Induction of the metabolism of etizolam by carbamazepine in humans

Objective To examine the effect of carbamazepine on the single oral dose pharmacokinetics of etizolam.Methods Eleven healthy male volunteers received carbamazepine 200 mg/day or placebo for 6 days in a double-blind, randomized, crossover manner, and on the sixth day they received a single oral 1-mg dose of etizolam. Blood samplings and evaluation of psychomotor function by the Digit Symbol Substitution Test and Stanford Sleepiness Scale were conducted up to 24 h after etizolam dosing. Plasma concentration of etizolam was measured using high-performance liquid chromatography.Results Carbamazepine treatment significantly decreased the peak plasma concentration (17.5±4.1 ng/ml versus 13.9±4.1 ng/ml; P<0.05), total area under the plasma concentration–time curve (194.8±88.9 ng h/ml versus 105.9±33.0 ng h/ml; P<0.001), and elimination half-life (11.1±4.6 h versus 6.8±2.8 h; P<0.01) of etizolam. No significant change was induced by carbamazepine in the two pharmacodynamic parameters.Conclusions The present study suggests that carbamazepine induces the metabolism of etizolam.     

Fluconazole but not itraconazole decreases the metabolism of losartan to E-3174

Objective: Losartan is metabolised to its active metabolite E-3174 by CYP2C9 and CYP3A4 in vitro. Itraconazole is an inhibitor of CYP3A4, whereas fluconazole affects CYP2C9 more than CYP3A4. We wanted to study the possible interaction of these antimycotics with losartan in healthy volunteers. Methods: A randomised, double-blind, three-phase crossover study design was used. Eleven healthy volunteers ingested orally, once a day for 4 days, either itraconazole 200 mg, fluconazole (400 mg on day 1 and 200 mg on days 2–4) or placebo (control). On day 4, a single 50-mg oral dose of losartan was ingested. Plasma concentrations of losartan, E-3174, itraconazole, hydroxy-itraconazole and fluconazole were determined over 24 h. The blood pressure and heart rate were also recorded over 24 h. Results: The mean peak plasma concentration (C_max) and area under the curve [AUC(0∞)] of E-3174 were significantly decreased by fluconazole to 30% and to 47% of their control values, respectively, and the t_1/2 was increased to 167%. Fluconazole caused only a nonsignificant increase (23–41%) in the AUC and t_1/2 of the unchanged losartan. Itraconazole had no significant effect on the pharmacokinetic variables of losartan or E-3174. The ratio AUC(0∞)_E-3174/AUC(0∞)_losartan was 60% smaller during the fluconazole than during the placebo and itraconazole phases. No clinically significant changes in the effects of losartan on blood pressure and heart rate were observed between fluconazole, itraconazole and placebo phases. Conclusion: Fluconazole but not itraconazole interacts with losartan by inhibiting its metabolism to the active metabolite E-3174. This implicates that, in man, CYP2C9 is a major enzyme for the formation of E-3174 from losartan. The clinical significance of the fluconazole–losartan interaction is unclear, but the possibility of a decreased therapeutic effect of losartan should be kept in mind. Received: 4 June 1997 / Accepted in revised form: 10 September 1997     

The effect of itraconazole on the pharmacokinetics and pharmacodynamics of bromazepam in healthy volunteers

Rationale and objective Bromazepam, an anti-anxiety agent, has been reported to be metabolized by cytochrome P ₄₅₀ (CYP). However, the enzyme responsible for the metabolism of bromazepam has yet to be determined. The purpose of this study was to examine whether the inhibition of CYP3A4 produced by itraconazole alters the pharmacokinetics and pharmacodynamics of bromazepam.Methods Eight healthy male volunteers participated in this randomized double-blind crossover study. The subjects received a 6-day treatment of itraconazole (200 mg daily) or its placebo. On day 4 of the treatment, each subject received a single oral dose of bromazepam (3 mg). Blood samplings for drug assay were performed up to 70 h after bromazepam administration. The time course of the pharmacodynamic effects of bromazepam on the central nervous system was assessed using a subjective rating of sedation, continuous number addition test and electroencephalography up to 21.5 h after bromazepam administration.Results Itraconazole caused no significant changes in the pharmacokinetics and pharmacodynamics of bromazepam. The mean (±SD) values of area under the plasma concentration–time curve and elimination half-life for placebo versus itraconazole were 1328±330 ng h/ml versus 1445±419 ng h/ml and 32.1±9.3 h versus 31.1±8.4 h, respectively.Conclusion The pharmacokinetics and pharmacodynamics of bromazepam were not affected by itraconazole, suggesting that CYP3A4 is not involved in the metabolism of bromazepam to a major extent. It is likely that bromazepam can be used in the usual doses for patients receiving itraconazole or other CYP3A4 inhibitors.     

