Transfection and functional expression of CYP4A1 and CYP4A2 using bicistronic vectors in vascular cells and tissues

20-hydroxyeicosatetraenoic acid (20-HETE), a CYP4A-derived arachidonic acid metabolite, is a potent vasoconstrictor and a modulator of vascular reactivity. We have shown that CYP4A1 and CYP4A2 are the major CYP4A isoforms expressed in the rat renal microcirculation. In the present study, we constructed two bicistronic vectors, pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2, and examined their functional efficacy in COS-1 and vascular smooth muscle (A7r5) cells and in microdissected rat interlobar arteries. Immunocytochemistry coupled with fluorescence microscopy of pIRES2-EGFP-4A1- or pIRES2-EGFP-4A2-transfected COS-1 and A7r5 cells indicated that both enhanced green fluorescence protein (EGFP) and CYP4A1/4A2 were expressed in 80 to 90% of the cells. Western blot analysis showed a 3- to 5-fold increase of CYP4A1 and CYP4A2 proteins in pIRES2-EGFP-4A1- and pIRES2-EGFP-4A2-transfected cells as compared with control pIRES2-transfected cells. Cells transfected with pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2 catalyzed arachidonic acid omega-hydroxylation to 20-HETE at rates of 0.85 +/- 0.29 and 0.27 +/- 0.04 nmol/10(7) cells/h, respectively. Transfection of interlobar arteries with either plasmid yielded EGFP immunofluorescence that was localized to the intima, media, and adventitia. Arteries transfected with pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2 showed increased vasoreactivity displaying EC50 to phenylephrine of 0.24 +/- 0.07 and 0.11 +/- 0.03 microM, respectively, as compared with arteries transfected with pIRES2-EGFP (1.11 +/- 0.21 microM; n=6, p <0.05). The increased vasoreactivity to phenylephrine was inhibited by N-methylsulfonyl-12,12-dibromododec-11-enamide, an inhibitor of CYP4A-catalyzed reactions, suggesting that a product of CYP4A1 and CYP4A2 catalytic activity contributed to the increased constrictor responsiveness. Removal of the endothelium did not prevent the sensitization to phenylephrine in vessels transfected with the plasmid containing the CYP4A1 cDNA, suggesting that the CYP4A product responsible for the sensitizing effect, presumably 20-HETE, is not of endothelial cell origin.     

Role of hepatic and intestinal cytochrome P450 3A and 2B6 in the metabolism, disposition, and miotic effects of methadone

BACKGROUND: The disposition of the long-acting opioid methadone, used to prevent opiate withdrawal and treat short- and long-lasting pain, is highly variable. Methadone undergoes N -demethylation to the primary metabolite 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium (EDDP), catalyzed in vitro by intestinal, hepatic, and expressed cytochrome P450 (CYP) 3A4. However, the role of CYP3A4 in human methadone disposition in vivo is unclear.This investigation tested the hypothesis that CYP3A induction (or inhibition) would increase (or decrease) methadone metabolism and clearance in humans. METHODS: Healthy volunteers were studied in a randomized, balanced, 4-way crossover study. They received intravenous (IV) midazolam (to assess CYP3A4 activity) and then simultaneous oral deuterium-labeled and IV unlabeled methadone after pretreatment with rifampin (INN, rifampicin) (hepatic/intestinal CYP3A induction), troleandomycin (hepatic/intestinal CYP3A inhibition), grapefruit juice (selective intestinal CYP3A inhibition), or nothing. Methadone effects were measured by dark-adapted pupil diameter. CYP isoforms catalyzing methadone metabolism by human liver microsomes and expressed CYPs in vitro were also evaluated. RESULTS: Methadone had high oral bioavailability (70%) and low intestinal (22%) and hepatic (9%) extraction, and there was a significant correlation ( r = 0.94, P <.001) between oral bioavailability and intestinal (but not hepatic) availability. Rifampin decreased bioavailability and oral and IV methadone plasma concentrations and increased IV clearance (4.42 +/- 1.00 mL. kg -1. min -1 versus 1.61 +/- 0.67 mL. kg -1. min -1, P <.05) and oral clearance (8.50 +/- 3.68 mL. kg -1. min -1 versus 2.05 +/- 0.92 mL. kg -1. min -1, P <.05), EDDP/methadone area under the curve (AUC) ratios, EDDP formation clearances, and hepatic extraction (0.27 +/- 0.06 versus 0.09 +/- 0.04, P <.05). Troleandomycin and grapefruit juice decreased the EDDP/methadone AUC ratio after oral methadone (0.17 +/- 0.10 and 0.14 +/- 0.06 versus 0.27 +/- 0.20, P <.05) but not IV methadone and had no effect on methadone plasma concentrations, IV clearance (1.29 +/- 0.41 mL. kg -1. min -1 and 1.48 +/- 0.55 mL. kg -1. min -1 ) or oral clearance (2.05 +/- 1.52 mL. kg -1. min -1 and 1.89 +/- 1.07 mL. kg -1. min -1 ), or other kinetic parameters. There was no correlation between methadone clearance and hepatic CYP3A4 activity. Pupil diameter changes reflected plasma methadone concentrations. In vitro experiments showed a predominant role for both CYP3A4 and CYP2B6 in liver microsomal methadone N -demethylation. CONCLUSION: First-pass intestinal metabolism is a determinant of methadone bioavailability. Intestinal and hepatic CYP3A activity only slightly affects human methadone N -demethylation but has no significant effect on methadone concentrations, clearance, or clinical effects. Greater rifampin effects, compared with troleandomycin and grapefruit juice, on methadone disposition suggest a major role for intestinal transporters and for other CYPs, such as CYP2B6. Interindividual variability and drug interactions affecting intestinal transporter and hepatic CYP3A and CYP2B6 activity may alter methadone disposition.     

