Substrate-dependent modulation of the catalytic activity of CYP3A by erlotinib

Aim:

To ascertain the effects of erlotinib on CYP3A, to investigate the amplitude and kinetics of erlotinib-mediated inhibition of seven major CYP isoforms in human liver microsomes (HLMs) for evaluating the magnitude of erlotinib in drug-drug interaction in vivo.

Methods:

The activities of 7 major CYP isoforms (CYP1A2, CYP2A6, CYP3A, CYP2C9, CYP2D6, CYP2C8, and CYP2E1) were assessed in HLMs using HPLC or UFLC analysis. A two-step incubation method was used to examine the time-dependent inhibition of erlotinib on CYP3A.

Results:

The activity of CYP2C8 was inhibited with an IC₅₀ value of 6.17±2.0 μmol/L. Erlotinib stimulated the midazolam 1′-hydroxy reaction, but inhibited the formation of 6β-hydroxytestosterone and oxidized nifedipine. Inhibition of CYP3A by erlotinib was substrate-dependent: the IC₅₀ values for inhibiting testosterone 6β-hydroxylation and nifedipine metabolism were 31.3±8.0 and 20.5±5.3 μmol/L, respectively. Erlotinib also exhibited the time-dependent inhibition on CYP3A, regardless of the probe substrate used: the value of K_I and k_inact were 6.3 μmol/L and 0.035 min⁻¹ for midazolam; 9.0 μmol/L and 0.045 min⁻¹ for testosterone; and 10.1 μmol/L and 0.058 min⁻¹ for nifedipine.

Conclusion:

The inhibition of CYP3A by erlotinib was substrate-dependent, while its time-dependent inhibition on CYP3A was substrate-independent. The time-dependent inhibition of CYP3A may be a possible cause of drug-drug interaction, suggesting that attention should be paid to the evaluation of erlotinib''s safety, especially in the context of combination therapy.     

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.     

Mechanism-based inhibition of cytochrome P450 (CYP)2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo

BACKGROUND AND PURPOSE

Chalepensin is a pharmacologically active furanocoumarin compound found in rue, a medicinal herb. Here we have investigated the inhibitory effects of chalepensin on cytochrome P450 (CYP) 2A6 in vitro and in vivo.

EXPERIMENTAL APPROACH

Mechanism-based inhibition was studied in vitro using human liver microsomes and bacterial membranes expressing genetic variants of human CYP2A6. Effects in vivo were studied in C57BL/6J mice. CYP2A6 activity was assayed as coumarin 7-hydroxylation (CH) using HPLC and fluorescence measurements. Metabolism of chalepensin was assessed with liquid chromatography/mass spectrometry (LC/MS).

KEY RESULTS

CYP2A6.1, without pre-incubation with NADPH, was competitively inhibited by chalepensin. After pre-incubation with NADPH, inhibition by chalepensin was increased (IC₅₀ value decreased by 98%). This time-dependent inactivation (k_inact 0.044 min⁻¹; K_I 2.64 µM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation. LC/MS analysis of chalepensin metabolites suggested an unstable epoxide intermediate was formed, identified as the corresponding dihydrodiol, which was then conjugated with glutathione. Compared with the wild-type CYP2A6.1, the isoforms CYP2A6.7 and CYP2A6.10 were less inhibited. In mouse liver microsomes, pre-incubation enhanced inhibition of CH activity. Oral administration of chalepensin to mice reduced hepatic CH activity ex vivo.

CONCLUSIONS AND IMPLICATIONS

Chalepensin was a substrate and a mechanism-based inhibitor of human CYP2A6. Formation of an epoxide could be a key step in this inactivation. ‘Poor metabolizers’ carrying CYP2A6*7 or *10 may be less susceptible to inhibition by chalepensin. Given in vivo, chalepensin decreased CYP2A activity in mice.     

Contribution of CYP2C19 and CYP3A4 to the formation of the active nortilidine from the prodrug tilidine

AIMS

To investigate in vivo the effect of the CYP2C19 genotype on the pharmacokinetics of tilidine and the contribution of CYP3A4 and CYP2C19 to the formation of nortilidine using potent CYP3A4 inhibition by ritonavir.

METHODS

Fourteen healthy volunteers (seven CYP2C19 poor and seven ultrarapid metabolizers) received ritonavir orally (300 mg twice daily) for 3 days or placebo, together with a single oral dose of tilidine and naloxone (100 mg and 4 mg, respectively). Blood samples and urine were collected for 72 h. Noncompartmental analysis was performed to determine pharmacokinetic parameters of tilidine, nortilidine, bisnortilidine and ritonavir.

