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Molecules (Basel, Switzerland) Mar 2022ARV-110, a novel proteolysis-targeting chimera (PROTAC), has been reported to show satisfactory safety and tolerability for prostate cancer therapy in phase I clinical...
ARV-110, a novel proteolysis-targeting chimera (PROTAC), has been reported to show satisfactory safety and tolerability for prostate cancer therapy in phase I clinical trials. However, there is a lack of bioanalytical assays for ARV-110 determination in biological samples. In this study, we developed and validated an LC-MS/MS method for the quantitation of ARV-110 in rat and mouse plasma and applied it to pharmacokinetic studies. ARV-110 and pomalidomide (internal standard) were extracted from the plasma samples using the protein precipitation method. Sample separation was performed using a C18 column and a mobile phase of 0.1% formic acid in distilled water-0.1% formic acid in acetonitrile (30:70, /). Multiple reaction monitoring was used to quantify ARV-110 and pomalidomide with ion transitions at m/z 813.4 → 452.2 and 273.8 → 201.0, respectively. The developed method showed good linearity in the concentration range of 2-3000 ng/mL with acceptable accuracy, precision, matrix effect, process efficiency, and recovery. ARV-110 was stable in rat and mouse plasma under long-term storage, three freeze-thaw cycles, and in an autosampler, but unstable at room temperature and 37 °C. Furthermore, the elimination of ARV-110 via phase 1 metabolism in rat, mouse, and human hepatic microsomes was shown to be unlikely. Application of the developed method to pharmacokinetic studies revealed that the oral bioavailability of ARV-110 in rats and mice was moderate (23.83% and 37.89%, respectively). These pharmacokinetic findings are beneficial for future preclinical and clinical studies of ARV-110 and/or other PROTACs.
Topics: Animals; Male; Mice; Rats; Chromatography, Liquid; Microsomes, Liver; Proteolysis; Reproducibility of Results; Tandem Mass Spectrometry
PubMed: 35335338
DOI: 10.3390/molecules27061977 -
Drug Metabolism and Disposition: the... Mar 2020The 20 uridine diphosphate glycosyl-transferases (UGTs) encoded in the human genome form an essential homeostatic network of overlapping catalytic functions that surveil...
The 20 uridine diphosphate glycosyl-transferases (UGTs) encoded in the human genome form an essential homeostatic network of overlapping catalytic functions that surveil and regulate the activity and clearance of scores of small molecule metabolites. Biochemical and biophysical UGT studies have been hampered by the inability to purify these membrane-bound proteins. Here, using cell-free expression and nanodisc technology, we assemble and purify to homogeneity the first UGT nanodisc-the human UGT2B7•nanodisc. The complex is readily isolated in milligram quantities. It is stable and its initial-rate parameters are identical within error to those associated with UGT2B7 in microsomal preparations (i.e., Supersomes). The high purity of the nanodisc preparation simplifies UGT assays, which allows complexities traditionally associated with microsomal assays (latency and the albumin effect) to be circumvented. Each nanodisc is shown to harbor a single UGT2B7 monomer. The methods described herein should be widely applicable to UGTs, and these findings are expected to set the stage for experimentalists to more freely explore the structure, function, and biology of this important area of phase II metabolism. SIGNIFICANCE STATEMENT: Lack of access to pure, catalytically competent human uridine diphosphate glucuronosyl-transferases (UGTs) has long been an impediment to biochemical and biophysical studies of this disease-relevant enzyme family. Here, we demonstrate this barrier can be removed using nanodisc technology-a human UGT2B7•nanodisc is assembled, purified to homogeneity, and shown to have activity comparable to microsomal UGT2B7.
Topics: Glucuronosyltransferase; Humans; Liver; Microsomes, Liver
PubMed: 31892527
DOI: 10.1124/dmd.119.089946 -
Drug Metabolism Reviews Aug 2022This year's review on bioactivation and reactivity began as a part of the annual review on biotransformation and bioactivation led by Cyrus Khojasteh (see references).... (Review)
Review
This year's review on bioactivation and reactivity began as a part of the annual review on biotransformation and bioactivation led by Cyrus Khojasteh (see references). Increased contributions from experts in the field led to the development of a stand alone edition for the first time this year focused specifically on bioactivation and reactivity. Our objective for this review is to highlight and share articles which we deem influential and significant regarding the development of covalent inhibitors, mechanisms of reactive metabolite formation, enzyme inactivation, and drug safety. Based on the selected articles, we created two sections: (1) reactivity and enzyme inactivation, and (2) bioactivation mechanisms and safety (Table 1). Several biotransformation experts have contributed to this effort from academic and industry settings.[Table: see text].
