Drug Metabolism Letters - Volume 4, Issue 3, 2010

In vitro inhibition assays are used to screen new chemical entities (NCEs) for inhibition studies by using human liver microsomes. High-throughput inhibition assays using pooling methods have been developed to keep pace with screening requirements at the lead ADME stage. This method can determine IC50 NCEs using microsomes from various organs from any species. A cassette analysis inhibition assay for five of the major CYP enzymes (phenacetin for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, bufurarol for CYP2D6 and midazolam, nifedipine and atorvastatin for CYP3A4) in pooled human liver microsomes using ultraperformance liquid chromatography tandem mass spectrometry (LC/MS/MS) were developed. The major metabolites of seven CYP-specific probe substrates for the five P450 isoforms were monitored and quantified to determine IC50 values. Human liver microsomal incubation samples at each test compound concentration were combined and analyzed simultaneously by the LC/MS/MS method. The incubation was performed using the selective CYP inhibitors for each isoform: fluvoxamine (CYP1A2), sulphaphenazole (CYP2C9), ticlopidine (CYP2C19), quinidine (CYP2D6), and Ketoconazole (CYP3A4). Similar IC50 results were obtained using the cassette analysis and discrete analysis method. The IC50 values determined for typical CYP inhibitors were reproducible and consistent with those reported in the literature. The assay was fully automated in 96 well plate formats using Microlab 4000 series (Hamilton) coupled with two termomixer comfort (Eppendorf). An overall 70% time savings was achieved by pooling four isoforms samples (1A2, 2C9, 2C19 and 2D6) into one sample and also by pooling three CYP 3A4 substrate samples (MDZ, ATR, NIF) into one sample. Cassette analysis minimized the number of injections on LC/MS/MS analysis which results in a decrease in the LC/MS/MS analysis time.

1-Aminobenzotriazole (ABT) has been widely used in drug development process as an irreversible inhibitor of CYP enzymes. One potential use of ABT is to potentiate pharmacological effects of rapidly-metabolized drugs on CYP expression by inhibiting their metabolism; however, ABT's own effects on CYP expression have been unknown. In this study, we show that ABT up-regulates expression of CYP2B6 and CYP3A4 potentially by activating nuclear receptor CAR. In freshly isolated human hepatocytes, ABT increased mRNA expression of CYP2B6 and CYP3A4 in a concentration- dependent manner. ABT also modulated CYP-inducing actions of CITCO and rifampin, the known inducers of CYP2B6 and CYP3A4. Results from luciferase reporter assays confirmed that ABT increases CYP2B6 promoter activity in CAR-expressing HepG2 cells. These results suggest that the use of ABT as a potentiator of pharmacological effects of rapidly-metabolized drugs is limited due to its own pharmacological actions on CYP expression as a CAR activator.

Schistosomiasis is one of the major health problems in many developing countries and causes liver damage. In addition, under the influence of schistosomaisis, most of the endogenous toxic compounds can be conjugated with glutathione via glutathione S-transferase. Therefore, the present study showed the effect of Schistosoma mansoni after 20, 30, 45, 60, and 75 days post-infection on the activity of glutathione-S-transferase (GST) and glutathione reductase (GR), and on the levels of glutathione [GSH] in the livers of male mice. In addition, anti-schistosomal drug (praziquantel) was administered orally [60 mg/kg body weight] for three consecutive days before decapitation of the infected mice at each time point. In the present, depletion of GSH levels was observed at 45, 60 and 75 days post-infection. However, treatment of infected mice at 45, 60, and 75 days post-infection with praziquantel for three consecutive days before decapitation at each time point restored the increased GSH levels to their normal values compared with control groups. Inhibition of GST and induction of GR activities in the livers of S. mansoni-infected mice at all time-points were restored to their normal levels after praziquantel treatment. It is concluded that S. mansoni infection changed the activities of GST, GR and GSH levels. Moreover, it has been found that praziquantel treatment of S. mansoni-infected mice restored such alterations to their normal values and this recovery could alleviate the deleterious effects of S. mansoni infection. In addition, the present study could provide new evidence to the damage occurred in livers of S. mansoni-infected hosts. Also, it is suggested that praziquantel is the best drug of choice for schistosoma mansoni treatment.

To determine in vivo if L-4F differentially alters plasma levels of oxidized fatty acids resulting in more antiinflammatory HDL. Injecting L-4F into apoE null mice resulted in a significant reduction in plasma levels of 15-HETE, 5- HETE, 13-HODE and 9-HODE. In contrast, plasma levels of 20-HETE were not reduced and plasma levels of 14,15-EET, which are derived from the cytochrome P450 pathway, were elevated after injection of L-4F. Injection of 13(S)-HPODE into wild-type C57BL/6J mice caused an increase in plasma levels of 13-HODE and 9-HODE and was accompanied by a significant loss in the anti-inflammatory properties of HDL. The response of atherosclerosis resistant C3H/HeJ mice to injection of 13(S)-HPODE was similar but much more blunted. Injection of L-4F at a site different from that at which the 13(S)-HPODE was injected resulted in significantly lower plasma levels of 13-HODE and 9-HODE and significantly less loss of HDL anti-inflammatory properties in both strains. i) L-4F differentially alters plasma levels of oxidized fatty acids in vivo. ii) The resistance of the C3H/HeJ strain to atherosclerosis may in part be mediated by a reduced reaction of this strain to these potent lipid oxidants. L-4F differentially alters plasma levels of oxidized fatty acids in mice and the resistance of C3H/HeJ mice to atherosclerosis may be mediated by a reduced reaction of this strain to these potent lipid oxidants.

