Drug Metabolism Studies Research Articles

Investigating the ability of the microbial model Cunninghamella elegans for the metabolism of synthetic tryptamines.

Tryptamines can occur naturally in plants, mushrooms, microbes, and amphibians. Synthetic tryptamines are sold as new psychoactive substances (NPS) because of their hallucinogenic effects. When it comes to NPS, metabolism studies are of crucial importance, due to the lack of pharmacological and toxicological data. Different approaches can be taken to study in vitro and in vivo metabolism of xenobiotica. The zygomycete fungus Cunninghamella elegans (C. elegans) can be used as a microbial model for the study of drug metabolism. The current study investigated the biotransformation of four naturally occurring and synthetic tryptamines [N,N-Dimethyltryptamine (DMT), 4-hydroxy-N-methyl-N-ethyltryptamine (4-HO-MET), N,N-di allyl-5-methoxy tryptamine (5-MeO-DALT) and 5-methoxy-N-methyl-N-isoporpoyltryptamine (5-MeO-MiPT)] in C. elegans after incubation for 72hours. Metabolites were identified using liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS) with a quadrupole time-of-flight (QqTOF) instrument. Results were compared to already published data on these substances. C. elegans was capable of producing all major biotransformation steps: hydroxylation, N-oxide formation, carboxylation, deamination, and demethylation. On average 63% of phase I metabolites found in the literature could also be detected in C. elegans. Additionally, metabolites specific for C. elegans were identified. Therefore, C. elegans is a suitable complementary model to other in vitro or in vivo methods to study the metabolism of naturally occurring or synthetic tryptamines.

Proteometabolomics of Melphalan Resistance in Multiple Myeloma

Although advancements in therapeutic regimens for treating multiple myeloma (MM) have prolonged patient survival, the disease remains incurable. Several classes of drugs have contributed to these improvements, such as proteasome inhibitors, immunomodulators, deacetylase inhibitors, monoclonal antibodies, and alkylating agents including melphalan. An expanded arsenal of diverse chemotherapy targets has improved patient care significantly, yet we still lack sufficient knowledge of how cellular metabolism and drug processing can contribute to drug resistance. To address this issue, we utilize cell line models to simulate naïve and drug resistant states, which identify drug modifications, endogenous metabolites, proteins, and acute metabolic profile alterations associated with therapeutic escape.Here, we specifically focus on melphalan; an alkylating agent that forms DNA interstrand crosslinks, inhibits cell division, and leads to cell death through apoptosis (Povirk & Shuker. Mutat. Res. 1994, 318, 205). Melphalan remains a critical component of high dose therapy in the context of stem cell transplant and induction therapy in transplant ineligible patients outside the US. Ineffectiveness of alkylating agents remains a critical problem and serves as an excellent model for investigation of cellular metabolism and its contribution to drug resistance.Two parental MM cell lines (8226 & U266) were obtained from ATCC and resistant derivatives of each cell line (8226-LR5 & U266-LR6) were selected after chronic drug exposure. To assess mechanisms of melphalan resistance, we use liquid chromatography-mass spectrometry-based metabolomics and proteomics approaches, including studies of drug metabolism, untargeted metabolomics, and activity based protein profiling (ABPP). Drug metabolism monitors the intracellular and extracellular drug modifications over a 24-hour period after acute treatment. Untargeted metabolomics is used to compare the steady state endogenous intracellular metabolites of naïve and drug resistant cells. Differences in endogenous metabolites between naïve and drug resistant cell lines are also examined in the acute treatment dataset. ABPP utilizes desthiobiotinylating probes to enrich for ATP-utilizing enzymes, which are identified and quantified to enable comparison.We initially compared acute melphalan treatment in drug naive and resistant isogenic cell line pairs. Predictably, melphalan was converted into monohydroxylated and dihydroxylated metabolites more quickly in cells than in media controls. Differences in the formation of these metabolites between the naïve and resistant cell lines were not observed. The untargeted metabolomics data indicated in the 8226-LR5 model, glutathione and xanthine levels are elevated, while guanine is suppressed relative to naive cells. ABPP demonstrated changes in several enzymes related to purine and glutathione metabolism (Figure 1). Interestingly, the U266/U266-LR6 cell line models exhibit higher baseline levels of glutathione when compared with 8226/8226-LR5, indicating heterogeneous means of drug resistance. Alterations in arginine biosynthesis and nicotinate/nicotinamide metabolism are observed in the untargeted metabolomics and ABPP of U266/U266-LR6. Common pathways (e.g. purine biosynthesis) are altered in both models, although the changes involve different molecules.In examining two models of acquired melphalan resistance, we demonstrate frank differences in metabolic pathways associated with steady state and acute drug response. These data demonstrate the potential heterogeneity in drug resistance mechanisms and the need for more biomarkers to personalize treatment. Ongoing studies involve introduction of enzyme inhibitors in targeted pathways and supplementation of metabolites to validate their role in resistance. Furthermore, we will examine expression of these metabolic pathways associated with ex vivo melphalan resistance in a cohort of over 100 patient samples with paired RNA sequencing. The long term goals are to elucidate mechanisms of therapeutic response, identify biomarkers of metabolism in melphalan resistance, enhance drug efficacy, predict personalized patient treatment, and improve overall MM patient care. DisclosuresNo relevant conflicts of interest to declare.