Exposure to oral oxycodone is increased by concomitant inhibition of CYP2D6 and 3A4 pathways, but not by inhibition of CYP2D6 alone

AIM

The aim of this study was to find out whether the inhibition of cytochrome P450 2D6 (CYP2D6) with paroxetine or concomitant inhibition of CYP2D6 and CYP3A4 with paroxetine and itraconazole, altered the pharmacokinetics and pharmacological response of orally administered oxycodone.

METHODS

A randomized placebo-controlled cross-over study design with three phases was used. Eleven healthy subjects ingested 10 mg of oral immediate release oxycodone on the fourth day of pre-treatment with either placebo, paroxetine (20 mg once daily) or paroxetine (20 mg once daily) and itraconazole (200 mg once daily) for 5 days. The plasma concentrations of oxycodone and its oxidative metabolites were measured for 48 h, and pharmacological (analgesic and behavioural) effects were evaluated.

RESULTS

Paroxetine alone reduced the area under concentration–time curve (AUC(0,0–48 h)) of the CYP2D6 dependent metabolite oxymorphone by 44% (P < 0.05), but had no significant effects on the plasma concentrations of oxycodone or its pharmacological effects when compared with the placebo phase. When both oxidative pathways of the metabolism of oxycodone were inhibited with paroxetine and itraconazole, the mean AUC(0,∞) of oxycodone increased by 2.9-fold (P < 0.001), and its C_max by 1.8-fold (P < 0.001). Visual analogue scores for subjective drug effects, drowsiness and deterioration of performance were slightly increased (P < 0.05) after paroxetine + itraconazole pre-treatment when compared with placebo.

CONCLUSIONS

Drug interactions arising from CYP2D6 inhibition most likely have minor clinical importance for oral oxycodone if the function of the CYP3A4 pathway is normal. When both CYP2D6 and CYP3A4 pathways are inhibited, the exposure to oral oxycodone is increased substantially.     

CYP2C8 but not CYP3A4 is important in the pharmacokinetics of montelukast

AIM

According to product information, montelukast is extensively metabolized by CYP3A4 and CYP2C9. However, CYP2C8 was also recently found to be involved. Our aim was to study the effects of selective CYP2C8 and CYP3A4 inhibitors on the pharmacokinetics of montelukast.

METHODS

In a randomized crossover study, 11 healthy subjects ingested gemfibrozil 600 mg, itraconazole 100 mg (first dose 200 mg) or both, or placebo twice daily for 5 days, and on day 3, 10 mg montelukast. Plasma concentrations of montelukast, gemfibrozil, itraconazole and their metabolites were measured up to 72 h.

RESULTS

The CYP2C8 inhibitor gemfibrozil increased the AUC(0,∞) of montelukast 4.3-fold and its t_1/2 2.1-fold (P < 0.001). Gemfibrozil impaired the formation of the montelukast primary metabolite M6, reduced the AUC and C_max of the secondary (major) metabolite M4 by more than 90% (P < 0.05) and increased those of M5a and M5b (P < 0.05). The CYP3A4 inhibitor itraconazole had no significant effect on the pharmacokinetic variables of montelukast or its M6 and M4 metabolites, but markedly reduced the AUC and C_max of M5a and M5b (P < 0.05). The effects of the gemfibrozil-itraconazole combination on the pharmacokinetics of montelukast did not differ from those of gemfibrozil alone.

CONCLUSIONS

CYP2C8 is the dominant enzyme in the biotransformation of montelukast in humans, accounting for about 80% of its metabolism. CYP3A4 only mediates the formation of the minor metabolite M5a/b, and is not important in the elimination of montelukast. Montelukast may serve as a safe and useful CYP2C8 probe drug.     

Effects of itraconazole on the pharmacokinetics and pharmacodynamics of intravenously and orally administered oxycodone

Background

The aim of this study was to investigate the effects of the cytochrome P450 3A4 (CYP34A) inhibitor itraconazole on the pharmacokinetics and pharmacodynamics of orally and intravenously administered oxycodone.

