Drug Metabolism Letters - Volume 4, Issue 2, 2010

The objectives of these studies were to characterize the pharmacokinetics (PK) of the nasal decongestant pseudoephedrine (PSE) in rats, dogs, and monkeys, and to evaluate its lower gastrointestinal tract regional bioavailability in rats. An LC-MS/MS assay with a lower limit of quantification (LLOQ) of 0.4 ng/mL of plasma was developed for the analysis of PSE in animal plasma. The total body clearance (CL) was the highest in rats (78 mL/min/kg), lowest in monkeys (15 mL/min/kg) and the dog averaged in between (33 mL/min/kg). The volume of distribution at steady state (Vdss) ranged from 3-5 L/kg in all species. In rats and dogs, the mean half-lives (t1/2) was ∼1.5 hr, while in monkeys the mean t1/2 was 4.6 hr, comparable to that observed in adult humans (4-8 hr). The oral bioavailability was 38, 58 and 87% in rats, dogs and monkeys. The bioavailability following intra-ileum or intra-colonic administration in rats was superior to that following oral dosing (66% and 78%, respectively) suggesting that colonic absorption may be compensating for the short half-life, thus enabling successful QD sustained release formulations of PSE. The pharmacokinetic/pharmacodynamic relationship (PK/PD) of PSE was also investigated in a feline model of nasal congestion to establish efficacious trough concentrations in cats for a comparison with that in humans. The PK/PD in the cat model followed a sigmoid Emax model with an EC50 (plasma concentration that elicits 50% of the maximum response) of 0.32 ± (0.05) (SD) μM consistent with human plasma concentrations required for efficacy.

To establish the zebrafish as a model to investigate drug metabolism through sulfation, we had previous cloned, expressed, and purified fourteen distinct zebrafish cytosolic sulfotransferases (SULTs). In the present study, we carried a systematic analysis of the sulfating activities of these fourteen zebrafish SULTs toward a panel of drug compounds. Results showed that four of the fourteen zebrafish SULTs showed no detectable activities toward any of the tested drugs. Among the other ten zebrafish SULTs, three SULT1 enzymes (SULT1 ST1, SULT1 ST2, and SULT1 ST3) displayed considerably stronger activities than the others toward the majority of the drug compounds tested. Specifically, SULT1 ST1, SULT1 ST2, and SULT1 ST3 showed the highest specific activities, at 26.9, 29.3, and 31.5 nmol/min/mg, toward aesculetin, 4-methylembelliferone, and dobutamine, respectively. To further investigate the sulfation of tested drugs by the responsible zebrafish SULT enzymes, the kinetics of the sulfation reactions were analyzed. Kinetic constants determined indicated that the sulfation of these drugs by the SULT enzymes tested is likely to be physiologically relevant. A metabolic labeling experiment using cultured zebrafish liver cells and HepG2 human hepatoma cells was performed. Results showed that zebrafish liver cells displayed a similar pattern of sulfation of the drugs tested as that of HepG2 cells, implying that human and zebrafish liver cells may share considerable similarities with regard to their constituent drug-sulfating SULT enzymes.

Clarithromycin is involved in a large number of clinically relevant drug-drug interactions. Discrepancies are observed between the magnitude of drug interactions predicted from in vitro competitive inhibition studies and changes observed clinically in the plasma levels of affected CYP3A substrates. The formation of metabolic-intermediate complexes has been proposed to explain these differences. The objectives of our study were: 1) to determine the competitive inhibition potency of clarithromycin on the metabolism of domperidone as a CYP3A probe drug using human recombinant CYP3A4 and CYP3A5 isoenzymes, human liver microsomes and cultured human hepatocytes; 2) to establish the modulatory role of cytochrome b5 on the competitive inhibition potency of clarithromycin; 3) to demonstrate the clarithromycin-induced formation of CYP450 metabolic-intermediate complexes in human liver microsomes; and 4) to determine the extent of CYP3A inhibition due to metabolic-intermediate complex formation using human liver microsomes and cultured human hepatocytes. At high concentrations (100 μM), clarithromycin had weak competitive inhibition potency towards CYP3A4 and CYP3A5. Inhibition potency was further decreased by the addition of cytochrome b5 (9- 19%). Clarithromycin-induced metabolic-intermediate complexes were revealed by spectrophotometry analysis using human liver microsomes while time- and concentration-dependent mechanism-based inhibitions were quantified using isolated hepatocytes. These results indicate that mechanism-based but not competitive inhibition of CYP3As is the major underlying mechanism of drug-drug interactions observed clinically with clarithromycin. Drug interactions between clarithromycin and several CYP3A substrates are predicted to be insidious; the risk of severe adverse events should increase over time and persist for a few days after cessation of the drug.

Imiloxan is an alpha2 adrenoceptor antagonist and was developed for depression in the 1980's. In Phase 1 clinical trials imiloxan dosing led to hypersensitivity reactions; the molecule's development was discontinued. The present study revisits the in vitro metabolism of imiloxan using modern analytical methods. Human and rat liver microsomes convert imiloxan into a variety of metabolites many of which are unstable and or reactive. Imiloxan also yields high protein covalent binding in microsomal assays. Imiloxan is a useful test molecule for defining the relationship between liver covalent binding and idiosyncratic toxicity.