Efficient Generation of Small Intestinal Epithelial-like Cells from Human iPSCs for Drug Absorption and Metabolism Studies.

SummaryThe small intestine plays an important role in the absorption and metabolism of oral drugs. In the current evaluation system, it is difficult to predict the precise absorption and metabolism of oral drugs. In this study, we generated small intestinal epithelial-like cells from human induced pluripotent stem cells (hiPS-SIECs), which could be applied to drug absorption and metabolism studies. The small intestinal epithelial-like cells were efficiently generated from human induced pluripotent stem cell by treatment with WNT3A, R-spondin 3, Noggin, EGF, IGF-1, SB202190, and dexamethasone. The gene expression levels of small intestinal epithelial cell (SIEC) markers were similar between the hiPS-SIECs and human adult small intestine. Importantly, the gene expression levels of colonic epithelial cell markers in the hiPS-SIECs were much lower than those in human adult colon. The hiPS-SIECs generated by our protocol exerted various SIEC functions. In conclusion, the hiPS-SIECs can be utilized for evaluation of drug absorption and metabolism.

Analysis of insulin and insulin analogs from dried blood spots by means of liquid chromatography-high resolution mass spectrometry.

While dried blood spot (DBS) analysis concerning low molecular mass molecules has become more and more established in various fields of analytical chemistry, the utility of DBS in determining peptides and proteins from DBS is yet comparably limited. In consideration of the fact that the apparent benefits of DBS sampling are similar for analytes of lower and higher molecular mass, dedicated (non-generic) sample preparation procedures are required that meet the needs for detecting peptidic drugs and hormones in DBS. The analysis of insulin and its synthetic analogs by mass spectrometry has received increased attention in several fields such as doping controls, forensics, and drug metabolism and pharmacokinetics studies. Hence, a strategy facilitating the analysis of insulin and its synthetic or animal analogs (human, Lispro, Aspart, Glulisine, Glargine, Detemir, Tresiba, and porcine and bovine insulin) from DBS was developed. The successful analysis of these substances at physiologically relevant concentrations was realized after ultrasonication-assisted extraction, immunoaffinity purification, and liquid chromatographic separation followed by high resolution mass spectrometric detection (with or without ion mobility). Assay validation demonstrated adequate sensitivity (LOD 0.5ng/mL for most insulins), as well as precise (< 25%) and reproducible results for all included target insulins. Additionally, proof-of-principle data were obtained by the analysis of DBS samples obtained from healthy volunteers in non-fasting state as well as a sample from a diabetic volunteer treated with the fast acting analog insulin Aspart.

Molecular and Functional Characterization of N-Acetyltransferases NAT1 and NAT2 in Cynomolgus Macaque.