Methods

Twelve healthy subjects were administered 200 mg itraconazole or placebo orally for 5 days in a four-session paired cross-over study. On day 4, oxycodone was administered intravenously (0.1 mg/kg) in the first part of the study and orally (10 mg) in the second part. Plasma concentrations of oxycodone and its oxidative metabolites were measured for 48 h, and pharmacodynamic effects were evaluated.

Results

Itraconazole decreased plasma clearance (Cl) and increased the area under the plasma concentration–time curve (AUC0–∞) of intravenous oxycodone by 32 and 51%, respectively (P?<?0.001) and increased the AUC(0–∞) of orally administrated oxycodone by 144% (P?<?0.001). Most of the pharmacokinetic changes in oral oxycodone were seen in the elimination phase, with modest effects by itraconazole on its peak concentration, which was increased by 45% (P?=?0.009). The AUC(0–48) of noroxycodone was decreased by 49% (P?<?0.001) and that of oxymorphone was increased by 359% (P?<?0.001) after the administration of oral oxycodone. The pharmacologic effects of oxycodone were enhanced by itraconazole only modestly.

Conclusions

Itraconazole increased the exposure to oxycodone by inhibiting its CYP3A4-mediated N-demethylation. The clinical use of itraconazole in patients receiving multiple doses of oxycodone for pain relief may increase the risk of opioid-associated adverse effects.     

Examining sex-related differences in enteric itraconazole metabolism in healthy adults using grapefruit juice

Objective To explore whether sex-related differences in intestinal itraconazole metabolism exist in healthy adults using grapefruit juice (GFJ) as a selective enteric cytochrome P450 3A4 (CYP3A4) inhibitor. Methods Twenty (ten female) subjects received 240 mL bottled water or single-strength GFJ from a frozen concentrate three times daily for 2 days. On day 3, the subjects received an itraconazole oral solution 200 mg with 240 mL of beverage followed 2 h later by 240 mL of the same beverage. Serial blood sampling for itraconazole and hydroxyitraconazole serum concentrations was performed over a 72-h period. After a 20-day washout, the subjects crossed over and repeated the study. Results Among the female subjects, GFJ reduced itraconazole weight-adjusted apparent oral clearance (Cl/F) (19%, p = 0.006) and increased (30%, p = 0.01), but produced no significant change in hydroxyitraconazole pharmacokinetics. In males, GFJ produced no significant change in either itraconazole, or hydroxyitraconazole pharmacokinetics. Grapefruit juice also significantly reduced the metabolite:parent ratio (12%, p = 0.047), in females, but not males. Itraconazole weight-adjusted oral Cl/F was significantly higher in females than males when itraconazole was administered with water (56%, p = 0.009), and although the extent to which GFJ altered itraconazole weight-adjusted oral CL/F was greater in females, it did not differ significantly between the sexes (p = 0.085). Results The influence of GFJ on the presystemic metabolism of itraconazole was greater in females than males. Repeated ingestion of GFJ significantly reduced itraconazole weight-adjusted oral CL/F and significantly increased exposure in females, but it produced no significant change among males. Although itraconazole weight-adjusted oral Cl/F was much higher in females than in males, the extent to which GFJ altered itraconazole weight-adjusted oral CL/F did not differ significantly between the sexes. This work was presented in part at the American College of Clinical Pharmacy Annual Meeting, October 25, 1999, Kansas City, Missouri and at the 40th Interscience Conference of Antimicrobial Agents and Chemotherapy, September 18, 2000, Toronto Canada.     

Influence of menthol on caffeine disposition and pharmacodynamics in healthy female volunteers

