Rate of onset of inhibition of gut-wall and hepatic CYP3A by clarithromycin.

AIMS: To determine the extent and time-course of hepatic and intestinal cytochrome P450 3A (CYP3A) inactivation due to the mechanism-based inhibitor clarithromycin. METHODS: Intestinal and hepatic CYP3A inhibition was examined in 12 healthy volunteers following the administration of single and multiple doses of oral clarithromycin (500 mg). Intestinal biopsies were obtained under intravenous midazolam sedation at baseline and after the first dose, on days 2-4, and on days 6-8 of the clarithromycin treatment. The formation of 1'-hydroxymidazolam in biopsy tissue and the serum 1'-hydroxymidazolam:midazolam ratio were indicators of intestinal and hepatic CYP3A activity, respectively. RESULTS: Intestinal CYP3A activity decreased by 64 % (p = 0.0029) following the first dose of clarithromycin, but hepatic CYP3A activity did not significantly decrease. Repeated dosing of clarithromycin caused a significant decrease in hepatic CYP3A activity (p = 0.005), while intestinal activity showed little further decline. The CYP3A5 or CYP3A4*1B genotype were unable to account for inter-individual variability in CYP3A activity. CONCLUSIONS: Following the administration of clarithromycin, the onset of hepatic CYP3A inactivation is delayed compared to that of intestinal CYP3A. The time-course of drug-drug interactions due to clarithromycin will vary with the relative contribution of intestinal and hepatic CYP3A to the clearance and bioavailability of a victim substrate.

Hydroxyitraconazole, formed during intestinal first-pass metabolism of itraconazole, controls the time course of hepatic CYP3A inhibition and the bioavailability of itraconazole in rats.

Itraconazole (ITZ) is a substrate of CYP3A and both ITZ and hydroxyitraconazole (OH-ITZ), a major metabolite formed by CYP3A, are potent inhibitors of CYP3A. The concentration- and time-dependent changes in the hepatic availability (F(H)) of ITZ were evaluated in rats after oral doses of 5 and 40 mg/kg. Simultaneous blood samples were obtained from the aorta, portal vein, and hepatic vein for 24 h following duodenal ITZ administration, and concentrations of ITZ and OH-ITZ determined by LC/MS. During the absorption phase, the F(H) of ITZ increased from 0.2 to 1.0, reflecting the time course of hepatic CYP3A inhibition. A counterclockwise hysteresis was observed between ITZ concentrations entering the liver (C(IN,ITZ)) and F(H), whereas there was no time delay observed between the change in F(H) and the OH-ITZ concentrations entering the liver (C(IN,OH-ITZ)). The direct relationship between C(IN,OH-ITZ) and F(H) suggested that OH-ITZ was mainly responsible for the inhibition of CYP3A. A positive portal venous-aortic gradient for OH-ITZ was measured after duodenal administration of ITZ, indicating intestinal formation of OH-ITZ. The in vivo Ki for OH-ITZ (38 +/- 3 nM) was estimated from C(IN,OH-ITZ) versus F(H) of ITZ, and is similar to values obtained from inhibition of midazolam hydroxylation in CYP3A4 supersomes (Drug Metab Dispos 32:1121-1131, 2004). The data suggest that OH-ITZ, formed by intestinal CYP3A, controls the time course of hepatic CYP3A inhibition and is mainly responsible for the observed increase in F(H) of ITZ.

The effect of short- and long-term administration of verapamil on the disposition of cytochrome P450 3A and P-glycoprotein substrates.

