Metabolism Of Probe Substrates Research Articles

Inhibitory mechanism of vortioxetine on CYP450 enzymes in human and rat liver microsomes.

Vortioxetine is a novel anti-major depression disorder drug with a high safety profile compared with other similar drugs. However, little research has been done on drug-drug interactions (DDI) about vortioxetine. In this paper, the inhibitory effect of vortioxetine on cytochrome P450 (CYP450) and the type of inhibitory mechanism were investigated in human and rat liver microsomes. We set up an in vitro incubation system of 200μL to measure the metabolism of probe substrates at the present of vortioxetine at 37°C. The concentrations of the metabolites of probe substrates were all measured by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. It was found no time-dependent inhibition (TDI) of vortioxetine through determination of half-maximal inhibitory concentration (IC50) shift values. The enzymes and metabolites involved in this experiment in human and rats were as follows: CYP3A4/CYP3A (midazolam); CYP2B6/CYP2B (bupropion); CYP2D6/CYP2D (dextromethorphan); CYP2C8/CYP2C-1 (amodiaquine); CYP2C9/CYP2C-2 (losartan); and CYP2C19/CYP2C-3 (mephenytoin). We found that vortioxetine competitively inhibited CYP2C19 and CYP2D6 in human liver microsomes (HLMs) with inhibition constant (Ki) values of 2.17μM and 9.37μM, respectively. It was noncompetitive inhibition for CYP3A4 and CYP2C8, and its Ki values were 7.26μM and 6.96μM, respectively. For CYP2B6 and CYP2C9, vortioxetine exhibited the mixed inhibition with Ki values were 8.55μM and 4.17μM, respectively. In RLMs, the type of vortioxetine inhibition was uncompetitive for CYP3A and CYP2D (Ki = 4.41 and 100.9μM). The inhibition type was competitive inhibition, including CYP2B and CYP2C-2 (Ki = 2.87 and 0.12μM). The inhibition types of CYP2C-1 and CYP2C-3 (Ki = 39.91 and 4.23μM) were mixed inhibition and noncompetitive inhibition, respectively. The study of the above mechanism will provide guidance for the safe clinical use of vortioxetine so that the occurrence of DDI can be avoided.

End-product inhibition of skatole-metabolising enzymes CYP1A, CYP2A19 and CYP2E1 in porcine and piscine hepatic microsomes

The hepatic cytochrome p450 enzymes 1 A, 2A19 and 2E1 is very important for the elimination of skatole from the body of pigs. Impaired skatole metabolism, results in skatole accumulation, which give rise to off flavor of the meat. Several metabolites of skatole has been identified, however the role of these metabolites in the inhibition of the skatole metabolizing enzymes are not documented. Using microsomes from pigs and fish, we determined the ability of several skatole metabolites to inhibit CYP1 A, CYP2A19 and CYP2E1 dependent activity. Our results show that 2-aminoacetophenone is an inhibitor of porcine CYP2A19 and CYP2E1 activity, but not the piscine orthologues. In conclusion, there is species specific differences in the inhibition of CYP1 A and CYP2A19 dependent metabolism of probe substrates. This is relevant to the evaluation of different model systems and to the reduction of off flavor of meat.

SiRNA-Mediated Knockdown of P450 Oxidoreductase in Rats: A Tool to Reduce Metabolism by CYPs and Increase Exposure of High Clearance Compounds

To develop a tool based on siRNA-mediated knockdown of hepatic P450 oxidoreductase (POR) to decrease the CYP-mediated metabolism of small molecule drugs that suffer from rapid metabolism in vivo, with the aim of improving plasma exposure of these drugs. siRNA against the POR gene was delivered using lipid nanoparticles (LNPs) into rats. The time course of POR mRNA knockdown, POR protein knockdown, and loss of POR enzyme activity was monitored. The rat livers were harvested to produce microsomes to determine the impact of POR knockdown on the metabolism of several probe substrates. Midazolam (a CYP3A substrate with high intrinsic clearance) was administered into LNP-treated rats to determine the impact of POR knockdown on midazolam pharmacokinetics. Hepatic POR mRNA and protein levels were significantly reduced by administering siRNA and the maximum POR enzyme activity reduction (~85%) occurred 2weeks post-dose. In vitro analysis showed significant reductions in metabolism of probe substrates due to POR knockdown in liver, and in vivo POR knockdown resulted in greater than 10-fold increases in midazolam plasma concentrations following oral dosing. Anti-POR siRNA can be used to significantly reduce hepatic metabolism by various CYPs as well as greatly increase the bioavailability of high clearance compounds following an oral dose, thus enabling it to be used as a tool to increase drug exposure in vivo.