Objectives The present study was undertaken to determine whether a single oral dose of menthol affects the metabolism of caffeine, a cytochrome P₄₅₀ 1A2 (CYP1A2) substrate, and pharmacological responses to caffeine in people.Methods Eleven healthy female subjects participated in a randomized, double-blind, two-way crossover study, comparing the kinetics and effects of a single oral dose of caffeine (200 mg) in coffee taken together with a single oral dose of menthol (100 mg) or placebo capsules. Serum caffeine concentrations and cardiovascular and subjective parameters were measured throughout the study.Results Co-administration of menthol resulted in an increase of caffeine t_max values from 43.6±20.6 min (mean±SD) to 76.4±28.0 min (P<0.05). The C_max values of caffeine were lower in the menthol phase than in the placebo phase, but this effect was not statistically significant (P=0.06). (AUC)_0–24, (AUC)_0–, terminal half-life and oral clearance were not affected by menthol. Only nine subjects' cardiovascular data were included in the analysis because of technical problems during the measurements. After caffeine, heart rate decreased in both treatment phases. The maximum decrease in heart rate was less in the menthol phase (–8.9±3.9 beats/min) than in the placebo phase (–13.1±2.1 beats/min) (P=0.024). There were no statistically significant differences in systolic and diastolic blood pressures between the two treatments.Conclusions We conclude that a single oral dose of pure menthol (100 mg) delays caffeine absorption and blunts the heart-rate slowing effect of caffeine, but does not affect caffeine metabolism. The possibility that menthol slows the absorption of other drugs should be considered.     

Single and multiple dose pharmacokinetics of etizolam in healthy subjects

Summary The pharmacokinetics of etizolam, a new thienodiazepine derivative, has been examined after single and multiple (0.5 mg tablet) (0.5 mg b.d for 1 week) oral therapeutic doses in healthy volunteers. The single-dose kinetic profile of etizolam suggested that absorption after oral dosage was reasonably rapid, the maximum plasma concentration (C_max) being attained within 0.5–2 h in all subjects. The mean elimination half-life (t_1/2) averaged 3.4 h. Consistent with this, steady-state concentration were rapidly achieved and accumulation was extremely limited. Predicted average plasma concentrations (Cp) did not differ significantly from those actually measured at steady-state, suggesting that the kinetics of etizolam was linear, at least at therapeutic doses. The mean wash-out t_1/2 was comparable to the elimination t_1/2 of the single dose, which means that the drug probably has no effect on hepatic microsomal enzymes and other kinetic variables after repeated dosing. At steady state plasma concentrations of the main metabolite, -hydroxyetizolam, were higher and disappeared more slowly (mean t_1/2 8.2 h) than those of the parent compound. Taken with the fact that in animals the metabolite shows almost the same potency of pharmacological action as etizolam, this suggests that it may contribute significantly to the clinical effects of the parent compound. Based on the kinetic characteristics of the parent drug and its metabolite, etizolam can be regarded as a short-acting benzodiazepine, with elimination kinetics between those of short-intermediate derivatives and ultra-rapidly eliminated benzodiazepines.     

Pharmacokinetics and pharmacodynamics of etizolam are influenced by polymorphic CYP2C19 activity

Objective To study the effect of erythromycin on metabolism of quetiapine in Chinese suffering from schizophrenia.Methods Nineteen patients received multiple doses of quetiapine (200 mg, twice daily) with or without co-administered erythromycin (500 mg, three times daily). Blood samples were collected at specified time intervals for determination of plasma concentrations of quetiapine and some of its metabolites.Results With erythromycin co-administration: for quetiapine, maximal plasma concentration (C _max), area under concentration–time curve of 0– h (AUC_0–) and terminal-phase elimination half-life time (t _1/2) increased 68, 129 and 92%, respectively, and clearance (CL) and terminal elimination rate constant (K _e) decreased 52% and 55%, respectively; for quetiapine sulfoxide (QTP-SF), C _max, AUC_0– and AUC ratio decreased 64, 23, and 70%, respectively, and t _1/2 increased 211%; for 7-hydroxy-quetiapine (QTP-H), K _e and AUC ratio decreased 61% and 45%, respectively, and t _1/2 increased 203%; for 7-hydroxy-N-desalkyl-quetiapine (QTP-ND), C _max, AUC_0– and AUC ratio decreased 36, 40 and 71%, respectively.Conclusion Erythromycin has a noticeable effect on the metabolism of quetiapine. When quetiapine is co-administered with CYP3A inhibitors such as erythromycin, the dosing regimen should be modified according to quetiapine TDM.     