RESULTS

Tilidine exposure increased sevenfold and terminal elimination half-life fivefold during ritonavir treatment, but no significant differences were observed between the CYP2C19 genotypes. During ritonavir treatment, nortilidine area under the concentration–time curve was on average doubled, with no differences between CYP2C19 poor metabolizers [2242 h ng ml⁻¹ (95% confidence interval 1811–2674) vs. 996 h ng ml⁻¹ (95% confidence interval 872–1119)] and ultrarapid metabolizers [2074 h ng ml⁻¹ (95% confidence interval 1353–2795) vs. 1059 h ng ml⁻¹ (95% confidence interval 789–1330)]. The plasma concentration–time curve of the secondary metabolite, bisnortilidine, showed a threefold increase of time to reach maximal observed plasma concentration; however, area under the concentration–time curve was not altered by ritonavir.

CONCLUSIONS

The sequential metabolism of tilidine is inhibited by the potent CYP3A4 inhibitor, ritonavir, independent of the CYP2C19 genotype, with a twofold increase in the exposure of the active nortilidine.     

A clinical study to assess CYP1A2 and CYP3A4 induction by AZD7325, a selective GABA(A) receptor modulator - an in vitro and in vivo comparison

AIM(S)

To investigate the potential of AZD7325 to induce CYP1A2 and CYP3A4 enzyme activities.

METHODS

Induction of CYP1A2 and CYP3A4 by AZD7325 was first evaluated using cultured human hepatocytes. The effect of multiple doses of 10 mg AZD7325 on the pharmacokinetics of midazolam and caffeine was then examined in healthy subjects.

RESULTS

The highest CYP1A2 and CYP3A4 induction responses were observed in human hepatocytes treated with 1 or 10 µm of AZD7325, in the range of 17.9%–54.9% and 76.9%–85.7% of the positive control responses, respectively. The results triggered the further clinical evaluation of AZD7325 induction potential. AZD7325 reached a plasma C_max of 0.2 µm after 10 mg daily dosing to steady-state. AZD7325 decreased midazolam geometric mean AUC by 19% (0.81-fold, 90% CI 0.77, 0.87), but had no effect on midazolam C_max (90% CI 0.82, 0.97). The mean CL/F of midazolam increased from 62 l h⁻¹ (midazolam alone) to 76 l h⁻¹ when co-administered with AZD7325. The AUC and C_max of caffeine were not changed after co-administration of AZD7325, with geometric mean ratios (90% CI) of 1.17 (1.12, 1.23) and 0.99 (0.95, 1.03), respectively.

CONCLUSIONS

While AZD7325 appeared to be a potent CYP3A4 inducer and a moderate CYP1A2 inducer from in vitro studies, the expected efficacious dose of AZD7325 had no effect on CYP1A2 activity and only a weak inducing effect on CYP3A4 activity. This comparison of in vitro and in vivo results demonstrates the critical role that clinical exposure plays in evaluating the CYP induction risk of a drug candidate.     

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.     

Effects of imatinib (Glivec) on the pharmacokinetics of metoprolol, a CYP2D6 substrate, in Chinese patients with chronic myelogenous leukaemia

AIMS

To investigate the effect of imatinib on the pharmacokinetics of a CYP2D6 substrate, metoprolol, in patients with chronic myeloid leukaemia (CML). The pharmacokinetics of imatinib were also studied in these patients.

METHODS

Patients (n = 20) received a single oral dose of metoprolol 100 mg on day 1 after an overnight fast. On days 2–10, imatinib 400 mg was administered twice daily. On day 8, another 100 mg dose of metoprolol was administered 1 h after the morning dose of imatinib 400 mg. Blood samples for metoprolol and α-hydroxymetoprolol measurement were taken on study days 1 and 8, and on day 8 for imatinib.

RESULTS

Of the 20 patients enrolled, six patients (30%) were CYP2D6 intermediate metabolizers (IMs), 13 (65%) extensive metabolizers (EMs), and the CYP2D6 status in one patient was unknown. In the presence of 400 mg twice daily imatinib, the mean metoprolol AUC was increased by 17% in IMs (from 1190 to 1390 ng ml⁻¹ h), and 24% in EMs (from 660 to 818 ng ml⁻¹ h). Patients classified as CYP2D6 IMs had an approximately 1.8-fold higher plasma metoprolol exposure than those classified as EMs. The oral clearance of imatinib was 11.0 ± 2.0 l h⁻¹ and 11.8 ± 4.1 l h⁻¹ for CYP2D6 IMs and EMs, respectively.

