The involvement of CYP1A2 and CYP3A4 in the metabolism of clozapine

Aims Clozapine (CLZ), an atypical neuroleptic with a high risk of causing agranulocytosis, is metabolized in the liver to desmethylclozapine (DCLZ) and clozapine N-oxide (CLZ-NO). This study investigated the involvement of different CYP isoforms in the formation of these two metabolites. Methods Human liver microsomal incubations, chemical inhibitors, specific antibodies, and different cytochrome P450 expression systems were used. Results K_m and V_max values determined in human liver microsomes were lower for the demethylation (61±21 μm, 159±42 pmol min⁻¹ mg protein⁻¹ mean±s.d.; n=4), than for the N-oxidation of CLZ (308±1.5 μm, 456±167pmol min⁻¹ mg protein⁻¹; n=3). Formation of DCLZ was inhibited by fluvoxamine (53±28% at 10 μm ), triacetyloleandomycin (33±15% at 10 μm ), and ketoconazole (51±28% at 2 μm ) and by antibodies against CYP1A2 and CYP3A4. CLZ-NO formation was inhibited by triacetyloleandomycin (34±16% at 10 μm ) and ketoconazole (51±13% at 2 μm ), and by antibodies against CYP3A4. There was a significant correlation between CYP3A content and DCLZ formation in microsomes from 15 human livers (r=0.67; P=0.04). A high but not significant correlation coefficient was found for CYP3A content and CLZ-NO formation (r=0.59; P=0.09). Using expression systems it was shown that CYP1A2 and CYP3A4 formed DCLZ and CLZ-NO. K_m and V_max values were lower in the CYP1A2 expression system compared to CYP3A4 for both metabolic reactions. Conclusions It is concluded that CYP1A2 and CYP3A4 are involved in the demethylation of CLZ and CYP3A4 in the N-oxidation of CLZ. Close monitoring of CLZ plasma levels is recommended in patients treated at the same time with other drugs affecting these two enzymes.     

Characterization of the cytochrome P450 isoenzymes involved in the in vitroN-dealkylation of haloperidol

Aims The present study was carried out to identify the cytochrome P450 isoenzyme(s) involved in the N-dealkylation of haloperidol (HAL). Methods In vitro studies were performed using human liver microsomes and c-DNA-expressed human P450 isoforms. N-dealkylation of HAL was assessed by measuring the formation of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP). Results There was a tenfold variation in the extent of CPHP formation amongst the nine human liver microsomal preparations. The CPHP formation rates as a function of substrate concentration, measured in three livers, followed monophasic enzyme kinetics. K_m and V_max values ranged respectively from 50 to 78 μm and from 180 to 412 pmol mg⁻¹ min⁻¹. CPHP formation rates in the nine liver preparations were significantly correlated with dextromethorphan N-demethylase activity (a marker of CYP3A4 activity), but not with the activity of dextromethorphan O-demethylase (CYP2D6), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). Ketoconazole, an inhibitor of CYP3A4, inhibited competitively CPHP formation (K_i=0.1 μm ), whereas sulphaphenazole (CYP2C9), furafylline (CYP1A2) and quinidine (CYP2D6) gave only little inhibition (IC₅₀>100 μm ). CPHP formation was, moreover, enhanced by α-naphtoflavone, an effect common to CYP3A4 mediated reactions. Anti-CYP3A4 antibodies strongly inhibited CPHP formation, whereas no inhibition was observed in the presence of CYP2D6 antibodies. Among the recombinant human CYP isoforms tested, CYP3A4 exhibited the highest activity with respect to CPHP formation rate, with no detectable effect of other CYP isoforms (CYP1A2, CYP2D6 and CYP2C9). HAL inhibited dextromethorphan O-demethylase (CYP2D6) with IC₅₀ values between 2.7 and 8.5 μm, but not (IC₅₀>100 μm ) dextromethorphan N-demethylase (CYP3A4), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). Conclusions These results strongly suggest that the N-dealkylation of HAL in human liver microsomal preparations is mediated by CYP3A4.     

Comparison of nitroprusside and nitroglycerin in inhibition of angiotensin II and other vasoconstrictor-mediated contraction in human coronary bypass conduits

Aims To compare the effect of nitroprusside (SNP) and nitroglycerin (NTG) on angiotensin II (ANGII), endothelin-1 (ET-1), and α₁-adrenoceptor (phenylephrine, PE)-mediated contraction in internal mammary artery (IMA). Methods Human IMA segments (n=120) taken from 37 patients were studied. Concentration-relaxation curves for SNP and NTG were established in IMA precontracted with these vasoconstrictors. Concentration-contraction curves were also constructed in IMA rings incubated with SNP and NTG (0.1 and 1 μm ) for 10 min. Results Both SNP and NTG caused full relaxation with similar EC₅₀ s except NTG was four-fold more potent than SNP in PE-induced contraction (−7.92±0.06 vs−7.32±0.2 log m, mean±s.e. mean, P<0.01; 95% confidence interval for the difference of the means: 0.19, 1.01 log m ). Pretreatment with SNP (0.1 and 1 μm ) significantly depressed the contraction by ANGII from 56.6±7.7% (of 100 mm K⁺-contraction) to 18.3±8.6% and 3.9±2.1% (P=0.0001). In four rings treated with SNP, the contraction to ANGII was abolished whereas NTG did not depress ANGII-mediated contraction. Pretreatment with SNP (1 μm ), but not NTG, significantly depressed the magnitude of the PE-induced contraction from 4.7±1.2 to 1.7±0.4 g (P<0.05). Treatment with both SNP and NTG significantly increased the EC₅₀ (−5.09±0.17 log m, P=0.0007 for SNP and −5.40±0.06 log M, P=0.02 for NTG). Pretreatment with SNP did not significantly change either the magnitude or the EC₅₀ of the ET-1-induced contraction. Conclusions SNP may be advantageous compared with NTG in preventing coronary arterial graft contraction. However, once grafts have constricted to ANGII, α₁-adrenoceptor agonists, and ET-1, NTG may be only marginally advantageous.     