BACKGROUND: Verapamil has the capability to inhibit and induce cytochrome P450 (CYP) 3A and P-glycoprotein (P-gp), but the relative extent and time course of these events in vivo are unclear. The effect of verapamil on CYP3A and P-gp activity was determined by examining its effect on its own disposition and on the disposition of fexofenadine, respectively. METHODS: Twelve healthy volunteers received 60 mg fexofenadine alone or after administration of 240 mg verapamil for 1, 10, and 38 days. The concentrations of verapamil and norverapamil, as well as their enantiomers, were quantified in serum by chiral HPLC. The concentrations of fexofenadine and its metabolite, azacyclonol, were quantified in serum and urine by liquid chromatography-mass spectrometry. RESULTS: The mean +/- SD maximum serum concentration (Cmax) and the area under the serum concentration-time curve of S-verapamil increased significantly on days 10 (40 +/- 21 ng/mL [P = .00044] and 433 +/- 316 ng.h.mL(-1) [P = .00047], respectively) and 38 (42 +/- 27 ng/mL [P = .019] and 433 +/- 256 ng.h.mL(-1) [P = .0081], respectively) compared with day 1 (21 +/- 12 ng/mL and 222 +/- 156 ng.h.mL(-1), respectively). The oral clearance (CLoral) of S-verapamil decreased significantly from 702 +/- 304 L/h on day 1 to 377 +/- 210 L/h on day 10 (P = .0029) and 449 +/- 419 L/h on day 38 (P = .05). Similar trends were observed for the Cmax and area under the serum concentration-time curve of R-verapamil and R- and S-norverapamil. All subjects showed a significant decrease in the CLoral of fexofenadine after a single dose (98 +/- 54 L/h, P = .00105) and 10-day dosing (102 +/- 40 L/h, P = .0011) of verapamil compared with the control value (156 +/- 69 L/h). The Cmax of fexofenadine was significantly increased by a single dose (165 +/- 42 ng/mL, P = .0005) and 10-day dosing (148 +/- 39 ng/mL, P = .0008) of verapamil compared with the control value (114 +/- 45 ng/mL). No significant difference in fexofenadine Cmax (P = .37) and CLoral (P = .43) was observed between the control values and values at 38 days of verapamil treatment. CONCLUSION: Verapamil inhibited CYP3A activity, with a maximum effect occurring within 10 days. Short-term administration of verapamil caused net inhibition of intestinal P-gp, whereas long-term administration of verapamil induced P-gp activity.

Dextromethorphan to dextrorphan urinary metabolic ratio does not reflect dextromethorphan oral clearance.

Dextromethorphan urinary metabolic ratio is widely used to determine the CYP2D6 phenotype, but its utility to reflect subtle differences in catalytic activity is unclear. We evaluated the capability of dextromethorphan urinary metabolic ratio to predict dextromethorphan oral clearance as a measure of CYP2D6 activity. Data from 10 healthy extensive metabolizers of CYP2D6 were given 30 mg of dextromethorphan hydrobromide orally on two occasions. Blood and urine samples were collected for 72 h. Dextromethorphan and dextrorphan were determined in urine by high-performance liquid chromatography with fluorescence detection and in serum by liquid chromatography-mass spectrometry. The urinary metabolic ratio was very weakly correlated with dextromethorphan oral clearance (r = 0.24; p = 0.04). In contrast, the dextromethorphan oral clearance was highly correlated with the dextromethorphan to dextrorphan area under the concentration-time curve ratio (r = 0.84; p = 0.005) and the 3-h (r = 0.60; p = 0.003), 4-h (r = 0.72, p < 0.001), 6-h (r = 0.67; p < 0.001), and 8-h (r = 0.74; p < 0.001) dextromethorphan to dextrorphan serum ratios. Assuming an effect size of 30%, the number of volunteers required for crossover and cross-sectional studies using the urinary metabolic ratio as the CYP2D6 index was calculated to be 56 and 524, respectively, whereas 14 and 60 subjects are needed if oral clearance is used. Considering the required sample size and the low correlation with oral clearance, urinary metabolic ratio is not recommended as the primary outcome variable in studies requiring the detection of modest changes in CYP2D6 activity.

Inhibition of human intestinal wall metabolism by macrolide antibiotics: effect of clarithromycin on cytochrome P450 3A4/5 activity and expression.

