Time-dependent inhibition and estimation of CYP3A clinical pharmacokinetic drug-drug interactions using plated human cell systems.

The current studies assessed the utility of freshly plated hepatocytes, cryopreserved plated hepatocytes, and cryopreserved plated HepaRG cells for the estimation of inactivation parameters k(inact) and K(I) for CYP3A. This was achieved using a subset of CYP3A time-dependent inhibitors (fluoxetine, verapamil, clarithromycin, troleandomycin, and mibefradil) representing a range of potencies. The estimated k(inact) and K(I) values for each time-dependent inhibitor were compared with those obtained using human liver microsomes and used to estimate the magnitude of clinical pharmacokinetic drug-drug interaction (DDI). The inactivation kinetic parameter, k(inact), was most consistent across systems tested for clarithromycin, verapamil, and troleandomycin, with a high k(inact) of 0.91 min(-1) observed for mibefradil in HepaRG cells. The apparent K(I) estimates derived from the various systems displayed a range of variability from 3-fold for clarithromycin (5.4-17.7 µM) to 6-fold for verapamil (1.9-12.6 µM). In general, the inactivation kinetic parameters derived from the cell systems tested fairly replicated what was observed in time-dependent inhibition studies using human liver microsomes. Despite some of the observed differences in inactivation kinetic parameters, the estimated DDIs derived from each of the tested systems generally agreed with the clinically reported DDI within approximately 2-fold. In addition, a plated cell approach offered the ability to conduct longer primary incubations (greater than 30 min), which afforded improved ability to identify the weak time-dependent inhibitor fluoxetine. Overall, results from these studies suggest that in vitro inactivation parameters generated from plated cell systems may be a practical approach for identifying time-dependent inhibitors and for estimating the magnitude of clinical DDIs.

Negligible pharmacokinetic interaction between oral DA-8159, a new erectogenic, and amlodipine in rats.

A pharmacokinetic interaction between oral DA-8159 and amlodipine was evaluated in male Sprague-Dawley rats. In rats pretreated with troleandomycin (a main inhibitor of CYP3A1/2 in rats), the AUC(0-6 h) of amlodipine was significantly greater than the controls (34.5+/-6.01 compared with 28.0+/-4.70 microg min/ml), indicating that amlodipine is metabolized via CYP3A1/2 in rats. It was reported that the metabolism of DA-8159 and the formation of DA-8164 (a metabolite of DA-8159) were mainly mediated via CYP3A1/2 in rats, and amlodipine significantly inhibited the CYP3A2 in rats. Therefore, a pharmacokinetic interaction between the two drugs could be expected. However, after oral administration of DA-8159 at a dose of 30 mg/kg with or without oral amlodipine at a dose of 5 mg/kg to rats, the pharmacokinetic parameters of DA-8159 and DA-8164 were not significantly different between the two groups of rats. Similar results were also obtained from amlodipine between with and without DA-8159. The above data indicated that the pharmacokinetic interaction between oral DA-8159 and amlodipine was almost negligible in rats.

Intravenous and oral alfentanil as in vivo probes for hepatic and first-pass cytochrome P450 3A activity: noninvasive assessment by use of pupillary miosis.

