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Viruses Mar 2021Small molecules targeting the PF74 binding site of the HIV-1 capsid protein (CA) confer potent and mechanistically unique antiviral activities. Structural modifications...
Small molecules targeting the PF74 binding site of the HIV-1 capsid protein (CA) confer potent and mechanistically unique antiviral activities. Structural modifications of PF74 could further the understanding of ligand binding modes, diversify ligand chemical classes, and allow identification of new variants with balanced antiviral activity and metabolic stability. In the current work, we designed and synthesized three series of PF74-like analogs featuring conformational constraints at the aniline terminus or the phenylalanine carboxamide moiety, and characterized them using a biophysical thermal shift assay (TSA), cell-based antiviral and cytotoxicity assays, and in vitro metabolic stability assays in human and mouse liver microsomes. These studies showed that the two series with the phenylalanine carboxamide moiety replaced by a pyridine or imidazole ring can provide viable hits. Subsequent SAR identified an improved analog which effectively inhibited HIV-1 (EC = 0.31 μM), strongly stabilized CA hexamer (ΔTm = 8.7 °C), and exhibited substantially enhanced metabolic stability (t = 27 min for 15 vs. 0.7 min for PF74). Metabolic profiles from the microsomal stability assay also indicate that blocking the C5 position of the indole ring could lead to increased resistance to oxidative metabolism.
Topics: Animals; Anti-HIV Agents; Binding Sites; Capsid Proteins; Cell Line; Drug Design; HEK293 Cells; HIV-1; Humans; Indoles; Liver; Mice; Microsomes; Models, Molecular; Molecular Conformation; Phenylalanine; Small Molecule Libraries; Virus Replication
PubMed: 33804121
DOI: 10.3390/v13030479 -
Pharmaceutical Biology Dec 2020Both nobiletin (NBL) and glycyrrhizin (GL) have anti-inflammatory and antitumor properties. These agents may be co-administered in the clinic. However, the drug-drug...
Both nobiletin (NBL) and glycyrrhizin (GL) have anti-inflammatory and antitumor properties. These agents may be co-administered in the clinic. However, the drug-drug interaction between them is not clear. The drug-drug interaction between GL and NBL was investigated, to clarify the effect of GL on the pharmacokinetics of NBL, and its main mechanism. The pharmacokinetic profiles of oral administration of NBL (50 mg/kg) in Sprague-Dawley rats of two groups with six each, with or without pre-treatment of GL (100 mg/kg/day for 7 days), were investigated. The effects of GL on the metabolic stability and transport of NBL were also investigated through the rat liver microsome and Caco-2 cell transwell models. The results showed that GL significantly decreased the peak plasma concentration (from 1.74 ± 0.15 to 1.12 ± 0.10 μg/mL) and the (7.44 ± 0.65 vs. 5.92 ± 0.68) of NBL, and the intrinsic clearance rate of NBL was increased by the pre-treatment with GL (39.49 ± 2.5 vs. 48.29 ± 3.4 μL/min/mg protein). The Caco-2 cell transwell experiments indicated that GL could increase the efflux ratio of NBL from 1.61 to 2.41. These results indicated that GL could change the pharmacokinetic profile of NBL, via increasing the metabolism and efflux of NBL in rats. It also suggested that the dose of NBL should be adjusted when co-administrated with GL in the clinic.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Administration, Oral; Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Biological Transport; Caco-2 Cells; Cytochrome P-450 CYP3A; Drug Interactions; Drugs, Chinese Herbal; Flavones; Glycyrrhizic Acid; Humans; Male; Metabolic Clearance Rate; Microsomes, Liver; Rats; Rats, Sprague-Dawley
PubMed: 32298152
DOI: 10.1080/13880209.2020.1751661 -
Molecules (Basel, Switzerland) Jun 2023Malaria continues to pose a significant health threat, causing thousands of deaths each year. The limited availability of vaccines and medications, combined with the...