The effect of omeprazole on the pharmacokinetics of metronidazole and hydroxymetronidazole in human plasma, saliva and gastric juice

Aims To evaluate the effect of omeprazole on the pharmacokinetics of metronidazole and hydroxymetronidazole in plasma, gastric juice and saliva following intravenous infusion or oral dosing of metronidazole. Methods Eight volunteers received single doses of metronidazole (400 mg) intravenously and orally, whilst taking placebo or omeprazole (40 mg, twice daily for 5 days) in a randomized 4-way crossover study. Metronidazole and hydroxymetronidazole concentrations in plasma, saliva and gastric juice samples were determined by h.p.l.c. Pharmacokinetic parameters for metronidazole and hydroxymetronidazole were calculated, and the significance of the mean differences in parameters between omeprazole and placebo co-administration was assessed using a two-tailed, paired t-test. Results There were no significant differences (P<0.05) in any of the plasma or saliva pharmacokinetic parameter values for metronidazole between volunteers receiving omeprazole or placebo when metronidazole was administered either as an intravenous infusion or orally. Following intravenous administration of metronidazole to the placebo group and omeprazole treated group respectively, the gastric transfer of metronidazole was significantly reduced from 15.5±10.4% to 2.6±1.0% of the dose (P=0.007; 95% CI of difference 4.8 to 21.0) with concomitant changes in the metronidazole AUC (from 77.5±18.0 μmol l⁻¹ h to 352.6±182.1 μmol l⁻¹ h; P=0.0003; 95% CI of difference 127.6 to 422.7), C_max (from 61.4±26.5 μmol l⁻¹ to 271.8±104.3 μmol l⁻¹; P=0.0001; 95% CI of difference 118.6 to 302.1). Similarly, the gastric juice AUC of hydroxymetronidazole was significantly reduced from 3.2±1.9 μmol l⁻¹ h to 1.5±0.8 μmol l⁻¹ h of the dose (P=0.0043; 95% CI of difference 0.4 to 3.0) with a concomitant change in C_max (from 5.0±2.5 μmol l⁻¹ to 3.0±1.2 μmol l⁻¹; P=0.0007; 95% CI of difference 0.7 to 3.4). Conclusions Omeprazole had little effect on the plasma and salivary pharmacokinetics of metronidazole (or its hydroxymetabolite) after intravenous or oral administration, but it did have a substantial effect on the pharmacokinetics of metronidazole and hydroxymetronidazole in gastric juice.     

Inhibition of vasoconstriction by potassium channel opener aprikalim in human conduit arteries used as bypass grafts

Aims Potassium channel openers (KCOs) are of potential therapeutic value. Little is known about the effect of these drugs on human conduit arteries used as coronary bypass grafts. The purpose of this study was to determine the effect of the KCO aprikalim (RP52891) on human arteries used as coronary bypass grafts with emphasis on the possible difference in the inhibitory effect on depolarizing agent-mediated rather than receptor-mediated contraction. Methods Human internal mammary artery segments (IMA, n=88) taken from 28 patients were studied. Concentration-relaxation curves for aprikalim were established in IMA precontracted with three vasoconstrictors (K⁺, U46619, and phenylephrine). In IMA rings incubated with aprikalim (1 or 30 μm ) for 10 min concentration-contraction curves for the three vasoconstrictors were constructed. Results Aprikalim-induced relaxation was less in K⁺ (37.3±6.4%) than in U46619 (80.2±7.7%, P=0.002), or phenylephrine (67.5±7.0%, P=0.038) -precontracted IMA. The EC₅₀ for K⁺-(−5.40±0.12 log m ) was significantly higher than that for phenylephrine (−6.43±0.30 log m, P=0.007) but not significant compared with that for U46619 (−5.81±0.11, P >0.05). Pretreatment with aprikalim depressed the contraction by phenylephrine from 140.6±27.6% to 49.3±14.1% (P=0.002) and shifted the EC₅₀ 11.0-fold higher in rings treated with 1 μm aprikalim (P=0.007). Treatment of aprikalim did not significantly reduce the K⁺ and U46619-induced contraction (P >0.05) but shifted the concentration-contraction curves rightward (2.8-fold higher for K⁺, P<0.05 and 2.2-fold higher for U46619, P<0.05). Conclusions This study demonstrates that aprikalim has vasorelaxant effects in human conduit arteries used as coronary artery bypass grafts contracted by a variety of vasoconstrictors and this effect is vasoconstrictor-selective with greater potency for α₁-adrenoceptor agonists than for depolarizing agent K⁺. These findings provide information on the possible use of this KCO in various clinical settings.     

