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.

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 Atabecestat (JNJ-54861911): A Thiazine-Based ß-Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor Advanced to the Phase 2b/3 EARLY Clinical Trial.

Accumulation of amyloid ß peptides (Aß) is thought to be one of the causal factors of Alzheimer's disease (AD). The aspartyl protease ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate-limiting protease for Aß production, and therefore, BACE1 inhibition is a promising therapeutic approach for the treatment of AD. Starting with a dihydro-1,3-thiazine-based lead, Compound J, we discovered atabecestat 1 (JNJ-54861911) as a centrally efficacious BACE1 inhibitor that was advanced into the EARLY Phase 2b/3 clinical trial for the treatment of preclinical AD patients. Compound 1 demonstrated robust and dose-dependent Aß reduction and showed sufficient safety margins in preclinical models. The potential of reactive metabolite formation was evaluated in a covalent binding study to assess its irreversible binding to human hepatocytes. Unfortunately, the EARLY trial was discontinued due to significant elevation of liver enzymes, and subsequent analysis of the clinical outcomes showed dose-related cognitive worsening.

Human mass balance, metabolism, and cytochrome P450 phenotyping of lusutrombopag.

The human mass balance of lusutrombopag, an orally bioavailable thrombopoietin (TPO) receptor agonist, was characterised in seven healthy male subjects after a single oral dose of [14C]-lusutrombopag (2 mg, 100 µCi) in solution. Lusutrombopag was the main component in plasma, accounting for 56% of plasma radioactivity AUC0-∞. In plasma, the half-life of radioactivity (70.7 h) was longer than that of lusutrombopag (25.7 h), suggesting the presence of long circulating metabolites. The main excretion pathway of lusutorombopag was feces, with a radioactivity recovery of approximately 83% within 336 h post-dose. M6 (lusutrombopag-O-propanol or lusutrombopag-O-acetic acid) and M7 (lusutrombopag-O-ethane-1,2-diol) were also identified as main components in feces, accounting for at most 17.9%, and 16.9% of the dose, respectively, and were ß-oxidation related metabolites. Our in vitro metabolism study of lusutrombopag indicated that ß-oxidation was a subsequent metabolism of ω-oxidation and CYP4 enzymes, including CYP4A11, were the major isozymes contributing to ω-oxidation. In conclusion, lusutrombopag is primarily eliminated via ω-oxidation and excreted in the feces, where CYP4 enzymes play an important role.

The Novel In Vitro Method to Calculate Tissue-to-Plasma Partition Coefficient in Humans for Predicting Pharmacokinetic Profiles by Physiologically-Based Pharmacokinetic Model With High Predictability.

Proper prediction of human pharmacokinetic (PK) profiles can accelerate the compound selection in drug discovery. Recently, we reported a robust bottom-up physiologically-based pharmacokinetic (PBPK) approach (J Pharm Sci. 2019 Aug; 108(8):2718-2727), which uses the in vivo rat distribution volume at the steady state (Vss) to determine human tissue-to-plasma partition coefficients (Kptissue). Here, we report on a bottom-up PBPK approach that can simulate the PK profile with both high-throughput and high-predictive accuracy only using in vitro data. In this study, as an alternative parameter of in vivo rat Vss which was used for the correction of human Kptissue, Vss, in vitro was obtained from protein binding data in rats, and the values of Vss, in vitro for 31 reference compounds showed good correlation with the observed rat Vss (R2 = 0.859). Next, rat and human PK profiles of reference compounds were predicted by the bottom-up PBPK approach using Kptissue corrected by rat Vss, in vitro. As a result, the absolute average fold errors for pharmacokinetic parameters were almost less than 2, showing that these PK profiles could be accurately predicted using in vitro data. This method enables the screening of promising compounds with good PK profiles in humans at an early stage of drug discovery.

Successful Prediction of Human Pharmacokinetics After Oral Administration by Optimized Physiologically Based Pharmacokinetics Approach and Permeation Assay Using Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Cells.

