYP1A2 activity by oltipraz.

PURPOSE: Oltipraz is currently undergoing clinical evaluation as a cancer chemopreventive agent, especially with respect to aflatoxin-associated hepatocarcinogenesis. The agent's ability to induce phase II xenobiotic enzymes that detoxify the ultimate carcinogen formed in vivo is thought to be an important mechanism by which disease risk may be attenuated. However, an additional mechanism could be a reduction in the activation of environmental procarcinogens by certain cytochrome P450 (CYP) isoforms. This hypothesis was tested with respect to CYP1A2, by using the clearance of caffeine by N-demethylation as a phenotypic trait measurement of the isoform's catalytic activity. METHODS: Subjects received a single oral dose of caffeine (200 mg) on five separate occasions: on the day prior to oltipraz administration (day 0), 2 h after the first (day 1) of eight daily oral doses of oltipraz (125 mg) and 2 h after the last dose (day 8). In addition, CYP1A2 activity was also measured 2 and 14 days (days 10 and 22, respectively) after discontinuation of oltipraz administration. Plasma concentrations of caffeine and its N-demethylated metabolite, paraxanthine, over 24 h after drug administration, were determined by HPLC. RESULTS: A single 125-mg dose of oltipraz markedly reduced CYP1A2 activity by 75 +/- 13% in nine healthy subjects, resulting in a higher caffeine plasma level and prolongation of the in vivo probe's elimination half-life. Daily administration of 125 mg oltipraz for 8 days resulted in further inhibition so that only 19 +/- 13% of the original baseline level of activity was present. However, 2 days after discontinuation of oltipraz treatment, CYP1A2 activity had returned to 66 +/- 33% of its original level and complete recovery was achieved within 14 days of the chemopreventive agent being stopped. CONCLUSIONS: These results demonstrate that oltipraz is a potent, in vivo inhibitor of CYP1A2 in humans and, because this isoform is importantly involved in procarcinogen activation, they also indicate that such inhibition probably contributes to oltipraz's cancer-chemopreventive effect. In addition, the findings also suggest the likelihood of significant drug interactions between oltipraz and drugs whose metabolism is mediated by CYP1A2.

Identification and functional characterization of a new CYP2C9 variant (CYP2C9*5) expressed among African Americans.

CYP2C9 is a polymorphic gene for which there are four known allelic variants; CYP2C9*1, CYP2C9*2, CYP2C9*3, and CYP2C9*4. In the present study, DNA from 140 European Americans and 120 African Americans was examined by single-strand conformational polymorphism and restriction fragment length polymorphism analyses, resulting in the identification of a new CYP2C9 variant, CYP2C9*5. This variant is derived from a C1080G transversion in exon 7 of CYP2C9 that leads to an Asp360Glu substitution in the encoded protein. The CYP2C9*5 variant was found to be expressed only in African Americans, such that approximately 3% of this population carries the CYP2C9*5 allele. The variant was expressed in, and purified from, insect cells infected with a recombinant baculovirus. Comparative kinetic studies using the purified wild-type protein CYP2C9*1; the Ile359Leu variant, CYP2C9*3; and the Asp360Glu variant, CYP2C9*5 were carried out using (S)-warfarin, diclofenac, and lauric acid as substrates. The major effect of the Asp360Glu mutation was to increase the K(m) value relative to that of CYP2C9*1 for all three substrates: 12-fold higher for (S)-warfarin 7-hydroxylation, 5-fold higher for the 4'-hydroxylation of diclofenac, and 3-fold higher for the omega-1 hydroxylation of lauric acid. V(max) values differed less than K(m) values between the CYP2C9*1 and CYP2C9*5 proteins. In vitro intrinsic clearances for CYP2C9*5, calculated as the ratio of V(max)/K(m), ranged from 8 to 18% of CYP2C9*1 values. The corresponding ratio for CYP2C9*3 was 4 to 13%. Accordingly, the in vitro data suggest that carriers of the CYP2C9*5 allele would eliminate CYP2C9 substrates at slower rates relative to persons expressing the wild-type protein.