Arylamine N-acetyltransferases (NATs) are drug-metabolizing enzymes essential for the metabolism of endogenous substrates and xenobiotics, and their molecular characteristics have been extensively investigated in humans, but not in cynomolgus macaques, nonhuman primate species important for drug metabolism studies. In this study, cynomolgus NAT1 and NAT2 cDNAs were isolated from livers. NAT1 and NAT2 were characterized by molecular analyses and drug-metabolizing assays. A total of 9 transcript variants were found for cynomolgus NAT1, similar to human NAT1, and contained 1-4 exons with the coding region largely conserved with human NAT1. Genomic organization was similar between cynomolgus macaques and humans. Cynomolgus NAT1 and NAT2 amino acid sequences showed high sequence homology (95% and 89%, respectively) and showed close relationships with human NAT1 and NAT2 in a phylogenetic tree. Cynomolgus NAT2 mRNA was predominantly expressed in liver among the 10 different tissues analyzed, followed by kidney and jejunum. In contrast, cynomolgus NAT1 mRNA showed more ubiquitous expression with relatively more abundant expression in liver, kidney, and jejunum, along with testis. Metabolic assays using recombinant proteins showed that cynomolgus NAT1 and NAT2 metabolized human NAT substrates, including p-aminobenzoic acid, sulfamethazine, isoniazid, and 2-aminofluorene. Interestingly, p-aminobenzoic acid and isoniazid were largely metabolized by NAT1 and NAT2, respectively, in cynomolgus macaques and humans; sulfamethazine, a human NAT2 substrate, was metabolized by both NAT enzymes in cynomolgus macaques. These results suggest molecular and enzymatic similarities of NAT1 and NAT2 between cynomolgus macaques and humans, despite some small differences in substrate specificity of the enzymes.

Impact of Drug Metabolism/Pharmacokinetics and their Relevance Upon Taxus-based Drug Development.

Drug Metabolism and Pharmacokinetic (DMPK) studies of Taxus natural products, their semi-synthetic derivatives and analogs are indispensable in the optimization of lead compounds and clinical therapy. These studies can lead to development of new drug entities with improved Absorption, Distribution, Metabolism, Excretion and Toxicity (ADME/T) profiles. To date, there have been no comprehensive reviews of the DMPK features of Taxus derived medicinal compounds. Natural and semi-synthetic taxanes may cause and could be affected by Drug-drug Interaction (DDI). Hence ADME/T studies of various taxane-containing formulations are important; to date these studies indicate that the role of cytochrome p450s and drug transporters is more prominent than phase II drug metabolizing enzymes. Mechanisms of taxane DMPK mediated by nuclear receptors, microRNAs, and single nucleotide polymorphisms are being revealed. Herein we review the latest knowledge on these topics, as well as the gaps in knowledge of the DMPK issues of Taxus compounds. DDIs significantly impact the PK/pharmacodynamics performance of taxanes and coadministered chemicals, which may inspire researchers to develop novel formula. While the ADME/T profiles of some taxanes are well defined, DMPK studies should be extended to more Taxus compounds, species, and Taxus - involved formulations, which would be streamlined by versatile omics platforms and computational analyses. Further biopharmaceutical investigations will be beneficial to the translation of bench findings to the clinical applications.

Microbial biotransformation – an important tool for the study of drug metabolism

Metabolite identification is an integral part of both preclinical and clinical drug discovery and development. Synthesis of drug metabolites is often required to support definitive identification, preclinical safety studies and clinical trials.Here we describe the use of microbial biotransformation as a tool to produce drug metabolites, complementing traditional chemical synthesis and other biosynthetic methods such as hepatocytes, liver microsomes and recombinant human drug metabolizing enzymes.A workflow is discussed whereby microbial strains are initially screened for their ability to form the putative metabolites of interest, followed by a scale-up to afford quantities sufficient to perform definitive identification and further studies.Examples of the microbial synthesis of several difficult-to-synthesize hydroxylated metabolites and three difficult-to-synthesize glucuronidated metabolites are described, and the use of microbial biotransformation in drug discovery and development is discussed.

Targeted LC-MS/MS Proteomics-Based Strategy To Characterize in Vitro Models Used in Drug Metabolism and Transport Studies.