CONCLUSIONS

Co-administration of a high dose of imatinib resulted in a small or moderate increase in metoprolol plasma exposure in all patients regardless of CYP2D6 status. The clearance of imatinib showed no difference between CYP2D6 IMs and EMs.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Imatinib, a tyrosine kinase inhibitor, exhibits a competitive inhibition on the CYP450 2D6 isozyme with a K_i value of 7.5 μm. However, the clinical significance of the inhibition and its relevance to 2D6 polymorphisms have not been evaluated. The pharmacokinetics of imatinib have been well studied in Caucasians, but not in a Chinese population.
• Metoprolol, a CYP2D6 substrate, has different clearances among patients with different CYP2D6 genotypes. It is often used as a CYP2D6 probe substrate for clinical drug–drug interaction studies.

WHAT THIS STUDY ADDS

• Co-administration of imatinib at 400 mg twice daily increased the plasma AUC of metoprolol by approximately 23% in 20 Chinese patients with chronic myeloid leukaemia (CML), about 17% increase in CYP2D6 intermediate metabolizers (IMs) (n = 6), 24% in extensive metabolizers (EMs) (n = 13), and 28% for the subject with unknown 2D6 status (n = 1) suggesting that imatinib has a weak to moderate inhibition on CYP2D6 in vivo.
• The clearance of imatinib in Chinese patients with CML showed no difference between CYP2D6 IMs and EMs, and no major difference from Caucasian patients with CML based on data reported in the literature.

     

Effects of CYP3A4 inhibition and induction on the pharmacokinetics and pharmacodynamics of tolvaptan, a non-peptide AVP antagonist in healthy subjects

AIMS

In vitro studies indicated CYP3A4 alone was responsible for tolvaptan metabolism. To determine the effect of a CYP3A4 inhibitor (ketoconazole) and a CYP3A4 inducer (rifampicin) on tolvaptan pharmacokinetics (PK) and pharmacodynamics (PD), two clinical trials were performed.

METHODS

For CYP3A4 inhibition, a double-blind, randomized (5:1), placebo-controlled trial was conducted in 24 healthy subjects given either a single 30 mg dose of tolvaptan (n = 19) or matching placebo (n = 5) on day 1 with a 72 h washout followed by a 3 day regimen of 200 mg ketoconazole, once daily with 30 mg tolvaptan or placebo also given on day 5. For CYP3A4 induction, 14 healthy subjects were given a single dose of 240 mg tolvaptan with 48 h washout followed by a 7 day regimen of 600 mg rifampicin, once daily, with 240 mg tolvaptan also given on the seventh day.

RESULTS

When co-administered with ketoconazole, mean C_max and AUC(0,∞) of tolvaptan were increased 3.48- and 5.40-fold, respectively. Twenty-four hour urine volume increased from 5.9 to 7.7 l. Erythromycin breath testing showed no difference following a single dose of tolvaptan. With rifampicin, tolvaptan mean C_max and AUC were reduced to 0.13- and 0.17-fold of tolvaptan administered alone. Twenty-four hour urine volume decreased from 12.3 to 8.8 l.

CONCLUSIONS

Tolvaptan is a sensitive CYP3A4 substrate with no inhibitory activity. Due to the saturable nature of tolvaptan''s effect on urine excretion rate, changes in the pharmacokinetic profile of tolvaptan do not produce proportional changes in urine output.     

Lack of effect of tofacitinib (CP-690,550) on the pharmacokinetics of the CYP3A4 substrate midazolam in healthy volunteers: confirmation of in vitro data

AIMS

To investigate inhibitive and inductive effects of tofacitinib (CP-690,550), a Janus kinase inhibitor, on CYP3A4 function via in vitro and in vivo studies.

METHODS

In vitro experiments were conducted to assess the inhibition and induction potential of tofacitinib for major drug metabolizing enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4). A phase 1, randomized, open-label, two-way crossover study ({"type":"clinical-trial","attrs":{"text":"NCT00902460","term_id":"NCT00902460"}}NCT00902460) was conducted to confirm the lack of inhibitive/inductive effect on a sensitive CYP3A4 substrate, midazolam, in healthy subjects. Midazolam pharmacokinetics were assessed over 24 h following single dose 2 mg administration prior to administering tofacitinib and after twice daily dosing of tofacitinib 30 mg for 6 days. The primary endpoint was midazolam area under the concentration–time profile, from time 0 to infinity (AUC(0,∞)).