CYP3A activity in European American and Japanese men using midazolam as an in vivo probe.

OBJECTIVE: To investigate putative differences in CYP3A activity between European American and Japanese subjects using midazolam as an in vivo probe. METHODS: Midazolam was administered orally (2 mg) to 22 young healthy Japanese men and, on a separate occasion, to 19 of these by the intravenous route (1 mg). The disposition of the drug and its 1'-hydroxy metabolite were determined and compared with data collected in a similar fashion in 20 young healthy European American men. RESULTS: Plasma concentrations of midazolam, especially those attained soon after drug administration, were higher after intravenous injection in Japanese subjects than those in European American men. This observation was associated with smaller initial (2.5-fold) and steady-state (1.8-fold) volumes of distribution for the drug; normalization for body weight only modestly reduced these differences. The systemic clearance value of midazolam was 25% lower (P < .03) in Japanese subjects, but this difference was not apparent after accounting for the smaller body weights of that group. No statistical differences were noted in the elimination half-life (t 1/2) of midazolam between European American and Japanese subjects. Much greater interindividual variability was observed after oral administration compared with intravenous administration, but significant differences were not found between the 2 groups with respect to the maximum midazolam plasma level or its oral clearance. Absolute oral bioavailability and its associated gastrointestinal and hepatic extraction ratios also showed no statistically significant interracial differences. CONCLUSIONS: On average, hepatic CYP3A, as measured by the metabolism of midazolam, is lower in young healthy Japanese men compared with similar European Americans. However, there is considerable interindividual variability, and body size appears to be an important determinant. After oral administration, even greater variability in the plasma level-time profile of midazolam is present, and no statistically significant or clinically important interracial/ethnic difference is present. Possibly because of smaller body mass and differences in body composition, midazolam has a smaller distribution volume(s) in Japanese men than in European American men that might be an important factor when drugs are administered intravenously.

Evidence of a source of HIV type 1 within the central nervous system by ultraintensive sampling of cerebrospinal fluid and plasma.

Defining the source of HIV-1 RNA in cerebrospinal fluid (CSF) will facilitate studies of treatment efficacy in the brain. Four antiretroviral drug-naive adults underwent two 48-hr ultraintensive CSF sampling procedures, once at baseline and again beginning on day 4 after initiating three-drug therapy with stavudine, lamivudine, and nelfinavir. At baseline, constant CSF HIV-1 RNA concentrations were maintained by daily entry of at least 10(4) to 10(6) HIV-1 RNA copies into CSF. Change from baseline to day 5 ranged from -0.38 to -1.18 log(10) HIV-1 RNA copies/ml in CSF, and from -0.80 to -1.33 log(10) HIV-1 RNA copies/ml in plasma, with no correlation between CSF and plasma changes. There was no evidence of genotypic or phenotypic viral resistance in either CSF or plasma. With regard to pharmacokinetics, mean CSF-to-plasma area-under-the-curve (AUC) ratios were 38.9% for stavudine and 15.3% for lamivudine. Nelfinavir and its active M8 metabolite could not be accurately quantified in CSF, although plasma M8 peak level and AUC(0-8hr) correlated with CSF HIV-1 RNA decline. This study supports the utility of ultraintensive CSF sampling for studying HIV-1 pathogenesis and therapy in the CNS, and provides strong evidence that HIV-1 RNA in CSF arises, at least in part, from a source other than plasma.

CYP3A activity in African American and European American men: population differences and functional effect of the CYP3A4*1B5'-promoter region polymorphism.