Pregnane X receptor (PXR), constitutive androstane receptor (CAR) and liver X receptor (LXR) are intracellular sensors for foreign chemicals and/or endogenous compounds. Docking of a ligand into the ligand-binding domain (LBD) of a nuclear receptor induces conformational changes and switches the nuclear receptor into an active conformation. In this study, we examined whether assembly assays to exploit the ligand-dependent interaction of N- and C-terminal regions of the LBD could be used for detection of ligands for PXR, CAR and LXR. Rifampicin, CITCO and T1317 significantly enhanced interactions for human PXR, human CAR and human LXR, respectively. The effects of ligands on the interaction of LBDs in PXR and CAR reflected the species differences in ligand response of PXR and CAR. In conclusion, it appears that the present assay, which exploits the interaction between N- and C-terminal regions of LBDs, is applicable to identify ligands.

Previous studies have indicated that CYP3As are involved in the metabolism of the prokinetic agent domperidone. The objectives of our study were to characterize further the role of specific CYP3A isoforms in the metabolism of domperidone and to compare the kinetic parameters of domperidone to those of the CYP3A probe drug midazolam. Intrinsic clearance for the formation of domperidone major metabolite (5-hydroxydomperidone) was the highest with rCYP3A4 (0.4 mL/min/nmol CYP450) compared to rCYP3A5 (0.04 mL/min/nmol CYP450). The addition of cytochrome b5 to recombinant enzymes (rCYP3A4 or rCYP3A5) increased up to 6-fold the Vmax for the formation of 5- hydroxydomperidone. In contrast, much similar intrinsic clearance values for rCYP3A4 and rCYP3A5 were determined in the respective formation of either 1-hydroxmidazolam (1.28 and 1.57 mL/min/nmol CYP450) or 4-hydroxymidazolam (0.04 and 0.06 mL/min/nmol CYP450). Vmax for the formation of midazolam metabolites was increased to a lesser extent (1.5-3-fold) by the addition of cytochrome b5. Ketoconazole more potently inhibited CYP3A4 than CYP3A5 for both domperidone and midazolam. However, the addition of cytochrome b5 to the incubation mixture appeared to decrease the inhibitory potency of ketoconazole towards CYP3A4 for domperidone but not for midazolam. Our results indicate that CYP3A4 plays major role in the metabolism of domperidone. We demonstrated a modulatory role of cytochrome b5 mostly for the metabolism of domperidone and confirmed selective inhibition of CYP3A4 over CYP3A5 by ketoconazole. Comparison of domperidone kinetic parameters to those of the CYP3A probe drug midazolam suggests that domperidone exhibits a much higher CYP3A4/CYP3A5 selectivity ratio than midazolam.

A higher throughput method of screening for the metabolism dependent inhibition of 56 marketed drugs was evaluated and compared data from the PHLM assay ( using midazolam as probe) with Cypex assay (using diethoxyfluoresin (DEF) as probe) for CYP3A4 by using 96 well plate. Also 27 marketed drugs were selected to evaluate the reproducibility of Cypex assay using 7-benzyloxyquinoline (7-BQ) as second probe substrate for CYP3A4. Furthermore Cypex CYP2D6 was used to evaluate the reproducibility of this system using 4-methylaminomethyl-7-methoxycoumarin (MMC) as probe substrate with 15 marketed drugs. The fold change was estimated using the fold change obtained from triplicates experiments in same day or different days. All replicates were in agreement (i.e. all positive or all negative) for >80% of compounds. The IC50 values for the two assays closely matched only for 13 compounds (23%). Only 5 of the variant 56 compounds had higher IC50 values with the recombinant enzymes, whereas 38 had lower IC50 values with the recombinant cypex CYP3A4 enzyme. The Cypex assay is comparable to PHLM assay in terms of predictivity and reproducibility. The Cypex assay therefore offers a higher throughput, reproducible alternative to PHLM for placement earlier in the lead optimisation process. In conclusion, the results obtained from a fluorescence-based method using Cypex CYP3A4 reflect mostly those obtained from conventional assay using human liver microsomes. This method provides more rapid and reliable detection of MDI inhibitors and may be useful in drug discovery.

Studies on the cellular disposition of targeted anticancer tyrosine kinases inhibitors (TKIs) have mostly focused on imatinib while the functional importance of P-glycoprotein (Pgp) the gene product of MDR1 remains controversial for more recent TKIs. By using RNA interference-mediated knockdown of MDR1, we have investigated and compared the specific functional consequence of Pgp on the cellular disposition of the major clinically in use TKIs imatinib, dasatinib, nilotinib, sunitinib and sorafenib. siRNA-mediated knockdown in K562/Dox cell lines provides a unique opportunity to dissect the specific contribution of Pgp to TKIs intracellular disposition. In these conditions, abrogating specifically Pgp-mediated efflux in vitro revealed the remarkable and statistically significant cellular accumulation of imatinib (difference in cellular levels between Pgp-expressing and silenced cells, at high and low incubation concentration, respectively: 6.1 and 6.6), dasatinib (4.9 and 5.6), sunitinib (3.7 and 7.3) and sorafenib (1.2 and 1.4)), confirming that these TKIs are all substrates of Pgp. By contrast, no statistically significant difference in cellular disposition of nilotinib was observed as a result of MDR1 expression silencing (differences: 1.1 and 1.5), indicating that differential expression and/or function of Pgp is unlikely to affect nilotinib cellular disposition. This study enables for the first time a direct estimation of the specific contribution of one transporter among the various efflux and influx carriers involved in the cellular trafficking of these major TKIs in vitro. Knowledge on the distinct functional consequence of Pgp expression for these various TKIs cellular distribution is necessary to better appreciate the efficacy, toxicity, and potential drug-drug interactions of TKIs with other classes of therapeutic agents, at the systemic, tissular and cellular levels.