Cytochrome P450s in Papilio multicaudatus and the transition from oligophagy to polyphagy in the Papilionidae

Although substrate-specific CYP6B1 and CYP6B3 enzymes in Papilio polyxenes contribute to specialization on furanocoumarin-containing host plants, CYP6B4 and CYP6B17 enzymes in the polyphagous Papilio glaucus and Papilio canadensis have a broader range of substrates. Papilio multicaudatus, an oligophage with one furanocoumarin-containing host, is putatively ancestral to polyphagous Papilio species. Furanocoumarin-inducible CYP6B33-CYP6B37 and CYP6AB6 were characterized from this species. Heterologous expression of CYP6B33 revealed furanocoumarin metabolism resembling that of CYP6B4-CYP6B17 enzymes from P. glaucus and P. canadensis. Molecular models of CYP6B33 and CYP6B4 indicate that seven conserved aromatic side chains stabilize their hydrophobic catalytic sites and that a Lys484-Ser484 substitution enlarges the CYP6B4 active site pocket to increase the predicted distance between the substrate and reactive oxygen relative to CYP6B1. Loss of specialization in this lineage may have resulted from relatively few mutational changes, allowing acquisition of broader catalytic activities without loss of ancestral furanocoumarin-metabolizing activities.     

Identification of novel CYP2A6*1B variants: the CYP2A6*1B allele is associated with faster in vivo nicotine metabolism

Cytochrome P450 2A6 (CYP2A6) is the human enzyme responsible for the majority of nicotine's metabolism. CYP2A6 genetic variants contribute to the interindividual and interethnic variation in nicotine metabolism. We examined the association between the CYP2A6*1B variant and nicotine's in vivo metabolism. Intravenous infusions of deuterium-labeled nicotine were administered to 292 volunteers, 163 of whom were White and did not have common CYP2A6 variants, other than CYP2A6*1B. We discovered three novel CYP2A6*1B variants in the 3'-flanking region of the gene that can confound genotyping assays. We found significant differences between CYP2A6*1A/*1A, CYP2A6*1A/*1B, and CYP2A6*1B/*1B groups in total nicotine clearance (17.2+/-5.2, 19.0+/-6.4, and 20.4+/-5.9, P<0.02), non-renal nicotine clearance (16.4+/-5.0, 18.5+/-6.2, and 19.8+/-5.7, P<0.01), and the plasma trans-3'-hydroxycotinine/cotinine ratio (0.26+/-0.1, 0.26+/-0.1, and 0.34+/-0.1, P<0.001). There were also differences in total nicotine (29.4+/-12.9, 25.8+/-0.12.9, and 22.4+/-12.4, P<0.01), cotinine (29.2+/-8.1, 32.2+/-9.1, and 33.0+/-6.6, P<0.01) and trans-3'-hydroxycotinine (32.4+/-9.1, 34.2+/-12.3, and 41.3+/-11.3, P<0.001) excreted in the urine. We report evidence that CYP2A6*1B genotype is associated with faster nicotine clearance in vivo, which will be important to future CYP2A6 genotype association studies.     