OBJECTIVE: Cytochrome P4503A (CYP3A) activity exhibits considerable interindividual variability. Possible differences in CYP3A activity were investigated in European American and African American men with the use of midazolam as an in vivo probe. METHODS: Midazolam was simultaneously administered intravenously (1 mg, [15N3]-labeled) and orally (2 mg, unlabeled in capsule form) to 15 young healthy European American men and a similar group of men of African American descent. Plasma concentration-time curves were measured. The subjects were subsequently genotyped with respect to the CYP3A4*B1 polymorphism (A-290G) in the 5'-promoter (nifedipine-specific element) region. RESULTS: The oral bioavailability of midazolam was about equally determined by intestinal and hepatic extraction with CYP3A activity at the former site exhibiting greater variability. Oral bioavailability was related to intestinal metabolism (r = 0.98), whereas hepatic CYP3A activity contributed little to the interindividual variability (r = 0.03). A lower systemic clearance (265+/-54 versus 310+/-56 mL/min; P = .04), but not oral clearance, was observed in African Americans. With one exception, the African Americans possessed a variant CYP3A4*1B allele (4 heterozygotes A/G and 10 homozygote G/G), whereas all of the European Americans were wild-type homozygotes (A/A). Hepatic CYP3A activity and the systemic clearance of midazolam were about 30% lower in G/G homozygotes than in A/A homozygotes (252+/-53 versus 310+/-54 mL/min; P = .02), and a gene-dose effect was present (P = .01). There was no genotype/phenotype relationship with respect to the oral clearance of midazolam. CONCLUSION: Comparison of CYP3A activity between populations is complicated by frequency distribution differences in the regulatory CYP3A4*1B polymorphism and lower hepatic CYP3A activity associated with the variant allele. However, this reduction is modest; therefore no major and clinically important difference in CYP3A activity is present between Americans of African or European descent.

Pharmacological inhibition of P-glycoprotein transport enhances the distribution of HIV-1 protease inhibitors into brain and testes.

HIV protease inhibitors have proven remarkably effective in treating HIV-1 infection. However, some tissues such as the brain and testes (sanctuary sites) are possibly protected from exposure to HIV protease inhibitors due to drug entry being limited by the membrane efflux transporter P-glycoprotein, located in the capillary endothelium. Intravenous administration of the novel and potent P-glycoprotein inhibitor LY-335979 to mice (1-50 mg/kg) increased brain and testes concentration of [(14)C]nelfinavir, up to 37- and 4-fold, respectively, in a dose-dependent fashion. Similar effects in brain levels were also observed with (14)C-labeled amprenavir, indinavir, and saquinavir. Because [(14)C]nelfinavir plasma drug levels were only modestly increased by LY-335979, the increase in brain/plasma and testes/plasma ratios of 14- to 17- and 2- to 5-fold, respectively, was due to increased tissue penetration. Less potent P-glycoprotein inhibitors like valspodar (PSC-833), cyclosporin A, and ketoconazole, as well as quinidine and verapamil, had modest or little effect on brain/plasma ratios but increased plasma nelfinavir concentrations due to inhibition of CYP3A-mediated metabolism. Collectively, these findings provide "proof-of-concept" for increasing HIV protease inhibitor distribution into pharmacologic sanctuary sites by targeted inhibition of P-glycoprotein using selective and potent agents and suggest a new therapeutic strategy to reduce HIV-1 viral replication.

Allelic, genotypic and phenotypic distributions of S-mephenytoin 4'-hydroxylase (CYP2C19) in healthy Caucasian populations of European descent throughout the world.

Impaired S-mephenytoin 4'-hydroxylation is a well-described genetic polymorphism affecting drug metabolism in humans. The reported population prevalence of the CYP2C19 poor metabolizer phenotype in Caucasians of European descent has been described as ranging from 0.9% to 7.7%. To address the question of whether the difference in the frequency of poor metabolizers represents an ethnic genetic microheterogeneity in the structure and expression of the CYP2C19 gene in Caucasian individuals, we performed a pooled analysis of available studies. Combined data from the 22 homogeneous studies showed that the frequency of poor metabolizers in healthy unrelated Caucasians determined by phenotyping was 2.8% (110 of 3990; 95% confidence interval 2.3-3.3). Data obtained from eight homogeneous studies that determined the frequency of poor metabolizers by genotyping showed that the genotypic frequency of poor metabolizers was 2.1% (28 of 1356; 95% confidence interval 1.3-2.8), consistent with the poor metabolizer frequency determined by phenotyping. In the extensive metabolizers, 26% (471 of 1786; 95% confidence interval 24.4-28.4) were heterozygotes. The observed frequencies of the three Mendelian genotypes were 73% for wt/wt, 26% for wt/m, and 2.1% for m/m. Based on the overall phenotypic poor metabolizer frequency of 2.8%, the expected genotypic frequencies were 69% for wt/wt, 28% for wt/m and 2.8% for m/m, which are in good agreement to the observed values. However, in the 84 Caucasian phenotyped and genotyped poor metabolizers, approximately 10% of the putative poor metabolizer alleles (17 of 168) were unknown. This study provides a systematic overview of the population distribution of the CYP2C19 poor metabolizer phenotype and CYP2C19 alleles and genotypes in healthy Caucasians living in different geographical areas, and shows a similar polymorphic pattern in the structure and expression of the CYP2C19 gene in the worldwide Caucasian populations.

