Inhibition effects of eight anti-coronavirus drugs on glycosides metabolism and glycosidases in human gut microflora.

The effects of eight oral anti-coronavirus drugs (lopinavir, ritonavir, chloroquine, darunavir, ribavirin, arbidol, favipiravir, oseltamivir) on the metabolism of four specific glycosides (polydatin, geniposide, quercitrin, glycyrrhizin) and on the activities of three major glycosidases (ß-glucosidase, α-rhamnosidase, ß-glucuronidase) from gut microflora were explored in vitro and determined by LC-MS/MS. The metabolism of polydatin, geniposide, quercitrin and glycyrrhizin was significantly inhibited by one or several anti-coronavirus drugs of 100 µM around 1 h and 4 h (P<0.05), among which darunavir could strongly reduce the production of genipin (70.6% reduction), quercitin (80.6% reduction) and glycyrrhetinic acid (37.9% reduction), which may cause a high risk of herb-drug interactions (HDI). Additionally, chloroquine reduced the production of genipin and quercitin by more than 75% (P<0.05), whereas arbidol had no significant influence on the metabolism of polydatin, quercitrin and glycyrrhizin (P>0.05) so that its risk may be lower. The inhibition of darunavir on ß-glucosidase was relatively strong (IC50 = 193±23 µM), and the inhibition became weaker on ß-glucuronidase and α-rhamnosidase (IC50>500 µM). The consistency between gut microflora and glycosidase system indicated that the inhibition of darunavir on the activity of ß-glucosidase and ß-glucuronidase may be the main reason for affecting the metabolism of geniposide, glycyrrhizin and polydatin in gut microflora. However, for the inhibition of darunavir and chloroquine on the metabolism of quercetrin, there was no correlation between gut microflora and α-rhamnosidase system. Assessing the risk of HDI mediated by glycosidases in gut microflora may be conducive to the safety and efficacy of combining traditional herbal and Western medicine for the treatment of patients with Covid-19.

Studies on oral bioavailability and first-pass metabolism of withaferin A in rats using LC-MS/MS and Q-TRAP.

Withaferin A (WA) is one of the major bioactive steroidal lactones with extensive pharmacological activities present in the plant Withania somnifera. The absolute oral bioavailability of WA remains unknown and human-related in vitro data are not available. Therefore, in the present study, the absolute oral bioavailability of WA in male rats and the in vitro screening of absorption factors by Q-trap and LC-MS/MS analysis were conducted to explore possible clinical properties of WA. The developed and validated analytical methods were successfully applied to the pharmacokinetic studies and in vitro measurement of WA. The oral bioavailability was determined to be 32.4 ± 4.8% based on intravenous (5 mg/kg) and oral (10 mg/kg) administrations of WA in male rats. The in vitro results showed that WA could be easily transported across Caco-2 cells and WA did not show as a substrate for P-glycoprotein. Moreover, the stability of WA was similar between male rat and human in simulated gastric fluid (stable), in intestinal microflora solution (slow decrease) and in liver microsomes (rapid depletion, with a half-life of 5.6 min). As such, the first-pass metabolism of WA was further verified by rat intestine-liver in situ perfusion, revealing that WA rapidly decreased and 27.1% remained within 1 h, while the content of three major metabolites (M1, M4, M5) identified by Q-trap increased. This perfusion result is consistent with the oral bioavailability results in vivo. The first-pass metabolism of WA might be the main barrier in achieving good oral bioavailability in male rats and it is predicted to be similar in humans. This study may hold clinical significance.

Advancing the predictivity of skin sensitization by applying a novel HMOX1 reporter system.

Reporter cell lines are a particularly useful tool to screen for the skin sensitization potential of chemicals. Current cell models based on Keap1-Nrf2 mimic induction by conducting antioxidant response element-luciferase plasmids. However, plasmid-based reporters may ignore comprehensive aspects of induction, thus affecting the accuracy of hazard identification. Herein, we developed a novel HaCaT-based reporter system, EndoSens, whereby luciferase was specifically inserted into the cassette for heme oxygenase (decycling) 1 (HMOX1, the most consistent marker induced by skin sensitizers) by CRISPR/Cas9. Testing data from 20 coded substances showed an accuracy of 90%, sensitivity of 91.7%, and specificity of 87.5%, which exceeded the OECD requirement. Among the 35 chemicals examined, predictivity was better than reported for the validated KeratinoSens™. These results indicate that the EndoSens assay could advance the predictivity of skin sensitization, thus making it a promising tool for in vitro skin sensitization testing.