Effects of garlic (Allium sativum L.) supplementation on cytochrome P450 2D6 and 3A4 activity in healthy volunteers

Garlic (Allium sativum L.) is a commonly used food and herbal supplement. The objective of this study was to assess in healthy volunteers (N = 14) the influence of a garlic extract on the activity of cytochrome P450 (CYP) 2D6 and 3A4. Probe substrates dextromethorphan (CYP2D6) and alprazolam (CYP3A4) were administered orally at baseline and again after treatment with garlic extract (3 x 600 mg twice daily) for 14 days. Urinary dextromethorphan/dextrorphan ratios and alprazolam plasma concentrations were determined by HPLC at baseline and after garlic extract treatment. The ratio of dextromethorphan to its metabolite was 0.044 +/- 0.48 at baseline and 0.052 +/- 0.095 after garlic supplementation. There were no significant differences between the baseline and garlic phases (P > or =.05). For alprazolam, there were no significant differences in pharmacokinetic parameters at baseline and after garlic extract treatment (all P values > or =.05; maximum concentration in plasma, 27.3 +/- 2.6 ng/mL versus 27.3 +/- 4.8 ng/mL; time to reach maximum concentration in plasma, 1.9 +/- 1.4 h versus 2.4 +/- 1.8 h; area under the time-versus-concentration curve, 537 +/- 94 h. ng. mL(-1) versus 548 +/- 159 h. ng. mL(-1); half-life of elimination, 13.7 +/- 4.4 h versus 14.5 +/- 4.3 h). Our results indicate that garlic extracts are unlikely to alter the disposition of coadministered medications primarily dependent on the CYP2D6 or CYP3A4 pathway of metabolism.     

Effect of interferon alpha-ribavirin bitherapy on cytochrome P450 1A2 and 2D6 and N-acetyltransferase-2 activities in patients with chronic active hepatitis C

BACKGROUND: Interferon alpha (IFN-alpha) is thought to be responsible for cytochrome P450 (CYP)-dependent drug interactions mediated by a decrease in CYP activities. OBJECTIVES: The objectives are to determine whether IFN-alpha and ribavirin can alter pretreatment CYP1A2, CYP2D6, CYP3A4 and N-acetyltransferase-2 activities after 1 month of treatment. METHODS: Enzymatic activities were determined among 14 patients with chronic active hepatitis C before IFN-alpha (3. 10(6) U, 3 times a week) and ribavirin introduction and after 1 month of treatment. During both study periods, subjects received 80 mg dextromethorphan and 140 mg caffeine (1,3,7-trimethylxanthine [137X]). CYP3A4, CYP2D6, and NAT2 activities were assessed by use of urinary metabolic ratios of 3-methoxymorphinan/dextromethorphan, dextrorphan/dextromethorphan, and 5-acetylamino-6-formylamino-3-methyluracil (AFMU)/1-methylxanthine(1X). The plasma paraxanthine/caffeine ratio was used to measure CYP1A2 activity. RESULTS: CYP3A4 and CYP2D6 activities tended to increase after 1 month of antiviral therapy, but the change did not reach statistical significance. CYP1A2 and NAT2 activities were not significantly modified after 1 month of antiviral treatment. Pretreatment activities were significantly lower than those previously observed in healthy volunteers for CYP2D6 (mean +/- SD, 148 +/- 139 versus 759 +/- 692; P =.0008) and CYP3A4 (0.18 +/- 0.06 versus 0.52 +/- 0.72; P =.0006). This difference was no longer statistically significant after 1 month of treatment, because CYP2D6 and CYP3A4 activities improved in 7 patients. CONCLUSION: In patients with chronic hepatitis C, pretreatment CYP3A4 and CYP2D6 activities were significantly lower than those observed in healthy volunteers. These differences disappeared after 1 month of antiviral treatment because of the restoration of these CYP activities in about half of the patients.     

