Cross-species drug metabolism and impact of metabolic stability testing under anaerobic condition on predicting pharmacokinetics of keto-enol containing compound in humans.

After oral administration of [14C]-S-1360 in rats and dogs, [14C]-S-1360 was absorbed rapidly and the bioavailability was 93.7% in rats and 75.1% in dogs. Based on the results in animals, good systemic exposure would be expected in humans. In contrast to the expectation, the exposure was low in healthy volunteers compared to the exposure expected. In addition, human mass balance study using [14C]-S1360 revealed that a large amount of metabolites existed in human plasma. The major metabolites in human plasma were reduced metabolite (HP1) and S-1360 N-glucuronide, and they respectively accounted for approximately 30% of total AUC. Unchanged S-1360 accounted for only 14% of total AUC. The results showed that a significant difference between humans and animals were observed in metabolism of S-1360. Although S-1360 was stable in human hepatocytes under aerobic condition (approximately 84% remaining at 1 h), S-1360 was labile under anaerobic condition (approximately 55% remaining at 1 h). The present study revealed that the reductive metabolism pathways are the key metabolic pathway of S-1360, especially the metabolic stability test under anaerobic condition is important to predict pharmacokinetics of keto-enol containing compound, such as S-1360.

Highly Potent and Oral Macrocyclic Peptides as a HIV-1 Protease Inhibitor: mRNA Display-Derived Hit-to-Lead Optimization.

Human immunodeficiency virus type-1 (HIV-1) protease is essential for viral propagation, and its inhibitors are key anti-HIV-1 drug candidates. In this study, we discovered a novel HIV-1 protease inhibitor (compound 16) with potent antiviral activity and oral bioavailability using a structure-based drug design approach via X-ray crystal structure analysis and improved metabolic stability, starting from hit macrocyclic peptides identified by mRNA display against HIV-1 protease. We found that the improvement of the proteolytic stability of macrocyclic peptides by introducing a methyl group to the α-position of amino acid is crucial to exhibit strong antiviral activity. In addition, macrocyclic peptides, which have moderate metabolic stability and solubility in solutions containing taurocholic acid, exhibited desirable plasma total clearance and oral bioavailability. These approaches may contribute to the successful discovery and development of orally bioavailable peptide drugs.

Improved Predictability of Hepatic Clearance with Optimal pH for Acyl-Glucuronidation in Liver Microsomes.

The purpose of this study was to investigate the optimal pH for acyl-glucuronidation formation with carboxylic acid-containing compounds in human and rat liver microsomes to improve the predictability of their hepatic clearance. The optimal pH for acyl-glucuronidation of all 17 compounds was around pH 6.0 in human and rat liver microsomes. Correlation analysis was done with the predicted in vitro intrinsic clearance (CLint,in vitro) and in vivo intrinsic clearance (CLint,in vivo) calculated from available reported data of total clearance (CLtot) of 11 compounds in humans. For 8 of the 11 compounds, under the pH 6.0 condition, the CLint,in vitro were within 1/3 to 3-fold error of the observed CLint,in vivo whereas, the error was within 1/3 to 3-fold of the observed CLint,in vivo for only 3 of the 11 under the pH 7.4 condition. The intracellular pH in human and rat hepatocytes decreased in the presence of a carboxylic acid-containing compound. These findings suggest that acyl-glucuronidation in liver microsomes at pH 6.0 is closer to physiological conditions in the presence of carboxylic acid compounds, and thus, use of this pH condition is important for physiological interpretation and predictability of intrinsic clearance.

Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. Here, we describe the discovery of S-217622, the first oral noncovalent, nonpeptidic SARS-CoV-2 3CL protease inhibitor clinical candidate. S-217622 was discovered via virtual screening followed by biological screening of an in-house compound library, and optimization of the hit compound using a structure-based drug design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel noncovalent inhibitor could be a potential oral agent for treating COVID-19.

The antiviral effects of baloxavir marboxil against influenza A virus infection in ferrets.