BACKGROUND: Clarithromycin increases both hepatic and intestinal availability of the selective cytochrome P450 (CYP) 3A probe midazolam. This study was designed to identify determinants of variability in the extent of intestinal wall CYP3A inhibition by clarithromycin, such as CYP3A5 genotype, and the mechanism of inhibition. METHODS: Ten healthy volunteers received 500 mg oral clarithromycin twice a day for 7 days. Before and after administration of clarithromycin, small-bowel mucosal biopsy specimens were obtained endoscopically. Intestinal CYP3A activity was determined from the rate of 1'-hydroxymidazolam and 4-hydroxymidazolam formation by incubation of small-bowel homogenate with midazolam (25 micromol/L) and NADPH for 5 minutes. Intestinal CYP3A4 and CYP3A5 messenger ribonucleic acid was quantified by real-time reverse transcriptase-polymerase chain reaction. Intestinal CYP3A4 and CYP3A5 protein concentrations were determined by immunoblotting. Serum and homogenate concentrations of midazolam, clarithromycin, and metabolites were determined by liquid chromatography-mass spectrometry. CYP3A5 genotype was determined by real-time polymerase chain reaction. RESULTS: The formation of 1'-hydroxymidazolam (1.36 +/- 0.46 pmol . min(-1) . mg(-1) at baseline versus 0.35 +/- 0.16 pmol . min(-1) . mg(-1) after administration) and 4-hydroxymidazolam (0.39 +/- 0.12 pmol . min(-1) . mg(-1) at baseline versus 0.12 +/- 0.05 pmol . min(-1) . mg(-1) after administration) was significantly (P < .001) reduced after clarithromycin administration. Clarithromycin administration did not result in a significant change in intestinal CYP3A4 and CYP3A5 messenger ribonucleic acid and protein expression. All subjects had detectable serum clarithromycin concentrations after 7 days of clarithromycin (3.71 +/- 2.43 micromol/L). The mean concentration of clarithromycin in the intestinal biopsy homogenate was 1.2 +/- 0.7 nmol/L (range, 0.42-2.39 nmol/L). Compared with CYP3A5 nonexpressers, subjects with at least 1 CYP3A5*1 allele (CYP3A5 expressers) had greater inhibition of intestinal CYP3A activity after treatment with clarithromycin. There was a strong linear relationship between the decrease in intestinal CYP3A activity and baseline catalytic activity (R(2) = 0.9). CONCLUSION: Baseline intestinal activity of CYP3A4 was a key determinant of variability of the inhibitory effect of clarithromycin among individuals. CYP3A5*1 alleles were associated with greater baseline intestinal CYP3A activity and, therefore, greater extent of inhibition. The primary in vivo mechanism was not rapidly reversible competitive or irreversible inhibition but was likely formation of metabolic intermediate complexes.

The effect of echinacea (Echinacea purpurea root) on cytochrome P450 activity in vivo.

BACKGROUND: Echinacea is a widely available over-the-counter herbal remedy. Tinctures of echinacea have been shown to inhibit cytochrome P450 (CYP) in vitro. The effect of echinacea (Echinacea purpurea root) on CYP activity in vivo was assessed by use of the CYP probe drugs caffeine (CYP1A2), tolbutamide (CYP2C9), dextromethorphan (CYP2D6), and midazolam (hepatic and intestinal CYP3A). METHODS: Twelve healthy subjects (6 men) completed this 2-period, open-label, fixed-schedule study. Caffeine, tolbutamide, dextromethorphan, and oral and intravenous midazolam were administered before and after a short course of echinacea (400 mg 4 times a day for 8 days) to determine in vivo CYP activities. RESULTS: Echinacea administration significantly increased the systemic clearance of midazolam by 34%, from 32 +/- 7 L/h to 43 +/- 16 L/h (P =.003; 90% confidence interval [CI], 116%-150%), and significantly reduced the midazolam area under the concentration-time curve by 23%, from 127 +/- 36 microg. h/L to 102 +/- 43 microg. h/L (P =.024; 90% CI, 63%-88%). In contrast, the oral clearance of midazolam was not significantly altered (P =.655; 90% CI, 75%-124%), 137 +/- 19 L/h compared with 146 +/- 71 L/h. The oral availability of midazolam after echinacea dosing was significantly increased (P =.028; 90% CI, 108%-153%), from 0.23 +/- 0.06 to 0.33 +/- 0.13. Hepatic availability (0.72 +/- 0.08 versus 0.61 +/- 0.16; P =.006; 90% CI, 73%-90%) and intestinal availability (0.33 +/- 0.11 versus 0.61 +/- 0.38; P =.015; 90% CI, 125%-203%) were significantly altered in opposite directions. Echinacea dosing significantly reduced the oral clearance of caffeine, from 6.6 +/- 3.8 L/h to 4.9 +/- 2.3 L/h (P =.049; 90% CI, 58%-96%). The oral clearance of tolbutamide was reduced by 11%, from 0.81 +/- 0.18 L/h to 0.72 +/- 0.19 L/h, but this change was not considered to be clinically relevant because the 90% CIs were within the 80% to 125% range. The oral clearance of dextromethorphan in 11 CYP2D6 extensive metabolizers was not affected by echinacea dosing (1289 +/- 414 L/h compared with 1281 +/- 483 L/h; P =.732; 90% CI, 89%-108%). CONCLUSIONS: Echinacea (E purpurea root) reduced the oral clearance of substrates of CYP1A2 but not the oral clearance of substrates of CYP2C9 and CYP2D6. Echinacea selectively modulates the catalytic activity of CYP3A at hepatic and intestinal sites. The type of drug interaction observed between echinacea and other CYP3A substrates will be dependent on the relative extraction of drugs at hepatic and intestinal sites. Caution should be used when echinacea is coadministered with drugs dependent on CYP3A or CYP1A2 for their elimination.

The interaction between St John's wort and an oral contraceptive.