Assessment of CYP2D6 and CYP2C19 activity in vivo in humans: a cocktail study with dextromethorphan and chloroguanide alone and in combination.

OBJECTIVES: Dextromethorphan and chloroguanide (INN, proguanil) are used as prototypic phenotyping substrates of polymorphically expressed CYP2D6 and CYP2C19 in humans. We determined whether the dextromethorphan/dextrorphan and chloroguanide/cycloguanil metabolic ratios, obtained after administration of the parent drugs either alone or in combination, are equivalent. METHODS: Thirty-six healthy male volunteers received single oral doses of 80 mg dextromethorphan and 200 mg chloroguanide during a three-period, randomized crossover study. Plasma and urine were collected to calculate metabolic ratios and analyze the disposition kinetics of the probe drugs. RESULTS: All subjects were extensive metabolizers for both CYP2D6 and CYP2C19. Chloroguanide kinetics and urinary metabolic ratio were not altered after dextromethorphan administration. Dextromethorphan urinary metabolic ratio increased from -2.52 +/- 0.67 to -2.03 +/- 0.58 (P < .001) in the presence of chloroguanide. This was caused by an increase of dextromethorphan without a significant change of dextrorphan in both urine and plasma. Inhibition of CYP3A-dependent biotransformation of dextromethorphan to methoxymorphinan did not appear to be responsible for this change because the log(dextromethorphan/methoxymorphinan) urinary ratio, an index of CYP3A activity, did not significantly change during chloroguanide coadministration. The chloroguanide and dextromethorphan metabolic ratio determined from urine collection correlated with the corresponding metabolic ratio determined from plasma obtained 3 hours after oral administration. CONCLUSION: When CYP2D6 and CYP2C19 activity are assessed, dextromethorphan and chloroguanide cannot be associated in a cocktail because chloroguanide increases the dextromethorphan metabolic ratio. CYP2D6 and CYP2C19 activity can be determined from a blood sample drawn 3 hours after oral administration of dextromethorphan and chloroguanide, respectively.

Modulation by drugs of human hepatic sodium-dependent bile acid transporter (sodium taurocholate cotransporting polypeptide) activity.

Adequate bile flow, maintained in part by the efficient enterohepatic recirculation of bile acids, is critical for normal liver function. One important component of this process is the uptake of bile acids from the portal circulation into hepatocytes by the bile acid uptake transporter sodium taurocholate cotransporting polypeptide (NTCP). Thus, the expression and functional activity of this transporter may affect the rate of bile acid removal from the portal circulation. Accordingly, we assessed NTCP mRNA expression from human livers using a sensitive RNase protection assay. In addition, the ability of various bile acids and drugs to inhibit NTCP activity was determined using a recombinant vaccinia expression system. A 40-fold interindividual variability was found in NTCP mRNA levels determined in eight liver samples of disease-free donors. Expressed NTCP exhibited high-affinity, sodium-dependent uptake of taurocholate, and as expected, this was markedly inhibited by bile acids and organic anions. A number of drugs, including peptidomimetic renin inhibitors, propranolol, cyclosporin, and progesterone, were found to be potent inhibitors, whereas antiarrhythmic agents, including bupivicaine, lidocaine, and quinidine, were found to enhance NTCP activity. Accordingly, these results indicate that large interindividual variability exists in NTCP mRNA level and that a number of drugs currently in clinical use have the potential to interact with and alter NTCP activity, thereby affecting hepatic bile acid uptake.