Effects of malnutrition on cytochrome P450 1A2 activity in elderly patients

Little is known of the influence of nutritional status on cytochrome P450 (CYP) 1A2 activity in elderly patients. Thirty elderly institutionalised patients with malnutrition (group A, aged 88 +/- 5 years) and 24 without (group B, aged 81 +/- 9 years) were included. Malnutrition was defined as weight loss of >10% over the previous 6 months and/or a body mass index (BMI) <21 kg/m2 and albuminaemia < or = 32 g/L. CYP1A2 activity was evaluated by the plasma paraxanthine/caffeine (PAX/CAF) metabolic ratio. The plasma PAX/CAF metabolic ratio was similar in both groups regardless of nutritional status (0.34 +/- 0.13 [A] versus 0.30 +/- 0.11 [B]; p = 0.11). The CYP1A2 metabolic ratio was not correlated to either BMI, serum albumin or renal clearance. CYPI A2 activity, as measured by the plasma PAX/CAF ratio, was not influenced by nutritional status in elderly patients.     

Potent cytochrome P450 2C19 genotype-related interaction between voriconazole and the cytochrome P450 3A4 inhibitor ritonavir

OBJECTIVES: Cytochrome P450 (CYP) 2C19 and CYP3A4 are the major enzymes responsible for voriconazole elimination. Because the activity of CYP2C19 is under genetic control, the extent of inhibition with a CYP3A4 inhibitor was expected to be modulated by the CYP2C19 metabolizer status. This study thus assessed the effect of the potent CYP3A4 inhibitor ritonavir after short-term administration on voriconazole pharmacokinetics in extensive metabolizers (EMs) and poor metabolizers (PMs) of CYP2C19. METHODS: In a randomized, placebo-controlled crossover study, 20 healthy participants who were stratified according to CYP2C19 genotype received oral ritonavir (300 mg twice daily) or placebo for 2 days. Together with the first ritonavir or placebo dose, a single oral dose of 400 mg voriconazole was administered. Voriconazole was determined in plasma and urine by liquid chromatography-mass spectrometry, and pharmacokinetic parameters were estimated by noncompartmental analysis. RESULTS: When given alone, the apparent oral clearance of voriconazole after single oral dosing was 26%+/-16% (P > .05) lower in CYP2C19*1/*2 individuals and 66%+/-14% (P < .01) lower in CYP2C19 PMs. The addition of ritonavir caused a major reduction in voriconazole apparent oral clearance (354+/-173 mL/min versus 202+/-139 mL/min, P = .0001). This reduction occurred in all CYP2C19 genotypes (463+/-168 mL/min versus 305+/-112 mL/min [P = .023] for *1/*1, 343+/-127 mL/min versus 190+/-93 mL/min [P = .008] for *1/*2, and 158+/-54 mL/min versus 22+/-11 mL/min for *2/*2) and is probably caused by inhibition of CYP3A4-mediated voriconazole metabolism. CONCLUSIONS: Coadministration of a potent CYP3A4 inhibitor leads to a higher and prolonged exposure with voriconazole that might increase the risk of the development of adverse drug reactions on a short-term basis, particularly in CYP2C19 PM patients.     

4-Hydroxylation of debrisoquine by human CYP1A1 and its inhibition by quinidine and quinine

A panel of 15 recombinant cytochromes P450 expressed in human B-lymphoblastoid cells was used to study debrisoquine 4-hydroxylation. Both CYP2D6 and CYP1A1 carried out the reaction. The apparent K(m) (micromolar) and V(max) (picomoles per minute per picomole of P450) for CYP2D6 were 12.1 and 18.2 and for CYP1A1 were 23.1 and 15.2, respectively. CYP1A1 debrisoquine 4-hydroxylase was inhibited by the CYP1A1 inhibitor alpha-naphthoflavone and the CYP1A1 substrate 7-ethoxyresorufin. Additionally and surprisingly, this reaction was also inhibited by quinidine and quinine, with respective IC(50) values of 1.38 +/- 0.10 and 3.31 +/- 0.14 microM, compared with those for CYP2D6 debrisoquine 4-hydroxylase of 0.018 +/- 0.05 and 3.75 +/- 2.07 microM, respectively. Anti-CYP1A1 monoclonal antibody (mAb) 1-7-1 abolished CYP1A1 debrisoquine hydroxylase and anti-CYP2D6 mAb 50-1-3 eradicated CYP2D6 debrisoquine 4-hydroxylase. Three further CYP2D6-specific reactions were tested: dextromethorphan O-demethylation, bufuralol 1'-hydroxylation, and sparteine dehydrogenation. The CYP2D6 specificity, judged by the CYP2D6/CYP1A1 activity ratios was 18.5, 7.0, 6.0, and 1.6 for dextromethorphan, bufuralol, sparteine, and debrisoquine, respectively. Thus, debrisoquine is not a specific CYP2D6 substrate and quinidine is not a specific CYP2D6 inhibitor. These findings have significant implications for the conduct of in vitro drug metabolism inhibition studies and underscore the fallacy of "specific chemical inhibitors" of a supergene family of enzymes that have overlapping substrate specificities. The use of highly specific mAbs in such studies is mandated. It is unclear as yet whether these findings have implications for the relationship between CYP2D6 genotype and in vivo debrisoquine 4-hydroxylase activity.     