Malaria continues to pose a significant health threat, causing thousands of deaths each year. The limited availability of vaccines and medications, combined with the emergence of drug resistance, further complicates the fight against this disease. In this study, we aimed to enhance the antimalarial potency of the previously reported hit compound BIPPO (pIC 5.9). Through systematic modification of pyrazolopyrimidinone analogs, we discovered the promising analog (NPD-3547), which exhibited approximately one log unit higher in vitro potency (pIC 6.8) against . Furthermore, we identified several other BIPPO analogs (, , and ) with potent antimalarial activity (pIC > 6.0) and favorable metabolic stability in mouse liver microsomes. These compounds can serve as new tools for further optimization towards the development of potential candidates for antimalarial studies.
Topics: Mice; Animals; Antimalarials; Malaria; Plasmodium falciparum; Microsomes, Liver; Drug Resistance; Folic Acid Antagonists
PubMed: 37446602
DOI: 10.3390/molecules28134939 -
Current Neuropharmacology Jul 2017Metabolite profiling of novel psychoactive substances (NPS) is critical for documenting drug consumption....
Metabolite profiling of novel psychoactive substances (NPS) is critical for documenting drug consumption. N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (ADB-FUBINACA) is an emerging synthetic cannabinoid whose toxicological and metabolic data are currently unavailable. We aimed to determine optimal markers for identifying ADB-FUBINACA intake. Metabolic stability was evaluated with human liver microsome incubations. Metabolites were identified after 1 and 3 h incubation with pooled human hepatocytes, liquid chromatography- high resolution mass spectrometry in positive-ion mode (5600+ TripleTOF®, Sciex) and several data mining approaches (MetabolitePilot™, Sciex). Metabolite separation was achieved on an Ultra Biphenyl column (Restek®); full-scan TOF-MS and information-dependent acquisition MS/MS data were acquired. ADB-FUBINACA microsomal half-life was 39.7 min, with a predicted hepatic clearance of 9.0 mL/min/kg and a 0.5 extraction ratio (intermediate-clearance drug). Twenty-three metabolites were identified. Major metabolic pathways were alkyl and indazole hydroxylation, terminal amide hydrolysis, subsequent glucuronide conjugations, and dehydrogenation. We recommend ADB-FUBINACA hydroxyalkyl, hydroxydehydroalkyl and hydroxylindazole metabolites as ADB-FUBINACA intake markers. N-dealkylated metabolites are not specific ADB-FUBINACA metabolites and should not be used as definitive markers of consumption. This is the first ADB-FUBINACA in vitro metabolism study; in vivo experiments enabling pharmacokinetic and pharmacodynamics studies or urine from authentic clinical/forensic cases are needed to confirm our results.
Topics: Biomarkers; Humans; In Vitro Techniques; Indazoles; Metabolome; Microsomes, Liver; Psychotropic Drugs
PubMed: 29403341
DOI: 10.2174/1570159X15666161108123419 -
European Journal of Pharmaceutical... Jan 2023Baicalin (BG) is a bioactive flavonoid extracted from the dried root of the medicinal plant, Scutellaria radix (SR) (dicotyledonous family, Labiatae), and has several...
Baicalin (BG) is a bioactive flavonoid extracted from the dried root of the medicinal plant, Scutellaria radix (SR) (dicotyledonous family, Labiatae), and has several biological activities. Polyethylene glycol 400 (PEG400) has been used as a suitable solvent for several traditional Chinese medicines (TCM) and is often used as an excipient for the compound preparation of SR. However, the drug-excipient interactions between BG and PEG400 are still unknown. Herein, we evaluated the effect of a single intravenous PEG400 administration on the BG levels of rats using pharmacokinetic and tissue distribution studies. A liver microsome and recombinant enzyme incubation system were used to further confirm the interaction mechanism between PEG400 and UDP-glucuronosyltransferases (UGTs) (UGT1A8 and UGT1A9). The pharmacokinetic study demonstrated that following the co-intravenous administration of PEG400 and BG, the total clearance (CL) of BG in the rat plasma decreased by 101.60% (p < 0.05), whereas the area under the plasma concentration-time curve (AUC) and AUC increased by 144.59% (p < 0.05) and 140.05% (p < 0.05), respectively. Additionally, the tissue distribution study showed that the concentration of BG and baicalein-6-O-β-D-glucuronide (B6G) in the tissues increased, whereas baicalein (B) in the tissues decreased, and the total amount of BG and its metabolites in tissues altered following the intravenous administration of PEG400. We further found that PEG400 induced the UGT1A8 and UGT1A9 enzyme activities by affecting the maximum enzymatic velocity (V) and Michaelis-Menten constant (K) values of UGT1A8 and UGT1A9. In conclusion, our results demonstrated that PEG400 interaction with UGTs altered the pharmacokinetic behaviors and tissue distribution characteristics of BG and its metabolites in rats.