Theophylline has no advantages over caffeine as a putative model drug for assessing CYP1A2 activity in humans

Aims The cytochrome P4501A2 (CYP1A2) catalyses the metabolism of a number of clinically used drugs, and thus there is an interest in determining the activity of CYP1A2 in patients before treatment with CYP1A2 substrates. Caffeine is the most commonly used model drug to assess CYP1A2 function, but due to the complex metabolism of caffeine, there is a need for an alternative drug to use as an index of CYP1A2 activity. In this study the CYP1A2 substrate theophylline was tested as a possible alternative to caffeine as a model drug for CYP1A2. Methods Twelve healthy volunteers ingested 200 mg of caffeine, and the caffeine metabolic ratios (CMR), CMR_urine=(AFMU+1MX+1MU)/17DMU and CMR_plasma=17DMX/137TMX were determined 6 h after drug intake. After a period of about 2 months the volunteers ingested 257 mg theophylline and blood samples were drawn and urine was collected during the following 48 h. The oral and partial clearances of theophylline were calculated via N-demethylation and 8-hydroxylation. The theophylline metabolic ratios, 1MU/13DMX and 3MX/13DMX being evaluated as indices of CYP1A2 catalysed N-demethylation and 13DMU/13DMX as an index of partly CYP1A2 catalysed 8-hydroxylation, were estimated in 0–12 h, 0–24 h and 0–48 h urine samples, and in plasma and spot urine samples 6 h after the intake of theophylline. Results The theophylline plasma ratios for the N-demethylation pathways correlated with the oral clearance of theophylline (r_s=0.881-0.934, P<0.001) and with the respective formation clearances of the metabolites (r_s=0.712-0.925, P<0.05). Furthermore, all of the theophylline plasma ratios correlated with the caffeine plasma ratio (r_s=0.645-0.663, P<0.05). None of the caffeine metabolic ratios and none of the 6 h urinary theophylline ratios correlated with the oral or the partial clearances of theophylline (r_s=0.042-0.556, P >0.05). The theophylline 0-12 h urine ratios correlated with the oral clearance of theophylline (r_s=0.677-0.757, P<0.05) and with the respective formation clearances of the metabolites (r_s=0.705-0.750, P<0.05). However, none of the theophylline urine ratios correlated with any of the caffeine metabolic ratios. Conclusions In summary the theophylline 6 h plasma and 0–12 h urine ratios 1MU/13DMX and 3MX/13DMX, both reflecting N-demethylation seem to be predictors of the CYP1A2 mediated metabolism of theophylline, whereas only the plasma ratio correlated with the caffeine plasma 17DMX/137TMX ratio. Thus, it would appear that the plasma theophylline N-demethylation ratios are superior to the urine ratios as indices of CYP1A2 activity. However, because in some individuals the concentrations of theophylline metabolites in plasma were close to the limit of detection, it is concluded that theophylline does not have marked advantages over caffeine as a model drug for assessing CYP1A2 activity.     

Effect of antidepressant drugs on cytochrome P450 2C11 (CYP2C11) in rat liver

BackgroundRat CYP2C11 (besides CYP2C6) can be regarded as a functional counterpart of human CYP2C9. The aim of the present study was to investigate the influence of classic and novel antidepressant drugs on the activity of CYP2C11, measured as a rate of testosterone 2α- and 16α-hydroxylation.MethodsThe reaction was studied in control liver microsomes in the presence of antidepressants, as well as in microsomes from rats treated intraperitoneally (ip) with pharmacological doses of the tested drugs (imipramine, amitriptyline, clomipramine, nefazodone – 10 mg/kg ip; desipramine, fluoxetine, sertraline - 5 mg/kg ip; mirtazapine - 3 mg/kg ip) for one day or two weeks (twice a day), in the absence of antidepressants in vitro.ResultsThe investigated antidepressant drugs added to control liver microsomes produced certain inhibitory effects on CYP2C11 activity, which were moderate (sertraline, nefazodone and clomipramine: K_i = 39, 56 and 66 μM, respectively), modest (fluoxetine and amitriptyline: K_i = 98 and 108 μM, respectively) or weak (imipramine and desipramine: K_i = 191 and 212 μM, respectively). Mirtazapine had no inhibitory effect on CYP2C11 activity. One-day exposure of rats to the antidepressant drugs did not significantly change the activity of CYP2C11 in liver microsomes; however, imipramine, desipramine and fluoxetine showed a tendency to diminish the activity of CYP2C11. Of the antidepressants studied, only desipramine and fluoxetine administered chronically elevated CYP2C11 activity; those effects were positively correlated with the observed increases in the enzyme protein level.ConclusionThree different mechanisms of the antidepressants-CYP2C11 interaction are postulated: 1) a direct inhibition of CYP2C11 shown in vitro by nefazodone, SSRIs and TADs; 2) in vivo inhibition of CYP2C11 produced by one-day treatment with imipramine, desipramine and fluoxetine, which suggests inactivation of the enzyme by reactive metabolites; 3) in vivo induction of CYP2C11 produced by chronic treatment with desipramine and fluoxetine, which suggests their influence on enzyme regulation.     

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.     