Subcellular fractionation of tissue homogenate provides enriched in vitro models (e.g., microsomes, cytosol, or membranes), which are routinely used in the drug metabolism or transporter activity and protein abundance studies. However, batch-to-batch or interlaboratory variability in the recovery, enrichment, and purity of the subcellular fractions can affect performance of in vitro models leading to inaccurate in vitro to in vivo extrapolation (IVIVE) of drug clearance. To evaluate the quality of subcellular fractions, we developed a simple, targeted, and sensitive LC-MS/MS proteomics-based strategy, which relies on determination of protein markers of various cellular organelles, i.e., plasma membrane, cytosol, nuclei, mitochondria, endoplasmic reticulum (ER), lysosomes, peroxisomes, cytoskeleton, and exosomes. Application of the quantitative proteomics method confirmed a significant effect of processing variables (i.e., homogenization method and centrifugation speed) on the recovery, enrichment, and purity of isolated proteins in microsomes and cytosol. Particularly, markers of endoplasmic reticulum lumen and mitochondrial lumen were enriched in the cytosolic fractions as a result of their release during homogenization. Similarly, the relative recovery and composition of the total membrane fraction isolated from cell vs tissue samples was quantitatively different and should be considered in IVIVE. Further, analysis of exosomes isolated from sandwich-cultured hepatocyte media showed the effect of culture duration on compositions of purified exosomes. Therefore, the quantitative proteomics-based strategy developed here can be applied for efficient and simultaneous determination of multiple protein markers of various cellular organelles when compared to antibody- or activity-based assays and can be used for quality control of subcellular fractionation procedures including in vitro model development for drug metabolism and transport studies.

Non-synonymous genetic variants of flavin-containing monooxygenase 3 (FMO3) in cynomolgus macaques

Polymorphic human flavin-containing monooxygenase (FMO) 3 is an important drug-metabolizing enzyme for nitrogen- or sulfur-containing compounds. Cynomolgus macaques, a non-human primate species widely used in drug metabolism studies, have corresponding FMO3 molecular and enzymatic similarities to humans; however, genetic polymorphisms have not been investigated in macaques. In this study, re-sequencing of FMO3 in 64 cynomolgus and 32 rhesus macaques found a total of 18 non-synonymous variants. Nine variants were unique to cynomolgus macaques, of which 4 (including Q506K) were found only in Indochinese, 4 (including V299I, E348H, and G530A) only in Indonesian lineages, and one was common. Other five variants (including S504T at >10% allele frequencies) were unique to rhesus macaques. By functional characterization using cynomolgus FMO3 proteins heterologously expressed in Escherichia coli, FMO3 R509H variant appeared to suppress methimazole and benzydamine S- or N-oxygenations. Seven variants showed substantially lower benzydamine N-oxygenation as compared with wild-type FMO3 protein. Further analysis indicated that two of these variants, FMO3 G530A and R417H, showed significantly lower benzydamine N-oxygenation in liver microsomes of the homozygotes as compared with wild-type animals. Therefore, inter-animal variability of FMO3-dependent drug metabolism is at least partly accounted for by genetic polymorphisms in cynomolgus and rhesus macaques, similar to humans.

Studies on phytoenzymes as mimics of mammalian drug metabolising enzymes

Drug metabolism and pharmacokinetics is an integral part of the drug discovery and development process. The pharmacokinetic (absorption, distribution, metabolism and excretion) properties of a drug allow the drug development scientist to understand the safety and efficacy data required for regulatory approval. Further, the knowledge of mammalian enzymes involved in the metabolism of a new chemical entity is of prime importance. In this regard, plant metabolism of organic compounds is similar to the mammalian metabolism in terms of the processes for detoxification of contaminants, the specific enzymatic pathways, the structure of the enzymes involved and the removal of the compounds from the susceptible organelles. In our studies, we isolated phytoenzymes (esterases and glutathione-S-transferases, GSTs) from plant cells and investigated their applicability as tools for drug metabolism studies. A total of 32 plants were screened for esterases activity and 14 plants for GSTs activity. In case of esterases, the activity was compared to human plasma esterases and also the effect of esterase inhibitor on the activity was studied. Overall, the presence of phytoenzymes and their catalytic similarities to the mammalian drug metabolising enzymes suggest that plants can be potential models for mammalian drug metabolism. Further, phytoenzymes can be considered as a potential surrogate for reducing the number of animals utilized in the drug discovery process.

Genetic Variants of Glutathione S-Transferase GSTT1 and GSTT2 in Cynomolgus Macaques: Identification of GSTT Substrates and Functionally Relevant Alleles