Effect of the CYP3A5 genotype on the pharmacokinetics of intravenous midazolam during inhibited and induced metabolic states

OBJECTIVE: Our objective was to investigate the effect of the CYP3A5 genotype on the systemic clearance of midazolam in constitutive, inhibited, and induced metabolic conditions. METHODS: Nineteen healthy volunteers were grouped with regard to the CYP3A5*3 allele, into homozygous wild-type (CYP3A5*1/*1, n = 6), heterozygous (CYP3A5*1/*3, n = 6), and homozygous variant-type (CYP3A5*3/*3, n = 7) subject groups. The pharmacokinetic profile of intravenous midazolam was characterized before and after itraconazole administration (200 mg once daily for 4 days) and also after rifampin (INN, rifampicin) pretreatment (600 mg once daily for 10 days), with a washout period of 2 weeks in between. RESULTS: The pharmacokinetic profiles of midazolam and of its hydroxy metabolites did not show differences between the genotype groups under basal and induced metabolic conditions. However, during the inhibited metabolic state, the CYP3A5*3/*3 group showed a greater decrease in systemic clearance than was seen in the CYP3A5*1/*1 group (8.5 +/- 3.8 L. h(-1). 70 kg(-1) versus 13.5 +/- 2.7 L. h(-1). 70 kg(-1), P =.027). The 1'-hydroxymidazolam-to-midazolam area under the plasma concentration-time curve ratio was also significantly lower in the CYP3A5*3/*3 group (0.58 +/- 0.35 versus 1.09 +/- 0.37 for the homozygous wild-type group, P =.026). CONCLUSIONS: The CYP3A5 genotype did not affect the pharmacokinetics of intravenous midazolam in the basal or induced states. However, during cytochrome P450 (CYP) 3A inhibition by itraconazole, individuals carrying the CYP3A5*1 allele were found to be less susceptible to changes in systemic clearance and showed higher 1'-hydroxymidazolam-to-midazolam area under the plasma concentration-time curve ratios, probably resulting from the relatively CYP3A4-specific inhibition caused by itraconazole.     

CYP2A6 genotype and the metabolism and disposition kinetics of nicotine

BACKGROUND AND OBJECTIVE: The liver enzyme cytochrome P450 (CYP) 2A6 is primarily responsible for the metabolism of nicotine. Variants in the CYP2A6 gene have been associated with altered nicotine metabolism and with effects on smoking behavior. Our objective was to determine the relationship between variant CYP2A6 genotypes and the disposition and metabolism of nicotine administered intravenously. METHODS: Intravenous infusions of deuterium-labeled nicotine and cotinine were administered to 278 healthy twin volunteers, most of whom were white. They were genotyped for CYP2A6*1, CYP2A6*2, CYP2A6*4, CYP2A6*7, CYP2A6*8, CYP2A6*9, CYP2A6*10, and CYP2A6*12. RESULTS: On the basis of the fractional clearance of nicotine to cotinine and on the plasma ratio of 3'-hydroxycotinine to cotinine, both shown to be indicators of CYP2A6 enzymatic activity, subjects were classified into 3 groups. Group 1 included wild-type variant CYP2A6*1/*1 (n=215) and was assumed to have 100% activity. Group 2 included *1/*9 (n=21) and *1/*12 (n=12), which averaged about 80% of normal activity. Group 3 included *1/*2 (n=10), *1/*4 (n=2), *9/*12 (n=3), *9/*4 (n=2), and *9/*9 (n=3), which averaged about 50% of normal activity. The mean total plasma clearance of nicotine (+/-SD) was 18.8+/-6.0, 15.5+/-4.9, and 11.7+/-5.1 mL.min-1.kg-1 in groups 1, 2, and 3, respectively, and group 1 had significantly faster clearance than group 2 (P<.05) and group 3 (P<.01). Overall, groups 2 and 3 also had lower total clearance of cotinine, had longer half-lives for nicotine and cotinine, and excreted in the urine a greater fraction of the nicotine dose as unchanged nicotine and nicotine glucuronide and excreted less as 3'-hydroxycotinine compared with group 1. CONCLUSIONS: We provide novel pharmacokinetic and metabolic data on nicotine after systemic dosing in relation to common CYP2A6 genotypes. Our data will enhance the interpretation of CYP2A6 genotypic data as used in association studies of smoking behavior and its health consequences.     