INTRODUCTION: Systemic clearance of intravenous (IV) alfentanil (ALF) is an in vivo probe for hepatic cytochrome P450 (CYP) 3A activity, miosis is a surrogate for plasma ALF concentrations, and IV ALF miosis is a noninvasive probe for hepatic CYP3A. This investigation characterized the bioavailability and first-pass metabolism of oral ALF and tested the hypotheses that (1) first-pass ALF clearance reflects first-pass CYP3A activity, (2) miosis after oral ALF will reflect intestinal and hepatic CYP3A activity, and (3) miosis can approximate plasma concentration-based pharmacokinetic measures for IV and oral ALF as a noninvasive in vivo probe for hepatic and first-pass CYP3A activity and drug interactions. Results were compared with those for midazolam (MDZ), an alternative CYP3A probe. METHODS: Ten volunteers were studied by use of a randomized, 9-way, crossover design after administration of rifampin (INN, rifampicin) (hepatic and intestinal CYP3A induction), troleandomycin (TAO) (hepatic and intestinal CYP3A inhibition), grapefruit juice (selective intestine CYP3A inhibition), or nothing (control). For each condition, they received 1 mg IV MDZ and then 15 microg/kg IV ALF, as well as 3 mg oral MDZ and then oral ALF (23 or 60 microg/kg) on another day. Plasma concentrations were determined by liquid chromatography-mass spectrometry. Dark-adapted pupil diameters were measured coincident with blood sampling. ALF effect was analyzed similarly to concentration to yield an effect "clearance" (Dose/Area under the pupil diameter change versus time curve). RESULTS: Bioavailability (Foral), hepatic extraction (EH), and intestinal availability (FG) were 0.26 +/- 0.08, 0.52 +/- 0.09, and 0.56 +/- 0.20, respectively, for MDZ and 0.42 +/- 0.15, 0.28 +/- 0.09, and 0.56 +/- 0.18, respectively, for ALF. Oral clearance (CL/F) was 34.7 +/- 12.8 and 10.9 +/- 3.5 mL.kg -1.min -1 , respectively, for MDZ and ALF. After rifampin, TAO, and grapefruit juice, ALF F oral was 0.04 +/- 0.02 (P <.05, versus control), 0.99 +/- 0.18 (P <.05, versus control), and 0.62 +/- 0.18 (P <.05, versus control), respectively; E H was 0.69 +/- 0.14 (P < .05, versus control), 0.04 +/- 0.01 (P <.05, versus control), and 0.26 +/- 0.08, respectively; F G was 0.16 +/- 0.10 (P <.05, versus control), 1.0 +/- 0.2 (P <.05, versus control), and 0.85 +/- 0.30 (P <.05, versus control), respectively; CL/F was 339 +/- 233 (P <.05, versus control), 0.62 +/- 0.26 (P <.05, versus control), and 6.7 +/- 2.5 (P <.05, versus control), respectively, and effect clearance was 2.1 +/- 1.1 (P <.05, versus control), 0.087 +/- 0.056 (P <.05, versus control), and 0.54 +/- 0.30 (0.73 +/- 0.43 mg.mm -1.h -1 in controls), respectively. There were significant correlations between ALF and MDZ systemic clearances (r2= 0.92), EH (r2=0.93), and CL/F (r2= 0.97), as well as between oral ALF effect (miosis) clearance and oral clearance (r2=0.59). CONCLUSIONS: ALF and MDZ have similar intestinal extraction but low and intermediate hepatic extraction, respectively. Systemic and oral clearances of ALF are excellent in vivo probes for hepatic and first-pass CYP3A activities and drug interactions. Miosis was an acceptable surrogate for plasma ALF. ALF miosis may be a suitable noninvasive in vivo probe for both hepatic and first-pass CYP3A.

Disposition of tacrolimus in isolated perfused rat liver: influence of troleandomycin, cyclosporine, and gg918.

The disposition of tacrolimus and the influence of cyclosporine, troleandomycin, and GF120918 (GG918, or N-[4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine) on its hepatic disposition were examined in the isolated perfused rat liver. Livers from groups of rats were perfused in a recirculatory manner following a bolus dose of tacrolimus (100 microg), a substrate for P-glycoprotein (P-gp) and CYP3A, or with felodipine (200 microg), a substrate only for CYP3A. Perfusions of each substrate were also examined in groups of rats in the presence of the inhibitors: troleandomycin (20 microM, CYP3A inhibitor), GG918 (1 microM, P-gp inhibitor), or cyclosporine (10 microM, CYP3A and P-gp inhibitor). In all experiments, perfusate and bile were collected for 60 min. Tacrolimus, felodipine, and their primary metabolites were determined in perfusate and bile by liquid chromatography/tandem mass spectrometry. The area under the curve (AUC) from 0 to 30 min was determined. For the dual CYP3A and P-gp substrate, tacrolimus, AUC +/- S.D. was decreased from control (2,260 +/- 430 ng. min/ml) by GG918 (1,730 +/- 270 ng. min/ml, P < 0.05) and was increased by troleandomycin (5,200 +/- 2,470 ng. min/ml, P < 0.05) and cyclosporine (4,390 +/- 2,080 ng. min/ml, P < 0.05). For the exclusive CYP3A substrate, felodipine, AUC was unchanged from control by GG918 but increased by troleandomycin and cyclosporine. It is concluded that GG918 increased the hepatic exposure of tacrolimus by inhibiting the canalicular P-gp transport, whereas GG918 has no effect on hepatic disposition of felodipine. These results support our hypothesis that the hepatic metabolic clearance of a dual substrate will be increased by inhibiting the efflux transporter.