RESULTS

In vitro studies demonstrated low potential for CYP inhibition (IC₅₀ estimates tofacitinib >30 µm), CYP3A4 mRNA induction (observed at tofacitinib concentrations ≥25 µm) and no effect on enzymatic activity of CYP substrates. In the human study, AUC(0,∞) adjusted geometric mean ratio for midazolam plus tofacitinib to midazolam alone was 103.97% [90% confidence interval (CI) 95.57, 113.12], wholly within the pre-specified acceptance region (80, 125). The 90% CI for the ratio of adjusted geometric means of maximum plasma concentration (C_max) (95.98, 108.87) was also wholly within this acceptance region.

CONCLUSIONS

These data confirm a lack of an inhibitive or inductive effect of tofacitinib on CYP3A activity in humans and, in conjunction with in vitro data, support the conclusion that tofacitinib is unlikely to influence the CYP enzyme system as a whole.     

Effect of danshen extract on the activity of CYP3A4 in healthy volunteers

AIMS

To assess the effect of danshen extract on CYP3A4 activity using midazolam as an in vivo probe.

METHODS

A sequential, open-label, two-period pharmacokinetic interaction study design was used to compare midazolam pharmacokinetic parameters before and after 14 days of administration of danshen tablets. Twelve healthy volunteers received a single oral dose (15 mg) of midazolam followed by danshen tablets (four tablets orally, three times a day) for 14 days. On the last day of the study they received four danshen tablets with a 15 mg midazolam tablet and plasma concentrations of midazolam and its corresponding metabolite 1–hydroxylmidazolam were measured prior to and after the administration of danshen tablets periodically for 12 h.

RESULTS

The 90% confidence intervals of C_max,t_1/2, CL/F and AUC(0,∞) of midazolam before and after administration of danshen tablets were (0.559, 0.849), (0.908, 1.142), (1.086, 1.688) and (0.592, 0.921), respectively; and those of C_max, t_1/2 and AUC(0,∞) of 1-hydroxylmidazolam after vs. before administration of danshen tablets were (0.633, 0.923), (0.801, 1.210) and (0.573, 0.980), respectively. Ratios of geometric LS means of C_max(1OHMid) : C_max(Mid) and AUC_max(1OHMid) : AUC_max(Mid) (after vs. before 14-day danshen) were 1.072 and 1.035, respectively.

CONCLUSIONS

Our findings suggest that multiple dose administration of danshen tablets may induce CYP3A4 in the gut. Accordingly, caution should be taken when danshen products are used in combination with therapeutic drugs metabolized by CYP3A.     

The effects of CYP2D6 and CYP3A activities on the pharmacokinetics of immediate release oxycodone

Background and purpose:

There is high interindividual variability in the activity of drug-metabolizing enzymes catalysing the oxidation of oxycodone [cytochrome P450 (CYP) 2D6 and 3A], due to genetic polymorphisms and/or drug–drug interactions. The effects of CYP2D6 and/or CYP3A activity modulation on the pharmacokinetics of oxycodone remains poorly explored.

Experimental approach:

A randomized crossover double-blind placebo-controlled study was performed with 10 healthy volunteers genotyped for CYP2D6 [six extensive (EM), two deficient (PM/IM) and two ultrarapid metabolizers (UM)]. The volunteers randomly received on five different occasions: oxycodone 0.2 mg·kg⁻¹ and placebo; oxycodone and quinidine (CYP2D6 inhibitor); oxycodone and ketoconazole (CYP3A inhibitor); oxycodone and quinidine+ketoconazole; placebo. Blood samples for plasma concentrations of oxycodone and metabolites (oxymorphone, noroxycodone and noroxymorphone) were collected for 24 h after dosing. Phenotyping for CYP2D6 (with dextromethorphan) and CYP3A (with midazolam) were assessed at each session.

Key results:

CYP2D6 activity was correlated with oxymorphone and noroxymorphone AUCs and C_max (−0.71 < Spearman correlation coefficient ρs < −0.92). Oxymorphone C_max was 62% and 75% lower in PM than EM and UM. Noroxymorphone C_max reduction was even more pronounced (90%). In UM, oxymorphone and noroxymorphone concentrations increased whereas noroxycodone exposure was halved. Blocking CYP2D6 (with quinidine) reduced oxymorphone and noroxymorphone C_max by 40% and 80%, and increased noroxycodone AUC_∞ by 70%. Blocking CYP3A4 (with ketoconazole) tripled oxymorphone AUC_∞ and reduced noroxycodone and noroxymorphone AUCs by 80%. Shunting to CYP2D6 pathway was observed after CYP3A4 inhibition.