Effect of probenecid on the pharmacokinetics of carbamazepine in healthy subjects

Objectives Carbamazepine (CBZ) undergoes biotransformation by CYP3A4 and CYP2C8, and glucuronide conjugation. There has been no clear demonstration to reveal the role of glucuronidation in the disposition of CBZ. We evaluated the effect of probenecid, a UDP-glucuronosyltransferase inhibitor, on the pharmacokinetics of CBZ in humans.Methods In a randomized, open-label, two-way crossover study, ten healthy male subjects were treated twice daily for 10 days with 500 mg probenecid or with a matched placebo. On day 6, a single dose of 200 mg CBZ was administered orally. Concentrations of CBZ and CBZ 10,11-epoxide (CBZ-E) in plasma and urine were measured.Results Probenecid decreased the area under the plasma concentration–time curve (AUC) of CBZ from 1253.9 mol h/l to 1020.7 mol h/l (P<0.001) while increasing that of CBZ-E from 137.6 mol h/l to 183.5 mol h/l (P=0.033). The oral clearance of CBZ was increased by probenecid by 26% (90% confidence interval, 17–34%; P<0.001). Probenecid increased the AUC ratio of CBZ-E/CBZ from 0.11 to 0.16 (P<0.001). However, probenecid had minimal effect on the recovery of the conjugated and free forms of CBZ and CBZ-E in urine.Conclusion Although probenecid showed a minimal effect on the glucuronidation of CBZ and CBZ-E, it increased CBZ biotransformation to CBZ-E, most likely reflecting the induction of CYP3A4 and CYP2C8 activities, in humans. These results demonstrate that glucuronide conjugation plays a minor role in the metabolism of CBZ and CBZ-E in humans, and that probenecid has an inducing effect on the disposition of CBZ.     

Pharmacokinetic interaction between itraconazole and metformin in rats: competitive inhibition of metabolism of each drug by each other via hepatic and intestinal CYP3A1/2

BACKGROUND AND PURPOSE

Fungal infection is prevalent in patients with diabetes mellitus. Thus, we investigated whether a pharmacokinetic interaction occurs between the anti-fungal agent itraconazole and the anti-glycaemic drug metformin, as both drugs are commonly administered together to diabetic patients and are metabolized via hepatic CYP3A subfamily in rats.

EXPERIMENTAL APPROACH

Itraconazole (20 mg·kg⁻¹) and metformin (100 mg·kg⁻¹) were simultaneously administered i.v. and p.o. to rats. Concentrations (I) of each drug in the liver and intestine, maximum velocity (V_max), Michaelis–Menten constant (K_m) and intrinsic clearance (CL_int) for the disappearance of each drug, apparent inhibition constant (K_i) and [I]/K_i ratios of each drug in the liver and intestine were determined. Also the metabolism of each drug in rat and human CYPs was measured in vitro.

KEY RESULTS

After simultaneous administration of both drugs, either i.v. or p.o., the total area under the plasma concentration–time curve from time zero to infinity (AUC)s of itraconazole and metformin were significantly greater than that of either drug administered alone. The metabolism of itraconazole and metformin was significantly inhibited by each other via CYP3A1 and 3A2 in rat and 3A4 in human microsomes.

CONCLUSIONS AND IMPLICATIONS

The significantly greater AUCs of itraconazole and metformin after i.v. administration of both drugs are probably due to competitive inhibition of the metabolism of each drug by each other via hepatic CYP3A1/2. Whereas after oral administration of both drugs, the significantly greater AUCs of each drug administered together than that of either drug alone is mainly due to competitive inhibition of intestinal metabolism of each drug by each other via intestinal CYP3A1/2.     

Effect of gemfibrozil on the pharmacokinetics of pioglitazone

Objective

Our objective was to study the effects of gemfibrozil on the pharmacokinetics of pioglitazone and the active compounds, which are all the substrates of CYP2C8 and CYP3A4.

Methods

In a randomized, two-phase crossover study, 10 healthy volunteers were pretreated for 2 days with either 600 mg oral gemfibrozil or placebo twice daily. On day 3, they received a single dose of 600 mg gemfibrozil or placebo, and 1 h later they received a single oral dose of 30 mg pioglitazone. Plasma concentrations of pioglitazone and both active metabolites M-III and M-IV were measured for up to 120 h.

Results

Gemfibrozil raised the mean total area under the concentration-time curve (AUC) of parent pioglitazone 3.4-fold (P<0.001). No statistically significant changes were seen in the total AUC of M-III or M-IV after gemfibrozil pretreatment. Gemfibrozil reduced the M-III/pioglitazone and M-IV/pioglitazone AUC_0–∞ ratio by 71% (P<0.001) and 65%(P<0.001), strikingly prolonging their t_½.