Glutathione S-transferase (GST) is a family of important drug-metabolizing enzymes, conjugating endogenous and exogenous compounds. Genetic polymorphisms result in the inter-individual variability of GST activity in humans. Especially, human GSTT1 and GSTT2 null alleles are associated with toxicity and various cancers derived from chemicals. Cynomolgus macaque, a nonhuman primate species widely used in drug metabolism studies, has molecular and enzymatic similarities of GSTs to the human orthologs; however, genetic polymorphisms have not been investigated in this species. In this study, resequencing of GSTT1 and GSTT2 in 64 cynomolgus and 32 rhesus macaques found 15 nonsynonymous variants and 1 nonsense variant for GSTT1 and 15 nonsynonymous variants for GSTT2. Some of these GSTT variants were distributed differently in Indochinese and Indonesian cynomolgus macaques and rhesus macaques. For analysis of functional relevance of the GSTT variants, 1-iodohexane and dibromomethane were determined to be suitable substrates for cynomolgus GSTT1 and GSTT2. However, the conjugation activities were roughly correlated with GSTT protein levels immunochemically quantified in cynomolgus liver samples with no statistical significances, implying the contributions of the GST genetic variants. Among the GSTT1 variants identified, the animals carrying R76C and D125G mutations showed lower conjugation activities toward dibromomethane than those of the wild-type in liver cytosolic fractions. Moreover, the recombinant R76C/D125G and D125G GSTT variant proteins showed significantly lower 1-iodohexane or dibromomethane conjugation activities than those of the wild-type protein. Therefore, inter-animal variability of GSTT-dependent drug metabolism is at least partly accounted for by GSTT1 and possibly GSTT2 variants in cynomolgus and rhesus macaques.

An Ex Vivo Fermentation Screening Platform to Study Drug Metabolism by Human Gut Microbiota.

Colon microbiota-based drug metabolism has received little attention thus far in the process of drug development, whereas the role of gut microbiota in clinical safety and efficacy of drugs has become more clear. Many of these studies have been performed using animal studies, but the translational value of these data with respect to drug pharmacokinetics, efficacy, and safety is largely unknown. To investigate human colon microbiota-mediated drug metabolism, we applied a recently developed ex vivo fermentation screening platform, in which human colonic microbiota conditions are simulated. A set of 12 drugs (omeprazole, simvastatin, metronidazole, risperidone, sulfinpyrazone, sulindac, levodopa, dapsone, nizatidine, sulfasalazine, zonisamide, and acetaminophen) was incubated with human colon microbiota under strictly anaerobic conditions, and samples were analyzed using high-performance liquid chromatograph-UV-high-resolution mass spectrometry analysis. The human microbiota in the fermentation assay consisted of bacterial genera regularly encountered in human colon and fecal samples and could be reproducibly cultured in independent experiments over time. In addition, fully anaerobic culture conditions could be maintained for 24 hours of incubation. Five out of the 12 included drugs (sulfasalazine, sulfinpyrazone, sulindac, nizatidine, and risperidone) showed microbiota-based biotransformation after 24 hours of incubation in the ex vivo fermentation assay. We demonstrated that drug metabolites formed by microbial metabolism can be detected in a qualitative manner and that the data are in accordance with those reported earlier for in vivo metabolism. In conclusion, we present a research tool to investigate human colon microbiota-based drug metabolism that may be applied to enable translatability of microbiota-based drug metabolism.

Cytochrome P450 1A2 Messenger RNA is a More Reliable Marker than Cytochrome P450 1A2 Activity, Phenacetin O-Deethylation, for Assessment of Induction Potential of Drug-Metabolizing Enzymes Using HepaRG Cells.

The HepaRG cells have key drug metabolism functionalities comparable to those of primary human hepatocytes. Many studies have reported that this cell line can be used as a reliable in vitro model for human drug metabolism studies, including the assessment of cytochrome P450 (CYP) induction. The objective of this study is to determine whether CYP mRNA level measurement is superior to the CYP enzyme activity measurement as a convenient high-throughput method for evaluating CYP induction potential using HepaRG cells. QuantiGene Plex 2.0 Assay and LC/MS/MS. mRNA expression levels and enzyme activities of CYP1A2, CYP2B6, and CYP3A in HepaRG cells treated with prototypical inducers of each CYP isoform [omeprazole (OME) for CYP1A2, phenobarbital (PB) for CYP2B6, and rifampicin (RIF) for CYP3A] were evaluated. Although the activities of CYP2B6 and CYP3A were induced by treatment with PB and RIF, we found that the activity of phenacetin O-deethylase (PHOD), which is known as a marker of the activity of CYP1A2, was also enhanced by treatment with these non-CYP1A2 inducers in HepaRG cells. Based on previously published reports, we hypothesized that the expression ratio of CYP3A to CYP1A2 is much higher in HepaRG cells than in human hepatocytes; this may result in a nonnegligible contribution of CYP3A to the PHOD reaction in HepaRG cells. Studies using CYP3A inhibitor and pregnane X receptor-knockout HepaRG cells supported this hypothesis. The measurement of mRNA serves as a higher reliable indicator for the evaluation of CYP induction potential when using HepaRG cells.