Assessment of drug-drug interaction potential of enfuvirtide in human immunodeficiency virus type 1-infected patients

INTRODUCTION: Enfuvirtide is the first drug to block human immunodeficiency virus type 1 (HIV-1) glycoprotein 41-mediated viral fusion to host cells. This study investigated whether enfuvirtide can influence the activities of cytochrome P450 (CYP) enzymes in HIV-1-infected patients. METHODS: An open-label, 1-sequence crossover study was conducted in 12 HIV-1-infected adults, by use of a 5-drug cocktail consisting of caffeine, chlorzoxazone, dapsone, debrisoquin (INN, debrisoquine), and mephenytoin to assess the activities of CYP1A2, CYP2E1, CYP3A4, CYP2D6, and CYP2C19, respectively. Dapsone was used to assess N-acetyltransferase activity. Patients received a single dose of the cocktail alone on day -15 and another together with enfuvirtide on day 6. Enfuvirtide (90 mg subcutaneously) was administered twice daily on days 1 to 7. Phenotypic index parameters were estimated and analyzed by ANOVA with factors subject and day (-15 and 6). RESULTS: The phenotypic index parameters, with and without enfuvirtide, for CYP3A4 (0.33 versus 0.34; 90% confidence interval [CI] for ratio of least squares means, 0.88-1.09), CYP2D6 (0.72 versus 0.71; 90% CI, 0.97-1.06), and N-acetyltransferase (0.35 versus 0.39; 90% CI, 0.82-0.98) were bioequivalent. The phenotypic index parameters, with and without enfuvirtide, for CYP1A2 (0.76 versus 0.81; 90% CI, 0.71-1.17), CYP2E1 (1.3 versus 1.2; 90% CI, 0.87-1.29), and CYP2C19 (93 versus 81.8; 90% CI, 0.98-1.28) were not bioequivalent but were not substantially different. CONCLUSIONS: Enfuvirtide had no clinically important effect on the metabolism of probe drugs mediated by CYP3A4, CYP2D6, or N-acetyltransferase and had little effect on the metabolism of drugs mediated by CYP1A2, CYP2E1, or CYP2C19. The potential for interactions between enfuvirtide and concomitantly administered drugs metabolized by the CYP enzymes tested in this study is low.     

Multiple doses of saw palmetto (Serenoa repens) did not alter cytochrome P450 2D6 and 3A4 activity in normal volunteers

Saw palmetto (Serenoa repens) is the most commonly used herbal preparation in the treatment of benign prostatic hyperplasia. The objective of this study was to determine whether a characterized saw palmetto product affects the activity of cytochrome P450 (CYP) 2D6 or 3A4 in healthy volunteers (6 men and 6 women). The probe substrates dextromethorphan (CYP2D6 activity) and alprazolam (CYP3A4 activity) were administered orally at baseline and again after exposure to saw palmetto (320-mg capsule once daily) for 14 days. Dextromethorphan metabolic ratios and alprazolam pharmacokinetics were determined at baseline and after saw palmetto treatment. The mean ratio of dextromethorphan to its metabolite was 0.038 +/- 0.044 at baseline and 0.048 +/- 0.080 after 14 days of saw palmetto administration (P =.704, not significant [NS]), indicating a lack of effect on CYP2D6 activity. The area under the plasma alprazolam concentration versus time curve was 476 +/- 178 h. ng. mL(-1) at baseline and 479 +/- 125 h. ng. mL(-1) after saw palmetto treatment (P =.923, NS), indicating a lack of effect on CYP3A4 activity. The elimination half-life of alprazolam was 11.4 +/- 3.1 hours at baseline and 11.6 +/- 2.7 hours after saw palmetto treatment (P =.770, NS), also indicating a lack of effect on CYP3A4 activity. Our results indicate that extracts of saw palmetto at generally recommended doses are unlikely to alter the disposition of coadministered medications primarily dependent on the CYP2D6 or CYP3A4 pathways for elimination. These conclusions must be weighed in the context of the study's limited assessments and regarded as only the initial investigation into the drug interaction potential of saw palmetto.     