An evaluation of a low-density DNA microarray using cytochrome P450 inducers.

The aim of this study was to validate a low-density DNA microarray "Rat HepatoChip", which contains 59 genes from a range of potential toxic markers and drug metabolism-related genes. Liver mRNA was isolated from rats dosed with six different chemicals, dexamethasone, troleandomycin, miconazole, clotrimazole, and methylclofanapate, which are all known to induce different cytochrome P450 genes, and isoniazid, which does not cause histopathological changes. Replicate microarrays were used to measure the variability in the chips and in the process. The average variability in signal between different chips observed in triplicate experiments was 33% ranging from 21 to 39% depending on genes. We also demonstrated a strong correlation between the liver histopathology and the gene expression profiles indicating that the gene expression profile reflects histopathological changes. These results suggest that the Rat HepatoChip microarray may provide a fast and effective tool for assessing the toxicity profile of developmental drug candidates during the drug discovery process.

Comparative studies of in vitro inhibition of cytochrome P450 3A4-dependent testosterone 6beta-hydroxylation by roxithromycin and its metabolites, troleandomycin, and erythromycin.

Roxithromycin has been shown to be a relatively weak inhibitor of cytochrome P450 (P450 or CYP)-dependent drug oxidations, compared with troleandomycin. The potential for roxithromycin and its major metabolites found in human urine [namely the decladinosyl derivative (M1), O-dealkyl derivative (M2), and N-demethyl derivative (M3)] to inhibit testosterone 6beta-hydroxylation after metabolic activation by CYP3A4 was examined and compared with inhibition by troleandomycin and erythromycin in vitro. Of roxithromycin and its studied metabolites, M3 was the most potent in inhibiting CYP3A4-dependent testosterone 6beta-hydroxylation by human liver microsomes and was activated to the inhibitory P450.Fe2+-metabolite complex to the greatest extent. Roxithromycin and its metabolites were N-demethylated by human liver microsomes, although the rates were slower than those measured with troleandomycin and erythromycin as substrates. Recombinant human CYP3A4 in a baculovirus system coexpressing NADPH-P450 reductase was very active in catalyzing the N-demethylation of roxithromycin, M1, and M2, as well as troleandomycin, erythromycin, and M3. The order for inhibition of CYP3A4-dependent testosterone 6beta-hydroxylation activities by these macrolide antibiotics in the recombinant CYP3A4 system was estimated to be troleandomycin > erythromycin >/= M3 >/= M2 > M1 >/= roxithromycin. Erythromycin, roxithromycin, and its metabolites all failed to inhibit CYP1A2-dependent (R)-warfarin 7-hydroxylation and CYP2C9-dependent (S)-warfarin 7-hydroxylation but did inhibit CYP3A4-dependent (R)-warfarin 7-hydroxylation. These results suggest that roxithromycin itself is not as potent an inhibitor of CYP3A4 activities as are troleandomycin and erythromycin, probably because of the slower metabolism of this compound to metabolites M1, M2, and M3 in humans.

Therapeutic drug monitoring of alprazolam in adolescents with asthma.