Conclusions and implications:

Drug–drug interactions via CYP2D6 and CYP3A affected oxycodone pharmacokinetics and its magnitude depended on CYP2D6 genotype.     

Prediction of tacrolimus metabolism and dosage requirements based on CYP3A4 phenotype and CYP3A5*3 genotype in Chinese renal transplant recipients

Aim:

To examine how the endogenous CYP3A4 phenotype and CYP3A5^*3 genotype of Chinese renal transplant recipients influenced the dose-corrected trough concentration (C₀/D) and weight-corrected daily dose (D/W) of tacrolimus.

Methods:

A total of 101 medically stable kidney transplant recipients were enrolled, and their blood and urine samples were gathered. The endogenous CYP3A4 phenotype was assessed by the ratio of 6β-hydroxycortisol and 6β-hydroxycortisone to cortisol and cortisone in urine. CYP3A5^*3 genotype was determined using PCR-RELP.

Results:

In overall renal transplant recipients, a multiple regression analysis including the endogenous CYP3A4 phenotype, CYP3A5^*3 genotype and post-operative period accounted for 60.1% of the variability in C₀/D ratio; a regression equation consisting of the endogenous CYP3A4 phenotype, post-operative period, body mass index, CYP3A5^*3 genotype, gender, total bilirubin and age explained 61.0% of the variability in D/W ratio. In CYP3A5^*3/^*3 subjects, a combination of the endogenous CYP3A4 phenotype, post-operative period and age was responsible for 65.3% of the variability in C₀/D ratio; a predictive equation including the endogenous CYP3A4 phenotype, post-operative period, body mass index, gender and age explained 61.2% of the variability in the D/W ratio. Base on desired target range of tacrolimus trough concentrations, individual daily dosage regimen was calculated, and all the observed daily doses were within the predicted range.

Conclusion:

This study provides the equations to predict tacrolimus metabolism and dosage requirements based on the endogenous CYP3A4 phenotype, CYP3A5^*3 genotype and other non-genetic variables.     

Lack of tacrolimus circadian pharmacokinetics and CYP3A5 pharmacogenetics in the early and maintenance stages in Japanese renal transplant recipients

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Circadian variations of tacrolimus pharmacokinetics are controversial.
• Also, the pharmacokinetics has time-dependent variability, such as a decrease in oral clearance and increase in the dose-adjusted AUC after transplantation.
• Although the CYP3A5 polymorphism is associated with tacrolimus pharmacokinetics, differences in the influence of this gene on the pharmacokinetics between the early and maintenance stages have not yet been clarified.

WHAT THIS STUDY ADDS

• Tacrolimus pharmacokinetics did not show circadian variation in either the early or maintenance stage with our designated-time administration strategy.
• Based on previous results and our own findings, the interval between food consumption and tacrolimus administration might influence the interindividual and interinstitutional variability of tacrolimus chronopharmacokinetics.
• The CYP3A5 polymorphism may be associated with the time-dependent changes in tacrolimus oral clearance.

AIMS

We investigated whether tacrolimus pharmacokinetics shows circadian variation and the influence of the CYP3A5 A6986G polymorphism on the pharmacokinetics in both the early and maintenance stages after renal transplantation.

METHODS

Tacrolimus was administered twice daily at specified times (09.00 and 21.00 h) throughout the pre- and post-transplant period according to the trough-targeting strategy. Fifty recipients with stable graft function were studied on day 28 and beyond 1-year post transplantation. Whole blood samples were collected prior to and 1, 2, 3, 4, 6, 9 and 12 h after both the morning and evening doses during hospitalization.

RESULTS

Tacrolimus pharmacokinetics did not show circadian variation in either the early or maintenance stage [AUC_0–12 197.1 (95% confidence interval 182.9, 212.3) in daytime vs. 203.6 ng h ml⁻¹ (189.8, 217.4) in the night-time at day 28, 102.0 (92.1, 111.9) vs. 107.7 (97.9, 117.5) at 1 year, respectively]. In CYP3A5 *1 allele carriers (CYP3A5 expressers), body weight-adjusted oral clearance was markedly decreased from the early stage to the maintenance stage [0.622 (0.534, 0.709) to 0.369 l h⁻¹ kg⁻¹ (0.314, 0425)] compared with a smaller decrease [0.368 (0.305, 0.430) to 0.305 (0.217, 0.393)] in CYP3A5 non-expressers; however, the CYP3A5 genetic variation did not influence tacrolimus chronopharmacokinetics.