OBJECTIVES: The popular herbal remedy St John's wort is an inducer of cytochrome P450 (CYP) 3A enzymes and may reduce the efficacy of oral contraceptives. Therefore we evaluated the effect of St John's wort on the disposition and efficacy of Ortho-Novum 1/35 (Ortho-McNeil Pharmaceutical, Inc, Raritan, NJ), a popular combination oral contraceptive pill containing ethinyl estradiol (INN, ethinylestradiol) and norethindrone (INN, norethisterone). METHODS: Twelve healthy premenopausal women who were using oral contraception (>3 months) received a combination oral contraceptive pill (Ortho-Novum 1/35) for 3 consecutive 28-day menstrual cycles. During the second and third cycles, the participants received 300 mg St John's wort 3 times a day. The serum concentrations of ethinyl estradiol (day 7), norethindrone (day 7), follicle-stimulating hormone (days 12-16), luteinizing hormone (days 12-16), progesterone (day 21), and intravenous and oral midazolam (days 22 and 23) were determined in serial blood samples. The incidence of breakthrough bleeding was quantified during the first and third cycles. RESULTS: Concomitant use of St John's wort was associated with a significant (P <.05) increase in the oral clearance of norethindrone (8.2 +/- 2.7 L/h to 9.5 +/- 3.4 L/h, P =.042) and a significant reduction in the half-life of ethinyl estradiol (23.4 +/- 19.5 hours to 12.2 +/- 7.1 hours, P =.023). The oral clearance of midazolam was significantly increased (109.2 +/- 47.9 L/h to 166.7 +/- 81.3 L/h, P =.007) during St John's wort administration, but the systemic clearance of midazolam was unchanged (37.7 +/- 11.3 L/h to 39.0 +/- 10.3 L/h, P =.567). Serum concentrations of follicle-stimulating hormone, luteinizing hormone, and progesterone were not significantly affected by St John's wort dosing (P >.05). Breakthrough bleeding occurred in 2 of 12 women in the control phase compared with 7 of 12 women in the St John's wort phase. The oral clearance of midazolam after St John's wort dosing was greater in women who had breakthrough bleeding (215.9 +/- 66.5 L/h) than in those who did not (97.5 +/- 37.2 L/h) (P =.005). CONCLUSION: St John's wort causes an induction of ethinyl estradiol-norethindrone metabolism consistent with increased CYP3A activity. Women taking oral contraceptive pills should be counseled to expect breakthrough bleeding and should consider adding a barrier method of contraception when consuming St Johns wort.

The effect of age, sex, and rifampin administration on intestinal and hepatic cytochrome P450 3A activity.

BACKGROUND: The relative susceptibility of intestinal and hepatic cytochrome P450 (CYP) 3A to induction by rifampin (INN, rifampicin), as a function of age and sex, was investigated with the CYP3A substrate midazolam. METHODS: Fourteen young women (mean age, 26 +/- 4 years), 14 young men (mean age, 27 +/- 4 years), 14 elderly women (mean age, 72 +/- 5 years), and 10 elderly men (mean age, 70 +/- 4 years) received simultaneous intravenous doses (0.05 mg/kg over a 30-minute period) and oral doses of midazolam (3-8 mg of a stable isotope, (15)N(3)-midazolam) before and after 7 days of rifampin dosing (600 mg once daily in the evening). Serum and urine samples were assayed for midazolam, (15)N(3)-midazolam, and metabolites by liquid chromatography-mass spectrometry. RESULTS: No significant difference (P > or =.05) in the baseline systemic and oral clearance of midazolam was observed between male and female or young and old volunteers. Rifampin significantly (P <.0001) increased the systemic and oral clearance of midazolam from 0.44 +/- 0.2 L. h/kg and 1.56 +/- 0.8 L x h/kg to 0.96 +/- 0.3 L x h/kg and 34.4 +/- 21.2 L x h/kg, respectively. Likewise, the oral clearance of midazolam was significantly (P <.0001) increased in women and men, from 1.64 +/- 0.87 L x kg/h and 1.46 +/- 0.7 L x kg/h to 28.4 +/- 13.2 L x kg/h and 41.6 +/- 26.5 L x kg/h, respectively. A significant (P =.0023) effect of sex was noted in the extent of induction of the oral clearance of midazolam, being greater in men than in women. In contrast, the extent of midazolam systemic clearance induction was greater in women than in men (P =.0107). Age did not influence the extent of intestinal and hepatic CYP3A induction as determined by the oral and systemic clearance of midazolam. Rifampin dosing significantly (P <.0001) reduced the oral availability by 88%, from 0.32 +/- 0.13 to 0.04 +/- 0.02. Correspondingly, hepatic and intestinal availabilities were significantly (P <.0001) reduced after rifampin administration. After rifampin, the correlation coefficient for the relationship between oral availability and intestinal availability was significantly (P <.0001) reduced from 0.96 to 0.67, which reflects the increasing contribution of hepatic extraction to the determination of midazolam oral availability. A significant nonlinear inverse relationship was observed between the percent change in systemic clearance of midazolam and the initial baseline midazolam systemic clearance (r = -0.68, N = 52, P <.0001). Likewise, a significant inverse relationship was observed between the percent change in oral clearance and the baseline oral clearance (r = -0.39, N = 52, P =.0041). A significant inverse relationship between the ratio of hepatic intrinsic clearance in the presence of rifampin to that in the absence of rifampin and the corresponding ratio of intestinal intrinsic clearance was observed (Spearman correlation coefficient [r] = -0.68, P <.0001) and indicates that in a given individual the extent of induction was high at either the hepatic or the intestinal site but not both. CONCLUSION: Sex-related differences exist in the extent of intestinal and hepatic CYP3A induction by rifampin. The extent of induction at hepatic and intestinal sites was inversely dependent and reflected the independent regulation of CYP3A expression at these sites. The large interindividual variation in the extent of induction is explained in part by the variation in baseline expression of CYP3A. Sex-related differences in response to CYP3A inducers will be substrate-dependent and reflect the relative contribution of hepatic and intestinal sites of metabolism.