Effects of oral and inhaled corticosteroid on lymphocyte β2-adrenoceptor function in asthmatic patients

Aims We have previously demonstrated that a single dose of oral prednisolone but not single doses of inhaled fluticasone had facilitatory effects on lymphocyte β₂-adrenoceptor (AR) function. To address possible differences in steady-state time-course, the aim of this study was to determine if repeated dosing with inhaled fluticasone would have facilitatory effects on lymphocyte β₂-AR. Plasma cortisol was also evaluated as a measure of systemic bioactivity. Methods Ten asthmatic subjects, mean (s.e.mean) age 29 (3) years, FEV₁ 89 (5) % predicted, were randomised in a double-blind crossover study to receive inhaled placebo (PL), inhaled fluticasone 1000 μg day⁻¹ (F1000) and inhaled fluticasone 2000 μg day⁻¹, each for 4 days and also a single dose of oral prednisolone 50 mg (PRED). Prednisolone was given as open medication. The last dose of study drug was taken at 22.00 h and subjects attended the laboratory at 08.00 h the following day. Results β₂-AR density (B_max; fmol/10⁶ cells) was significantly increased after PRED compared with PL and inhaled fluticasone. B_max (geometric mean) after each treatment were: PL 1.51, F1000 1.20, F2000 1.20 and PRED 2.14 (a 1.4 fold difference PRED vs PL; 95% CI 1.05 to 1.95; P<0.001). There was significant (P<0.001) suppression of plasma cortisol (nmol l⁻¹ ) following F2000 and PRED compared with PL: 393.8, F1000 302.1, F2000 205.0 (95% CI F2000 vs PL 58.1 to 319.4) and PRED 87.0 (95% CI PRED vs PL 176.2 to 437.5). The estimated milligram equivalence ratio for adrenal suppression was calculated at 1:11 for fluticasone vs prednisolone. Conclusions Repeated dosing with high-dose inhaled fluticasone did not up-regulate lymphocyte β₂-AR as compared with a single dose of oral prednisolone, despite having significantly suppressed early morning plasma cortisol. This study confirms our previous finding of a dissociation in sensitivity between effects of inhaled corticosteroid on adrenal suppression and lymphocyte β₂-AR regulation, at least for doses up to 2 mg day⁻¹ of fluticasone.     

Enzyme kinetic modelling as a tool to analyse the behaviour of cytochrome P450 catalysed reactions: application to amitriptyline N-demethylation

1To determine kinetic parameters (V_max, K_m) for cytochrome P450 (CYP) mediated metabolic pathways, nonlinear least squares regression is commonly used to fit a model equation (e.g., Michaelis Menten [MM]) to sets of data points (reaction velocity vs substrate concentration). This method can also be utilized to determine the parameters for more complex mechanisms involving allosteric or multi-enzyme systems. Akaike''s Information Criterion (AIC), or an estimation of improvement of fit as successive parameters are introduced in the model ( F-test), can be used to determine whether application of more complex models is helpful. To evaluate these approaches, we have examined the complex enzyme kinetics of amitriptyline (AMI) N-demethylation in vitro by human liver microsomes. 2For a 15-point nortriptyline (NT) formation rate vs substrate (AMI) concentration curve, a two enzyme model, consisting of one enzyme with MM kinetics (V_max=1.2 nmol min⁻¹ mg⁻¹, K_m=24 μm) together with a sigmoidal component (described by an equation equivalent to the Hill equation for cooperative substrate binding; V_max=2.1 nmol min⁻¹ mg⁻¹, K′=70 μm; Hill exponent n=2.34), was favoured according to AIC and the F-test. 3Data generated by incubating AMI under the same conditions but in the presence of 10 μm ketoconazole (KET), a CYP3A3/4 inhibitor, were consistent with a single enzyme model with substrate inhibition (V_max=0.74 nmol min⁻¹ mg⁻¹, K_m=186 μm, K₁=0.0028 μm⁻¹). 4Sulphaphenazole (SPA), a CYP2C9 inhibitor, decreased the rate of NT formation in a concentration dependent manner, whereas a polyclonal rat liver CYP2C11 antibody, inhibitory for S-mephenytoin 4′-hydroxylation in humans, had no important effect on this reaction. 5Incubation of AMI with 50 μm SPA resulted in a curve consistent with a two enzyme model, one with MM kinetics (V_max=0.72 nmol min⁻¹ mg⁻¹, K_m=54 μm) the other with ‘Hill-kinetics’ (V_max=2.1 nmol min⁻¹ mg⁻¹, K′=195 μm; n=2.38). 6A fourth data-set was generated by incubating AMI with 10 μm KET and 50 μm SPA. The proposed model of best fit describes two activities, one obeying MM-kinetics (V_max=0.048 nmol min⁻¹ mg⁻¹, K_m=7 μm) and the other obeying MM kinetics but with substrate inhibition (V_max=0.8 nmol min⁻¹ mg⁻¹, K_m=443 μm, K₁=0.0041 μm⁻¹). 7The combination of kinetic modelling tools and biological data has permitted the discrimination of at least three CYP enzymes involved in AMI N-demethylation. Two are identified as CYP3A3/4 and CYP2C9, although further work in several more livers is required to confirm the participation of the latter.     

Potent mechanism-based inhibition of CYP3A4 by imatinib explains its liability to interact with CYP3A4 substrates

BACKGROUND AND PURPOSE

Imatinib, a cytochrome P450 2C8 (CYP2C8) and CYP3A4 substrate, markedly increases plasma concentrations of the CYP3A4/5 substrate simvastatin and reduces hepatic CYP3A4/5 activity in humans. Because competitive inhibition of CYP3A4/5 does not explain these in vivo interactions, we investigated the reversible and time-dependent inhibitory effects of imatinib and its main metabolite N-desmethylimatinib on CYP2C8 and CYP3A4/5 in vitro.

EXPERIMENTAL APPROACH

Amodiaquine N-deethylation and midazolam 1′-hydroxylation were used as marker reactions for CYP2C8 and CYP3A4/5 activity. Direct, IC₅₀-shift, and time-dependent inhibition were assessed with human liver microsomes.

