Evaluation of species differences in the metabolism of the selective NaV1.7 inhibitor DS-1971a, a mixed substrate of cytochrome P450 and aldehyde oxidase.

Nonclinical metabolite profiling of DS-1971a, a potent selective NaV1.7 inhibitor, was performed to predict human metabolites.After the oral administration of radiolabelled DS-1971a, the predominant metabolite in mouse plasma was M4, a monoxide at the pyrimidine ring, while the major metabolites with the first and second highest exposure in monkey plasma were M2, a monoxide at the cyclohexane ring, and M11, a demethylated pyrazole metabolite.Incubation studies with liver cytosolic and microsomal fractions in the absence or presence of NADPH indicated that the metabolising enzyme responsible for M4 formation was aldehyde oxidase (AO), while cytochrome P450s (P450s) were responsible for M2 and M11 formation. These results suggest that DS-1971a is a substrate for both AO and P450.When DS-1971a was incubated with liver S9 fractions and NADPH, the most abundant metabolites were M4 in mice, and M2 and M11 in monkeys, indicating that the results of in vitro incubation studies could provide information reflecting the in vivo plasma metabolite profiles in mice and monkeys. The results obtained from the incubation with the human liver S9 fraction and NADPH suggested that a major circulating metabolite in humans is M1, a regioisomer of M2.

Identification of cytochrome P450s involved in the metabolism of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1) using human recombinant enzymes and rat liver microsomes in vitro.

1. The aim of this study was to identify the hepatic metabolic enzymes, which involved in the biotransformation of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1), a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) in rat and human in vitro. 2. The parent drug of W-1 was incubated with rat liver microsomes (RLMs) or recombinant CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5, respectively) in the presence or absence of nicotinamide adeninedinucleotide phosphate (NADPH)-regenerating system. The metabolites of W-1 were analyzed with liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS). 3. The parent drug of W-1 was metabolized in a NADPH-dependent manner in RLMs. The kinetic parameters of prototype W-1 including Km, Vmax, and CLint were 2.3 µM, 3.3 nmol/min/mg protein, and 1.4 mL/min/mg protein, respectively. Two metabolites M1 and M2 were observed in shorter retention times (2.988 and 3.188 min) with a higher molecular ion at m/z 463.0160 (both M1 and M2) than that of the W-1 parent drug (6.158 min with m/z 447.0218). The CYP selective inhibition and recombinant enzymes also showed that two hydroxyl metabolites M1 and M2 are mainly mediated by CYP2C19 and CYP3A4. 4. The identification of CYPs involved in W-1 biotransformation is important to understand and minimize, if possible, the potential of drug-drug interactions.

The relationship between CYP2C9 gene polymorphisms and azilsartan metabolism in vitro.

BACKGROUND: Thegene polymorphisms of the CYP2C9, as well as the substrate specificity of theenzyme, result in different clearances for different substrates by CYP2C9variants. RESEARCH DESIGNAND METHODS: The CYP2C9 wild type and 38 CYP2C9 variants, expressed in insectmicrosomes, were incubated with azilsartan. The resulting metabolite,O-desethyl azilsartan, was determined by HPLC-MS/MS. The enzyme kineticparameters of the 38 variants were calculated and compared with the wild type.Subsequently, we selected CYP2C9 × 1, *2, and * 3 as target proteins for moleculardocking with azilsartan to elucidate the mechanisms underlying changes inenzyme function. RESULTS: Comparedwith CYP2C9 × 1, three variants (CYP2C9 × 29, *39, and * 49) exhibited markedlyincreased CLint values (from 170%-275%, *p < 0.05), whereas 28variants exhibited significantly decreased CLint values (from3%-63%,*p < 0.05). The molecular docking results showed that the binding energy ofCYP2C9 × 2 and * 3 was lower than that of the wild type. CONCLUSION: Thisassessment revealed the effect of CYP2C9 gene polymorphisms on azilsartanmetabolism, establishing a theoretical basis for further in-vivo studies andclinical applications. This study will help expand the database of CYP2C9gene-drug pairs and identify appropriate treatment strategies for azilsartan,contributing to the field of precision medicine.

Stereoselective in vitro metabolism of cyproconazole in rat liver microsomes and identification of major metabolites.