Different binding sites of bovine organic anion-transporting polypeptide1a2 are involved in the transport of different fluoroquinolones.

Because of their wide distribution and capability of transporting a large variety of compounds, organic anion-transporting polypeptides (OATPs) have been extensively recognized as crucial players in absorption, distribution, and excretion of various drugs. OATP1A2 was the first cloned human OATP and has been found to transport wide range of endogenous and exogenous compounds. Bovine Oatp1a2 (bOatp1a2) shares high homology with human OATP1A2 and is considered the functional ortholog of the latter. Previous study in our laboratory demonstrated that bOatp1a2 transport of estrone-3-sulfate (ES) exhibited biphasic saturation kinetics. In the present study, we investigated the transport function of bOatp1a2 for four different quinolone antibacterial agents (enrofloxacin, levofloxacin, norfloxacin, and ciprofloxacin) and found that all the tested fluoroquinolones can be transported by bOatp1a2. Further studies showed that different binding sites are responsible for the transport of different fluoroquinolones. Both ciprofloxacin and norfloxacin exhibited biphasic saturation kinetics. The Kms of the high- and low-affinity components for ciprofloxacin were 3.80 ± 0.85 µM and 182 ± 31 µM, respectively, while those for norfloxacin were 24.7 ± 0.1 µM and 393 ± 79 µM, respectively. Enrofloxacin and levofloxacin showed an inhibitory effect on the uptake of only the high concentration of ES and thus may be transported by the low-affinity site for ES. Interestingly, enrofloxacin and levofloxacin demonstrated an activation effect on ES uptake at the high-affinity binding site. These results suggested that multiple binding sites within the structure of bOatp1a2 may be responsible for the uptake of different quinolone antimicrobial agents.

Metabolism of fenofibrate in beagle dogs: new metabolites identified and metabolic pathways revealed.

Fenofibrate is a prototypical agonist of peroxisome proliferator-activated receptor alpha (PPARalpha) which is well known to be associated with species related carcinogenesis. Important species differences have been reported in its metabolism and elimination pattern. Its new metabolites have been revealed in Cynomolgus monkeys and Sprague-Dawley rats. However in beagle dogs, several polar metabolites of fenofibrate have not been identified yet. In this study, beagle dogs were orally dosed with fenofibrate mixed with feeds. Urine and plasma samples were collected and subject to LC-MS/MS by comparison with authentic compounds and confirmed using an API 4000 Q-TRAP system. In vitro cultured primary hepatocytes were used to reveal metabolic pathways and confirm the data in vivo. Seven new metabolites of fenofibrate in dogs were identified, and their metabolic pathways were revealed. Fenofibrate in beagle dogs was found to be more prone to be metabolized into other secondary metabolites than fenofibric acid, compared with that in rats.

Study of Pharmacokinetics for Ivermectin B1a from Beagle Dogs.

Ivermectin has been widely used for antiparasitic drug, and has recently shown a broad-spectrum antiviral activity, including anti-Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the pharmacokinetic property of ivermectin has not been fully investigated yet. During the plasma preparation, ~32-46% of ivermectin was found in the precipitation. An Liquid Chromatograph-Mass Spectrometer (LC-MS/MS) method for ivermectin in the whole blood samples from beagle dogs was developed and validated. The specificity, accuracy, precision (intra-day and inter-day), matrix effect, recovery and stability of analyte reported here are satisfied with the criteria of Food and Drug Administration (FDA)-Bioanalysis guideline. The oral administrations pharmacokinetics of ivermectin in beagle dogs under fasting and after high-fat meal were studied, and the following parameters were obtained: fasting Cmax, 104 ± 35 µg·L-1; area under the concentration-time curve (AUC0-∞), 2,555 ± 941 h·µg·L-1; and high-fat meal Cmax, 147 ± 35 µg·L-1; AUC0-∞, 4,198 ± 1,279 h·µg·L-1. When the P-gp inhibitor curcumin was also coadministrated orally, Cmax and AUC0-∞ were found to be 177 ± 57 and 4,213 ± 948 h·µg·L-1, respectively. With the comparison to fasting treatment, coadministration of P-gp inhibitor curcumin resulted in increase of the exposure of ivermectin by 1.6-fold, while the exposure after the high-fat diet versus fasting was increased approximately in 1.4-fold, indicating that alternative absorption might play an important role for increasing the exposure of ivermectin for future clinic applications.