Conclusion

Gemfibrozil greatly increased the plasma concentration of parent pioglitazone and also inhibited the further metabolism of M-III and M-IV. Careful blood glucose monitoring and dosage adjustments are suggested during coadministration of pioglitazone and gemfibrozil.     

The effect of itraconazole on the pharmacokinetics and pharmacodynamics of oral prednisolone

Objective: To examine the possible effect of itraconazole on the pharmacokinetics and pharmacodynamics of orally administered prednisolone. Methods: In this double-blind, randomised, two-phase cross-over study, ten healthy subjects received either 200 mg itraconazole or placebo orally once a day for 4 days. On day 4, 20 mg prednisolone was given orally. Plasma concentrations of prednisolone, cortisol, itraconazole, and hydroxyitraconazole were determined by means of high-performance liquid chromatography up to 47 h. Results: Itraconazole increased the total area under the plasma prednisolone concentration–time curve by 24% (P < 0.001) and the elimination half-life of prednisolone by 29% (P < 0.001) compared with placebo. The peak plasma concentration and time to the peak of prednisolone were not affected by itraconazole. The mean morning plasma cortisol concentration, measured 23 h after the ingestion of prednisolone, during the itraconazole phase was 73% of that during the placebo phase (P < 0.001). Conclusions: The observed minor interaction between itraconazole and oral prednisolone is probably of limited clinical significance. The susceptibility of prednisolone to interact with CYP3A4 inhibitors is considerably smaller than that of methylprednisolone, and itraconazole and probably also other inhibitors of CYP3A4 can be used concomitantly with prednisolone without marked changes in the effects of this corticosteroid. Received: 4 October 1999 / Accepted in revised form: 29 November 1999     

Impact of CYP2D6*10 on mexiletine pharmacokinetics in healthy adult volunteers

Objective In vitro studies with human liver microsomes have suggested that the oxidative conversion of mexiletine (MX) to its metabolites is catalyzed by CYP2D6 and is significantly impaired in microsomes with the CYP2D6*10/*10 genotype. Therefore, we examined the influence of the CYP2D6*10 allele on MX pharmacokinetics in Japanese subjects.Methods Subjects with CYP2D6*1/*1 (group*1/*1; n=5), CYP2D6*10/*10 (group*10/*10; n=6) and CYP2D6*5/*10 (group*5/*10; n=4) genotypes received a single 200-mg dose of MX. Plasma and urinary levels of MX and its metabolites (p-hydroxymexiletine (PHM), hydroxymethylmexiletine (HMM) and N-hydroxymexiletine (NHM)) were determined by means of high-performance liquid chromatography.Results Mean area under the concentration–time curve (AUC) and t_1/2 of MX were significantly (P<0.05) higher in the CYP2D6*10/*5 group (AUC 11.23±3.05 µg·h/ml; t_1/2 15.5±3.2 h) than in the CYP2D6*1/*1 (AUC 5.53±1.01 µg·h/ml; t_1/2 8.1±1.6 h) and CYP2D6*10/*10 (AUC 7.32±2.36 µg·h/ml; t_1/2 10.8±2.8 h) groups, but there was no significant difference between the CYP2D6*1/*1 and CYP2D6*10/*10 groups. The maximum plasma concentration of MX was not significantly different among the three groups. The values of urinary excretion of PHM and HMM in the CYP2D6*1/*1 group were significantly (P<0.05) higher than those in the CYP2D6*10/*10 and CYP2D6*5/*10 groups, but there was no significant difference in that of NHM among the three groups. Clearance of MX in the CYP2D6*5/*10 subjects was comparable to that in the poor metabolizers described previously.Conclusion The present findings demonstrated that carriers of the CYP2D6*10 allele showed a decreased clearance of MX. Subjects with CYP2D6*5/*10 showed significantly (P<0.05) increased plasma levels of MX, and homozygotes for CYP2D6*10 also showed an increase, although to a lesser extent. Thus, the CYP2D6*10 allele plays an important role in MX pharmacokinetics.     