KEY RESULTS

Inhibition of CYP3A4 activity by imatinib was pre-incubation time-, concentration- and NADPH-dependent, and the time-dependent inactivation variables K_I and k_inact were 14.3 µM and 0.072 min⁻¹ respectively. In direct inhibition experiments, imatinib and N-desmethylimatinib inhibited amodiaquine N-deethylation with a K_i of 8.4 and 12.8 µM, respectively, and midazolam 1′-hydroxylation with a K_i of 23.3 and 18.1 µM respectively. The time-dependent inhibition effect of imatinib was predicted to cause up to 90% inhibition of hepatic CYP3A4 activity with clinically relevant imatinib concentrations, whereas the direct inhibition was predicted to be negligible in vivo.

CONCLUSIONS AND IMPLICATIONS

Imatinib is a potent mechanism-based inhibitor of CYP3A4 in vitro and this finding explains the imatinib–simvastatin interaction and suggests that imatinib could markedly increase plasma concentrations of other CYP3A4 substrates. Our results also suggest a possibility of autoinhibition of CYP3A4-mediated imatinib metabolism leading to a less significant role for CYP3A4 in imatinib biotransformation in vivo than previously proposed.     

Effect of simvastatin on cyclosporine unbound fraction and apparent blood clearance in heart transplant recipients

Aims To investigate the effects of lipid lowering therapy on the fraction unbound and dosage requirement of cyclosporine in heart transplant recipients. Methods Cyclosporine fraction unbound (fu) was measured ex vivo in plasma obtained from heart transplant recipients (n=12) before and after lipid lowering treatment, using equilibrium dialysis. Cyclosporine trough concentration data were also collected from cardiac transplant recipients (n=32) who received simvastatin for the treatment of hyperlipidaemia. Cyclosporine daily dosage and total concentration (monoclonal FPIA method) were recorded for periods up to 6 months before and after simvastatin administration. The total number of dose rate-concentration observations was 172 before and 135 after simvastatin administration respectively. Using a population pharmacokinetic approach (implemented in P-PHARM software) the ratio of dose rate to trough concentration at steady state (DR/C_sstrough ), an estimation of apparent clearance, was determined. The posterior Bayesian estimate of DR/C_sstrough was calculated for each patient before and after simvastatin administration. Results The mean fu increased by 29%, from 1.40±0.1% (mean±s.d.) to 1.82±0.22% after simvastatin administration (P<0.01). Mean trough concentrations of cyclosporine in whole blood were 349 μg l⁻¹ before and 242 μg l⁻¹ after simvastatin administration (P<0.0001). The mean cyclosporine daily dosage was 2.87 mg kg⁻¹ and 2.33 mg kg⁻¹ (NS), before and after simvastatin administration respectively. The average cyclosporine DR/C_sstrough was significantly increased from 24.5 l h⁻¹ before to 28.9 l h⁻¹ after simvastatin administration (P<0.05). Furthermore the median increase in cyclosporine DR/C_sstrough was 18 l h⁻¹ (−3.1 to 42.1 l h⁻¹, interquartile range). Conclusions Cyclosporine fraction unbound and clearance are increased following co-administration of lipid lowering agents, necessitating closer monitoring of cyclosporine total blood concentration when lipid lowering agents are administered concomitantly with cyclosporine.     

Pharmacokinetics of β-adrenoceptor blockers in obese and normal volunteers

Aims Obesity can modify the pharmacokinetics of lipophilic drugs. As β-adrenoceptor blockers (BB) are often prescribed for obese patients suffering from hypertension or coronary heart disease, this study compares the pharmacokinetics of lipophilic β-adrenoceptor blockers in obese and control subjects. Methods Nine obese (157±24% of ideal body weight (IBW) mean±s.d.) and nine non-obese healthy volunteers (98±10% IBW), aged 32±9 years, were included in the study. Subjects were randomly given a single i.v. infusion of one of the following racemic β-adrenoceptor blockers, whose doses (expressed as base per kg of IBW) were: propranolol (0.108 mg), labetalol (0.99 mg) and nebivolol (0.073 mg). The plasma concentrations of unchanged drugs were measured by h.p.l.c. The ionisation constants and lipophilicity parameters of β-adrenoceptor blockers were assessed. Results The pharmacokinetic data for the three drugs were qualitatively similar. There was a trend towards a greater total distribution volume (V_ss ) in obese patients than in controls. However, V_ss expressed per kg body weight was slightly smaller in obese patients. The relationship between V_ss and lipophilicity of five β-adrenoceptor was studied by combining the current results with those previously obtained with a moderately lipophilic drug (bisoprolol) and a hydrophilic one (sotalol). The V_ss of the five drugs was positively and well-correlated (r²=0.90; P<0.01) with their distribution coefficient at pH 7.4 (log D^7.4 ), but not with their partition coefficients. The linear regression coefficients for lean and obese subjects were very similar. Conclusions Lipophilic β-adrenoceptor blockers seem to diffuse less into adipose than into lean tissues. All electrical forms of the drugs (i.e. cations, neutral forms, or zwitterions) present at physiological pH contribute to their tissue distribution, in both obese and lean subjects.Their tissue distribution in obese patients could be restricted by the sum of hydrophobic forces and hydrogen bonds they elicit with macromolecules in lean tissues.     

