Scott J. Grossman

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.

Comparative Metabolism of 14C-Labeled Apixaban in Mice, Rats, Rabbits, Dogs, and Humans

The metabolism and disposition of [14C]apixaban, a potent, reversible, and direct inhibitor of coagulation factor Xa, were investigated in mice, rats, rabbits, dogs, and humans after a single oral administration and in incubations with hepatocytes. In plasma, the parent compound was the major circulating component in mice, rats, dogs, and humans. O-Demethyl apixaban sulfate (M1) represented approximately 25% of the parent area under the time curve in human plasma. This sulfate metabolite was present, but in lower amounts relative to the parent, in plasma from mice, rats, and dogs. Rabbits showed a plasma metabolite profile distinct from that of other species with apixaban as a minor component and M2 (O-demethyl apixaban) and M14 (O-demethyl apixaban glucuronide) as prominent components. The fecal route was a major elimination pathway, accounting for >54% of the dose in animals and >46% in humans. The urinary route accounted for <15% of the dose in animals and 25 to 28% in humans. Apixaban was the major component in feces of every species and in urine of all species except rabbit. M1 and M2 were common prominent metabolites in urine and feces of all species as well as in bile of rats and humans. In vivo metabolite profiles showed quantitative differences between species and from in vitro metabolite profiles, but all human metabolites were found in animal species. After intravenous administration of [14C]apixaban to bile duct-cannulated rats, the significant portion (approximately 22%) of the dose was recovered as parent drug in the feces, suggesting direct excretion of the drug from gastrointestinal tracts of rats. Overall, apixaban was effectively eliminated via multiple elimination pathways in animals and humans, including oxidative metabolism, and direct renal and intestinal excretion.

Synthesis and structure-activity relationships of 8-(pyrid-3-yl)pyrazolo[1,5-a]-1,3,5-triazines: potent, orally bioavailable corticotropin releasing factor receptor-1 (CRF1) antagonists.

This report describes the syntheses and structure-activity relationships of 8-(substituted pyridyl)pyrazolo[1,5-a]-1,3,5-triazine corticotropin releasing factor receptor-1 (CRF(1)) receptor antagonists. These CRF(1) receptor antagonists may be potential anxiolytic or antidepressant drugs. This research resulted in the discovery of compound 13-15, which is a potent, selective CRF(1) antagonist (hCRF(1) IC(50) = 6.1 +/- 0.6 nM) with weak affinity for the CRF-binding protein and biogenic amine receptors. This compound also has a good pharmacokinetic profile in dogs. Analogue 13-15 is orally effective in two rat models of anxiety: the defensive withdrawal (situational anxiety) model and the elevated plus maze test. Analogue 13-15 has been advanced to clinical trials.

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.

The chimpanzee (Pan troglodytes) as a pharmacokinetic model for selection of drug candidates: Model characterization and application

The chimpanzee (CHP) was evaluated as a pharmacokinetic model for humans (HUMs) using propranolol, verapamil, theophylline, and 12 proprietary compounds. Species differences were observed in the systemic clearance of theophylline (∼5-fold higher in CHPs), a low clearance compound, and the bioavailability of propranolol and verapamil (lower in CHPs), both high clearance compounds. The systemic clearance of propranolol (∼1.53 l/h/kg) suggested that the hepatic blood flow in CHPs is comparable to that in humans. No substantial differences were observed in the in vitro protein binding. A preliminary attempt was made to characterize cytochrome P450 (P450) activities in CHP and HUM liver microsomes. Testosterone 6β-hydroxylation and tolbutamide methylhydroxylation activities were comparable in CHP and HUM liver microsomes. In contrast, dextromethorphan O-demethylation and phenacetin O-deethylation activities were ∼10-fold higher (per mg protein) in CHP liver microsomes. Intrinsic clearance estimates in CHP liver microsomes were higher for propranolol (∼10-fold) and theophylline (∼5-fold) and similar for verapamil. Of the 12 proprietary compounds, 3 had oral clearances that differed in the two species by more than 3-fold, an acceptable range for biological variability. Most of the observed differences are consistent with species differences in P450 enzyme activity. Oral clearances of proprietary compounds in HUMs were significantly correlated to those from CHPs (r = 0.68; p = 0.015), but not to estimates from rat, dog, and monkey. In summary, the chimpanzee serves as a valuable surrogate model for human pharmacokinetics, especially when species differences in P450 enzyme activity are considered.

8-(4-Methoxyphenyl)pyrazolo[1,5-a]-1,3,5-triazines: Selective and Centrally Active Corticotropin-Releasing Factor Receptor-1 (CRF1) Antagonists

This report describes the syntheses and structure-activity relationships of 8-(4-methoxyphenyl)pyrazolo[1,5-a]-1,3,5-triazine corticotropin releasing factor receptor-1 (CRF(1)) receptor antagonists. CRF(1) receptor antagonists may be potential anxiolytic or antidepressant drugs. This research culminated in the discovery of analogue 12-3, which is a potent, selective CRF(1) antagonist (hCRF(1) IC(50) = 4.7 +/- 2.0 nM) with weak affinity for the CRF-binding protein and biogenic amine receptors. This compound also has a good pharmacokinetic profile in dogs. Analogue 12-3 is orally effective in two rat models of anxiety: the defensive withdrawal (situational anxiety) model and the elevated plus maze test. Analogue 12-3 has been advanced to clinical trials.