None of the physiologically based pharmacokinetic (PBPK) approaches using preclinical data show high predictability of human pharmacokinetic (PK) profiles for drugs affected by the intestinal first-pass effect. Here we report a novel PBPK approach that incorporated the findings of a permeation study using human induced pluripotent stem cell-derived intestinal epithelial cells (hiPSC-IECs) to predict human PK profiles after oral administration of drugs. In hiPSC-IECs, gene expression levels of cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) are enhanced by rifampicin and 1,25-dihydroxyvitamin D3. The permeability of 24 drugs (10 test drugs and 14 reference drugs), including CYP3A4 and P-gp substrates, correlated highly with gastrointestinal availability (Fa × Fg), and could be converted to the apparent absorption rate constant (ka, app) based on the correlation between Fa × Fg and in vivo ka of 27 drugs. The ka, app was input into the PBPK model which contained the optimized calculation processes of metabolism and tissue distribution. The absolute average fold error of PK parameters such as maximum plasma concentration and bioavailability for test drugs was less than 2, suggesting that human PK profiles could be predicted with high accuracy. Our robust PBPK approach enables quick decision-making in drug discovery based on human PK profiles.

Metabolite profiling of guanfacine in plasma and urine of healthy Japanese subjects after oral administration of guanfacine extended-release tablets.

Guanfacine is used for the treatment of attention-deficit/hyperactivity disorder (ADHD). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), metabolite profiling of guanfacine was performed in plasma and urine collected from healthy Japanese adults following repeated oral administration of guanfacine extended-release formulation. Unchanged guanfacine was the most abundant component in both plasma and urine (from the MS signal intensity). In plasma, the M3 metabolite (a sulfate of hydroxy-guanfacine) was the prominent metabolite; the M2 metabolite (a glucuronide of a metabolite formed by monooxidation of guanfacine), 3-hydroxyguanfacine and several types of glucuronide at different positions on guanfacine were also detected. In urine, the M2 metabolite and 3-hydroxyguanfacine were the principal metabolites. From metabolite analysis, the proposed main metabolic pathway of guanfacine is monooxidation on the dichlorobenzyl moiety, followed by glucuronidation or sulfation. A minor pathway is glucuronidation at different positions on guanfacine. As the prominent metabolites in plasma were glucuronide and sulfate of hydroxyguanfacine, which have no associated toxicity concerns, further toxicity studies of the metabolites, for example in animals, were not deemed necessary.

Application of Intestinal Epithelial Cells Differentiated from Human Induced Pluripotent Stem Cells for Studies of Prodrug Hydrolysis and Drug Absorption in the Small Intestine.

Cell models to investigate intestinal absorption functions, such as those of transporters and metabolic enzymes, are essential for oral drug discovery and development. The purpose of this study was to generate intestinal epithelial cells from human induced pluripotent stem cells (hiPSC-IECs) and then clarify whether the functions of hydrolase and transporters in them reflect oral drug absorption in the small intestine. The hiPSC-IECs showed the transport activities of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and peptide transporter 1 (PEPT1), revealed by using their probe substrates ([3H]digoxin, sulfasalazine, and [14C]glycylsarcosine), and the metabolic activities of CYP3A4, CES2, and CES1, which were clarified using their probe substrates (midazolam, irinotecan, and temocapril). The intrinsic clearance by hydrolysis of six ester prodrugs into the active form in hiPSC-IECs was correlated with the plasma exposure (Cmax , AUC, and bioavailability) of the active form after oral administration of these prodrugs to rats. Also, the permeability coefficients of 14 drugs, containing two substrates of P-gp (doxorubicin and [3H]digoxin), one substrate of BCRP (sulfasalazine), and 11 nonsubstrates of transporters (ganciclovir, [14C]mannitol, famotidine, sulpiride, atenolol, furosemide, ranitidine, hydrochlorothiazide, acetaminophen, propranolol, and antipyrine) in hiPSC-IECs were correlated with their values of the fraction of intestinal absorption (Fa) in human clinical studies. These findings suggest that hiPSC-IECs would be a useful cell model to investigate the hydrolysis of ester prodrugs and to predict drug absorption in the small intestine.

Therapeutic efficacy of peramivir against H5N1 highly pathogenic avian influenza viruses harboring the neuraminidase H275Y mutation.

High morbidity and mortality associated with human cases of highly pathogenic avian influenza (HPAI) viruses, including H5N1 influenza virus, have been reported. The purpose of the present study was to evaluate the antiviral effects of peramivir against HPAI viruses. In neuraminidase (NA) inhibition and virus replication inhibition assays, peramivir showed strong inhibitory activity against H5N1, H7N1 and H7N7 HPAI viruses with sub-nanomolar activity in enzyme assays. In H5N1 viruses containing the NA H275Y mutation, the antiviral activity of peramivir against the variant was lower than that against the wild-type. Evaluation of the in vivo antiviral activity showed that a single intravenous treatment of peramivir (10 mg/kg) prevented lethality in mice infected with wild-type H5N1 virus and also following infection with H5N1 virus with the H275Y mutation after a 5 day administration of peramivir (30 mg/kg). Furthermore, mice injected with peramivir showed low viral titers and low levels of proinflammatory cytokines in the lungs. These results suggest that peramivir has therapeutic activity against HPAI viruses even if the virus harbors the NA H275Y mutation.

