In vitro evaluations for pharmacokinetic drug-drug interactions of a novel serotonin-dopamine activity modulator, brexpiprazole.

Brexpiprazole, a serotonin-dopamine activity modulator, is indicated for the treatment of schizophrenia and also adjunctive therapy to antidepressants for the treatment of Major Depressive Disorder. To determine the drug-drug interaction risk for cytochrome P450, and SLC and ABC transporters, brexpiprazole and its metabolite, DM-3411 were assessed in this in vitro investigation.Brexpiprazole exhibited weak inhibitory effects (IC50 >13 µmol/L) on CYP2C9, CYP2C19, CYP2D6 and CYP3A4 activities, but had moderate inhibitor activity on CYP2B6 (IC50 8.19 µmol/L). The ratio of systemic unbound concentration (3.8 nmol/L) to the Ki value was sufficiently low. DM-3411 had comparable inhibitory potentials with brexpiprazole only for CYP2D6 and CYP3A4. The mRNA expressions of CYP1A2, CYP2B6 and CYP3A4 were not changed by the exposure of brexpiprazole to human hepatocytes.Brexpiprazole and DM-3411 exhibited weak or no inhibitory effects for hepatic and renal transporters (OATPs, OATs, OCTs, MATE1, and BSEP), except for MATE-2K (0.156 µmol/L of DM-3411), even for which the ratio to systemic unbound concentration (5.3 nmol/L) was sufficiently low.Brexpiprazole effected the functions of P-gp and BCRP with IC50 values of 6.31 and 1.16 µmol/L, respectively, however, the pharmacokinetic alteration was not observed in the clinical concomitant study on P-gp and BCRP substrates.These in vitro data suggest that brexpiprazole is unlikely to cause clinically relevant drug interactions resulting from the effects on CYPs or transporters mediating the absorption, metabolism, and/or disposition of co-administered drugs.

Cytochromes P450 catalyze the reduction of α,ß-unsaturated aldehydes.

The metabolism of α,ß-unsaturated aldehydes, e.g., 4-hydroxynonenal, involves oxidation to carboxylic acids, reduction to alcohols, and glutathionylation to eventually form mercapturide conjugates. Recently, we demonstrated that P450s can oxidize aldehydes to carboxylic acids, a reaction previously thought to involve aldehyde dehydrogenase. When recombinant cytochrome P450 3A4 was incubated with 4-hydroxynonenal, O(2), and NADPH, several products were produced, including 1,4-dihydroxynonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), and an unknown metabolite. Several P450s catalyzed the reduction reaction in the order (human) P450 2B6 ≅ P450 3A4 > P450 1A2 > P450 2J2 > (mouse) P450 2c29. Other P450s did not catalyze the reduction reaction (human P450 2E1 and rabbit P450 2B4). Metabolism by isolated rat hepatocytes showed that HNA formation was inhibited by cyanamide, while DHN formation was not affected. Troleandomycin increased HNA production 1.6-fold while inhibiting DHN formation, suggesting that P450 3A11 is a major enzyme involved in rat hepatic clearance of 4-HNE. A fluorescent assay was developed using 9-anthracenealdehyde to measure both reactions. Feeding mice a diet containing t-butylated hydroxyanisole increased the level of both activities with hepatic microsomal fractions but not proportionally. Miconazole (0.5 mM) was a potent inhibitor of these microsomal reduction reactions, while phenytoin and α-naphthoflavone (both at 0.5 mM) were partial inhibitors, suggesting the role of multiple P450 enzymes. The oxidative metabolism of these aldehydes was inhibited >90% in an Ar or CO atmosphere, while the reductive reactions were not greatly affected. These results suggest that P450s are significant catalysts of the reduction of α,ß-unsaturated aldehydes in the liver.

Direct and cytokine-mediated effects of albumin-fused growth hormone, TV-1106, on CYP enzyme expression in human hepatocytes in vitro.