Objective: This study investigated the absorption, distribution, metabolism and excretion (ADME) of nebicapone [BIA 3-202; 1-(3,4-dihydroxy-5-nitrophenyl)-2-phenyl-ethanone], a reversible catechol-O-methyltransferase (COMT) inhibitor, in 4 healthy male subjects. Methods; This was a single center, open, non-placebo-controlled, single-group, and a single 200 mg dose study of [14C]-nebicapone (2.5 MBq). Blood, urine and faeces were collected up to 264 hours post-dose. Results: Collectively more than 22 metabolites were identified in plasma, urine and faeces, with 3-Onebicapone- glucuronide (BIA 3-476) identified as the major metabolite. Plasma concentration-time profiles of [14C]- nebicapone demonstrated Tmax (h) 1.25±0.65, t1/2 (h) 134.55±25.67, Cmax (ng-eq/g) 19647.02±4930.20, AUC0-t (h.ng-eq/g) 161735.51±9224.66, AUC0-∞ (h.ng-eq/g) 199603.30±16854.08, and for whole blood Tmax 1.00±0.41, t1/2 32.98±22.82, AUC0-t 35539.23±13664.87, AUC0-∞ 36970.64±14559.17. Plasma pharmacokinetics of nebicapone demonstrated Tmax (h) 1.00±0.41, t1/2 (h) 2.34±0.51; Cmax (ng-eq/g) 12650.00±2898.85, AUC0-t (h.ng-eq/g) 18719.96±734.18, AUC0-∞ (h.ngeq/ g) 18392.12±753.81; BIA 3-476 demonstrated Tmax 1.25±0.50, t1/2 3.47±0.68; Cmax 15250±2563.20, AUC0-t 53810.61 ±7358.81, AUC0-∞ 54541.21±7135.70; 3-O-methyl-nebicapone (BIA 3-270) demonstrated Tmax 21.01±6.01 , t1/2 103.43± 6.01; Cmax 286.25±20.48, AUC0-t 27641.89±4569.99, AUC0-∞ 36968.12±4294.42. Conclusions: Nebicapone and BIA 3- 476 accounted for most early phase circulating nebicapone-derived moieties, have limited circulating cell association, peak concentrations shortly after dosing, and short body residence. In longer terminal half-life phases low concentrations of BIA 3-270 predominate. While about 70% of the dose was eliminated in the urine as BIA 3-476, < 1% of the dose was excreted as unchanged nebicapone. Faecal excretion accounted for 17.3% administered dose. On average, the total recovery of 88.6% of the radioactivity suggested no worrisome retention of drug derived material following a single 200 mg administration of nebicapone to healthy volunteers. The treatment was very well tolerated with no reported adverse events.

The quinuclidine PHA-0568487(1) is an agonist of the alpha7 nicotinic acetylcholine receptor that was designed to mitigate the bioactivation associated with the core scaffold and subsequently remove associated liabilities with in vivo tolerability. The drug metabolites of 1 in nonclinical species were identified in plasma and urine of rats, dogs and monkeys receiving oral administrations of 1. The in vitro biotransformation of 1 was subsequently investigated in multiple species employing cryopreserved hepatocytes, hepatic subcellular fractions and recombinantly-expressed human P450 enzymes. In addition, in vitro metabolism of synthetically prepared metabolite precursors were instrumental in the elucidation of several secondary metabolites. The results indicated that the principal biotransformation of 1 was oxidation of the benzo[1,4]dioxane moiety (M8, M10) followed by subsequent oxidation to a range of secondary metabolites (M1-7, M9,M11,M13-15, and M17-18). The carboxylic acids M1 and M2 resulting from the oxidative cleavage of the dioxane ring were the principal metabolites observed in the plasma, urine and hepatocyte incubations across all species (M1 & M2). Quinuclidine oxidation was another pathway of importance, yielding an N-oxide (M12) which was also observed in all species.P450 2D6 and FMO1 catalyze the oxidation of the quinuclidine nitrogen. The N-oxidation of the quinuclidine moiety is consistent with previously published accounts of this scaffold's metabolism and, interestingly, may implicate the uncommon quinuclidine moiety as an entity directing the metabolism of this scaffold (e.g., 1) via FMO1 and P450 2D6 oxidation.

The effect of licorice root drink (aqueous extract of Glycyrrhiza glabra Fabaceae) on plasma concentration of verapamil, using rabbits as animal model, was investigated. Two groups of locally inbred Newzeland male rabbits were used. The first group was given a single dose of licorice drink (4 ml/kg body weight) concomitantly with 30 mg/kg verapamil, and the second group was given a daily dose of licorice drink (4 ml/kg body weight) for two weeks, with single doses of verapamil on days, 7 and 14. Single dose treatment resulted in a nonsignificant decrease in mean Cmax by 33.2% (P = 0.41), but in a significant decrease of AUC0-24 and AUC0-∞ by 60.5% and 63.6%, respectively (P = 0.01). First period of multiple dose treatment study (7 days), resulted in a significant reduction in mean Cmax, AUC0-24 and AUC0-∞ by 55.0%, 47.0% and 45.7%, respectively (P = 0.02, 0.03 and 0.03, respectively). A more pronounced effect was seen at second period of multiple dose treatment study (14 days), where the corresponding decrease was, 85.4%, 76.8% and 73.3%, respectively (P < 0.01). Mean Tmax was significantly increased 4.2-fold over control period at day 14 of multiple dose study (P = 0.02). In conclusion, licorice root drink decreased verapamil systemic exposure both after single dose and after daily doses for 14 days.

Morphological evaluation of humanized chimeric mouse livers from the PhoenixBio® (uPA+/+/SCID) mouse model show robust replacement and expansion with human hepatocytes, however areas of human hepatocytes had prominent steatosis and a variable lack of sinusoids which was consistent with decreased hepatocellular perfusion and lacked bile canalicular formation between human and mouse hepatocytes.