Kan He

In Vitro Assessment of Metabolic Drug-Drug Interaction Potential of Apixaban through Cytochrome P450 Phenotyping, Inhibition, and Induction Studies

Apixaban is an oral, direct, and highly selective factor Xa inhibitor in late-stage clinical development for the prevention and treatment of thromboembolic diseases. The metabolic drug-drug interaction potential of apixaban was evaluated in vitro. The compound did not show cytochrome P450 inhibition (IC50 values >20 μM) in incubations of human liver microsomes with the probe substrates of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4/5. Apixaban did not show any effect at concentrations up to 20 μM on enzyme activities or mRNA levels of selected P450 enzymes (CYP1A2, 2B6, and 3A4/5) that are sensitive to induction in incubations with primary human hepatocytes. Apixaban showed a slow metabolic turnover in incubations of human liver microsomes with formation of O-demethylation (M2) and hydroxylation products (M4 and M7) as prominent in vitro metabolites. Experiments with human cDNA-expressed P450 enzymes and P450 chemical inhibitors and correlation with P450 activities in individual human liver microsomes demonstrated that the oxidative metabolism of apixaban for formation of all metabolites was predominantly catalyzed by CYP3A4/5 with a minor contribution of CYP1A2 and CYP2J2 for formation of M2. The contribution of CYP2C8, 2C9, and 2C19 to metabolism of apixaban was less significant. In addition, a human absorption, distribution, metabolism, and excretion study showed that more than half of the dose was excreted as unchanged parent (fm CYP <0.5), thus significantly reducing the overall metabolic drug-drug interaction potential of apixaban. Together with a low clinical efficacious concentration and multiple clearance pathways, these results demonstrate that the metabolic drug-drug interaction potential between apixaban and coadministered drugs is low.

Bergamottin contribution to the grapefruit juice–felodipine interaction and disposition in humans

Our objectives were to evaluate the contribution of bergamottin to the grapefruit juice–felodipine interaction and to characterize bergamottin disposition.

Tissue Distribution and Elimination of [14C]Apixaban in Rats

Apixaban, a potent and highly selective factor Xa inhibitor, is currently under development for treatment of arterial and venous thrombotic diseases. The distribution, metabolism, and elimination of [14C]apixaban were investigated in male, female, pregnant, and lactating rats after single oral doses. Tissue distribution of radioactivity in rats was measured using quantitative whole-body autoradiography. After a single oral administration, radioactivity distributed quickly in rats with Cmax at 1 h for most tissues. The elimination t1/2 of radioactivity in blood was 1.7 to 4.2 h. The blood area under the plasma concentration-time curve of radioactivity was similar between male and female rats and was slightly higher in pregnant rats and lower in lactating rats. The radioactivity concentration in tissues involved in elimination was greater than that in blood with the highest concentration in the gastrointestinal tract, liver, and urinary bladder/contents and lowest level in brains. In pregnant rats, the whole-body autoradiogram showed that low levels of radioactivity were present in fetal blood, liver, and kidney and were much lower than the radioactivity in the respective maternal organs. The fecal route was the major pathway (74% of dose), and the urinary route was the minor pathway (14%) for apixaban elimination. After single oral doses of [14C]apixaban to lactating rats, apixaban exhibited extensive lacteal excretion with apixaban as the major component. In summary, tissue distribution of apixaban in rats was extensive but with limited transfer to fetal and brain tissues and extensive secretion into rat milk with the parent drug as the major component. Milk excretion could account for 10% of apixaban dose, which was comparable to urinary elimination in rats. Tissue distribution and drug excretion of apixaban are consistent with those for a moderately permeable drug that is a substrate for P-glycoprotein and breast cancer resistance protein efflux transporters.

Synthesis and biological evaluation of 6′,7′-dihydroxybergamottin (6,7-DHB), a naturally occurring inhibitor of cytochrome P450 3A4

Abstract The recently isolated inhibitor of cytochrome P450 CYP3A4, 6′,7′-dihydroxybergamottin (6,7-DHB), was synthesized by an efficient route that is readily adaptable to the production of analogues. The compound was evaluated as an inhibitor of 3A4 in a purified enzyme preparation, as well as against human liver microsomes and human 3A4 expressed in Escherichia coli membrane. In each case, 6,7-DHB proved to be a potent NADPH- and time dependent inactivator of 3A4.

Inhibition of MDR3 Activity in Human Hepatocytes by Drugs Associated with Liver Injury

MDR3 dysfunction is associated with liver diseases. We report here a novel MDR3 activity assay involving in situ biosynthesis in primary hepatocytes of deuterium (d9)-labeled PC and LC-MS/MS determination of transported extracellular PC-d9. Several drugs associated with DILI such as chlorpromazine, imipramine, itraconazole, haloperidol, ketoconazole, sequinavir, clotrimazole, ritonavir, and troglitazone inhibit MDR3 activity. MDR3 inhibition may play an important role in drug-induced cholestasis and vanishing bile duct syndrome. Several lines of evidence demonstrate that the reported assay is physiologically relevant and can be used to assess the potential of chemical entities and their metabolites to modulate MDR3 activity and/or PC biosynthesis in hepatocytes.

In Vitro Drug-Induced Liver Injury Prediction: Criteria Optimization of Efflux Transporter IC50 and Physicochemical Properties

Drug-induced liver injury (DILI) is a severe drug adverse response, which cannot always be reliably predicted in preclinical or clinical studies. Lack of observation of DILI during preclinical and clinical drug development has led to DILI being a leading cause of drug withdrawal from the market. As DILI is potentially fatal, pharmaceutical companies have been developing in vitro tools to screen for potential liver injury. Screens for physicochemical properties, mitochondrial function, and transport protein inhibition have all been employed to varying degrees of success. In vitro inhibition of the bile salt export pump (BSEP) has become a major risk factor for in vivo DILI predictions, yet discrepancies exist in which methods to use and the extent to which BSEP inhibition predicts clinical DILI. The presented work focuses on optimizing DILI predictions by comparing BSEP inhibition via the membrane vesicle assay and the hepatocyte-based BSEPcyte assay, as well as dual and triple liabilities. BSEP transport inhibition of taurcholic acids and glycocholic acids were similar for up to 29 drugs tested, in both the vesicle and hepatocyte-based assays. Positive and negative DILI predictions were optimized at a 50-µM cutoff value for 50 drugs using both NIH Livertox and PharmaPendium databases. Additionally, dual inhibition of BSEP and other efflux transporters (multidrug resistance-associated protein [MRP]2, MRP3, or MRP4) provided no observable predictive benefit compared with BSEP inhibition alone. Eighty-five percent of drugs with high molecular weight (>600 Da), high cLogP (>3), or a daily dose >100 mg and BSEP inhibition were associated with DILI. Triple liability of BSEP inhibition, high molecular weight, and high cLogP attained a 100% positive prediction rate.