Topics: Animals; Rats; Flavonoids; Microsomes, Liver; Polyethylene Glycols; Tissue Distribution; Injections, Intravenous; UDP-Glucuronosyltransferase 1A9
PubMed: 36379359
DOI: 10.1016/j.ejps.2022.106328 -
Archives of Toxicology May 2022The present study compares two approaches to evaluate the effects of inter-individual differences in the biotransformation of chlorpyrifos (CPF) on the sensitivity...
Inter-individual variation in chlorpyrifos toxicokinetics characterized by physiologically based kinetic (PBK) and Monte Carlo simulation comparing human liver microsome and Supersome cytochromes P450 (CYP)-specific kinetic data as model input.
The present study compares two approaches to evaluate the effects of inter-individual differences in the biotransformation of chlorpyrifos (CPF) on the sensitivity towards in vivo red blood cell (RBC) acetylcholinesterase (AChE) inhibition and to calculate a chemical-specific adjustment factor (CSAF) to account for inter-individual differences in kinetics (HK). These approaches included use of a Supersome cytochromes P450 (CYP)-based and a human liver microsome (HLM)-based physiologically based kinetic (PBK) model, both combined with Monte Carlo simulations. The results revealed that bioactivation of CPF exhibits biphasic kinetics caused by distinct differences in the Km of CYPs involved, which was elucidated by Supersome CYP rather than by HLM. Use of Supersome CYP-derived kinetic data was influenced by the accuracy of the intersystem extrapolation factors (ISEFs) required to scale CYP isoform activity of Supersome to HLMs. The predicted dose-response curves for average, 99th percentile and 1st percentile sensitive individuals were found to be similar in the two approaches when biphasic kinetics was included in the HLM-based approach, resulting in similar benchmark dose lower confidence limits for 10% inhibition (BMDL) and HK values. The variation in metabolism-related kinetic parameters resulted in HK values at the 99th percentile that were slightly higher than the default uncertainty factor of 3.16. While HK values up to 6.9 were obtained when including also the variability in other influential PBK model parameters. It is concluded that the Supersome CYP-based approach appeared most adequate for identifying inter-individual variation in biotransformation of CPF and its resulting RBC AChE inhibition.
Topics: Acetylcholinesterase; Chlorpyrifos; Cytochrome P-450 Enzyme System; Humans; Kinetics; Liver; Microsomes, Liver; Models, Biological; Monte Carlo Method; Toxicokinetics
PubMed: 35294598
DOI: 10.1007/s00204-022-03251-z -
FASEB Journal : Official Publication of... Jun 2018Alcohol acts through numerous pathways leading to alcoholic liver disease (ALD). Cytochrome P450 (CYP2E1), an ethanol-inducible enzyme, metabolizes ethanol-producing...