Interaction between sotalol and an antacid preparation

Aims The aim of the study was to investigate the pharmacokinetic interaction between sotalol and antacids, and its pharmacodynamic relevance. Methods In a randomized cross-over design with three treatment groups, six healthy volunteers received orally either 160 mg of sotalol alone (phase 1), or 160 mg sotalol plus 20 ml of a suspension of an antacid (MAH; magnesium hydroxide (1200 mg) and aluminium oxide (1800 mg)) (phase 2) or 160 mg sotalol plus the antacid given 2 h after sotalol administration (phase 3). Heart rate and plasma sotalol concentrations were measured before and 1, 2, 3, 4, 6, 8, 12 and 24 h after sotalol administration. Urinary sotalol excretion was measured for 24 h after sotalol application. Results C_max of sotalol decreased from 1.22±0.22 mg l⁻¹ (phase 1) to 0.89±0.29 mg l⁻¹ (phase 2) and increased again to 1.27±0.18 mg l⁻¹ in phase 3. A similar significant change was noted in AUC (15.6±2.75 mg l⁻¹ , 12.3±3.04 mg h l⁻¹ and 15.0±2.06 mg l⁻¹ ) and in the amount of cumulative urinary excretion (79.2±11.1 mg, 72.1±11.2 mg and 80.6±7.9 mg), respectively. t_max and elimination half-life (t_1/2,z) of sotalol remained unchanged in the presence of MAH. After combined administration with MAH, the area under the heart rate curve of sotalol was reduced between 0 and 4 h when compared across treatments. Conclusions Combined administration of sotalol and MAH decreased the serum sotalol levels. The interaction can be avoided by a two hour interval between application of these drugs.     

A pharmacokinetic and pharmacodynamic interaction study between nebivolol and the H2-receptor antagonists cimetidine and ranitidine

Aims The study was designed to investigate the effects of the H₂-receptor antagonists, cimetidine and ranitidine on the pharmacokinetics and pharmacodynamics of nebivolol in healthy volunteers. Methods Twelve healthy volunteers took part in a randomized placebo-controlled cross-over study. Each subject received on three separate occasions placebo, cimetidine (400 mg twice daily) or ranitidine (150 mg twice daily) for 24 h before and 48 h after a single oral dose of nebivolol (5 mg). Nebivolol and its individual (+) and (−) enantiomers were determined tby h.p.l.c. Results Ranitidine had no significant effect on nebivolol pharmacokinetics. Cimetidine, however, resulted in a 21–23% increase in C_max of unchanged nebivolol and of each enantiomer plus its hydroxylated metabolites. Cimetidine significantly (P<0.05) increased the AUC [mean±s.d. (95% C.I. of differences in mean)] for unchanged (±)-nebivolol [7.76±3.07 ng ml⁻¹h with placebo; 11.50±5.40 (1.75, 8.76) ng ml⁻¹h with cimetidine], (+)-nebivolol plus its hydroxylated metabolites [73.0±18.0 ng ml⁻¹h with placebo; 91.5±25.7 (1.0, 23.1) ng ml⁻¹h with cimetidine] and (−)-nebivolol plus its hydroxylated metabolites [101±32 ng ml⁻¹h with placebo; 123±38 (3.3, 27.0) ng ml⁻¹h with cimetidine]. Statistical analysis of the resting blood pressure and heart rate and exercise data did not suggest any consistent effects of ranitidine or cimetidine upon the pharmacodynamic effects of nebivolol. Conclusions There was no interaction between ranitidine and nebivolol. Although cimetidine inhibited nebivolol metabolism, it did not have a significant influence on the pharmacodynamics of the drug.     

The influence of the sparteine/debrisoquine genetic polymorphism on the disposition of dexfenfluramine

1 To determine whether dexfenfluramine is a substrate of cytochrome P450 2D6 (CYP2D6), its disposition has been studied in nine extensive (EM) and eight poor metabolizers (PM) of debrisoquine. 2 Following a 30 mg dose of dexfenfluramine hydrochloride, urine was collected in all subjects for 96 h post-dose and plasma samples were collected in 11 subjects (six EMs and five PMs). Dexfenfluramine and nordexfenfluramine were measured in urine by h.p.l.c. and in plasma by g.c. 3 Urinary recovery of dexfenfluramine was greater in PMs than EMs (4136±1509 μg vs 1986±792 μg; 95% CI of difference 926–3374; P<0.05) whereas that of nordexfenfluramine was similar in both phenotypes (PM: 1753±411 μg vs 1626±444 μg). 4 Dexfenfluramine AUC was higher in PMs (677±348 μg l⁻¹ h) than EMs (359±250 μg l⁻¹ h). The apparent oral clearance of dexfenfluramine was greater in EMs than PMs (93.6±42.4 l h⁻¹vs 45.6±19.5 l h⁻¹; 95% CI of difference 1.2–94.7; P<0.05). The renal clearance was similar in both phenotypes (EMs: 5.88±2.83 l h⁻¹; PMs 6.60±2.01 l h⁻¹), indicating that the higher urinary recovery of dexfenfluramine in PMs reflects higher plasma concentrations, rather than phenotype differences in the renal handling, of dexfenfluramine. 5 The apparent nonrenal clearance of dexfenfluramine was substantially lower (P<0.05; 95% CI of difference 3.0–94.1) in PMs (39.0±19.5 l h⁻¹) than EMs (87.6±41.2 l h⁻¹). 6 There was a significant inverse correlation (r_s=−0.776 95% CI −0.31–−0.94; n=11; P=0.005) between the debrisoquine metabolic ratio and the apparent nonrenal clearance of dexfenfluramine. 7 PMs had a higher incidence of adverse effects (nausea and vomiting) than EMs. 8 In conclusion, the metabolism of dexfenfluramine is impaired in PMs. Thus CYP2D6, the isoenzyme deficient in poor metabolizers of debrisoquine, must catalyse at least one pathway of dexfenfluramine biotransformation.     