CONCLUSION

Equivalent daytime and night-time tacrolimus pharmacokinetics were achieved during both the early and maintenance stages with our specified-time administration strategy. The CYP3A5 polymorphism may be associated with the time-dependent changes in the oral clearance of tacrolimus, suggesting that genotyping of this polymorphism is useful for determining the appropriate dose of tacrolimus in both the early and maintenance stages after renal transplantation.     

Metabolism of novel anti-HIV agent 3-cyanomethyl-4-methyl-DCK by human liver microsomes and recombinant CYP enzymes

Aim:

To investigate the metabolism of 3-cyanomethyl-4-methyl-DCK (CMDCK), a novel anti-HIV agent, by human liver microsomes (HLMs) and recombinant cytochrome P450 enzymes (CYPs).

Methods:

CMDCK was incubated with HLMs or a panel of recombinant cytochrome P450 enzymes including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5. LC-ion trap mass spectrometry was used to separate and identify CMDCK metabolites. In the experiments with recombinant cytochrome P450 enzymes, specific chemical inhibitors combined with CYP antibodies were used to identify the CYP isoforms involved in CMDCK metabolism.

Results:

CMDCK was rapidly and extensively metabolized by HLMs. Its intrinsic hepatic clearance estimated from the in vitro data was 19.4 mL·min⁻¹·kg⁻¹, which was comparable to the mean human hepatic blood flow rate (20.7 mL·min⁻¹·kg⁻¹). The major metabolic pathway of CMDCK was oxidation, and a total of 14 metabolites were detected. CYP3A4 and 3A5 were found to be the principal CYP enzymes responsible for CMDCK metabolism.

Conclusion:

CMDCK was metabolized rapidly and extensively in human hepatic microsomes to form a number of oxidative metabolites. CYP3A4 and 3A5 were the predominant enzymes responsible for the oxidation of CMDCK.     

Effects of drug interactions on biotransformation and antiplatelet effect of clopidogrel in vitro

BACKGROUND AND PURPOSE

The conversion of clopidogrel to its active metabolite, R-130964, is a two-step cytochrome P450 (CYP)-dependent process. The current investigations were performed to characterize in vitro the effects of different CYP inhibitors on the biotransformation and on the antiplatelet effect of clopidogrel.

EXPERIMENTAL APPROACH

Clopidogrel biotransformation was studied using human liver microsomes (HLM) or specific CYPs and platelet aggregation using human platelets activated with ADP.

KEY RESULTS

Experiments using HLM or specific CYPs (3A4, 2C19) revealed that at clopidogrel concentrations >10 µM, CYP3A4 was primarily responsible for clopidogrel biotransformation. At a clopidogrel concentration of 40 µM, ketoconazole showed the strongest inhibitory effect on clopidogrel biotransformation and clopidogrel-associated inhibition of platelet aggregation with IC₅₀ values of 0.03 ± 0.07 µM and 0.55 ± 0.06 µM respectively. Clarithromycin, another CYP3A4 inhibitor, impaired clopidogrel biotransformation and antiplatelet activity almost as effectively as ketoconazole. The CYP3A4 substrates atorvastatin and simvastatin both inhibited clopidogrel biotransformation and antiplatelet activity, less potently than ketoconazole. In contrast, pravastatin showed no inhibitory effect. As clopidogrel itself inhibited CYP2C19 at concentrations >10 µM, the CYP2C19 inhibitor lansozprazole affected clopidogrel biotransformation only at clopidogrel concentrations ≤10 µM. The carboxylate metabolite of clopidogrel was not a CYP substrate and did not affect platelet aggregation.

CONCLUSIONS AND IMPLICATIONS

At clopidogrel concentrations >10 µM, CYP3A4 is mainly responsible for clopidogrel biotransformation, whereas CYP2C19 contributes only at clopidogrel concentrations ≤10 µM. CYP2C19 inhibition by clopidogrel at concentrations >10 µM may explain the conflicting results between in vitro and in vivo investigations regarding drug interactions with clopidogrel.     

The effect on sotrastaurin pharmacokinetics of strong CYP3A inhibition by ketoconazole

AIMS

Sotrastaurin is an immunosuppressant that reduces T-lymphocyte activation via protein kinase C inhibition. The effect of CYP3A4 inhibition by ketoconazole on the pharmacokinetics of sotrastaurin, a CYP3A4 substrate, was investigated.