Alcohol acts through numerous pathways leading to alcoholic liver disease (ALD). Cytochrome P450 (CYP2E1), an ethanol-inducible enzyme, metabolizes ethanol-producing toxic reactive oxygen species (ROS) and is regulated at the posttranslational level. Small ubiquitin-like modifier (SUMO)ylation is a posttranslational modification that involves the addition of SUMOs, which modulate protein stability, activity, and localization. We demonstrated that ubiquitin-conjugation enzyme 9, the SUMO-conjugating enzyme, is induced in the livers of an intragastric ethanol mouse model. Our aim is to examine whether SUMOylation could regulate ethanol-induced CYP2E1 expression in ALD and to elucidate the molecular mechanism(s). CYP2E1 and UBC9 expression in vitro and in vivo was detected by real-time PCR and immunoblotting/immunostaining. SUMOylation was assayed by mass spectrometry and coimmunoprecipitation. Ubc9 expression was induced in ethanol-fed mouse livers, and silencing inhibited ethanol-mediated CYP2E1 microsomal retention and enzymatic activity. CYP2E1 SUMOylation was found to be induced by ethanol in vitro and in vivo. Ubc9 silencing prevents ethanol-induced lipid accumulation and ROS production. UBC9 was highly expressed in human ALD livers. Finally, we found that lysine 410 is a key SUMOylated residue contributing to CYP2E1 protein stability and activity preventing CYP2E1 SUMOylation. Ethanol-mediated up-regulation of CYP2E1 via SUMOylation enhancing its protein stability and activity and may have important implications in ALD.-Tomasi, M. L., Ramani, K., Ryoo, M., Cossu, C., Floris, A., Murray, B. J., Iglesias-Ara, A., Spissu, Y., Mavila, N. SUMOylation regulates cytochrome P450 2E1 expression and activity in alcoholic liver disease.
Topics: Animals; Cytochrome P-450 CYP2E1; Enzyme Stability; Ethanol; Gene Expression Regulation, Enzymologic; Liver Diseases, Alcoholic; Mice; Microsomes, Liver; Reactive Oxygen Species; Sumoylation; Ubiquitin-Conjugating Enzymes
PubMed: 29401608
DOI: 10.1096/fj.201701124R -
Drug Metabolism and Disposition: the... Jun 2023Cannabidiol (CBD) is available as a prescription oral drug that is indicated for the treatment of some types of epilepsy in children and adults. CBD is also available...
Cannabidiol (CBD) is available as a prescription oral drug that is indicated for the treatment of some types of epilepsy in children and adults. CBD is also available over-the-counter and is used to self-treat a variety of other ailments, including pain, anxiety, and insomnia. Accordingly, CBD may be consumed with other medications, resulting in possible CBD-drug interactions. Such interactions can be predicted in healthy and hepatically-impaired (HI) adults and in children through physiologically based pharmacokinetic (PBPK) modeling and simulation. These PBPK models must be populated with CBD-specific parameters, including the enzymes that metabolize CBD in adults. In vitro reaction phenotyping experiments showed that UDP-glucuronosyltransferases (UGTs, 80%), particularly UGT2B7 (64%), were the major contributors to CBD metabolism in adult human liver microsomes. Among the cytochrome P450s (CYPs) tested, CYP2C19 (5.7%) and CYP3A (6.5%) were the major CYPs responsible for CBD metabolism. Using these and other physicochemical parameters, a CBD PBPK model was developed and validated for healthy adults. This model was then extended to predict CBD systemic exposure in HI adults and children. Our PBPK model successfully predicted CBD systemic exposure in both populations within 0.5- to 2-fold of the observed values. In conclusion, we developed and validated a PBPK model to predict CBD systemic exposure in healthy and HI adults and children. This model can be used to predict CBD-drug or CBD-drug-disease interactions in these populations. SIGNIFICANCE STATEMENT: Our PBPK model successfully predicted CBD systemic exposure in healthy and hepatically-impaired adults, as well as children with epilepsy. This model could be used in the future to predict CBD-drug or CBD-drug-disease interactions in these special populations.
Topics: Humans; Adult; Child; Cannabidiol; Cytochrome P-450 Enzyme System; Drug Interactions; Microsomes, Liver; Models, Biological
PubMed: 36972999
DOI: 10.1124/dmd.122.001128 -
The AAPS Journal Mar 2016In 2014, FDU-PB-22 and FUB-PB-22, two novel synthetic cannabinoids, were detected in herbal blends in Japan, Russia, and Germany and were quickly added to their...