Validation of visualized transgenic zebrafish as a high throughput model to assay bradycardia related cardio toxicity risk candidates.

Drug-induced QT prolongation usually leads to torsade de pointes (TdP), thus for drugs in the early phase of development this risk should be evaluated. In the present study, we demonstrated a visualized transgenic zebrafish as an in vivo high-throughput model to assay the risk of drug-induced QT prolongation. Zebrafish larvae 48 h post-fertilization expressing green fluorescent protein in myocardium were incubated with compounds reported to induce QT prolongation or block the human ether-a-go-go-related gene (hERG) K⁺ current. The compounds sotalol, indapaminde, erythromycin, ofoxacin, levofloxacin, sparfloxacin and roxithromycin were additionally administrated by microinjection into the larvae yolk sac. The ventricle heart rate was recorded using the automatic monitoring system after incubation or microinjection. As a result, 14 out of 16 compounds inducing dog QT prolongation caused bradycardia in zebrafish. A similar result was observed with 21 out of 26 compounds which block hERG current. Among the 30 compounds which induced human QT prolongation, 25 caused bradycardia in this model. Thus, the risk of compounds causing bradycardia in this transgenic zebrafish correlated with that causing QT prolongation and hERG K⁺ current blockage in established models. The tendency that high logP values lead to high risk of QT prolongation in this model was indicated, and non-sensitivity of this model to antibacterial agents was revealed. These data suggest application of this transgenic zebrafish as a high-throughput model to screen QT prolongation-related cardio toxicity of the drug candidates.

Drug-drug interactions between ketoconazole and berberine in rats: pharmacokinetic effects benefit pharmacodynamic synergism.

Ketoconazole (KTZ), a clinical antifungal agent, is a known inhibitor of CYP3A and permeability glycoprotein (P-gp). Berberine (BBR), a natural plant-derived product used for gastroenteritis, is a substrate of P-gp. Recently, a synergistic antifungal effect of KTZ combined with BBR has been revealed. In this study, we performed both in vivo and in vitro experiments to explore whether pharmacokinetic interactions between KTZ and BBR would benefit their pharmacodynamic synergism. After oral co-administration of 10 mg/kg KTZ with 60 mg/kg BBR, the average area under the curve (AUC) and the maximum concentration (C(max) ) for KTZ increased to 215% and 449% (p < 0.05), respectively, in male rats and 157% and 172% (p < 0.05), respectively, in female rats, compared with those administered KTZ alone. Area under the curve and C(max) for BBR increased to 173% and 142%, respectively, compared with those administered BBR alone. After intravenous co-administration of 0.5 mg/kg KTZ and 0.8 mg/kg BBR, the pharmacokinetic properties of KTZ remained the same, but AUC of BBR increased to 254% (p < 0.05) compared with those administered BBR alone. In rat liver microsomes, inhibitory concentration (IC)(50) of BBR inhibiting KTZ depletion was determined to be 103 µm. These resulting pharmacokinetic interactions may benefit their pharmacodynamic synergism to a certain extent.

Biphasic regulation of intracellular calcium by gemfibrozil contributes to inhibiting L6 myoblast differentiation: implications for clinical myotoxicity.

Gemfibrozil is the most myotoxic fibrate drug commonly used for dyslipidemia, but the mechanism is poorly understood. The current study revealed that gemfibrozil inhibits myoblast differentiation through the regulation of intracellular calcium ([Ca(2+)]i) as revealed in L6 myoblasts by use of laser scan confocal microscopy and flow cytometry using Fluo-4 AM as a probe. Gemfibrozil at 20-400 µM, could regulate [Ca(2+)]i in L6 cells in a biphasic manner, and sustained reduction was observed when the concentration reached 200 µM. Inhibition of L6 differentiation by gemfibrozil was concentration-dependent with maximal effect noted between 200 and 400 µM, as indicated by creatine kinase activities and the differentiation index, respectively. In differentiating L6 myoblasts, gemfibrozil at concentrations below 400 µM led to no significant signs of apoptosis or cytotoxicity, whereas differentiation, inhibited by 200 µM gemfibrozil, was only partially recovered. A good correlation was noted between gemfibrozil concentrations that regulate [Ca(2+)]i and inhibit L6 myoblasts differentiation, and both are within the range of total serum concentrations found in the clinic. These data suggest a potential pharmacodynamic effect of gemfibrozil on myogenesis as a warning sign, in addition to the complex pharmacokinetic interactions. It is also noteworthy that mobilization of [Ca(2+)]i by gemfibrozil may trigger complex biological responses besides myocyte differentiation. Information revealed in this study explores the mechanism of gemfibrozil-induced myotoxicity through the regulation of intracellular calcium.