Advances in Engineered Human Liver Platforms for Drug Metabolism Studies.

Metabolism in the liver often determines the overall clearance rates of many pharmaceuticals. Furthermore, induction or inhibition of the liver drug metabolism enzymes by perpetrator drugs can influence the metabolism of victim drugs (drug-drug interactions). Therefore, determining liver-drug interactions is critical during preclinical drug development. Unfortunately, studies in animals are often of limited value because of significant differences in the metabolic pathways of the liver across different species. To mitigate such limitations, the pharmaceutical industry uses a continuum of human liver models, ranging from microsomes to transfected cell lines and cultures of primary human hepatocytes (PHHs). Of these models, PHHs provide a balance of high-throughput testing capabilities together with a physiologically relevant cell type that exhibits all the characteristic enzymes, cofactors, and transporters. However, PHH monocultures display a rapid decline in metabolic capacity. Consequently, bioengineers have developed several tools, such as cellular microarrays, micropatterned cocultures, self-assembled and bioprinted spheroids, and perfusion devices, to enhance and stabilize PHH functions for ≥2 weeks. Many of these platforms have been validated for drug studies, whereas some have been adapted to include liver nonparenchymal cells that can influence hepatic drug metabolism in health and disease. Here, we focus on the design features of such platforms and their representative drug metabolism validation datasets, while discussing emerging trends. Overall, the use of engineered human liver platforms in the pharmaceutical industry has been steadily rising over the last 10 years, and we anticipate that these platforms will become an integral part of drug development with continued commercialization and validation for routine screening use.

Critical differences in drug metabolic properties of human hepatic cellular models, including primary human hepatocytes, stem cell derived hepatocytes, and hepatoma cell lines

Primary human hepatocytes (PHH), HepaRG™, HepG2, and two sources of induced pluripotent stem cell (iPSC) derived hepatocytes were characterized regarding gene expression and function of key hepatic proteins, important for the metabolic fate of drugs. The gene expression PCA analysis showed a distance between the two iPSC derived hepatocytes as well as the HepG2 and HepaRG™ cells to the three PHH donors and PHH pool, which were clustered more closely together. Correlation-based hierarchical analysis clustered HepG2 close to the stem cell derived hepatocytes both when the expression of 91 genes related to liver function or only cytochrome P450 (P450) genes were analyzed indicating the non-liver feature and a similar low P450 profile in these cell models. The specific P450 activities and the metabolic pattern of well-characterized drug substances in the cell models demonstrated that iPSC derived hepatocytes had modest levels of CYP3A and CYP2C9, while CYP1A2, 2B6, 2C8, 2C9, 2C19, and 2D6 were barely detectable. High expression of several extrahepatic P450s such as CYP1A1 and 1B1 detected in the stem cell derived hepatocytes may have significant effects on metabolite profiles. However, one of the iPSC derived hepatocytes demonstrated significant combined P450 and conjugating enzyme activity of certain drugs. HepaRG™ cells showed many metabolic properties similar to PHHs and will in many respects be a good model in studies of metabolic pathways and induction of drug metabolism whereas there is still ground to cover before iPSC derived hepatocytes will be seen as a substitute to PHH in drug metabolism studies.

Synthesis of 13 C6 -labeled, dual-target inhibitor of cannabinoid-1 receptor (CB1 R) and inducible nitric oxide synthase (iNOS).

Cannabinoid-1 receptor (CB1 R) antagonists/inverse agonists have great potential in the treatment of metabolic disorders like dyslipidemia, type 2 diabetes, and nonalcoholic steatohepatitis. Cannabinoid-1 receptor inverse agonists have also been reported to be effective in mitigating fibrotic disorders in murine models. Inducible nitric oxide synthase is another promising target implicated in fibrotic and inflammatory disorders. We have disclosed MRI-1867 as a potent and selective, peripherally acting dual-target inhibitor of the CB1 R and inducible nitric oxide synthase (iNOS). Herein, we report the synthesis of [13 C6 ]-MRI-1867 as a racemate from commercially available chlorobenzene-13 C6 as the starting, stable-isotope label reagent. The racemic [13 C6 ]-MRI-1867 was further processed to the stable-isotope-labeled enantiopure compounds using chiral chromatography. Both racemic [13 C6 ]-MRI-1867 and S-13 C6 -MRI-1867 will be used to quantitate unlabeled S-MRI-1867 during clinical drug metabolism and pharmacokinetics studies and will be used as a liquid chromatography-tandem mass spectrometry bioanalytical standard.