Failure of erythromycin breath test to correlate with midazolam clearance as a probe of cytochrome P4503A.

BACKGROUND: Cytochrome P4503A (CYP3A) activity exhibits considerable interindividual variability, and an in vivo probe to measure such differences would serve several purposes. The erythromycin breath test (ERBT) is an established approach that has proven useful in this regard, but it has several limitations. More recently, the hydroxylation of midazolam has been suggested as an alternative in vivo probe approach, because it is possible to estimate CYP3A activity in the intestinal epithelium as well as in the liver. The purpose of this study was to investigate the relationship, if any, between the ERBT and midazolam's CYP3A-mediated metabolism. METHODS: Twenty healthy, medication-free young (24 to 46 years) European Americans (10 women) each received on separate days, in random order, either 3 microCi [14C-N-methyl]-erythromycin intravenously, 1 mg midazolam intravenously, or 2 mg midazolam orally. An ERBT value was determined 60 minutes after administration, and clearances were estimated after midazolam administration. In addition, an endogenous 0- to 4-hour urinary 6beta-hydroxycortisol/cortisol ratio was measured. RESULTS: All three measured drug trait values varied approximately threefold to fivefold, whereas the endogenous phenotype measure exhibited far greater variability (>100-fold). No statistically significant (P < .05) correlations existed between any of the trait values, including the ERBT value, obtained after intravenous administration of the radiolabeled probe and the systemic clearance of midazolam, expressed in terms of either total or unbound drug, or on an absolute or a body weight-corrected basis (r = 0.03 to r = 0.24; P = .08 to P = .90). Substratification according to sex generally did not improve such relationships. CONCLUSION: Although both erythromycin N-demethylation and the metabolism of midazolam by hydroxylation are mediated by CYP3A, the phenotypic trait measures associated with these two in vivo probe drugs do not provide the same information about the catalytic activity of the enzyme. An indirect measure such as the ERBT may reflect CYP3A activity and be useful for some purposes, but the estimation of the oral and intravenous clearance of midazolam has additional advantages, and they may be more applicable and have broader usefulness as quantitative estimates of CYP3A activity.

Genetic polymorphism of (S)-mephenytoin 4'-hydroxylation in populations of African descent.

AIMS: The frequency of CYP2C19 poor metabolizers (PMs) in populations of African descent has been reported to range from 1.0% to 35.4%. In order to determine with greater certainty the frequency of CYP2C19 PMs in such black populations we have performed a meta-analysis of the studies. METHODS: Relevant data on the frequency of both the PM phenotype of probe drugs (mephenytoin, omeprazole, and proguanil), and the distribution frequencies of CYP2C19 alleles and genotypes in black populations were summarized and reanalysed using a meta-analytical approach. RESULTS: Of nine reported studies two were excluded because of significant heterogeneity (chi2=115, P<0.0001). The combined data from the remaining seven studies showed that the frequency of the PM phenotype in 922 healthy unrelated black Africans and black Americans ranged from 1.0% to 7.5% (n=7 for combined data) with an overall frequency being 3.9% (36 of 922; 95%CI: 2.7%-5.2%). The frequency of the PM genotypes in blacks was 3.7% (36 of 966; 95%CI: 2.5%-4.9%), in agreement with the frequency of the PM phenotype. In the extensive metabolizers (EMs) 29% (271 of 930) were heterozygotes (wt/m ). The observed frequencies of the three Mendelian genotypes were 0.68 for wt/wt, 0.28 for wt/m, and 0.04 for m/m. The allelic distribution was appropriate at 82.3% (95%CI: 80.5%-83.9%) for CYP2C19*1, 17.3% (95%CI:15.7%-19.0%) for CYP2C19*2 (m1 ), and 0.4% (95%CI: 0.1%-0.7%) for CYP2C19*3 (m2 ) in these populations. CONCLUSIONS: We conclude that subjects of African ancestry have a low frequency of the CYP2C19 PM phenotype and genotype; that the defective CYP2C19 alleles are uncommon, and that a small proportion of heterozygotes exists in the EM subpopulation.