The inhibition study of human UDP-glucuronosyltransferases with cytochrome P450 selective substrates and inhibitors.

Human uridine-5'-diphosphoglucuronosyltransferases (UGTs) are the major phase II metabolizing enzymes. In the present study, five human UGTs (UGT1A1, 1A4, 1A6, 2B7, and 2B10) were individually expressed and used to examine the inhibition IC(50) values of 20 selective substrates and inhibitors of major cytochromes P450 (CYPs). The inhibition kinetics of UGT1A1 was also analyzed. The results showed that some compounds like α-naphthoflavone, paclitaxel, midazolam, cyclosporine A, and ketoconazole displayed strong inhibitions on UGT activities with their IC(50) values in a range of 4.1-26 µM. Especially, the IC(50) values were 4.1 ± 0.8 µM for ketoconazole in inhibiting UGT1A1-mediated ß-estradiol-3-glucuronidation, and 4.9 ± 0.3 µM for paclitaxel towards UGT1A4-mediated midazolam-N-glucuronidation. Additionally, the IC(50) values of bupropion, tolbutamide, and testosterone in inhibiting UGT-mediated metabolisms were similar with the K(m) values of respective CYPs. Some kinetic behaviours of UGTs were following Michaelis-Menten kinetics, while some were not.

Influence of two-period cross-over design on the bioequivalence study of gefitinib tablets in beagle dogs.

Generally, two-period cross-over design is used in bioequivalence (BE) study. High intra-subject variability of gefitinib was reported in a clinical BE study, and significant changes in gefitinib exposures were observed among different periods in our previous BE study in dogs. Therefore, commercial gefitinib tablets from the same batch were used in the present study and assigned to two groups: the testing drug (GF1) group and the reference drug (GF2) group. A single-oral-dose, two-period cross-over study with a 7-day washout (approximately 24 half-lives) under fasting condition was conducted in 12 dogs to explore the factors. The results showed that the mean values of AUC(0-t), AUC(0-∞), Cmax and Tmax of these two GF1 and GF2 groups were similar. However, the GF1 and GF2 did not meet the acceptance criteria of bioequivalence with 90% confidence intervals, since the values obtained were 76.22%-117.43% for AUC(0-t) and 87.55%-131.59% for Cmax. ANOVA revealed a significant difference between the two periods (P < 0.05). Interestingly, the mean AUC(0-t) of gefitinib in the period 2 was 2.3-fold greater than that in the period 1, while Cmax in the period 2 was 1.7-fold higher than that in the period 1. However, the volume of distribution was significantly decreased, becoming 0.4-fold lower in the period 2. No statistically significant difference in the half-life and Tmax was observed between the two periods (P < 0.05). The pharmacokinetic alteration might come from the different physiological absorption and/or metabolism between periods. Since a 7-day washout interval was applied, DDI risk from P450s and/or P-gp would not play a significant role in the non-bioequivalence. As regard the variability, the intra-subject variation crossing the periods was much larger than the inter-subject variation within each period. The absorption and/or metabolism function of the gut bacteria might play an important role in the increasing exposure of gefitinib in the second period, especially with the comparison with the analysis from the high-fat-diet treatments in humans. Therefore, further studies might be needed to evaluate whether the assessment of bioequivalence could be facilitated by a much longer washout interval allowing the recovery of gut bacteria.

Roles of diclofenac and its metabolites in immune activation associated with acute hepatotoxicity in TgCYP3A4/hPXR-humanized mice.