The vast usage of agrochemicals enhances food security globally but may pose challenge to understand the risk assessment to non-target organisms and human beings, and liver microsomes are responsible for metabolism of these agrochemicals in vivo. In this study, stereoselective metabolism of chiral triazole fungicide cyproconazole in rat liver microsomes has been investigated through chiral LC-MS/MS technique. The half-lives of four cyproconazole stereoisomers were different ranging from 95 to 187 min, and (2S, 3R)-cyproconazole preferentially metabolized in rat liver microsomes. In addition, the results from metabolism kinetic study indicated that rat liver microsomes showed the stronger potency to deplete (2S, 3R)-cyproconazole than the others. Then, homology modeling and molecular docking results revealed that the docking energy between (2S, 3R)-cyproconazole and the cytochrome P450 CYP3A1 (-7.46 kcal⋅mol-1) was higher than the others, meaning that (2S, 3R)-cyproconazole exhibited the strongest binding ability to this enzyme. Moreover, two main metabolites of cyproconazole coming from hydroxylation and dehydration were observed, and possible metabolic reactions of cyproconazole in rat liver microsomes were identified through using an LCQ ion trap mass spectrometer. This kind of systematic metabolic investigation of cyproconazole at chiral level would provide valuable information for ecological and human health risk assessment of chiral pesticides.

Design, synthesis and biological evaluation of novel O-carbamoyl ferulamide derivatives as multi-target-directed ligands for the treatment of Alzheimer's disease.

A novel series of O-carbamoyl ferulamide derivatives were designed by multitarget-directed ligands (MTDLs) strategy, the derivatives were synthesized and evaluated to treat Alzheimer's disease (AD). In vitro biological evaluation demonstrated that compound 4f was the best pseudo-irreversible hBChE (human butyrylcholinesterase) inhibitor with an IC50 value of 0.97 µM 4f was a potent selective MAO-B (monoamine oxidase-B) inhibitor (IC50 = 5.3 µM), and could inhibit (58.2%) and disaggregate (43.3%) self-mediated Aß aggregation. 4f also could reduce the levels of pathological tau and APP clearance, and displayed a wide safe range hepatotoxicity on LO2 cells. The in vivo studies revealed that 4f exhibited fascinating dyskinesia recovery rate and response efficiency on AlCl3-mediated zebrafish, and demonstrated significant protective effect on vascular injury caused by Aß1-40. PET-CT imaging demonstrated that [11C]4f exhibited high BBB penetration (especially could reach to hippocampus and striatum of brain) and had a fast brain uptake after intravenous bolus injection. Furthermore, compound 4f could improve scopolamine-induced cognitive impairment. Further, the metabolism in vitro of 4f was also investigated, and presented 3 metabolites in rat liver microsome metabolism, 4 metabolites in human liver microsome, and 4 metabolites in rat intestinal flora, providing previous data for the preclinical study. Therefore, these results implied that compound 4f was an advanced multi-function agent and deserved further preclinical study against mild-to-serve Alzheimer's disease.

Apigenin-rivastigmine hybrids as multi-target-directed liagnds for the treatment of Alzheimer's disease.

Here we reported novel apigenin-rivastigmine hybrids were rationally designed and synthesized by the multi-target-directed ligands (MTDLs) strategy, their activity in vitro results revealed that compound 3d showed significant antioxidant potency (ORAC = 1.3 eq), and it was a reversible huAChE (IC50 = 6.8 µM) and huBChE (IC50 = 16.1 µM) inhibitor. 3d also served as a selective metal chelator, and it significantly inhibited and disaggregated self-mediated and Cu2+-mediated Aß1-42 aggregation, and also inhibited hAChE-mediated induced Aß1-40 aggregation. Compound 3d exhibited remarkable neuroprotective effect and hepatoprotective activity. In addition, compound 3d presented favourable blood-brain barrier penetration in vitro and drug-like property. Further, the in vivo assay displayed that 3d indicated remarkable dyskinesia recovery rate and response efficiency on AD zebrafish, and exhibited surprising protective effect on Aß1-40-mediated zebrafish vascular injury. More importantly, 3d did not indicate obvious acute toxicity at dose up to 2000 mg/kg, and could improve scopolamine-induced memory impairment. Subsequently, the regulation of multi-targets for 3d were further confirmed through transcriptome sequencing of brain hippocampi, which also offered novel potential targets and opened a new way to treat Alzheimer's disease. More interestingly, the metabolism of 3din vitro indicated that 4 metabolites in rat liver microsome metabolism, 2 metabolites in human liver microsome metabolism, and 4 metabolites in intestinal flora metabolism, which offered supports for the preclinical study of 3d. Overall, this study exhibited that compound 3d was a promising advanced compound targeted multiple factors associated with AD.