BACKGROUND: Baloxavir marboxil (BXM), the oral prodrug of baloxavir acid (BXA), greatly reduces virus titers as well as influenza symptoms of uncomplicated influenza in patients. OBJECTIVES: To investigate the pharmacokinetic profiles of BXA and its efficacy against influenza A virus infection in ferrets. METHODS: Ferrets were dosed orally with BXM (10 and 30 mg/kg twice daily for 1 day), oseltamivir phosphate (OSP) (5 mg/kg twice daily for 2 days) or vehicle to measure the antiviral effects of BXM and OSP. The pharmacokinetic parameters of BXA was determined after single oral dosing of BXM. RESULTS: The maximum plasma concentrations of BXA were observed at 1.50 and 2.00 hours with the two BXM doses, which then declined with an elimination half-life of 6.91 and 4.44 hours, respectively. BXM at both doses remained detectable in the plasma in ferrets, which may be due to higher stability in liver microsomes. BXM (10 and 30 mg/kg twice daily) treatment at Day 1 post-infection (p.i.) reduced virus titers by ≥3 log10 of the 50% tissue culture infective doses by Day 2, which was significantly different compared with vehicle or OSP. Body temperature drops over time were significantly greater with BXM than with vehicle or OSP. Significant reduction in virus titers was also demonstrated when BXM was administrated after symptom onset at Day 2 p.i. compared with vehicle and OSP, although body temperature changes largely overlapped between Day 2 and Day 4. CONCLUSIONS: The results highlight the rapid antiviral action of BXM with post-exposure prophylaxis or therapeutic dosing in ferrets and offer support for further research on prevention of influenza virus infection and transmission.

Improved Human Pharmacokinetic Prediction of Hepatically Metabolized Drugs With Species-Specific Systemic Clearance.

Accurate prediction of human pharmacokinetics (PK) is important for the choice of promising compounds in humans. As the predictability of human PK by an empirical approach is low for drugs with species-specific PK, the utility of a physiologically based pharmacokinetic (PBPK) model was verified using 16 hepatically metabolized reference drugs. After the prediction method for total clearance (CLtot) and distribution volume at steady state (Vdss) in the conventional PBPK model had been optimized, plasma concentrations following a single oral administration of each reference drug to healthy volunteers were simulated, and the prediction accuracy for human PK was compared between empirical approaches and the optimized PBPK model. In the drugs with low species-specific CLtot, there was little difference in predictability for maximum concentration (Cmax), time to maximum plasma concentration (Tmax), and area under the curve (AUC) (absolute average fold error: 1.3-2.4). In contrast, the optimized PBPK model predicted Cmax and AUC of the drugs with high species-specific CLtot with lower absolute average fold error (Cmax and AUC: 2.8 and 3.2, respectively) than those of the empirical approach (Cmax and AUC: 2.6-4.9 and 3.9-10.7, respectively). Therefore, the optimized PBPK model is useful for human PK prediction of drugs with species-specific CLtot.

Potent estrogenic metabolites of bisphenol A and bisphenol B formed by rat liver S9 fraction: their structures and estrogenic potency.

We previously demonstrated that the estrogenicity of either bisphenol A [BPA; 2,2-bis(4-hydroxyphenyl)propane] or bisphenol B [BPB; 2,2-bis(4-hydroxyphenyl)butane] was increased several times after incubation with rat liver S9 fraction (Yoshihara et al., 2001). This metabolic activation, requiring both microsomal and cytosolic fractions, was observed with not only rat liver, but also human, monkey, and mouse liver S9 fractions. To characterize the active metabolites of BPA and BPB, we investigated the structures of the isolated active metabolites by negative mode LC/MS/MS and GC/MS. The active metabolite of BPA gave a negative mass peak at [M-H](-) 267 on LC/MS and a single daughter ion at m/z 133 on MS/MS analysis, suggesting an isopropenylphenol dimer structure. Finally, this active metabolite was confirmed to be identical with authentic 4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene (MBP) by means of various instrumental analyses. The corresponding peaks of the BPB metabolite were [M-H](-) 295 and m/z 147, respectively, suggesting an isobutenylphenol dimer structure. Further, coincubation of BPA and BPB with rat liver S9 afforded an additional active metabolite(s), which gave a negative mass peak at [M-H](-) 281 and two daughter ion peaks at m/z 133 and m/z 147 on MS/MS analysis. These results strongly suggest that the active metabolite of either BPA or BPB might be formed by recombination of a radical fragment, a one-electron oxidation product of carbon-phenyl bond cleavage. It is noteworthy that the estrogenic activity of MBP, the active metabolite of BPA, is much more potent than that of the parent BPA in several assays, including two reporter assays using a recombinant yeast expressing human estrogen receptor alpha and an MCF-7-transfected firefly luciferase plasmid.