P-glycoprotein and cytochrome P-450 3A inhibition: dissociation of inhibitory potencies.

Many P-glycoprotein (P-gp) inhibitors studied in vitro and in vivo are also known or suspected to be substrates and/or inhibitors of cytochrome P-450 3A (CYP3A). Such overlap raises the question of whether CYP3A inhibition is an intrinsic characteristic of P-gp inhibitors, a matter of concern in the development and rational use of such agents. Thus, the purpose of the present study was to determine whether the ability to inhibit P-gp and CYP3A is, in fact, linked and whether specific P-gp inhibitors with limited ability to inhibit CYP3A can be identified. Therefore, the potency of a series of 14 P-gp inhibitors was assessed by measuring their inhibition of the transepithelial flux across Caco-2 cells of digoxin, a prototypical P-gp substrate. CYP3A inhibition was determined from the impairment of nifedipine oxidation by human liver microsomes. Determination of the apparent Ki values for CYP3A inhibition and the IC50s for P-gp and CYP3A inhibition allowed comparison of the relative inhibitory potency of the compounds on the two proteins' function. The IC50s for P-gp inhibition ranged from 0.04 to 3.8 microM. All compounds inhibited CYP3A with apparent Ki values of between 0.3 and 76 microM and IC50s between 1.5 and 50 microM. However, no correlation was found between the extent of P-gp inhibition and CYP3A inhibition, and the ratio of the IC50 for CYP3A inhibition to the IC50 for P-gp inhibition varied from 1.1 to 125. These results demonstrate that, although many P-gp inhibitors are potent inhibitors of CYP3A, a varying degree of selectivity is present. The development and use of P-gp inhibitors with minimal or absent CYP3A inhibitory effects should decrease the impact of drug interactions on the therapeutic use of such compounds.

OATP and P-glycoprotein transporters mediate the cellular uptake and excretion of fexofenadine.

Fexofenadine, a nonsedating antihistamine, does not undergo significant metabolic biotransformation. Accordingly, it was hypothesized that uptake and efflux transporters could be importantly involved in the drug's disposition. Utilizing a recombinant vaccinia expression system, members of the organic anion transporting polypeptide family, such as the human organic anion transporting polypeptide (OATP) and rat organic anion transporting polypeptides 1 and 2 (Oatp1 and Oatp2), were found to mediate [(14)C]fexofenadine cellular uptake. On the other hand, the bile acid transporter human sodium taurocholate cotransporting polypeptide (NTCP) and the rat organic cation transporter rOCT1 did not exhibit such activity. P-glycoprotein (P-gp) was identified as a fexofenadine efflux transporter, using the LLC-PK1 cell, a polarized epithelial cell line lacking P-gp, and the derivative cell line (L-MDR1), which overexpresses P-gp. In addition, oral and i.v. administration of [(14)C]fexofenadine to mice lacking mdr1a-encoded P-gp resulted in 5- and 9-fold increases in the drug's plasma and brain levels, respectively, compared with wild-type mice. Also, a number of drug inhibitors of P-gp were found to be effective inhibitors of OATP. Because OATP transporters and P-gp colocalize in organs of importance to drug disposition such as the liver, their activity provides an explanation for the heretofore unknown mechanism(s) responsible for fexofenadine's disposition and suggests potentially similar roles in the disposition of other xenobiotics.

A novel transversion in the intron 5 donor splice junction of CYP2C19 and a sequence polymorphism in exon 3 contribute to the poor metabolizer phenotype for the anticonvulsant drug S-mephenytoin.