Effect of fluvoxamine on the pharmacokinetics of mexiletine in healthy Japanese men

BACKGROUND AND OBJECTIVES: Fluvoxamine, a selective serotonin reuptake inhibitor, is known to inhibit several hepatic cytochrome P450 (CYP) isozymes, in particular CYP1A2. Mexiletine is mainly catalyzed by CYP2D6 and partially catalyzed by CYP1A2. Our objective was to study the potential pharmacokinetic interaction between fluvoxamine and mexiletine. METHODS: A randomized crossover design with two phases was used. A 7-day washout period separated the two treatment conditions. In the one phase, 6 healthy Japanese men received an oral dose of 200 mg of mexiletine alone (study 1); in the other phase, the men received fluvoxamine (50 mg twice a day) for 7 days, and on the eighth day they received oral mexiletine (200 mg) and fluvoxamine concomitantly (study 2). The concentrations of mexiletine were measured with HPLC. RESULTS: The area under the concentration-time curve and serum peak concentration of mexiletine in study 2 were significantly increased compared with those in study 1 (10.4 +/- 4.85 versus 6.70 +/- 3.21 microg x h/mL, P =.006 and 0.623 +/- 0.133 versus 0.536 +/- 0.164 microg/mL, P =.008, respectively). CONCLUSION: The effect of fluvoxamine on the mexiletine disposition is comparatively large, and when mexiletine and fluvoxamine are coadministered careful monitoring of mexiletine is needed.     

CYP2C9, but not CYP2C19, polymorphisms affect the pharmacokinetics and pharmacodynamics of glyburide in Chinese subjects

BACKGROUND: Although cytochrome P450 (CYP) 2C9 was thought to be the main pathway for glyburide (INN, glibenclamide) metabolism in vivo, studies in vitro indicated that CYP2C19 had a more dominant effect. This study investigated the relative influence of CYP2C9 and CYP2C19 genotypes on the pharmacokinetics and pharmacodynamics of glyburide in Chinese subjects. METHODS: Three groups of healthy male Chinese subjects (n=6 per group) were enrolled, as follows: group I, CYP2C9*1/*1 and CYP2C19 extensive metabolizers (EMs); group II, CYP2C9*1/*1 and CYP2C19 poor metabolizers (PMs); and group III, CYP2C9*1/*3 and CYP2C19 EMs. Subjects received single oral doses of 5 mg glyburide. Multiple blood samples were collected, and the plasma glyburide concentrations were determined by an HPLC method. The plasma glucose and insulin concentrations were also measured up to 2 hours after dosing. RESULTS: No significant differences in glyburide pharmacokinetics were observed between CYP2C19 EM and PM subjects who had the CYP2C9*1/*1 genotype (group I versus group II). Their respective values for area under the plasma concentration-time curve from time 0 to infinity (AUC0-infinity) and elimination half-life (t1/2) were 0.46+/-0.13 microg.h/mL versus 0.57+/- 0.11 microg.h/mL (P=.569) and 2.09+/-0.22 hours versus 2.24+/- 0.27 hours (P=.721). However, significant increases in AUC(0-infinity) (125% and 82%; P=.008 and .024, respectively) and t1/2 (71% and 60%; P=.003 and .007, respectively) were observed when CYP2C9*1/*3 subjects (group III) were compared with CYP2C9*1/*1 subjects in group I or II. Blood glucose reductions at 2 hours after dosing were 41.8%, 23.9%, and 27.7% in groups I, II, and III, respectively (P=.029), and hypoglycemia developed in 3 of 6 CYP2C9*1/*3 carriers and 2 of 12 CYP2C9*1/*1 carriers. CONCLUSION: CYP2C9, but not CYP2C19, polymorphism appears to exert a dominant influence on glyburide pharmacokinetics and pharmacodynamics in vivo. Further studies in diabetic patients with long-term dosing are warranted to confirm these findings.     