Diclofenac (DCF) is a widely used nonsteroidal anti-inflammatory drug, but it comes with a high risk of drug-induced liver injury (DILI). Despite the quinone-imine adduct pathways, the immunotoxicity is recently considered as another factor for DILI. However, such immune responses are still elusive. In the present study, investigation of the immune response in the acute hepatotoxicity model of TgCYP3A4/hPXR-humanized mice was conducted by administration of DCF and DCF metabolites, respectively. In a single dose intraperitoneal injection of 80 mg/kg DCF, the pharmacokinetic results showed the major DCF metabolites, including 4'-hydroxy-diclofenac (4'-OH-DCF), 5-hydroxy-diclofenac (5-OH-DCF) and diclofenac glucuronide (DCF-G) were generated after DCF treatment. Not only DCF, but those DCF metabolites could also directly cause different degrees of acute liver injury as significantly increased the serum ALT levels in a short time period in the TgCYP3A4/hPXR-humanized mice. Furthermore, the three DCF metabolites could directly stimulate the significant elevation of serum immune-related factors in varying degrees. Transcriptome analysis revealed the differentially expressed genes in the liver of DCF-G treated mice were mostly involved with the "immune system process" and "cell death" and related to "IL-17 signaling pathway" and "TNF-α signaling pathway", but 5-OH-DCF had little effect on the expressions of those genes. These results indicate that the metabolite DCF-G plays an important role in the activation of the hepatic immune system, which might be involved in the pathogenesis of DCF-induced acute liver injury.

Fenofibrate metabolism in the cynomolgus monkey using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry-based metabolomics.

Fenofibrate, widely used for the treatment of dyslipidemia, activates the nuclear receptor, peroxisome proliferator-activated receptor alpha. However, liver toxicity, including liver cancer, occurs in rodents treated with fibrate drugs. Marked species differences occur in response to fibrate drugs, especially between rodents and humans, the latter of which are resistant to fibrate-induced cancer. Fenofibrate metabolism, which also shows species differences, has not been fully determined in humans and surrogate primates. In the present study, the metabolism of fenofibrate was investigated in cynomolgus monkeys by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS)-based metabolomics. Urine samples were collected before and after oral doses of fenofibrate. The samples were analyzed in both positive-ion and negative-ion modes by UPLC-QTOFMS, and after data deconvolution, the resulting data matrices were subjected to multivariate data analysis. Pattern recognition was performed on the retention time, mass/charge ratio, and other metabolite-related variables. Synthesized or purchased authentic compounds were used for metabolite identification and structure elucidation by liquid chromatographytandem mass spectrometry. Several metabolites were identified, including fenofibric acid, reduced fenofibric acid, fenofibric acid ester glucuronide, reduced fenofibric acid ester glucuronide, and compound X. Another two metabolites (compound B and compound AR), not previously reported in other species, were characterized in cynomolgus monkeys. More importantly, previously unknown metabolites, fenofibric acid taurine conjugate and reduced fenofibric acid taurine conjugate were identified, revealing a previously unrecognized conjugation pathway for fenofibrate.

Myotoxicity of gemfibrozil in cynomolgus monkey model and its relationship to pharmacokinetic properties.

Fibrate drugs are PPARalpha agonists prescribed for the treatment of dyslipidemia. Severe myotoxicity has been reportedly associated with their use albeit at a low frequency, especially for gemfibrozil. Few studies have investigated the mechanism of fibrate-induced myotoxicity in vivo. Considering the apparent species-related differences in PPARalpha agonist-induced hepatotoxicity, we studied the myotoxicity of gemfibrozil in a Cynomolgus monkey model and explored the relationship between myotoxicity and pharmacokinetics. Six Cynomolgus monkeys were dosed with gemfibrozil twice daily at 600 mg/kg/day for the first two periods (P1 and P2, 8 days and 9 days respectively) and 300 mg/kg/day for the third period (P3, 14 days). Creatine kinase and myoglobin were measured, together with hepatotoxicity and nephrotoxicity markers. Behavioral responses were recorded for indication of toxicity. Pharmacokinetics was carried out following the 16th dosage of P1 and 17th dosage of P2 when myotoxicity was identified. Multivariable data analysis was employed to explore the relationship between pharmacokinetic parameters and myotoxicity markers. Consequently, myotoxicity occurred in monkey #2 (M2) and M6 in P1, M3 and M4 in P2, M3 and M6 in P3. Data analysis showed T80-150 (sustained time above the given concentration) contributed for myotoxicity discriminance and correlated with myotoxicity risk. This study revealed Cynomolgus monkey may be a good animal model for myotoxicity evaluation with sensitivity, reproducibility and similarities to humans. More interestingly, they exhibited a much higher incidence of myotoxicity than that of humans. Sustained high drug concentration plays an important role for the occurrence of myotoxicity. This may suggest an influence of drug transport and metabolism on myotoxicity.

An inhibition study of beauvericin on human and rat cytochrome P450 enzymes and its pharmacokinetics in rats.