Dopamine natriuresis in salt-repleted, water-loaded humans: a dose-response study

Aims The purpose of the present study was to define the dose-response relationship between exogenous dopamine and systemic haemodynamics, renal haemodynamics, and renal excretory function at infusion rates in the range 0 to 12.5 μg kg⁻¹ min⁻¹ in normal volunteers. Methods While undergoing water diuresis, eight subjects were infused with 0, 1, 2, 3, 5, 7.5, 10 or 12.5 μg of dopamine kg⁻¹ min⁻¹ over 2 h in a randomized and double-blind fashion. On each study day, renal clearance studies were performed during a 1 h baseline period and subsequently during the second 1 h infusion period. Lithium clearance (CL_Li ) was used to estimate proximal tubular outflow. Results Cardiac output increased with the four highest doses. Mean arterial pressure followed a biphasic pattern with a decrease during the two lowest doses and a dose-dependent increase from the 7.5 μg kg⁻¹ min⁻¹ dose onwards. Effective renal plasma flow increased with all doses of dopamine, but peaked with the 3 μg kg⁻¹ min⁻¹ infusion rate [ from 617 (585–649) ml min⁻¹ with placebo to 915 (824–1006) ml min⁻¹ (means with 95% CI, P<0.001)]. None of the doses changed glomerular filtration rate (GFR). Sodium clearance (CL_Na ) and CL_Li were elevated with the four lowest doses but increased further from 7.5 μg kg⁻¹ min⁻¹ onwards. Compared with placebo, the percentage increase in CL_Na with increasing dose was 77 (5–159), 93 (13–172), 107 (24–190), 121 (60–181), 253 (65–441), 284 (74–494), and 212 (111–312) %, respectively. There were only small, inconsistent decreases in absolute proximal reabsorption rate (APR=GFR-CL_Li ). Fractional distal reabsorption of sodium (FDR_Na=(CL_Li-CL_Na )/CL_Li ) decreased with all doses, reaching its nadir with 7.5 μg kg⁻¹ min⁻¹ [from 95.9 (94.6–97.2) % with placebo to 91.5 (90.0–93.0) % (P<0.01)] whereafter a flat dose-response curve was observed. Conclusions In conclusion, the renal vasodilating effect of dopamine was maximal with 3 μg kg⁻¹ min⁻¹. The dose-dependent attenuation seen with higher doses is consistent with an increased α-adrenergic stimulation opposing the effect on dopaminergic receptors. The present CL_Li studies confirm that dopamine increases proximal tubular outflow. The results suggest that the natriuretic effect of depressor doses of dopamine was primarily caused by attenuation of the increase in distal sodium reabsorption normally seen after an increase in proximal tubular outflow. Pressor doses further increased sodium excretion, indicating the presence of pressure natriuresis at these high doses.     

The effects of combined treatment with β1-selective receptor antagonists and lipid-lowering drugs on fat metabolism and measures of fatigue during moderate intensity exercise: a placebo-controlled study in healthy subjects

Aims We examined the effects of different combinations of β₁-selective adrenoceptor blockers and lipid-lowering drugs, on fat metabolism and fatigue during moderate intensity exercise in 14 healthy young volunteers. Methods The study was a randomized crossover design, each subject completing 5, 90 min walks at 50% of predetermined maximal oxygen uptake (VO₂ max), one following each 3 day treatment period with either: atenolol 100 mg and bezafibrate 400 mg, atenolol 100 mg and fluvastatin 40 mg, metoprolol CR 100 mg and bezafibrate 400 mg, metoprolol CR 100 mg and fluvastatin 40 mg, or placebo. Results Plasma free fatty acid (FFA) concentration during exercise was significantly reduced on all treatments, in comparison with placebo, P=0.0001. Following 90 min of exercise FFA levels were as follows: placebo 573 μmol l⁻¹ (105–1041), metoprolol CR+fluvastatin 277 μmol l⁻¹ (0–647), metoprolol CR+bezafibrate 182 μmol l⁻¹ (0–396), atenolol+fluvastatin 211 μmol l⁻¹ (0–511), and atenolol+bezafibrate 123 μmol l⁻¹ (0–352). Total fat oxidation during exercise was also reduced on all treatments in comparison with placebo: 38.1% (2–74), compared with 29.1% (0–61) on metoprolol CR+fluvastatin, P=0.02, 26.2% (2–51) on metoprolol CR+bezafibrate, P=0.002, 25.5% (3–48) on atenolol+fluvastatin, P=0.009, and 22.8% (0–47) on atenolol+bezafibrate treatment, P=0.0002. Plasma ammonia concentration was elevated on all treatments during exercise in comparison with placebo. After 90 min of exercise, plasma ammonia levels were as follows: placebo 37 μmol l⁻¹ (0–84), metoprolol CR+fluvastatin 56 μmol l⁻¹ (2–110), metoprolol CR+bezafibrate 79 μmol l⁻¹ (0–167), atenolol+fluvastatin 90 μmol l⁻¹ (10–170), and atenolol+bezafibrate 100 μmol l⁻¹ 26–174). In comparison with placebo, metoprolol CR+fluvastatin had the least adverse impact on measures of perceived exertion and the ‘feeling scale’ during exercise. Metoprolol CR+bezafibrate, atenolol+fluvastatin, and atenolol+bezafibrate treatments had greater adverse effects, particularly on perceived ‘cardiorespiratory effort’ and ‘feeling scale’ scores. Conclusions In healthy volunteers, combinations of β₁-selective blockers and lipid-lowering drugs were associated with significant reductions in fat metabolism, increased plasma ammonia levels, and raised the perception of effort during exercise, in comparison with placebo. Metoprolol CR+fluvastatin had the least effect, combinations metoprolol CR+bezafibrate and atenolol+fluvastatin had intermediate effects, and atenolol+bezafibrate had the most adverse effect.     