In vitro inhibition and enhancement of liver microsomal S-777469 metabolism by long-chain fatty acids and serum albumin: insight into in vitro and in vivo discrepancy of metabolite formation in humans.

1. It was previously demonstrated that 10% of S-777469, a cannabinoid receptor 2 selective agonist, is metabolized to its carboxylic acid metabolite (S-777469 5-carboxylic acid, 5-CA) in humans in vivo, while the formation of 5-CA is extremely low in human cryopreserved hepatocytes and liver microsomes (HLMs). In this study, factors causing the different metabolite formation rates of S-777469 in vitro and in vivo were investigated. 2. Formation of 5-CA and S-777469 5-hydroxymethyl (5-HM), a precursor metabolite of 5-CA, was catalyzed by CYP2C9. Arachidonic acid, α-linolenic acid, oleic acid and myristic acid, which have been reported to exist in liver microsomes, inhibited S-777469 oxidation by CYP2C9, but serum albumin enhanced this reactions. 3. The IC50 values of these fatty acids for 5-CA formation from 5-HM were lower than those of 5-HM formation from S-777469. Serum albumin extensively enhanced 5-CA formation from 5-HM in comparison to 5-HM formation from S-777469. 4. CYP2C9 was the enzyme responsible for S-777469 oxidation in human livers. The suppressive effects of several fatty acids and enhancing action of serum albumin in vitro are likely to be the causal factors for the apparently different rates of in vitro and in vivo metabolite formation of S-777469.

The metabolism and pharmacokinetics of [14C]-S-777469, a new cannabinoid receptor 2 selective agonist, in healthy human subjects.

1. The metabolism and pharmacokinetics of S-777469 were investigated after a single oral administration of [14C]-S-777469 to healthy human subjects. 2. Total radioactivity was rapidly and well absorbed in humans, with Cmax of 11,308 ng eq. of S-777469/ml at 4.0 h. The AUCinf ratio of unchanged S-777469 to total radioactivity was approximately 30%, indicating that S-777469 was extensively metabolized in humans. 3. The metabolite profiling in human plasma showed that S-777469 5-carboxymethyl (5-CA) and S-777469 5-hydroxymethyl (5-HM) were the main circulating metabolites, and the AUCinf ratio of 5-CA and 5-HM to total radioactivity were 24 and 9.1%, respectively. These data suggest that S-777469 was subsequently metabolized to 5-CA in humans although the production amount of 5-CA was extremely low in human hepatocytes. 4. Total radioactivity was mainly excreted via the feces, with 5-CA and 5-HM being the main excretory metabolites in feces and urine. Urinary excretion of 5-CA was comparable with that of 5-HM, whereas fecal excretion of 5-CA was lower than that of 5-HM. 5. In conclusion, the current mass balance study revealed the metabolic and pharmacokinetic properties of S-777469 in humans. These data should be useful to judge whether or not the safety testing of metabolite of S-777469 is necessary.

The relationship between in vivo antiviral activity and pharmacokinetic parameters of peramivir in influenza virus infection model in mice.

The purpose of this study was to investigate the relationship between pharmacokinetic (PK) parameters of intravenous (IV) peramivir and in vivo antiviral activity pharmacodynamic (PD) outcomes in a mouse model of influenza virus infection. Peramivir was administrated to mice in three dosing schedules; once, twice and four times after infection of A/WS/33 (H1N1). The survival rate at day 14 after virus infection was employed as the antiviral activity outcome for analysis. The relationship between day 14 survival and PK parameters, including area under the concentration-time curve (AUC), maximum concentration (Cmax) and time that drug concentration exceeds IC95 (T(>IC95)), was estimated using a logistic regression model, and model fitness was evaluated by calculation of the Akaike information criterion (AIC) index. The AIC indices of AUC, Cmax and T(>IC95) were about 114, 151 and 124, respectively. The AIC of AUC and T(>IC95) were smaller than that of Cmax. Therefore, both AUC and T(>IC95) were the PK parameters that correlated best with the antiviral activity of peramivir IV against influenza virus infection in mice.