Children and adolescents with severe asthma frequently experience anxiety or depression with anxiety, which can undermine their response to treatment. In addition, these patients often receive theophylline and a variety of adrenergic stimulants, which can exacerbate or worsen anxiety. Such children occasionally are candidates for treatment with anxiolytic therapy. There is a paucity of drug disposition data in adolescents for benzodiazepines, the most frequently used antianxiety drugs. The authors monitored the steady state alprazolam plasma concentration in six children with severe asthma who were administered standard doses of alprazolam. In one patient administered concurrent therapy with troleandomycin, a recognized cytochrome 3A4 inhibitor, alprazolam plasma concentration was markedly elevated. Overall, the disposition data of alprazolam was consistent with data previously reported in adults. Alprazolam appeared to be safe and effective for use in adolescents with asthma.

Effects of roxithromycin, erythromycin and troleandomycin on their N-demethylation by rat and human cytochrome P450 enzymes.

1. The effects of treatment of rat with roxithromycin, erythromycin and troleandomycin as well as other chemicals including typical cytochrome P450 inducers were examined in rat and human liver microsomes. 2. Erythromycin and troleandomycin but not roxithromycin caused slight increases in CYP3A1 levels and the N-demethylation of roxithromycin, erythromycin and troleandomycin and oxidation of nifedipine in rat, but none of these chemicals induced significantly CYP2B1 levels or benzphetamine N-demethylation activities. 3. Dexamethasone and pregnenolone 16 alpha-carbonitrile induced CYP3A1 levels and N-demethylation of roxithromycin, erythromycin and troleandomycin but not of benzphetamine, in rat liver microsomes. Treatment of rat with phenobarbital caused increases in both CYP2B1 and 3A1 levels and all of the N-demethylation activities examined. Phenytoin and metyrapone produced increases in contents of 2B1 and activities of benzphetamine N-demethylation as well as of roxithromycin, erythromycin and troleandomycin, although these two inducers did not induce 3A1 protein significantly. 4. In man, a liver sample that was high in CYP3A4 and nifedipine oxidation activity was found to be the most active in N-demethylation activities towards these substrates examined. In addition, recombinant 3A4 catalysed very efficiently the N-demethylation of roxithromycin, erythromycin and troleandomycin in reconstituted monooxygenase systems. 5. These data suggest that erythromycin and troleandomycin, but not roxithromycin, were able to induce CYP3A1 in rat liver microsomes, and that N-demethylation of roxithromycin, erythromycin and troleandomycin were catalysed mainly by 3A1 (and partly by 2B1) in rat and by 3A4 in man.

Cytochrome P-450 complex formation in rat liver by the antibiotic tiamulin.

Tiamulin is a semisynthetic diterpene antibiotic frequently used in farm animals. The drug has been shown to produce clinically important--often lethal--interactions with other compounds. It has been suggested that this is caused by a selective inhibition of oxidative drug metabolism via the formation of a cytochrome P-450 metabolic intermediate complex. In the present study, rats were treated orally for 6 days with tiamulin at two different doses: 40 and 226 mg/kg of body weight. For comparison, another group received 300 mg of triacetyloleandomycin (TAO) per kg, which is equivalent to the 226-mg/kg tiamulin group. Subsequently, microsomal P-450 contents, P-450 enzyme activities, metabolic intermediate complex spectra, and P-450 apoprotein concentrations were assessed. In addition, effects on individual microsomal P-450 activities were studied in control microsomes at different tiamulin and substrate concentrations. In the rats treated with tiamulin, a dose-dependent complex formation as evidenced by its absorption spectrum and an increase in cytochrome P-4503A1/2 contents as assessed by Western blotting (immunoblotting) were found. The effects were comparable to those of TAO. Tiamulin induced microsomal P-450 content, testosterone 6 beta-hydroxylation rate, erythromycin N-demethylation rate, and the ethoxyresorufin O-deethylation activity. Other activities were not affected or decreased. When tiamulin was added to microsomes of control rats, the testosterone 6 beta-hydroxylation rate and the erythromycin N-demethylation were strongly inhibited. It is concluded that tiamulin is a potent and selective inducer-inhibitor of cytochrome P-450. Though not belonging to the macrolides, the compound produces an effect on P-450 similar to those of TAO and related compounds.