In vivo effects of interleukin-10 on human cytochrome P450 activity

BACKGROUND: Injection of lipopolysaccharide into human volunteers leads to an increase in serum interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha and a significant decrease in cytochrome P450 (CYP)-mediated drug metabolism. The in vivo effects of the noninflammatory cytokine interleukin-10 (IL-10) on CYP-mediated drug metabolism was examined. METHODS: IL-10 (8 microg/kg) and placebo were administered for 6 days to 12 healthy volunteers in a double-blind crossover study. Tolbutamide (CYP2C9), caffeine (CYP1A2), dextromethorphan (CYP2D6 and CYP3A), and midazolam (CYP3A) were administered on days 4 and 5 to determine individual CYP activities. RESULTS: Few clinically apparent side effects were observed after administration of IL-10; however, blood chemistries reflected an acute-phase response. A significant drop in serum albumin (mean percentage change +/- SD between groups; 4.7% +/- 6.0%, P < or = .02), a significant increase in serum ferritin (736% +/- 717%, P < or = .001), and a significant reduction in platelet count (49% +/- 12%, P < or = .0001) was observed after administration of IL-10. IL-10 significantly (P < or = .02) decreased CYP3A activity 12% +/- 17%, as reflected by midazolam clearance. CYP2C9 activity was significantly (P < or = .005) increased by 38% +/- 35%, as reflected by the tolbutamide urinary metabolic ratio and oral clearance. However, administration of IL-10 resulted in a 40% increase in the fraction unbound of tolbutamide. Therefore no difference in the unbound clearance of tolbutamide was observed between placebo (23.3 +/- 9.7 L/h) or IL-10 (23.5 +/- 11.4 L/h) administration. No significant changes in either CYP1A2 or CYP2D6 activities were observed between placebo and treatment arms of the study. CONCLUSION: IL-10 administration resulted in an acute-phase response. Administration of IL-10 did not alter CYP1A2, CYP2C9, and CYP2D6 activities. CYP3A-mediated biotransformation was reduced by administration of IL-10.     

CYP2D6 genotype and induction of intestinal drug transporters by rifampin predict presystemic clearance of carvedilol in healthy subjects

BACKGROUND: Clinical trials have indicated that the combined beta- and alpha-adrenergic receptor blocker carvedilol improves the survival rate in patients with advanced chronic heart failure. The objective of our study was the identification and quantification of factors that modulate steady-state serum concentrations of carvedilol and its enantiomers and that may influence therapeutic efficacy and safety. METHODS: The influence of genetic variants of cytochrome P450 (CYP) 2D6 and CYP2C9 and of transporter proteins (P-glycoprotein, multidrug resistance protein 2 [MRP2]) on the disposition of carvedilol and its enantiomers after intravenous (5 mg) and long-term oral administration (25 mg for 7 days) was assessed in 12 healthy subjects. The intestinal expression of P-glycoprotein and MRP2 was analyzed by quantitative real-time polymerase chain reaction and immunohistochemical techniques. RESULTS: The area under the serum concentration-time curve (AUC) values of carvedilol were significantly (P <.05) increased in 6 subjects with CYP2D6 deficiency, with effects being more pronounced for R(+)-carvedilol (230 +/- 72.6 ng. h/mL versus 93.9 +/- 64.6 ng. h/mL in extensive metabolizers) than for S(-)-carvedilol (62.9 +/- 21.1 ng. h/mL versus 32.7 +/- 14.5 ng. h/mL). The AUC and fecal excretion of intravenous carvedilol were correlated with the intestinal expression of MDR1 messenger ribonucleic acid (mRNA) (r = -0.67, P =.001; r = 0.83, P =.002) and MRP2 mRNA (r = -0.74, P <.001; r = 0.70, P =.025). Furthermore, we measured the disposition of long-term oral carvedilol after comedication of the pregnane X receptor ligand rifampin (INN, rifampicin) (600 mg, 9 days), which up-regulates both P-glycoprotein and MRP2 but not CYP2D6. Rifampin decreased the AUC of carvedilol to an extent independent of the CYP2D6 genotype (poor metabolizers, 341 +/- 147 ng. h/mL versus 126 +/- 41.7 ng. h/mL; extensive metabolizers, 173 +/- 102 ng. h/mL versus 74 +/- 41.4 ng. h/mL; both P <.05). The AUC was significantly correlated with intestinal expression of MDR1 mRNA (r = -0.671, P =.001) and MRP2 mRNA (r = -0.595, P <.006). CONCLUSIONS: Variable plasma concentrations of carvedilol during long-term administration are predicted by CYP2D6 genotype and intestinal expression of P-glycoprotein and MRP2.