Comparative metabolic capabilities of CYP3A4, CYP3A5, and CYP3A7.

The human cytochromes P450 (P450) CYP3A contribute to the biotransformation of 50% of oxidatively metabolized drugs. The predominant hepatic form is CYP3A4, but recent evidence indicates that CYP3A5 contributes more significantly to the total liver CYP3A than was originally thought. CYP3A7 is the major fetal form and is rarely expressed in adults. To compare the metabolic capabilities of CYP3A forms for 10 substrates, incubations were performed using a consistent molar ratio (1:7:9) of recombinant CYP3A, P450 reductase, and cytochrome b5. A wide range of substrate concentrations was examined to determine the best fit to kinetic models for metabolite formation. In general, K(m) or S(50) values for the substrates were 3 to 4 times lower for CYP3A4 than for CYP3A5 or CYP3A7. For a more direct comparison of these P450 forms, clearance to the metabolites was determined as a linear relationship of rate of metabolite formation for the lowest substrate concentrations examined. The clearance for 1'-hydroxy midazolam formation at low substrate concentrations was similar for CYP3A4 and CYP3A5. For CYP3A5 versus CYP3A4, clearance values at low substrate concentrations were 2 to 20 times lower for the other biotransformations. The clearance values for CYP3A7-catalyzed metabolite formation at low substrate concentrations were substantially lower than for CYP3A4 or CYP3A5, except for clarithromycin, 4-OH triazolam, and N-desmethyl diltiazem (CYP3A5 - CYP3A7). The CYP3A forms demonstrated regioselective differences in some of the biotransformations. These results demonstrate an equal or reduced metabolic capability for CYP3A5 compared with CYP3A4 and a significantly lower capability for CYP3A7.

Effect of St John's wort on the pharmacokinetics of fexofenadine.

BACKGROUND: St John's wort is a popular over-the-counter dietary supplement and herbal remedy that has been implicated in drug interactions with several substrates of P-glycoprotein. The effect of St John's wort on P-glycoprotein activity in vivo was examined with use of fexofenadine as selective probe drug. METHODS: A 3-period, open-label, fixed-schedule study design was used. Fexofenadine, 60 mg, was administered orally before administration of St John's wort, with a single dose of St John's wort (900 mg), and after 2 weeks of treatment with St John's wort (300 mg 3 times a day) to determine P-glycoprotein activity. RESULTS: A single dose of St John's wort significantly (P <.05) increased the maximum plasma concentration of fexofenadine by 45% and significantly (P <.05) decreased the oral clearance by 20%, with no change in half-life or renal clearance. Long-term administration of St John's wort did not cause a significant change in fexofenadine disposition relative to the untreated phase. Compared with the single-dose treatment phase, long-term St John's wort caused a significant 35% decrease (P <.05) in maximum plasma concentration and a significant 47% increase (P <.05) in fexofenadine oral clearance. CONCLUSIONS: A single dose of St John's wort resulted in a significant inhibition of intestinal P-glycoprotein. Long-term treatment with St John's wort reversed the changes in fexofenadine disposition observed with single-dose administration.