Comprehensive Nonclinical Safety Assessment of Nirmatrelvir Supporting Timely Development of the SARS-COV-2 Antiviral Therapeutic, Paxlovid™.

COVID-19 is a potentially fatal infection caused by the SARS-CoV-2 virus. The SARS-CoV-2 3CL protease (Mpro) is a viral enzyme essential for replication and is the target for nirmatrelvir. Paxlovid (nirmatrelvir co-administered with the pharmacokinetic enhancer ritonavir) showed efficacy in COVID-19 patients at high risk of progressing to hospitalization and/or death. Nonclinical safety studies with nirmatrelvir are essential in informing benefit-risk of Paxlovid and were conducted to support clinical development. In vivo safety pharmacology assessments included a nervous system/pulmonary study in rats and a cardiovascular study in telemetered monkeys. Potential toxicities were assessed in repeat dose studies of up to 1 month in rats and monkeys. Nirmatrelvir administration (1,000 mg/kg, p.o.) to male rats produced transient increases in locomotor activity and respiratory rate but did not affect behavioral endpoints in the functional observational battery. Cardiovascular effects in monkeys were limited to transient increases in blood pressure and decreases in heart rate, observed only at the highest dose tested (75 mg/kg per dose b.i.d; p.o.). Nirmatrelvir did not prolong QTc-interval or induce arrhythmias. There were no adverse findings in repeat dose toxicity studies up to 1 month in rats (up to 1,000 mg/kg daily, p.o.) or monkeys (up to 600 mg/kg daily, p.o.). Nonadverse, reversible clinical pathology findings without clinical or microscopic correlates included prolonged coagulation times at ≥60 mg/kg in rats and increases in transaminases at 600 mg/kg in monkeys. The safety pharmacology and nonclinical toxicity profiles of nirmatrelvir support clinical development and use of Paxlovid for treatment of COVID-19.

Future of Biotransformation Science in the Pharmaceutical Industry.

Over the past decades, the number of scientists trained in departments dedicated to traditional medicinal chemistry, biotransformation and/or chemical toxicology have seemingly declined. Yet, there remains a strong demand for such specialized skills in the pharmaceutical industry, particularly within drug metabolism/pharmacokinetics (DMPK) departments. In this position paper, the members of the Biotransformation, Mechanisms, and Pathways Focus Group (BMPFG) steering committee reflect on the diverse roles and responsibilities of scientists trained in the biotransformation field in pharmaceutical companies and contract research organizations. The BMPFG is affiliated with the International Society for the Study of Xenobiotics (ISSX) and was specifically created to promote the exchange of ideas pertaining to topics of current and future interest involving the metabolism of xenobiotics (including drugs). The authors also delve into the relevant education and diverse training skills required to successfully nurture the future cohort of industry biotransformation scientists and guide them toward a rewarding career path. The ability of scientists with a background in biotransformation and organic chemistry to creatively solve complex drug metabolism problems encountered during research and development efforts on both small and large molecular modalities is exemplified in five relevant case studies. Finally, the authors stress the importance and continued commitment to training the next generation of biotransformation scientists who are not only experienced in the metabolism of conventional small molecule therapeutics, but are also equipped to tackle emerging challenges associated with new drug discovery modalities including peptides, protein degraders, and antibodies. SIGNIFICANCE STATEMENT: Biotransformation and mechanistic drug metabolism scientists are critical to advancing chemical entities through discovery and development, yet the number of scientists academically trained for this role is on the decline. This position paper highlights the continuing demand for biotransformation scientists and the necessity of nurturing creative ways to train them and guarantee the future growth of this field.

Comparison of the circulating metabolite profile of PF-04991532, a hepatoselective glucokinase activator, across preclinical species and humans: potential implications in metabolites in safety testing assessment.

A previous report from our laboratory disclosed the identification of PF-04991532 [(S)-6-(3-cyclopentyl-2-(4-trifluoromethyl)-1H-imidazol-1-yl)propanamido)nicotinic acid] as a hepatoselective glucokinase activator for the treatment of type 2 diabetes mellitus. Lack of in vitro metabolic turnover in microsomes and hepatocytes from preclinical species and humans suggested that metabolism would be inconsequential as a clearance mechanism of PF-04991532 in vivo. Qualitative examination of human circulating metabolites using plasma samples from a 14-day multiple ascending dose clinical study, however, revealed a glucuronide (M1) and monohydroxylation products (M2a and M2b/M2c) whose abundances (based on UV integration) were greater than 10% of the total drug-related material. Based on this preliminary observation, mass balance/excretion studies were triggered in animals, which revealed that the majority of circulating radioactivity following the oral administration of [¹4C]PF-04991532 was attributed to an unchanged parent (>70% in rats and dogs). In contrast with the human circulatory metabolite profile, the monohydroxylated metabolites were not detected in circulation in either rats or dogs. Available mass spectral evidence suggested that M2a and M2b/M2c were diastereomers derived from cyclopentyl ring oxidation in PF-04991532. Because cyclopentyl ring hydroxylation on the C-2 and C-3 positions can generate eight possible diastereomers, it was possible that additional diastereomers may have also formed and would need to be resolved from the M2a and M2b/M2c peaks observed in the current chromatography conditions. In conclusion, the human metabolite scouting study in tandem with the animal mass balance study allowed early identification of PF-04991532 oxidative metabolites, which were not predicted by in vitro methods and may require additional scrutiny in the development phase of PF-04991532.