Single-dose pharmacokinetics of ampicillin and tobramycin administered by hypodermoclysis in young and older healthy volunteers

1To test the feasibility of administering antibiotics by subcutaneous infusion to the elderly, we compared the pharmacokinetics of tobramycin (single dose of 80 mg) given by hypodermoclysis (HDC) with the kinetics of the antibiotic injected intravenously (i.v.) in 10 young (<50 years old) and 10 elderly (>65 years old) healthy volunteers. Similar studies were performed with ampicillin (single dose of 1 g) in 12 young and 10 older healthy volunteers. 2Compared with the i.v. route, HDC delayed the time to reach the maximal plasma concentration (t_max) of tobramycin in young volunteers: 32±6 (s.d.) min vs 88±46, P<0.005, and older volunteers: 27±4 min vs 89±15, P<0.005. Administration of the antibiotics by HDC was well tolerated. The plasma concentration of tobramycin 30 min after the end of infusion (C₆₀) was lower (P<0.05) following HDC than after the i.v. route in both young, 2.2±0.7 vs 3.5±0.8 μg ml⁻¹, and elderly subjects, 2.2±0.8 vs 3.8±0.9. μg ml⁻¹. 3The area under the curve (AUC) of tobramycin given by HDC was slightly smaller than when given i.v., i.e. in young subjects: 740±225 (s.d.) vs 893±223 μg ml⁻¹ min, NS, and in the elderly: 980±228 vs 1056±315 μg ml⁻¹ min, NS. 4When ampicillin was administered by HDC, the t_max was also delayed in young volunteers: 45±18 vs 23±6 min, and in the elderly: 49±18 vs 27±4 min, P<0.005, the AUC was greater by HDC than i.v. in the young volunteers: 4527±1658 μg ml⁻¹ min vs 3810±1033 μg ml⁻¹ min and in the elderly: 6795±2094 μg ml⁻¹ min vs 4217±1518 μg ml⁻¹ min, and the C₆₀ was higher by HDC in the young: 27±7 vs 24±9 μg ml⁻¹, and in the elderly: 32±9 vs 23±11 μg ml⁻¹, P<0.05. 5In conclusion, HDC delays the entry of the antibiotic into the systemic circulation, but did not affect the amount available. HDC was well tolerated and could become an adequate method for antibiotic administration to the elderly.     

Direct and indirect interactions between antidepressant drugs and CYP2C6 in the rat liver during long-term treatment

The aim of the present study was to investigate the influence of tricyclic antidepressants (TADs: imipramine, amitriptyline, clomipramine, desipramine), selective serotonin reuptake inhibitors (SSRIs: fluoxetine, sertraline) and novel antidepressant drugs (mirtazapine, nefazodone) on the activity of CYP2C6 measured as a rate of warfarin 7-hydroxylation. The reaction was studied in control liver microsomes in the presence of the antidepressants, as well as in microsomes of rats treated intraperitoneally (i.p.) for one day or two weeks with pharmacological doses of the drugs (imipramine, amitriptyline, clomipramine, nefazodone at 10 mg/kg i.p.; desipramine, fluoxetine, sertraline at 5 mg/kg i.p.; mirtazapine at 3 mg/kg i.p.), in the absence of the antidepressants in vitro. Some of the investigated antidepressant drugs added to liver microsomes of control rats inhibited the rate of 7-hydroxylation of warfarin. The obtained K_i values indicated that nefazodone and fluoxetine were the most potent inhibitors of the studied reaction (K_i = 13 and 23 μM, respectively), while tricyclic antidepressants and sertraline were weak in this respect (K_i = 70–127 μM). A one-day (i.e. 24 h) exposure to fluoxetine and mirtazapine resulted in a significant increase in the rate of the 7-hydroxylation of warfarin in rat liver microsomes. The other studied antidepressants did not significantly affect the rate of the CYP2C6-specific reaction. After two-week treatment with the investigated antidepressants, the increase in CYP2C6 activity observed after 24-h exposure to fluoxetine and mirtazapine was more pronounced. Moreover, unlike after one-day exposure, imipramine and sertraline significantly increased the activity of the enzyme. The other tricyclic antidepressants or nefazodone did not produce any significant effect when administered in vivo. The above-described enhancement of CYP2C6 activity correlated positively with the simultaneously observed increases in the enzyme protein level, which indicates the enzyme induction. The studied antidepressants increased the CYP2C6 protein level in the liver microsomes of rats after chronic treatment: imipramine to 174.6 ± 18.3%, fluoxetine to 159.1 ± 13.7%, sertraline to 135.3 ± 11.2% and mirtazapine to 138.4 ± 10.2% of the control. In summary, two different mechanisms of the antidepressant–CYP2C6 interaction have been found to operate in the rat liver: 1) direct inhibition of CYP2C6 shown in vitro mainly for nefazodone and fluoxetine, with their inhibitory effects being somewhat more potent than their action on human CYP2C9; 2) the in vivo induction of CYP2C6 by imipramine, fluoxetine, sertraline and mirtazapine.