Topics: Biotransformation; Humans; Microsomes, Liver
PubMed: 35876116
DOI: 10.1080/03602532.2022.2097254 -
Nature Chemical Biology Aug 2021Multiple Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase...
Multiple Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M (Palm M) block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared with Palm M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MAP kinase kinase (MEK) inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes as regulators of the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers.
Topics: Cell Membrane; Cell Proliferation; Cells, Cultured; Humans; Hydrolases; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Lipoylation; Microsomes, Liver; Molecular Structure; ras Proteins
PubMed: 33927411
DOI: 10.1038/s41589-021-00785-8 -
Current Drug Metabolism 2021MIDD0301 is an oral asthma drug candidate that binds GABAA receptors on airway smooth muscle and immune cells. (Comparative Study)
Comparative Study
BACKGROUND
MIDD0301 is an oral asthma drug candidate that binds GABAA receptors on airway smooth muscle and immune cells.
OBJECTIVE
The objective of this study is to identify and quantify MIDD0301 metabolites in vitro and in vivo and determine the pharmacokinetics of oral, IP, and IV administered MIDD0301.
METHODS
In vitro conversion of MIDD0301 was performed using liver and kidney microsomes/S9 fractions followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A LC-MS/MS method was developed using synthesized standards to quantify MIDD0301 and its metabolites in urine and feces. Blood, lung, and brain were harvested from animals that received MIDD0301 by oral, IP, and IV administration, followed by LCMS/ MS quantification. Imaging mass spectrometry was used to demonstrate the presence of MIDD0301 in the lung after oral administration.
RESULTS
MIDD0301 is stable in the presence of liver and kidney microsomes and S9 fractions for at least two hours. MIDD0301 undergoes conversion to the corresponding glucuronide and glucoside in the presence of conjugating cofactors. For IP and IV administration, unconjugated MIDD0301 together with significant amounts of MIDD0301 glucoside and MIDD0301 taurine were found in urine and feces. Less conjugation was observed following oral administration, with MIDD0301 glucuronide being the main metabolite. Pharmacokinetic quantification of MIDD0301 in blood, lung, and brain showed very low levels of MIDD0301 in the brain after oral, IV, or IP administration. The drug half-life in these tissues ranged between 4-6 hours for IP and oral and 1-2 hours for IV administration. Imaging mass spectrometry demonstrated that orally administered MIDD0301 distributes uniformly in the lung parenchyma.
CONCLUSION
MIDD0301 undergoes no phase I and moderate phase II metabolism.
Topics: Administration, Intravenous; Administration, Oral; Animals; Anti-Asthmatic Agents; Azepines; Chromatography, Liquid; Dogs; Female; Humans; Imidazoles; Injections, Intraperitoneal; Kidney; Lung; Mice; Microsomes; Microsomes, Liver; Rats; Tandem Mass Spectrometry; Tissue Distribution
PubMed: 34856893
DOI: 10.2174/1389200222666211202093841 -
Planta Medica Sep 2023Oral preparations of (guacatonga) are used as antacid, analgesic, anti-inflammatory, and antiulcerogenic medicines. The clerodane diterpenes casearin B and...
Oral preparations of (guacatonga) are used as antacid, analgesic, anti-inflammatory, and antiulcerogenic medicines. The clerodane diterpenes casearin B and caseargrewiin F are major active compounds and . The oral bioavailability and metabolism of casearin B and caseargrewiin F were not previously investigated. We aimed to assess the stability of casearin B and caseargrewiin F in physiological conditions and their metabolism in human liver microsomes. The compounds were identified by UHPLC-QTOF-MS/MS and quantified by validated LC-MS methods. The stability of casearin B and caseargrewiin F in physiological conditions was assessed . Both diterpenes showed a fast degradation (p < 0.05) in simulated gastric fluid. Their metabolism was not mediated by cytochrome P-450 enzymes, but the depletion was inhibited by the esterase inhibitor NaF. Both diterpenes and their dialdehydes showed a octanol/water partition coefficient in the range of 3.6 to 4.0, suggesting high permeability. Metabolism kinetic data were fitted to the Michaelis-Menten profile with K values of 61.4 and 66.4 µM and V values of 327 and 648 nmol/min/mg of protein for casearin B and caseargrewiin F, respectively. Metabolism parameters in human liver microsomes were extrapolated to predict human hepatic clearance, and suggest that caseargrewiin F and casearin B have a high hepatic extraction ratio. In conclusion, our data suggest that caseargrewiin F and casearin B present low oral bioavailability due to extensive gastric degradation and high hepatic extraction.