Some biologics can modulate cytokines that may lead to changes in expression of drug-metabolizing enzymes and cause drug-drug interactions (DDI). DDI potential of TV-1106-an albumin-fused growth hormone (GH)-was investigated. In this study, human blood was exposed to recombinant human growth hormone (rhGH) or TV-1106, followed by isolation of the plasma and its application to human hepatocytes. While the treatment of blood with rhGH increased multiple cytokines, treatment of blood with TV-1106 had no effect on any of the nine cytokines tested. The interleukin (IL)-6 concentration was higher in the rhGH then in the TV-1106-treated plasma (P < .05). While rhGH had little or no effect on CYP1A2 or CYP2C19 mRNA but increased CYP3A4 mRNA twofold, TV-1106 had little or no effect on cytochrome P450 (CYP) mRNAs in hepatocytes. Although the plasma from rhGH-treated blood lowered CYP1A2 activity, the TV-1106 plasma had no effect on CYP activities. The CYP1A2 activity was lower in the rhGH- then in the TV-1106-plasma treated hepatocytes (P < .05). The results indicated that fusing GH with albumin made TV-1106 an unlikely participant of CYP1A2, CYP2C19 or CYP3A4-facilitated, direct or cytokine-driven DDI.

Aldehyde reduction by cytochrome P450.

This protocol describes the procedure for measuring the relative rates of metabolism of the α,ß-unsaturated aldehydes 9-anthracene aldehyde (9-AA) and 4-hydroxy-trans-2-nonenal (4-HNE). More specifically, these assays measure the aldehyde reduction reactions of cytochrome P450s (CYPs). They can be performed using liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method for reduction of 9-AA (a model α,ß-unsaturated aldehyde) by CYPs was adapted from an assay for 9-anthracene oxidation published by Marini et al. (2003). For reduction of the endogenous aldehyde 4-HNE, the substrate was incubated with CYP in the presence of oxygen and NADPH, and the metabolites were separated by HPLC, using an adaptation of the method by Srivastava et al. (2010). For both 9-AA and 4-HNE, the first step involves incubation of the substrate with the CYP in an appropriate medium. This is followed by quantification of metabolites through by spectrofluorometry (9-AA) or HPLC coupled with a radiometric assay (4-HNE). Metabolite identification can be achieved by HPLC GC/MS analysis. Inhibitors of cytochrome P450 can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction of CYPs is not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These characteristics are similar to those reported for some cytochrome P450-catalyzed azo reduction reactions.

Cytochromes P450 catalyze oxidation of alpha,beta-unsaturated aldehydes.

We sought to establish whether heme-thiolate monooxygenases oxidize, alpha,beta-unsaturated aldehydes generated during lipid peroxidation. Several recombinant P450s co-expressed with NADPH:P450 oxidoreductase were surveyed for aldehyde oxidation activity with anthracene-9-carboxaldehyde and 4-hydroxy-trans-2-nonenal (HNE). Murine P4502c29, human P4503A4, human P4502B6, and rabbit P4502B4 were good catalysts of aldehyde oxidation to carboxylic acids. Other P450s (e.g., P4501A2, 2E1, and 2J2) did not oxidize these aldehydes. P4502c29 and P4503A4 displayed K(m)/S(0.5) values of approx. 1-20microM. The product measured by HPLC that co-migrates with authentic 4-hydroxynonenoic acid (HNA) had a mass spectrum identical to the standard. Using P4502c29, HNE was a mixed-competitive inhibitor of anthracene-9-carboxaldehyde oxidation, suggesting that both aldehydes are substrates for P4502c29. Specific inhibitors of aldehyde dehydrogenases and P450 were used to assess their role in the metabolism of HNE in primary rat hepatocytes. Inhibitors of aldehyde dehydrogenase (cyanamide) inhibited HNA formation by 60% and together cyanamide and miconazole (P450) caused over 85% inhibition of HNA formation. P450s are significant participants in metabolism of endogenous and exogenous unsaturated aldehydes in primary rat hepatocytes.