Protective mechanism of rhubarb anthraquinone glycosides in rats with cerebral ischaemia-reperfusion injury: interactions between medicine and intestinal flora.

BACKGROUND: Anthraquinone glycosides extracted from rhubarb have been proven to have significant therapeutic effects on ischaemic stroke. It is well known that anthraquinone glycosides are not easily absorb. Thus, how can rhubarb anthraquinone glycosides (RAGs) exert protective effects on the brain? Is this protective effect related to interactions between RAGs and intestinal flora? METHODS: The model used in this study was established by middle cerebral artery occlusion (MCAO) and reperfusion. Twenty-seven adult male Sprague-Dawley (SD) rats were randomly divided into 3 groups: the normal group (A) (non-MCAO + 0.5% sodium carboxymethyl cellulose (CMC-Na)), model group (B) (MCAO + 0.5% CMC-Na) and medicine group (C) (MCAO + RAGs (15 mg/(kg day)). The rats were fed by gavage once a day for 7 days. Fresh faeces were collected from the normal group to prepare the intestinal flora incubation liquid. Add RAGs, detect the RAGs and the corresponding anthraquinone aglycones by HPLC-UV at different time points. On the 8th day, the rats were euthanized, and the colonic contents were collected and analysed by high-throughput sequencing. In addition, 12 adult male SD rats were randomly divided into 2 groups: the normal group (D) (non-MCAO + RAGs (15 mg/(kg day)) and model group (E) (MCAO + RAGs (15 mg/(kg day)). The rats were fed by gavage immediately after reperfusion. Blood was collected from the orbital venous plexus, and the RAGs and anthraquinone aglycones were detected by HPLC-UV. RESULTS: The abundance and diversity of the intestinal flora in rats decreased after cerebral ischaemia-reperfusion injury (CIRI). RAGs could effectively improve the abundance of the intestinal flora. In addition, in vitro metabolism studies showed that RAGs were converted into anthraquinone aglycones by intestinal flora. In the in vivo metabolism studies, RAGs could not be detected in the plasma; in contrast, the corresponding anthraquinone aglycones could be detected. Absorption of RAGs may be inhibited in rats with CIRI. CONCLUSIONS: CIRI may lead to intestinal flora disorder in rats, and after the administration of RAGs, the abundance of intestinal flora can be improved. RAGs can be metabolized into their corresponding anthraquinone aglycones by intestinal flora so that they can be absorbed into the blood.

On-line incubation and real-time detection by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry for rapidly analyzing metabolites of anthraquinones in rat liver microsomes.

The traditional studies on metabolism in liver microsomes were carried out in off-line form. In this paper, a rapid and convenient method for the study of metabolism of substrates in liver microsomes was established by means of ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS): on-line incubation and real-time detection of substrates in liver microsomes. The liver microsomal incubation system was placed in a sample chamber at 37 °C. On-line solid phase extraction (SPE) column was used for on-line sample treatment, its function was to enrich the drug prototype and its metabolites with weak polarity, and elute the phosphate in the samples. The incubation samples were analyzed by setting appropriate injection time, liquid phase elution procedure and mass spectrometry acquisition time. The phase I metabolites of anthraquinone compounds, aloe-emodin (A), rhein (R), emodin (E), chrysophanol (CP), physcion (PS) and their glucosides, were analyzed through this method. The results showed that 8 anthraquinone compounds underwent metabolic reactions such as hydrolysis, oxidation, reduction and hydroxylation in liver microsomal incubation system. In addition, a certain degree of mutual transformation of anthraquinones in liver microsomal incubation system was found. The results provide a reference for in vivo metabolism of anthraquinones in rhubarb. On-line incubation and real-time detection is a feasible, convenient and rapid method for the analysis of drug metabolism in vitro.