In 2014, FDU-PB-22 and FUB-PB-22, two novel synthetic cannabinoids, were detected in herbal blends in Japan, Russia, and Germany and were quickly added to their scheduled drugs list. Unfortunately, no human metabolism data are currently available, making it challenging to confirm their intake. The present study aims to identify appropriate analytical markers by investigating FDU-PB-22 and FUB-PB-22 metabolism in human hepatocytes and confirm the results in authentic urine specimens. For metabolic stability, 1 μM FDU-PB-22 and FUB-PB-22 was incubated with human liver microsomes for up to 1 h; for metabolite profiling, 10 μM was incubated with human hepatocytes for 3 h. Two authentic urine specimens from FDU-PB-22 and FUB-PB-22 positive cases were analyzed after β-glucuronidase hydrolysis. Metabolite identification in hepatocyte samples and urine specimens was accomplished by high-resolution mass spectrometry using information-dependent acquisition. Both FDU-PB-22 and FUB-PB-22 were rapidly metabolized in HLM with half-lives of 12.4 and 11.5 min, respectively. In human hepatocyte samples, we identified seven metabolites for both compounds, generated by ester hydrolysis and further hydroxylation and/or glucuronidation. After ester hydrolysis, FDU-PB-22 and FUB-PB-22 yielded the same metabolite M7, fluorobenzylindole-3-carboxylic acid (FBI-COOH). M7 and M6 (hydroxylated FBI-COOH) were the major metabolites. In authentic urine specimens after β-glucuronidase hydrolysis, M6 and M7 also were the predominant metabolites. Based on our study, we recommend M6 (hydroxylated FBI-COOH) and M7 (FBI-COOH) as suitable urinary markers for documenting FDU-PB-22 and/or FUB-PB-22 intake.
Topics: Cannabinoids; Hepatocytes; Humans; Microsomes, Liver; Plant Preparations
PubMed: 26810398
DOI: 10.1208/s12248-016-9867-4 -
Molecules (Basel, Switzerland) Nov 2022CYP-mediated fast metabolism may lead to poor bioavailability, fast drug clearance and significant drug interaction. Thus, metabolic stability screening in human liver...
CYP-mediated fast metabolism may lead to poor bioavailability, fast drug clearance and significant drug interaction. Thus, metabolic stability screening in human liver microsomes (HLM) followed by metabolic soft-spot identification (MSSID) is routinely conducted in drug discovery. Liver microsomal incubations of testing compounds with fixed single or multiple incubation time(s) and quantitative and qualitative analysis of metabolites using high-resolution mass spectrometry are routinely employed in MSSID assays. The major objective of this study was to develop and validate a simple, effective, and high-throughput assay for determining metabolic soft-spots of testing compounds in liver microsomes using a single variable incubation time and LC/UV/MS. Model compounds (verapamil, dextromethorphan, buspirone, mirtazapine, saquinavir, midazolam, amodiaquine) were incubated at 3 or 5 µM with HLM for a single variable incubation time between 1 and 60 min based on predetermined metabolic stability data. As a result, disappearances of the parents were around 20-40%, and only one or a few primary metabolites were generated as major metabolite(s) without notable formation of secondary metabolites. The unique metabolite profiles generated from the optimal incubation conditions enabled LC/UV to perform direct quantitative estimation for identifying major metabolites. Consequently, structural characterization by LC/MS focused on one or a few major primary metabolite(s) rather than many metabolites including secondary metabolites. Furthermore, generic data-dependent acquisition methods were utilized to enable Q-TOF and Qtrap to continuously record full MS and MS/MS spectral data of major metabolites for post-acquisition data-mining and interpretation. Results from analyzing metabolic soft-spots of the seven model compounds demonstrated that the novel MSSID assay can substantially simplify metabolic soft-spot identification and is well suited for high-throughput analysis in lead optimization.
Topics: Humans; Microsomes, Liver; Tandem Mass Spectrometry; Chromatography, Liquid; Metabolic Clearance Rate; High-Throughput Screening Assays
PubMed: 36432161
DOI: 10.3390/molecules27228058