Imidazo[4,5-c]pyridines as corticotropin releasing factor receptor ligands

A series of high affinity CRF receptor ligands with an imidazo[4,5-c]pyridine core is described. Individual analogues were synthesized and tested in vitro in rat brain receptors to determine binding affinity. The best compound was further tested in the dog N-in-1 pharmacokinetic model to assess oral bioavailability at 1 mg/kg po.

Neuroactive Steroids. 1. Positive Allosteric Modulators of the (γ-Aminobutyric Acid)A Receptor: Structure–Activity Relationships of Heterocyclic Substitution at C-21

Neuroactive steroids (NASs) have been shown to impact central nervous system (CNS) function through positive allosteric modulation of the GABA(A) receptor (GABA(A)-R). Herein we report the effects on the activity and pharmacokinetic properties of a series of nor-19 pregnanolone analogues bearing a heterocyclic substituent at C-21. These efforts resulted in the identification of SGE-516, a balanced synaptic/extrasynaptic GABA(A) receptor modulator, and SGE-872, a selective extrasynaptic GABA(A) receptor modulator. Both molecules possess excellent druglike properties, making them advanced leads for oral delivery of GABA(A) receptor modulators.

In Vitro and In Vivo Metabolism and Pharmacokinetics of BMS-562086, a Potent and Orally Bioavailable Corticotropin-Releasing Factor-1 Receptor Antagonist

The absorption, distribution, metabolism, and excretion (ADME) and the pharmacokinetic characteristics of BMS-562086 [pexacerfont; 8-(6-methoxy-2-methyl-3-pyridinyl)-2,7-dimethyl-N-[(1R)-1-methylpropyl]pyrazolo(1,5-a)-1,3,5-triazin-4-amine (DPC-A69448)] were investigated in vitro and in animals to support its clinical development. BMS-562086 was orally bioavailable in rats, dogs, and chimpanzees, with an absolute oral bioavailability of 40.1, 58.8, and 58.5%, respectively. BMS-562086 was extensively metabolized in hepatocytes from all species and completely metabolized in rats. The primary biotransformation pathways found for BMS-562086 in both liver microsomal and hepatocyte preparations and in rats were similar. These included O-demethylation, hydroxylation at the N-alkyl side chain and N-dealkylation. Multiple cytochromes P450 including CYP3A4/5 were involved in the metabolic clearance of BMS-562086. Both renal and biliary excretion played a significant role in elimination of the metabolites of BMS-562086. The involvement of other metabolic enzymes in addition to CYP3A4/5 in elimination of BMS-562086 suggests a reduced potential for drug-drug interaction through modulation of CYP3A4/5. Chimpanzees proved to be a good animal model in predicting BMS-562086 human clearance. Virtual clinical trials performed with a population-based ADME simulator suggested that a minimal dose of 100 mg daily would provide sufficient drug exposure to achieve plasma concentrations above the projected human efficacious plasma concentration of BMS-562086 (>500 nM). In summary, BMS-562086 exhibited favorable ADME and pharmacokinetic properties for further development.

Glucuronidation in the chimpanzee (Pan troglodytes): Studies with acetaminophen, oestradiol and morphine

The chimpanzee has recently been characterized as a surrogate for oxidative drug metabolism in humans and as a pharmacokinetic model for the selection of drug candidates. In the current study, the glucuronidation of acetaminophen, morphine and oestradiol was evaluated in the chimpanzee to extend the characterization of this important animal model. Following oral administration of acetaminophen (600 mg) to chimpanzees (n = 2), pharmacokinetics were comparable with previously reported human values, namely mean oral clearance 0.91 vs. 0.62 ± 0.05 l h−1 kg−1, apparent volume of distribution 2.29 vs. 1.65 ± 0.25 l kg−1, and half-life 1.86 vs. 1.89 ± 0.27 h, for chimpanzee vs. human, respectively. Urinary excretions (percentage of dose) of acetaminophen, acetaminophen glucuronide and acetaminophen sulfate were also similar between chimpanzees and humans, namely 2.3 vs. 5.0, 63.1 vs. 54.7, and 25.0 vs. 32.3%, respectively. Acetaminophen, oestradiol and morphine glucuronide formation kinetics were investigated using chimpanzee (n = 2) and pooled human liver microsomes (n = 10). and (or ) for acetaminophen glucuronide, morphine 3- and 6-glucuronide, and oestradiol 3- and 17-glucuronide formation were comparable in both species. Eadie–Hofstee plots of oestradiol 3-glucuronide formation in chimpanzee microsomes were characteristic of autoactivation kinetics. Western immunoblot analysis of chimpanzee liver microsomes revealed a single immunoreactive band when probed with anti-human UGT1A1, anti-human UGT1A6, and anti-human UGT2B7. Taken collectively, these data demonstrate similar glucuronidation characteristics in chimpanzees and humans.