Beauvericin is a secondary metabolite natural product from microorganisms and has been shown to have a new potential antifungal activity. In this study, the metabolism and inhibition of beauvericin in human liver microsomes (HLM) and rat liver microsomes (RLM) were investigated. The apparent K(m) and V(max) of beauvericin in HLM were determined by substrate depletion approach and its inhibitory effects on cytochromes P450 (CYP) activities were evaluated using probe substrates, with IC(50) and the (K(i)) values were 1.2 microM (0.5 microM) and 1.3 microM (1.9 microM), respectively for CYP3A4/5 (midazolam) and CYP2C19 (mephenytoin). Similarly, beauvericin was also a potent inhibitor for CYP3A1/2 (IC(50): 1.3 microM) in RLM. Furthermore, the pharmacokinetics of beauvericin in the rat were studied after p.o administration alone and co-administration with ketoconazole, which indicated a pharmacodynamic function may play a role in the synergistic effect on antifungal activity.

Comparison of in vitro/in vivo blood distribution and pharmacokinetics of artemisinin, artemether and dihydroartemisinin in rats.

Artemisinin and its derivatives have been widely used for treatment of malaria and the therapeutic targets are considered within the red blood cells. In the recent studies on the erythrocytes' uptake of artemisinin-derivatives in vitro, applying the radioisotope-labeled technology, it was trying to predict the in vivo disposition properties, but different distribution results were revealed from a preliminary study in one human. The pharmacokinetic differences among blood cells and plasma still remain unclear. To explore the therapeutic related pharmacokinetics and compare the in vitro-in vivo blood distribution in rats, an improving blood sample preparation and LC-MS/MS detection method was developed and successfully validated. The lower limit of quantification was smaller than the previous studies. In the in vitro blood distribution studies, the content ratios from blood cells to plasma were compared in the concentrations from 20 ng/mL to 1000 ng/mL. Such ratios were determined to be 1.1-1.6 for artemisinin, 0.9-1.2 for artemether, and around 0.7 for dihydroartemisinin. In the oral administration pharmacokinetic studies in rats, the concentration ratios from blood cells to plasma were from high (2.6-3.6) to medium (1.3-2.5), and low (0.5-1.5) for artemisinin, artemether, and dihydroartemisinin respectively in all measuring time points, displaying the similar affinity order toward blood cells in artemisinin > artemether > dihydroartemisinin as the in vitro measurements. The dosages of 10 mg/kg for intravenous administrations of artemisinin and 200 mg/kg for oral administrations of artemisinin or artemether were used for the pharmacokinetic study in rats. The geometric mean exposures (AUC(0-t)) of artemisinin, artemether and dihydroartemisinin in blood cells were determined to be 2.6 folds, 1.7 folds, or 1.2 folds greater than those in plasma, respectively. Referring to the in vitro distribution, the AUC(0-t) ratios from the blood cells measurements to the plasma measurements of these three antimalarial drugs were also in a similar trend as the in vitro distribution measurements. Furthermore, the half-life (t1/2) of artemether in blood cells was even longer than that in plasma, while the clearance of artemisinin, artemether, or dihydroartemisinin in blood cells was slower than that in plasma. Particularly, it was found that the concentrations of artemisinin and artemether were presented in blood cells over longer time period than in plasma above their antimalarial IC50, which might result from both the affinity toward blood cells and the drugs clearance differences between blood cells and plasma. These results were indicated that the exposures and pharmacokinetic properties in the whole blood or the blood cells should be taken into account for the drug candidates with higher distribution affinity toward blood cells especially for the antimalarial drugs.

Studies of pharmacokinetics in beagle dogs and drug-drug interaction potential of a novel selective ZAK inhibitor 3h for hypertrophic cardiomyopathy treatment.

