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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 -
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 -
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 -
Pharmacology Research & Perspectives Feb 2023Scientific literature describes that sumatriptan is metabolized by oxidative deamination of its dimethylaminoethyl residue by monoamine oxidase A (MAO A) and not by...
Scientific literature describes that sumatriptan is metabolized by oxidative deamination of its dimethylaminoethyl residue by monoamine oxidase A (MAO A) and not by cytochrome P450 (CYP)-mediated demethylation, as is usual for such structural elements. Using recombinant human enzymes and HPLC-MS analysis, we found that CYP enzymes may also be involved in the metabolism of sumatriptan. The CYP1A2, CYP2C19, and CYP2D6 isoforms converted this drug into N-desmethyl sumatriptan, which was further demethylated to N,N-didesmethyl sumatriptan by CYP1A2 and CYP2D6. Otherwise, sumatriptan and its two desmethyl metabolites were metabolized by recombinant MAO A but not by MAO B to the corresponding acetaldehyde, with sumatriptan being only a poor substrate for MAO A compared to the N-demethylated and the N,N-didemethylated derivatives.
Topics: Humans; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP2D6; Cytochrome P-450 Enzyme System; Microsomes, Liver; Monoamine Oxidase; Sumatriptan
PubMed: 36655303
DOI: 10.1002/prp2.1051 -
BioDrugs : Clinical Immunotherapeutics,... Jun 2020Proteins are the main source of drug targets and some of them possess therapeutic potential themselves. Among them, membrane proteins constitute approximately 50% of the... (Review)
Review
Proteins are the main source of drug targets and some of them possess therapeutic potential themselves. Among them, membrane proteins constitute approximately 50% of the major drug targets. In the drug discovery pipeline, rapid methods for producing different classes of proteins in a simple manner with high quality are important for structural and functional analysis. Cell-free systems are emerging as an attractive alternative for the production of proteins due to their flexible nature without any cell membrane constraints. In a bioproduction context, open systems based on cell lysates derived from different sources, and with batch-to-batch consistency, have acted as a catalyst for cell-free synthesis of target proteins. Most importantly, proteins can be processed for downstream applications like purification and functional analysis without the necessity of transfection, selection, and expansion of clones. In the last 5 years, there has been an increased availability of new cell-free lysates derived from multiple organisms, and their use for the synthesis of a diverse range of proteins. Despite this progress, major challenges still exist in terms of scalability, cost effectiveness, protein folding, and functionality. In this review, we present an overview of different cell-free systems derived from diverse sources and their application in the production of a wide spectrum of proteins. Further, this article discusses some recent progress in cell-free systems derived from Chinese hamster ovary and Sf21 lysates containing endogenous translocationally active microsomes for the synthesis of membrane proteins. We particularly highlight the usage of internal ribosomal entry site sequences for more efficient protein production, and also the significance of site-specific incorporation of non-canonical amino acids for labeling applications and creation of antibody drug conjugates using cell-free systems. We also discuss strategies to overcome the major challenges involved in commercializing cell-free platforms from a laboratory level for future drug development.
Topics: Animals; CHO Cells; Cell-Free System; Cricetulus; Drug Development; Membrane Proteins; Microsomes; Protein Biosynthesis; Protein Folding; Receptors, G-Protein-Coupled
PubMed: 32198631
DOI: 10.1007/s40259-020-00417-y -
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 -
International Journal of Molecular... Jan 2021The major determinants of drug or, al bioavailability are absorption and metabolism in the digestive tract. Genetic variations can cause significant differences in... (Review)
Review
The major determinants of drug or, al bioavailability are absorption and metabolism in the digestive tract. Genetic variations can cause significant differences in transporter and enzyme protein expression and function. The racial distribution of selected efflux transporter (i.e., Pgp, BCRP, MRP2) and metabolism enzyme (i.e., UGT1A1, UGT1A8) single nucleotide polymorphisms (SNPs) that are highly expressed in the digestive tract are reviewed in this paper with emphasis on the allele frequency and the impact on drug absorption, metabolism, and in vivo drug exposure. Additionally, preclinical and clinical models used to study the impact of transporter/enzyme SNPs on protein expression and function are also reviewed. The results showed that allele frequency of the major drug efflux transporters and the major intestinal metabolic enzymes are highly different in different races, leading to different drug disposition and exposure. The conclusion is that genetic polymorphism is frequently observed in different races and the related protein expression and drug absorption/metabolism function and drug in vivo exposure can be significantly affected, resulting in variations in drug response. Basic research on race-dependent drug absorption/metabolism is expected, and FDA regulations of drug dosing adjustment based on racial disparity are suggested.
Topics: ATP-Binding Cassette Transporters; Alleles; Animals; Biological Availability; Biological Transport; Biomarkers; Gastrointestinal Absorption; Gastrointestinal Tract; Gene Frequency; Humans; Inactivation, Metabolic; Metabolic Clearance Rate; Microsomes; Polymorphism, Single Nucleotide; Race Factors
PubMed: 33494365
DOI: 10.3390/ijms22031038 -
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 -
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