Atomoxetine hydrochloride: clinical drug-drug interaction prediction and outcome.

In the studies reported here, the ability of atomoxetine hydrochloride (Strattera) to inhibit or induce the metabolic capabilities of selected human isoforms of cytochrome P450 was evaluated. Initially, the potential of atomoxetine and its two metabolites, N-desmethylatomoxetine and 4-hydroxyatomoxetine, to inhibit the metabolism of probe substrates for CYP1A2, CYP2C9, CYP2D6, and CYP3A was evaluated in human hepatic microsomes. Although little inhibition of CYP1A2 and CYP2C9 activity was observed, inhibition was predicted for CYP3A (56% predicted inhibition) and CYP2D6 (60% predicted inhibition) at concentrations representative of high therapeutic doses of atomoxetine. The ability of atomoxetine to induce the catalytic activities of CYP1A2 and CYP3A in human hepatocytes was also evaluated; however, atomoxetine did not induce either isoenzyme. Based on the potential of interaction from the in vitro experiments, drug interaction studies in healthy subjects were conducted using probe substrates for CYP2D6 (desipramine) in CYP2D6 extensive metabolizer subjects and CYP3A (midazolam) in CYP2D6 poor metabolizer subjects. Single-dose pharmacokinetic parameters of desipramine (single dose of 50 mg) were not altered when coadministered with atomoxetine (40 or 60 mg b.i.d. for 13 days). Only modest changes (approximately 16%) were observed in the plasma pharmacokinetics of midazolam (single dose of 5 mg) when coadministered with atomoxetine (60 mg b.i.d. for 12 days). Although at high therapeutic doses of atomoxetine inhibition of CYP2D6 and CYP3A was predicted, definitive in vivo studies clearly indicate that atomoxetine administration with substrates of CYP2D6 and CYP3A does not result in clinically significant drug interactions.

Pharmacogenetics of warfarin elimination and its clinical implications.

Warfarin is one of the most widely prescribed oral anticoagulants. However, optimal use of the drug has been hampered by its >10-fold interpatient variability in the doses required to attain therapeutic responses. Pharmacogenetic polymorphism of cytochrome P450 (CYP) may be associated with impaired elimination of warfarin and exaggerated anticoagulatory responses to the drug in certain patients. Clinically available warfarin is a racemic mixture of (R)- and (S)-warfarin, and the (S)-enantiomer has 3 to 5 times greater anticoagulation potency than its optical congener. Both enantiomers are eliminated extensively via hepatic metabolism with low clearance relative to hepatic blood flow. CYP2C9 is almost exclusively responsible for the metabolism of the pharmacologically more active (S)-enantiomer. Several human allelic variants of CYP2C9 have been cloned, designated as CYP2C9*1 (reference sequence or wild-type allele), CYP2C9*2, CYP2C9*3 and CYP2C9*4, respectively. The allelic frequencies for these variants differ considerably among different ethnic populations. Caucasians appear to carry the CYP 2C9*2 (8 to 20%) and CYP2C9*3 (6 to 10%) variants more frequently than do Asians (0% and 2 to 5%, respectively). The metabolic activities of the CYP2C9 variants have been investigated in vitro. The catalytic activity of CYP2C9*3 expressed from cDNA was significantly less than that of CYP2C9*1. Human liver microsomes obtained from individuals heterozygous for CYP2C9*3 showed significantly reduced (S)-warfarin 7-hydroxylation as compared with those obtained from individuals genotyped as CYP2C9*1. The influence of the CYP2C9*3 allele on the in vivo pharmacokinetics of (S)-warfarin has been studied in Japanese patients. Patients with the homozygous CYP2C9*3 genotype, as well as those with the heterozygous CYP2C9*1/*3 genotype, had significantly reduced clearance of (S)-warfarin (by 90 and 60%, respectively) compared with those with homozygous CYP2C9*1. The maintenance dosages of warfarin required in Japanese patients with heterozygous and homozygous CYP2C9*3 mutations were significantly lower than those in patients with CYP2C9*1/*1. In addition, 86% of British patients exhibiting adequate therapeutic responses with lower maintenance dosages of warfarin (<1.5 mg/day) had either the CYP2C9*2 or CYP2C9*3 mutation singly or in combination, whereas only 38% of randomly selected patients receiving warfarin carried the corresponding mutations. Furthermore, the former group showed more frequent episodes of major bleeding associated with warfarin therapy. These data indicate that the CYP2C9*3 allele may be associated with retarded elimination of (S)-warfarin and the resulting clinical effects. However, relationships between CYP2C9 genotype, enzyme activity, metabolism of probe substrates, dosage requirements and bleeding complications should be interpreted with caution, and further studies are required.