Identification of cytochrome P450 isoforms involved in the metabolism of loperamide in human liver microsomes

Objective: The purpose of the present study was to elucidate the cytochrome P450 (P450) isoform(s) involved in the metabolism of loperamide (LOP) to N-demethylated LOP (DLOP) in human liver microsomes. Methods: Three established approaches were used to identify the P450 isoforms responsible for LOP N-demethylation using human liver microsomes and cDNA-expressed P450 isoforms: (1) correlation of LOP N-demethylation activity with marker P450 activities in a panel of human liver microsomes, (2) inhibition of enzyme activity by P450-selective inhibitors, and (3) measurement of DLOP formation by cDNA-expressed P450 isoforms. The relative contribution of P450 isoforms involved in LOP N-demethylation in human liver microsomes were estimated by applying relative activity factor (RAF) values. Results: The formation rate of DLOP showed biphasic kinetics, suggesting the involvement of multiple P450 isoforms. Apparent K_m and V_max values were 21.1 M and 122.3 pmol/min per milligram of protein for the high-affinity component and 83.9 M and 412.0 pmol/min per milligram of protein for the low-affinity component, respectively. Of the cDNA-expressed P450 s tested, CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyzed LOP N-demethylation. LOP N-demethylation was significantly inhibited when coincubated with quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4 inhibitor) by 40 and 90%, respectively, but other chemical inhibitors tested showed weak or no significant inhibition. DLOP formation was highly correlated with CYP3A4-catalyzed midazolam 1-hydroxylation (r_s=0.829; P<0.01), CYP2B6-catalzyed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (r_s=0.691; P<0.05), and CYP2C8-catalyzed paclitaxel 6-hydroxylation (r_s=0.797; P<0.05). Conclusion: CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyze LOP N-demethylation in human liver microsomes, and among them, CYP2C8 and CYP3A4 may play a crucial role in LOP metabolism at the therapeutic concentrations of LOP. Coadministration of these P450 inhibitors may cause drug interactions with LOP. However, the clinical significance of potential interaction of LOP metabolism by CYP2C8 and CYP3A4 inhibitors should be studied further.     

Effects of fluvastatin and cigarette smoking on CYP2C9 activity measured using the probe S-warfarin

Objective The effect of cigarette smoking on CYP2C9 activity is unknown. We conducted a study to evaluate whether there is a difference in CYP2C9 activity in smokers versus non-smokers by examining S-warfarin AUC after CYP2C9 inhibition with fluvastatin. In addition, the effect of the CYP2C9 inhibitor fluvastatin was evaluated using S-warfarin as a probe.Methods A randomized, single dose, two-treatment crossover study of warfarin with a washout period of 21 days was performed. Eighteen healthy Caucasian smokers and non-smokers, genotyped as CYP2C9*1/*1 or CYP2C9*1/*2, received warfarin 10 mg plus vitamin K 10 mg to measure baseline CYP2C9 activity. Warfarin dosing was repeated after 18 days of fluvastatin 40 mg twice daily to evaluate CYP2C9 activity after inhibition.Results The S-warfarin between smokers and non-smokers did not differ by >25% after inhibition. There was no difference in S-warfarin during baseline (p=0.45) or inhibition (p=0.19) periods for smokers versus non-smokers. Fluvastatin increased the AUC of S-warfarin by 42±29% and 26±18% in smokers and nonsmokers, respectively. Linear regression analyses showed significant but weak correlations between peak concentrations (C_{at 1 h}) or (−) 3S,5R-fluvastatin AUC_{0–12 h} and extent of warfarin inhibition. For (+) 3R,5S-fluvastatin, a weak correlation was found between C_{at 1 h} and extent of warfarin inhibition.Conclusions Cigarette smoking does not affect CYP2C9 activity as evaluated using S-warfarin as a CYP2C9 probe. Fluvastatin is a weak inhibitor of CYP2C9 activity in both smokers and non-smokers.     

A case of etizolam dependence

Etizolam is a thienodiazepine anxiolytic which is said to have lower dependence potential than other benzodiazepines. We report a case of etizolam dependence in a young male with social anxiety disorder and moderate depression. This case report highlights the fact that the same caution be exercised while prescribing etizolam with respect to its potential to cause dependence as with any other benzodiazepine.KEY WORDS: Addiction, dependence, etizolam, thienodiazepine     