Metabolites in safety testing assessment in early clinical development: a case study with a glucokinase activator.

The present article summarizes Metabolites in Safety Testing (MIST) studies on a glucokinase activator, N,N-dimethyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (PF-04937319), which is under development for the treatment of type 2 diametes mellitus. Metabolic profiling in rat, dog, and human hepatocytes revealed that PF-04937319 is metabolized via oxidative (major) and hydrolytic pathways (minor). N-Demethylation to metabolite M1 [N-methyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide] was the major metabolic fate of PF-04937319 in human (but not rat or dog) hepatocytes, and was catalyzed by CYP3A and CYP2C isoforms. Qualitative examination of circulating metabolites in humans at the 100- and 300-mg doses from a 14-day multiple dose study revealed unchanged parent drug and M1 as principal components. Because M1 accounted for 65% of the drug-related material at steady state, an authentic standard was synthesized and used for comparison of steady-state exposures in humans and the 3-month safety studies in rats and dogs at the no-observed-adverse-effect level. Although circulating levels of M1 were very low in beagle dogs and female rats, adequate coverage was obtained in terms of total maximal plasma concentration (∼7.7× and 1.8×) and area under the plasma concentration-time curve (AUC; 3.6× and 0.8× AUC) relative to the 100- and 300-mg doses, respectively, in male rats. Examination of primary pharmacology revealed M1 was less potent as a glucokinase activator than the parent drug (compound PF-04937319: EC50 = 0.17 µM; M1: EC50 = 4.69 µM). Furthermore, M1 did not inhibit major human P450 enzymes (IC50 > 30 µM), and was negative in the Salmonella Ames assay, with minimal off-target pharmacology, based on CEREP broad ligand profiling. Insights gained from this analysis should lead to a more efficient and focused development plan for fulfilling MIST requirements with PF-04937319.

Assessment of the contributions of CYP3A4 and CYP3A5 in the metabolism of the antipsychotic agent haloperidol to its potentially neurotoxic pyridinium metabolite and effect of antidepressants on the bioactivation pathway.

As a plausible explanation for the large interindividual variability in the pharmacokinetics of the neuroleptic agent haloperidol, the contributions of CYP3A isozymes (CYP3A4 and the polymorphic CYP3A5) predominantly involved in haloperidol bioactivation to the neurotoxic pyridinium species 4-(4-Chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-pyridinium (HPP(+)) were assessed in human liver microsomes and heterologously expressed enzymes. Based on recent reports on drug-drug interactions between haloperidol and antidepressants including selective serotonin reuptake inhibitors, the inhibitory effects of antidepressants on the CYP3A4/5-mediated haloperidol bioactivation were also evaluated. HPP(+) formation followed Michaelis-Menten kinetics in microsomes, recombinant CYP3A4, and CYP3A5 with K(m) values of 24.4 +/- 8.9 microM, 18.3 +/- 4.9 microM, and 200.2 +/- 47.6 microM, respectively, and V(max) values of 157.6 +/- 13.2 pmol/min/mg of protein, 10.4 +/- 0.6 pmol/min/pmol P450, and 5.16 +/- 0.6 pmol/min/pmol P450, respectively. The similarity in K(m) values between human liver microsomal and recombinant CYP3A4 incubations suggests that polymorphic CYP3A5 may not be an important genetic contributor to the interindividual variability in CYP3A-mediated haloperidol clearance pathways. Besides HPP(+), a novel 4-fluorophenyl-ring-hydroxylated metabolite of haloperidol in microsomes/CYP3A enzymes was also detected. Its formation was consistent with previous reports on the detection of O-sulfate and -glucuronide conjugates of a fluorophenyl ring-hydroxylated metabolite of haloperidol in human urine. Finally, all antidepressants except buspirone inhibited the CYP3A4/5-catalyzed oxidation of haloperidol to HPP(+) in a concentration-dependent manner. Based on the estimated IC(50) values for inhibition of HPP(+) formation in microsomes, the antidepressants were ranked in the following order: fluoxetine, nefazodone, norfluoxetine, trazodone, and fluvoxamine. These inhibition results suggest that clinically observed drug-drug interactions between haloperidol and antidepressants may arise via the attenuation of CYP3A4/5-mediated 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-4-piperidinol biotransformation pathways.