Non-clinical evaluation of the metabolism, pharmacokinetics and excretion of S-777469, a new cannabinoid receptor 2 selective agonist.

1. The drug metabolism and pharmacokinetics of S-777469 were investigated in in vitro (rat, dog and human) and in in vivo (rats and dogs). 2. S-777469 was rapidly and well absorbed, with bioavailability values ranging from 50 to 70% in rats and dogs, almost all drug radioactivity was excreted into the feces via bile within 48 h. Thus, good pharmacokinetics of S-777469 (e.g. systemic exposure and excretion rate) would be anticipated in humans. 3. In vitro metabolism of S-777469 was qualitatively similar in rat, dog and human hepatocytes. S-777469 acyl glucuronide, S-777469 5-hydroxymethyl and S-777469 4-hydroxycyclohexane were the main metabolites in rats, dogs and humans. In vivo metabolism in rats and dogs showed good qualitative agreement with in vitro metabolism, and no metabolites exceeded 10% of total radioactivity in rat and dog plasma. 4. No unique metabolites were observed in human hepatocytes. Therefore, rats and dogs were thought to be appropriate species for non-clinical toxicity studies. 5. In conclusion, these data should be useful for the characterization of the pharmacokinetic properties of S-777469 and the estimation of its pharmacokinetic fate in humans.

Efficacy of repeated intravenous injection of peramivir against influenza A (H1N1) 2009 virus infection in immunosuppressed mice.

The efficacy of intravenous peramivir against influenza A (H1N1) 2009 virus infection was evaluated in mice in which the immune system was suppressed by cyclophosphamide (CP) treatment. The mortality rate of the vehicle control group was 100%, and the mice lost 20% of their body weight on average by day 13 postinfection (p.i.). Repeated administration of peramivir (40 mg/kg of body weight once a day, given intravenously for 20 days), starting at 1 h p.i., significantly reduced mortality, body weight loss, viral titers, and cytokine production in infected mice compared with results for administration of vehicle (P < 0.01). In addition, repeated administration of peramivir, starting at 24 h, 48 h, or 72 h p.i., also resulted in increases in survival rates and reduction of viral titers in the lungs (P < 0.01). The mean days to death (MDD) of the vehicle group was 14.5 days, while in the groups treated with peramivir starting at 24 h, 48 h, and 72 h p.i., the MDDs were >23.0, 20.9, and 21.8 days, respectively. In comparison, repeated administration of oseltamivir phosphate (5 mg/kg twice a day, given orally for 20 days), starting at 24 h, 48 h, and 72 h p.i., also significantly prevented body weight loss, whereas no significant differences in mortality rates and viral titers in the lungs were observed compared with results for the vehicle group. These data indicated that repeated administration of peramivir was effective in promoting the survival and reducing virus replication in immunosuppressed mice infected with influenza A (H1N1) 2009 virus.

Investigation of drug-drug interaction via mechanism-based inhibition of cytochrome P450 3A by macrolides in dexamethasone-treated female rats.

The in vitro and in vivo inhibition of cytochrome P450 (CYP) 3A with mechanism-based inhibition (MBI) by macrolides was investigated using dexamethasone-treated female rats (DEX-female rats). In the in vitro CYP inhibition studies using erythromycin (ERM) and clarithromycin (CAM), similar inhibition responses were observed between human and DEX-female rat liver microsomes, however, there were fewer effects in intact male rats. The ex vivo study showed that midazolam (MDZ) metabolism in liver microsomes of DEX-female rats was reduced by ERM administration and the inhibitory effect was increased with increasing ERM doses, indicating that metabolite intermediate complex formation caused irreversible inhibition of CYP3A activity in DEX-female rats as well as in humans. In the in vivo studies, ERM and CAM significantly increased the area under the plasma concentration-time curve of MDZ and decreased the total clearance in DEX-female rats. It was concluded that the DDIs via MBI of CYP3A following macrolide administration in humans could be reproduced in female rats, suggesting that DEX-female rats can serve as an in vivo model for assessing this DDI in humans.

Efficacy of single intravenous injection of peramivir against influenza B virus infection in ferrets and cynomolgus macaques.