Topics: Humans; Diterpenes, Clerodane; Tandem Mass Spectrometry; Liver; Microsomes, Liver
PubMed: 37084791
DOI: 10.1055/a-2078-5920 -
Pharmacology Research & Perspectives Jun 2022Fenfluramine (FFA) has potent antiseizure activity in severe, pharmacoresistant childhood-onset developmental and epileptic encephalopathies (e.g., Dravet syndrome). To... (Review)
Review
Fenfluramine (FFA) has potent antiseizure activity in severe, pharmacoresistant childhood-onset developmental and epileptic encephalopathies (e.g., Dravet syndrome). To assess risk of drug interaction affecting pharmacokinetics of FFA and its major metabolite, norfenfluramine (nFFA), we conducted in vitro metabolite characterization, reaction phenotyping, and drug transporter-mediated cellular uptake studies. FFA showed low in vitro clearance in human liver S9 fractions and in intestinal S9 fractions in all three species tested (t > 120 min). Two metabolites (nFFA and an N-oxide or a hydroxylamine) were detected in human liver microsomes versus six in dog and seven in rat liver microsomes; no metabolite was unique to humans. Selective CYP inhibitor studies showed FFA metabolism partially inhibited by quinidine (CYP2D6, 48%), phencyclidine (CYP2B6, 42%), and furafylline (CYP1A2, 32%) and, to a lesser extent (<15%), by tienilic acid (CYP2C9), esomeprazole (CYP2C19), and troleandomycin (CYP3A4/5). Incubation of nFFA with rCYP1A2, rCYP2B6, rCYP2C19, and rCYP2D6 resulted in 10%-20% metabolism and no clear inhibition of nFFA metabolism by any CYP-selective inhibitor. Reaction phenotyping showed metabolism of FFA by recombinant human cytochrome P450 (rCYP) enzymes rCYP2B6 (10%-21% disappearance for 1 and 10 µM FFA, respectively), rCYP1A2 (22%-23%), rCYP2C19 (49%-50%), and rCYP2D6 (59%-97%). Neither FFA nor nFFA was a drug transporter substrate. Results show FFA metabolism to nFFA occurs through multiple pathways of elimination. FFA dose adjustments may be needed when administered with strong inhibitors or inducers of multiple enzymes involved in FFA metabolism (e.g., stiripentol).
Topics: Animals; Cytochrome P-450 Enzyme System; Dogs; Drug Interactions; Fenfluramine; Humans; Microsomes, Liver; Norfenfluramine; Rats
PubMed: 35599345
DOI: 10.1002/prp2.958 -
Journal of Medicinal Chemistry Apr 2021Development of new chemical entities is costly, time-consuming, and has a low success rate. Accurate prediction of pharmacokinetic properties is critical to progress... (Review)
Review
Development of new chemical entities is costly, time-consuming, and has a low success rate. Accurate prediction of pharmacokinetic properties is critical to progress compounds with favorable drug-like characteristics in lead optimization. Of particular importance is the prediction of hepatic clearance, which determines drug exposure and contributes to projection of dose, half-life, and bioavailability. The most commonly employed methodology to predict hepatic clearance is termed to extrapolation (IVIVE) that involves measuring drug metabolism , scaling-up this intrinsic clearance to a prediction of intrinsic clearance by reconciling the enzymatic content between the incubation and an average human liver, and applying a model of hepatic disposition to account for limitations of protein binding and blood flow to predict clearance. This manuscript reviews common techniques used to predict hepatic clearance as well as current challenges and recent theoretical advancements in IVIVE.
Topics: Hepatocytes; Humans; Kinetics; Liver; Metabolic Clearance Rate; Microsomes, Liver; Models, Biological; Pharmaceutical Preparations; Pharmacokinetics
PubMed: 33765384
DOI: 10.1021/acs.jmedchem.0c01930 -
Journal of Proteomics Jul 2022Model-based assessment of drug pharmacokinetics in liver disease requires quantification of abundance and disease-related changes in hepatic enzymes and transporters....