METHODS

This was a two-period, single-sequence crossover study in 18 healthy subjects. They received a single 50 mg oral dose of sotrastaurin in period 1 followed by a 14-day inter-treatment phase. In period 2 they received ketoconazole 200 mg twice daily for 6 days and a single 50 mg dose of sotrastaurin on the fourth day of ketoconazole administration.

RESULTS

Co-administration of single-dose sotrastaurin during steady-state ketoconazole increased sotrastaurin C_max by 2.5-fold (90% confidence interval 2.2, 2.9) from 285 ± 128 to 678 ± 189 ng ml⁻¹ and increased AUC by 4.6-fold (4.1, 5.2) from 1666 ± 808 to 7378 ± 3011 ng ml⁻¹ h. Sotrastaurin half-life was nearly doubled from 5.9 ± 1.7 to 10.6 ± 2.5 h. The AUC of the active metabolite N-desmethyl-sotrastaurin was increased by 6.8-fold. Sotrastaurin did not alter ketoconazole steady-state predose plasma concentrations.

CONCLUSIONS

The strong CYP3A4 inhibitor ketoconazole increased sotrastaurin AUC by 4.6-fold. A compensatory reduction in the dose of sotrastaurin is warranted when strong CYP3A4 inhibitors are co-administered.     

The recovery time-course of CYP3A after induction by St John's wort administration

AIMS

To examine the recovery time course of CYP3A after enzyme induction by St John''s wort administration.

METHODS

The subjects were 12 healthy men, aged 20–33 years. On the first day, they received an oral dose of midazolam 5 mg without St John''s wort (day −14). From the next day, they took St John''s wort for 14 days. On the last day of St John''s wort treatment (day 0) and 3 and 7 days after completion of St John''s wort treatment (days 3 and 7), they received the same dose of midazolam. On each day, blood samples were obtained until 8 h after midazolam administration. Plasma concentrations of midazolam were measured by HPLC. Pharmacokinetic parameters of midazolam were determined using noncompartmental analysis.

RESULTS

Apparent oral clearance of midazolam was significantly increased after St John''s wort administration from 65.3 ± 8.4 l h⁻¹ (day −14) to 86.8 ± 17.3 l h⁻¹ (day 0). It returned to the control level 7 days after the completion of St John''s wort (day 7, 59.7 ± 3.8 l h⁻¹). No significant difference in the elimination half-life between the four periods of the study was observed. The changes in apparent oral clearance after St John''s wort discontinuation indicated that CYP3A activity recovers from enzyme induction with an estimated half-life of 46.2 h.

CONCLUSIONS

CYP3A activity induced by St John''s wort administration progressively returns to the basal level after approximately 1 week. This finding may provide useful information to avoid clinically significant interactions of St John''s wort with CYP3A substrates.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• St John''s wort causes the induction of CYP3A. Little is known about how long the effect remains after cessation of St John''s wort.

WHAT THIS STUDY ADDS

• The in vivo CYP3A activity returns progressively to the basal level approximately 1 week after cessation of St John''s wort administration

     

Impact of CYP2D6, CYP3A5, CYP2C9 and CYP2C19 polymorphisms on tamoxifen pharmacokinetics in Asian breast cancer patients

AIM

To investigate the impact of genetic polymorphisms in CYP2D6, CYP3A5, CYP2C9 and CYP2C19 on the pharmacokinetics of tamoxifen and its metabolites in Asian breast cancer patients.

METHODS

A total of 165 Asian breast cancer patients receiving 20 mg tamoxifen daily and 228 healthy Asian subjects (Chinese, Malay and Indian; n = 76 each) were recruited. The steady-state plasma concentrations of tamoxifen and its metabolites were quantified using high-performance liquid chromatography. The CYP2D6 polymorphisms were genotyped using the INFINITI™ CYP450 2D6I assay, while the polymorphisms in CYP3A5, CYP2C9 and CYP2C19 were determined via direct sequencing.

RESULTS

The polymorphisms, CYP2D6*5 and *10, were significantly associated with lower endoxifen and higher N-desmethyltamoxifen (NDM) concentrations. Patients who were *1/*1 carriers exhibited 2.4- to 2.6-fold higher endoxifen concentrations and 1.9- to 2.1-fold lower NDM concentrations than either *10/*10 or *5/*10 carriers (P < 0.001). Similarly, the endoxifen concentrations were found to be 1.8- to 2.6-times higher in *1/*5 or *1/*10 carriers compared with *10/*10 and *5/*10 carriers (P≤ 0.001). Similar relationships were observed between the CYP2D6 polymorphisms and metabolic ratios of tamoxifen and its metabolites. No significant associations were observed with regards to the polymorphisms in CYP3A5, CYP2C9 and CYP2C19.