We evaluated the efficacy of a single intravenous dose peramivir for treatment of influenza B virus infection in ferrets and cynomolgus macaques in the present study. A single dose of peramivir (60 mg/kg of body weight) given to ferrets on 1 day postinfection with influenza B virus significantly reduced median area under the curve (AUC) virus titers (peramivir, 8.3 log(10) 50% tissue culture infective doses [TCID(50)s] · day/ml; control, 10.7 log(10) TCID(50)s · day/ml; P < 0.0001). Furthermore, nasal virus titers on day 2 postinfection in ferrets receiving a single injection of peramivir (30 mg/kg) and AUCs of the body temperature increase in ferrets receiving a single injection of peramivir (30 and 60 mg/kg) were lower than those in ferrets administered oral oseltamivir phosphate (30 and 60 mg/kg/day twice daily for 3 days). In macaques infected with influenza B virus, viral titers in the nasal swab fluid on days 2 and 3 postinfection and body temperature after a single injection of peramivir (30 mg/kg) were lower than those after oral administration of oseltamivir phosphate (30 mg/kg/day for 5 days). The two animal models used in the present study demonstrated that inhibition of viral replication at the early time point after infection was critical in reduction of AUCs of virus titers and interleukin-6 production, resulting in amelioration of symptoms. Our results shown in animal models suggest that the early treatment with a single intravenous injection of peramivir is clinically recommended to reduce symptoms effectively in influenza B virus infection.

Glucuronidation converting methyl 1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921) to a potent apical sodium-dependent bile acid transporter inhibitor, resulting in a hypocholesterolemic action.

Methyl 1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921) is a novel inhibitor of the ileal apical sodium-dependent bile acid transporter (ASBT/SLC10A2) developed for the treatment of hypercholesterolemia. The present study investigated the hypocholesterolemic action of S-8921 glucuronide (S-8921G) in rats. The plasma concentration of S-8921G was higher than that of S-8921 after single oral administration of S-8921 in normal rats, and S-8921G was excreted into the bile (13% dose). Oral administration of either S-8921 or S-8921G reduced the serum total cholesterol, particularly nonhigh-density lipoprotein cholesterol, in hypercholesterolemic normal rats. In Gunn rats devoid of UDP glucuronosyltransferase-1A activity, S-8921G was undetectable both in the plasma and bile specimens, and only S-8921G administration significantly reduced the serum nonhigh-density lipoprotein cholesterol. An in vitro inhibition study showed that glucuronidation converts S-8921 to a 6000-fold more potent inhibitor of human ASBT (K(i) = 18 nM versus 109 microM). S-8921G was detected both in the portal plasma and loop when S-8921 was administered into the loop of the rat jejunum, although the cumulative amount of S-8921G recovered in the bile was 5-fold greater than that in the loop. The uptake of S-8921G by freshly prepared rat hepatocytes was saturable, and sodium-dependent and -independent systems were involved. Organic anions, such as bromosulfophthalein, estrone 3-sulfate, and taurocholic acid, inhibited the uptake. These results suggest that UDP glucuronosyltransferase-1 isoforms play a critical role in the hypocholesterolemic action of S-8921 by converting S-8921 to a more potent ASBT inhibitor, and organic anion transporter(s) are also involved in its pharmacological action through the biliary excretion of S-8921G.

Assessment of the hepatic and intestinal first-pass metabolism of midazolam in a CYP3A drug-drug interaction model rats.

In the current study, to understand the characteristics of dexamethasone (DEX)-treated female rats as an animal model for drug-drug interactions, a double-cannulation method was applied and separately assessed for the intestinal and hepatic first-pass metabolism of midazolam. Midazolam was administered intravenously or orally to the animals, and midazolam concentrations in the portal and systemic plasma were simultaneously determined. Next, the rates of elimination from the intestine and liver were estimated using the AUC values. After oral administration of midazolam, the entire drug was absorbed without intestinal first-pass metabolism, and 93% of the administered midazolam was extracted in the liver of the DEX-treated female rats. Seven per cent of the midazolam administered reached the systemic circulation. When ketoconazole was given orally to the animals, in conjunction with midazolam, the extraction ratio in the liver decreased from 93% to 77% in the control rats, and the bioavailability of midazolam increased to 23%. On the other hand, after intravenous administration, the elimination half-life of midazolam was not changed by ketoconazole pretreatment. These results indicated that midazolam is only extracted in the liver of DEX-treated female rats and that ketoconazole inhibits the hepatic first-pass metabolism, but not the systemic metabolism. In conclusion, DEX-treated female rats can be used as a drug-drug interaction model via CYP3A4 enzyme inhibition, especially for the hepatic first-pass metabolism of orally administered drugs.