Model-based assessment of drug pharmacokinetics in liver disease requires quantification of abundance and disease-related changes in hepatic enzymes and transporters. This study aimed to assess performance of three label-free methods [high N (HiN), intensity-based absolute quantification (iBAQ) and total protein approach (TPA)] against QconCAT-based targeted data in healthy and diseased (cancer and cirrhosis) liver tissue. Measurements were compared across methods and disease-to-control ratios provided a 'disease perturbation factor' (DPF) for each protein. Mean label-free measurements of targets correlated well (Pearson's coefficient, r = 0.91-0.98 p < 0.001) and with targeted data (r = 0.65-0.95, p < 0.001). Concordance with targeted data was generally moderate (Lin's concordance coefficient, ρ = 0.46-0.92), depending on methodology. Moderate precision and accuracy were observed for label-free methods (average fold error, AFE = 1.44-1.68; absolute average fold error, AAFE = 2.44-3.23). The DPF reconciled the data and indicated downregulated expression in cancer and cirrhosis, consistent with an inflammatory effect. HiN estimated perturbation consistently with targeted data (AFE = 1.07, AAFE = 1.57), whereas iBAQ overestimated (AFE = 0.81, AAFE = 1.67) and TPA underestimated (AFE = 1.37, AAFE = 1.65) disease effect. Progression from mild to severe cirrhosis was consistent with progressive decline in expression, reproduced by HiN but overestimated by iBAQ and underestimated by TPA (AFE = 0.98, AFE = 0.60, AFE = 1.24). DPF data confirmed non-uniform disease effect on drug-elimination pathways and progressive impact of disease severity. SIGNIFICANCE: This study demonstrated good correlation and moderate concordance between intensity-based label-free proteomic methods (HiN, iBAQ and TPA) and targeted data. Label-free measurements tended to overestimate abundance, but differences were reconciled using a disease perturbation factor (DPF) for each protein. With targeted data as a reference, HiN defined disease perturbation and the impact of disease progression consistently, indicating that the use of 'razor' peptides for quantification against an exogenous standard provides biologically sensible quantitative fingerprints of disease. Disease-driven perturbations in expression relative to healthy baseline are incorporated into drug kinetic models used to predict drug exposure in disease populations where clinical studies may not be feasible.
Topics: Humans; Liver Cirrhosis; Membrane Transport Proteins; Microsomes, Liver; Proteomics
PubMed: 35537666
DOI: 10.1016/j.jprot.2022.104601 -
Pharmaceutical Biology Dec 2023Poziotinib and vonoprazan are two drugs mainly metabolized by CYP3A4. However, the drug-drug interaction between them is unknown.
CONTEXT
Poziotinib and vonoprazan are two drugs mainly metabolized by CYP3A4. However, the drug-drug interaction between them is unknown.
OBJECTIVE
To study the interaction mechanism and pharmacokinetics of poziotinib on vonoprazan.
MATERIALS AND METHODS
experiments were performed with rat liver microsomes (RLMs) and the contents of vonoprazan and its metabolite were then determined with UPLC-MS/MS after incubation of RLMs with vonoprazan and gradient concentrations of poziotinib. For the experiment, rats in the poziotinib treated group were given 5 mg/kg poziotinib by gavage once daily for 7 days, and the control group was only given 0.5% CMC-Na. On Day 8, tail venous blood was collected at different time points after the gavage administration of 10 mg/kg vonoprazan, and used for the quantification of vonoprazan and its metabolite. DAS and SPSS software were used for the pharmacokinetic and statistical analyses.
RESULTS
experimental data indicated that poziotinib inhibited the metabolism of vonoprazan (IC = 10.6 μM) in a mixed model of noncompetitive and uncompetitive inhibition. The inhibitory constant K was 0.574 μM and the binding constant αK was 2.77 μM. experiments revealed that the AUC (15.05 90.95 μg/mL·h) and AUC (15.05 91.99 μg/mL·h) of vonoprazan increased significantly with poziotinib pretreatment. The MRT of vonoprazan increased from 2.29 to 5.51 h, while the CLz/F value decreased from 162.67 to 25.84 L/kg·h after pretreatment with poziotinib.
CONCLUSIONS
Poziotinib could significantly inhibit the metabolism of vonoprazan and more care may be taken when co-administered in the clinic.
Topics: Rats; Animals; Chromatography, Liquid; Tandem Mass Spectrometry; Drug Interactions; Microsomes, Liver
PubMed: 36728978
DOI: 10.1080/13880209.2023.2173253