Identification of P450 enzymes involved in metabolism of verapamil in humans

Summary The calcium channel blocker verapamil[2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] is widely used in the treatment of hypertension, angina pectoris and cardiac arrythmias. The drug undergoes extensive and variable hepatic metabolism in man with the major metabolic steps comprising formation of D-617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile] and norverapamil [2,8-bis-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoxtanitrile]. The enzymes involved in metabolism of verapamil have not been characterized so far. Identification of these enzymes would enable estimation of both interindividual variability in verapamil metabolism introduced by the respective pathway and potential for metabolic interactions. We therefore characterized the enzymes involved in formation of D-617 and norverapamil.The maximum rate of formation of D-617 and norverapamil was determined in the microsomal fraction of 21 human livers which had been previously characterized for the individual expression of various P450 enzymes (CYP1A2, CYP2C, CYP2D6, CYP2E1 and CYP3A3/4) by means of Western blotting. Specific antibodies directed against CYP3A were used to inhibit formation of D-617 and norverapamil. Finally, formation of both metabolites was investigated in microsomes obtained from yeast cells which were genetically engineered for stable expression of human P450.Formation of D-617 was correlated with the expression of CYP3A (r=0.85; P<0.001) and CYP1A2 (r=0.57; P<0.01) in the microsomal fraction of 21 human livers after incubation with racemic verapamil. Formation of norverapamil was correlated with the expression of CYP3A (r=0.58; P<0.01) and CYP1A2 (r=0.5; P<0.05) in the same preparations after incubation with racemic verapamil. Antibodies against CYP3A reduced maximum rate of formation of D-617 (to 37.1±11% and 40.6±6.801o of control after incubation with S- and R-verapamil, respectively) and norverapamil (to 38.2±4.5% and 29.2±5.5% of control after incubation with S- and R-verapamil, respectively). Both D-617 and norverapamil were formed by stable expressed CYP3A4 (16.6 pmol/mg protein/min and 22.6 pmol/mg protein/min, respectively). In summary, formation of D-617 and norverapamil is catalyzed mainly by CYP3A4. D-617 is also formed by CYP1A2. Veraparnil therefore has the potential to interact with other drugs which are substrates or inducers of CYP3A and CYP1A2.Part of this work has been presented at the 32 Annual Spring Meeting of the German Society for Pharmacology and Toxicology, 1991, The abstract was published in Naunyn-Schmiedeberg's Archives of Pharmacology (1991) 343:R124 Correspondence to H. K. Kroemer at the above address     

The CYP2C8 inhibitor gemfibrozil does not increase the plasma concentrations of zopiclone

Objective Zopiclone is a short acting hypnotic, which is metabolised by cytochrome P450 (CYP) 3A4 and 2C8 in vitro. We studied the possible effect of gemfibrozil, an inhibitor of CYP2C8, on the pharmacokinetics and pharmacodynamics of zopiclone.Methods In a randomised 2-phase crossover study, 10 healthy volunteers took 600 mg gemfibrozil or placebo orally twice daily for 3 days. On day 3, each ingested a 7.5 mg dose of zopiclone. Plasma concentrations and urinary excretion of zopiclone and its two primary metabolites, plasma gemfibrozil, and psychomotor performance were measured. The effects of CYP2C8, CYP2C9 and CYP3A4 inhibitors on the depletion of zopiclone (500 nM) were studied in vitro in human liver microsomes.Results The pharmacokinetic variables of the parent zopiclone were not significantly affected by gemfibrozil. However, gemfibrozil raised the mean peak plasma concentration (C_max) of N-oxide-zopiclone (1.6-fold; P<0.001) and that of N-desmethyl-zopiclone (1.2-fold; P<0.001). The mean area under the plasma concentration-time curve () values of N-oxide-zopiclone and N-desmethyl-zopiclone were raised 2-fold (P<0.001) and 1.2-fold (P<0.01), respectively. The renal clearance of N-oxide-zopiclone was reduced by 48% by gemfibrozil (P<0.001). The pharmacodynamic effects of zopiclone, measured using psychometric tests, were not affected by gemfibrozil. In vitro, ketoconazole (1 μM) and itraconazole (8 μM) decreased the elimination rate of zopiclone enantiomers by about 65–95%, while montelukast (16 μM), gemfibrozil (200 μM) and sulfaphenazole (10 μM) had no appreciable effect.Conclusions Gemfibrozil does not increase the plasma concentrations of the parent zopiclone. Accordingly, CYP2C8 does not significantly metabolise zopiclone in vivo. However, as gemfibrozil raises the concentrations of two potentially active metabolites of zopiclone, slightly enhanced effects of zopiclone by gemfibrozil can not be excluded.