CONCLUSIONS

The present study in Asian breast cancer patients showed that CYP2D6*5/*10 and *10/*10 genotypes are associated with significantly lower concentrations of the active metabolite of tamoxifen, endoxifen. Identifying such patients before the start of treatment may be useful in optimizing therapy with tamoxifen. The role of CYP3A5, CYP2C9 and CYP2C19 seem to be minor.     

Methadone inhibits CYP2D6 and UGT2B7/2B4 in vivo: a study using codeine in methadone- and buprenorphine-maintained subjects

AIMS

To compare the O-demethylation (CYP2D6-mediated), N-demethylation (CYP3A4-mediated) and 6-glucuronidation (UGT2B4/7-mediated) metabolism of codeine between methadone- and buprenorphine-maintained CYP2D6 extensive metabolizer subjects.

METHODS

Ten methadone- and eight buprenorphine-maintained subjects received a single 60 mg dose of codeine phosphate. Blood was collected at 3 h and urine over 6 h and assayed for codeine, norcodeine, morphine, morphine-3- and -6-glucuronides and codeine-6-glucuronide.

RESULTS

The urinary metabolic ratio for O-demethylation was significantly higher (P = 0.0044) in the subjects taking methadone (mean ± SD, 2.8 ± 3.1) compared with those taking buprenorphine (0.60 ± 0.43), likewise for 6-glucuronide formation (0.31 ± 0.24 vs. 0.053 ± 0.027; P < 0.0002), but there was no significant difference (P = 0.36) in N-demethylation. Similar changes in plasma metabolic ratios were also found. In plasma, compared with those maintained on buprenorphine, the methadone-maintained subjects had increased codeine and norcodeine concentrations (P < 0.004), similar morphine (P = 0.72) and lower morphine-3- and -6- and codeine-6-glucuronide concentrations (P < 0.008).

CONCLUSION

Methadone is associated with inhibition of CYP2D6 and UGTs 2B4 and 2B7 reactions in vivo, even though it is not a substrate for these enzymes. Plasma morphine was not altered, owing to the opposing effects of inhibition of both formation and elimination; however, morphine-6-glucuronide (analgesically active) concentrations were substantially reduced. Drug interactions with methadone are likely to include drugs metabolized by various UGTs and CYP2D6.     

Progressive decline in tacrolimus clearance after renal transplantation is partially explained by decreasing CYP3A4 activity and increasing haematocrit

Aims

The long-term disposition of tacrolimus following kidney transplantation is characterized by a gradual decrease in dose requirements and increase in dose-corrected exposure. This phenomenon has been attributed to a progressive decline in cytochrome P450 3A4 (CYP3A4) activity, although this has never been demonstrated in vivo.

Methods

Sixty-five tacrolimus- and 10 cyclosporine-treated renal transplant recipients underwent pharmacokinetic testing at day 7 and months 1, 3, 6 and 12 after transplantation, including 8-h area under the concentration-time curve (AUC) for tacrolimus or cyclosporine and assessment of CYP3A4 activity using oral and intravenous midazolam (MDZ) drug probes.

Results

Tacrolimus clearance decreased gradually throughout the entire first year but only in CYP3A5*3/*3 homozygous recipients (25.6 ± 11.1 l h^–1 at day 7; 17 ± 9.1 l h^–1 at month 12; P < 0.001). In mixed model analysis, decreasing CYP3A4 activity, measured by apparent oral MDZ clearance (924 ± 443 ml min^–1 at day 7 vs. 730 ± 344 ml min^–1 at month 1; P < 0.001), explained 55.4% of the decline in tacrolimus clearance in the first month. CYP3A4 activity decreased by 18.9 ml min^–1 for every milligram of methylprednisolone dose tapering within the first month; beyond this point it remained stable. A gradual rise in haematocrit throughout the entire first year explained 31.7% of the decrease in tacrolimus clearance in the first month and 23.6% of the decrease between months 1 and 12. Cyclosporine clearance did not change over time.

Conclusions

The maturation of tacrolimus disposition in the first year after renal transplantation observed in CYP3A5*3/*3 homozygous patients can partly be explained by a (steroid tapering-related) decline in CYP3A4 activity and a progressive increase in haematocrit.