Cytochrome P-450 (CYP) 2C19 is responsible for the metabolism of a number of therapeutic agents such as S-mephenytoin, omeprazole, proguanil, certain barbiturates, diazepam, propranolol, citalopram and imipramine. Genetic polymorphisms in this enzyme are responsible for the poor metabolizers (PM) of mephenytoin, which represent approximately 13-23% of Asians and 3-5% of Caucasians. Several polymorphisms contribute to this phenotype. We have isolated two new allelic variants that contribute to the PM phenotype in Caucasians. CYP2C19*7 contained a single T --> A nucleotide transversion in the invariant GT at the 5' donor splice site of intron 5. The second PM allele, CYP2C19*8, consisted of a T358C nucleotide transition in exon 3 that results in a Trp120Arg substitution. In a bacterial expression system, CYP2C198 protein exhibited a dramatic (approximately 90% and 70%) reduction in the metabolism of S-mephenytoin and tolbutamide, respectively, when compared with the wild-type CYP2C191B protein. Restriction fragment length polymerase chain reaction tests were developed to identify the new allelic variants.

Genetic and dietary predictors of CYP2E1 activity: a phenotyping study in Hawaii Japanese using chlorzoxazone.

Cytochrome P4502E1 (CYP2E1) is considered to play an important role in the metabolic activation of procarcinogens such as N-nitrosoamines and low molecular weight organic compounds. An RsaI polymorphism is present in the 5'-flanking region of the CYP2E1 gene, which could possibly affect its transcription. However, the relationship between genotype and the phenotypic catalytic activity of the enzyme has not been defined. Also, the effects in humans of specific dietary factors, other than ethanol, which have been shown in animal and in vitro studies to modulate CYP2E1 activity, are unknown. Accordingly, the CYP2E1-mediated metabolism of chlorzoxazone to its 6-hydroxy metabolite was investigated in 50 healthy Japanese of both sexes in Hawaii. The oral clearance of the in vivo probe, the trait measure of CYP2E1 activity, was smaller than that reported in European-Americans. Significantly, after adjustment for age and sex, the oral clearance of chlorzoxazone decreased with the number of variant c2 alleles, and its mean in the c2/c2 genotype (147 ml/min) was statistically lower (P < or = 0.05) than that for either the homozygous wild-type (238 ml/min) or the heterozygote (201 ml/min) genotypes. Stepwise multiple regression analysis indicated that body weight was a major contributor to the interindividual variability in the oral clearance of chlorzoxazone, accounting for 43% of the variance. Consumption of lettuce, broccoli, and black tea explained additional components of the variability (7, 5, and 6%, respectively), as did medication use (3%), age (4%), and CYP2E1 genotype (5%). Overall, 73% of the variance could be accounted for by these variables. Body weight, lettuce, and use of medications were associated with increased CYP2E1 activity, and the other covariates were associated with reduced enzyme function. Because of the role that CYP2E1 plays in procarcinogen activation, especially of N-nitrosamines involved in lung cancer, the identified factors may account in part for observed differences in individual susceptibility to disease and may also have implications for cancer prevention.

Interrelationship between substrates and inhibitors of human CYP3A and P-glycoprotein.

PURPOSE: CYP3A and P-gp both function to reduce the intracellular concentration of drug substrates, one by metabolism and the other by transmembrane efflux. Moreover, it has been serendipitously noted that the two proteins have many common substrates and inhibitors. In order to test this notion more fully, systematic studies were undertaken to determine the P-gp-mediated transport and inhibitory characteristics of prototypical CYP substrates. METHODS: L-MDR1, LLC-PK1, and Caco-2 cells were used to evaluate established CYP substrates as potential P-gp substrates and inhibitors in vitro, and mdr1a deficient mice were used to assess the in vivo relevance of P-gp-mediated transport. RESULTS: Some (terfenadine, erythromycin and lovastatin) but not all (nifedipine and midazolam) CYP3A substrates were found to be P-gp substrates. Except for debrisoquine, none of the prototypical substrates of other common human CYP isoforms were transported by P-gp. Studies in mdr1a disrupted mice confirmed that erythromycin was a P-gp substrate but the CYP3A-inhibitor ketoconazole was not. In addition, CYP3A substrates and inhibitors varied widely in their ability to inhibit the P-gp-mediated transport of digoxin. CONCLUSIONS: These results indicate that the overlap in substrate specificities of CYP3A and P-gp appears to be fortuitous rather than indicative of a more fundamental relationship.