Overexpression of leucine-zipper and sterile-α motif kinase (ZAK) in heart has been closely associated with the development of hypertrophic cardiomyopathy (HCM). N-(3-(1H-pyrazolo[3,4-b]pyridin-5-yl)ethynyl) benzene-sulfonamides, novel highly selective ZAK inhibitors, had exhibited reasonable orally therapeutic effects on HCM in spontaneous hypertensive rat models. In the present study, a rapid and sensitive HPLC-MS/MS method for determining ZAK inhibitor 3h in beagle dog plasma was developed and validated. Meanwhile, the pharmacokinetics in beagle dog and drug-drug interaction potential of 3h had been conducted. The pharmacokinetic results showed that the absolute oral bioavailability for 3h in beagle dogs was determined to be 61.9%, which was significantly higher than that in the previous determination in Spragur-Dawley rats (F = 20%). The Cytochrome P450 enzymes and P-glycoprotein mediated drug-drug interactions by 3h were also investigated using dog and human liver microsomes and Caco-2 cells. The results demonstrated that only CYP2C9 was obviously inhibited (IC50 = 1.66 µM). Besides, 3h could significantly decrease digoxin efflux ratio in Caco-2 experiments in a dose-dependent manner (IC50 = 13.3 µM). Considering 3h strongly suppressed the ZAK kinase activity with an IC50 of 3.3 nM, there are significantly differences between this IC50 value for ZAK inhibition and the present determinations of IC50 values. In general, the clinical drug-drug interaction potential for 3h could be well monitored during the treatment of HCM.

Improved defined approaches for predicting skin sensitization hazard and potency in humans.

Since the EU banned animal testing for cosmetic products and ingredients in 2013, many defined approaches (DA) for skin sensitization assessment have been developed. Machine learning models were shown to be effective in DAs, but the predictivity might be affected by data imbalance (i.e. more numbers of sensitizers than non-sensitizers) and limited information in the databases. To improve the predictivity of DAs, here we attempted to apply data-rebalancing ensemble learning (bagging with support vector machine (SVM)) and a novel and comprehensive Cosmetics Europe database. For predicting human hazard and three-class potency, 12 models were built for each using a training set of 96 substances and a test set of 32 substances from the database. The model with the highest accuracy for predicting hazard (90.63% for the test set and 88.54% for the training set, named hazard-DA) used the SVM-bagging with combinations of all variables (V6), while the model with the highest accuracy for predicting potency (68.75% for the test set and 82.29% for the training set, named potency-DA) used SVM alone. Both DAs showed higher performance than LLNA and other machine-learning-based DAs, and the potency-DA could provide more in-depth assessment. Those findings indicated that SVM-bagging-based DAs provided enhanced predictivity for hazard assessment by further data rebalancing. Meanwhile, the effect of imbalanced data might be offset by more detailed categorization of sensitizers for potency assessment, thus SVM-based DA without bagging could provide sufficient predictivity. The improved DAs in this study could be promising tools for skin sensitization assessment without animal testing.

Development and full validation of six inhibition assays for five major cytochrome P450 enzymes in human liver microsomes using an automated 96-well microplate incubation format and LC-MS/MS analysis.

Substrate inhibition assays for five of the major CYP enzymes (phenacetin for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam and testosterone for CYP3A4) in human liver microsomes were developed. Fully automated incubations were conducted in a 96-well format under optimized enzyme kinetic conditions. Metabolites of probe substrates were analyzed with rapid LC-MS/MS methods. The assays were fully validated following the procedure for validating bioanalytical methods recommended by regulatory agencies. Quality control samples and a positive control CYP inhibitor were included in each assay. The IC(50) values determined for typical CYP inhibitors were reproducible and consistent with those reported in the literature. The high quality and throughput of these assays make them ideally suited for providing information for decision making in late drug discovery and early development and for providing labeling input for new drug registrations.

Determination of a novel dipeptidyl peptidase IV inhibitor in monkey plasma by HPLC-MS/MS and its application in a pharmacokinetics study.

A lot of attention has been given to novel diabetes treatment, which is used to replace injectable insulin. A novel dipeptidyl peptidase IV inhibitor (HWH-10d) for treating type 2 diabetes was previously determined to have comparable efficacy to the marketed drug (alogliptin) in ICR male mice. Therefore, a sensitive and rapid liquid chromatography-tandem mass spectrometric method was developed and validated for the further evaluation of HWH-10d in monkey. The analytes were extracted through a liquid-liquid extraction with ethyl acetate. The linear detection range for HWH-10d in monkey plasma was from 0.5 to 2000 ng/mL with the lower limit of quantification of 0.5 ng/mL. The relative standard deviation was measured to be less than 10.4% for determination of inter- and intra-day precisions, and the relative error was determined to be within ±7.2% for all accuracy measurements. The simple and rapid LC-MS/MS method for HWH-10d in monkey plasma could be used for the pharmacokinetics studies of HWH-10d in monkeys. The oral bioavailability of HWH-10d in monkeys is 57.8%.