Molecular and functional characterization of UDP-glucuronosyltransferase 1A in cynomolgus macaques

UDP-glucuronosyltransferases (UGTs) are drug-metabolizing enzymes essential for the metabolism of endogenous substrates and xenobiotics. Molecular characteristics of UGTs have been extensively investigated in humans, but in cynomolgus macaques, a non-human primate species widely used in drug metabolism studies, remain to be investigated. In this study, 12 UGT1A cDNAs (UGT1A1, 1A2, 1A4A, 1A4B, 1A5A, 1A5B, 1A5C, 1A6, 1A7, 1A8, 1A9, and 1A10) were isolated and characterized in cynomolgus macaques. UGT1A5C cDNA did not contain a complete coding region due to nonsense mutations, and was excluded from further analysis. Amino acid sequences of all 11 cynomolgus UGT1As had high sequence identities (92–95%) with human UGT1As and were phylogenetically close to human UGT1As. These cynomolgus UGT1A genes shared exons 2–5, and contained a variable exon 1 unique to each gene, similar to human UGT1A genes. Moreover, cynomolgus and human UGT1A gene clusters were located in corresponding regions in the genome. Among the 10 tissue types analyzed, cynomolgus UGT1A mRNAs were most abundantly expressed in the liver, jejunum, and/or kidney, the drug-metabolizing organs, similar to human UGT1As. Among these 11 cynomolgus UGT1A mRNAs, cynomolgus UGT1A2, UGT1A9, and UGT1A10 mRNAs were most abundantly expressed in the liver, kidney, and jejunum, respectively. Cynomolgus liver microsomes and UGT1A proteins catalyzed glucuronidation of the substrates human UGT1As catalyze, including 4-methylumbelliferone, 4-nitrophenol, estradiol, trifluoperazine, serotonin, and propofol, although trifluoperazine glucuronidation was not catalyzed by any cynomolgus UGT1A proteins. These results suggest that cynomolgus UGT1As are functional enzymes with molecular similarities to human UGT1As.

Redox Biotransformation and Delivery of Anthracycline Anticancer Antibiotics: How Interpretable Structure-activity Relationships of Lethality Using Electrophilicity and the London Formula for Dispersion Interaction Work.

Quantum chemical methods and molecular mechanics approaches face a lot of challenges in drug metabolism study because of either insufficient accuracy, huge computational cost, or lack of clear molecular level pictures for building computational models. Low-cost QSAR methods can often be carried out, even though molecular level pictures are not well defined; however, they show difficulty in identifying the mechanisms of drug metabolism and delineating the effects of chemical structures on drug toxicity because a certain amount of molecular descriptors are difficult to be interpreted. In order to make a breakthrough of QSAR, mechanistically interpretable molecular descriptors were used to correlate with biological activity to establish structure-activity plots. The biological activity is the lethality of anthracycline anticancer antibiotics denoted as log LD50. The mechanistically interpretable molecular descriptors include electrophilicity and the mathematical function in the London formula for dispersion interaction. The descriptors were calculated using quantum chemical methods. The plots for electrophilicity, which is interpreted as redox reactivity of anthracyclines, can describe oxidative degradation for detoxification and reductive bioactivation for toxicity induction. The plots for the dispersion interaction function, which represents the attraction between anthracyclines and biomolecules, can describe efflux from and influx into the target cells of toxicity. The plots can also identify three structural scaffolds of anthracyclines that have different metabolic pathways, resulting in their different toxicity behavior. This structure-dependent toxicity behavior revealed in the plots can provide perspectives on drug design and drug metabolism study.

Use of hepatocytes isolated from a liver-humanized mouse for studies on the metabolism of drugs: application to the metabolism of fentanyl and acetylfentanyl

PurposeThe usefulness of hepatocytes isolated from a liver-humanized mouse (PXB-cells) as a model in vitro system for the prediction of the in vivo metabolism of new drugs of abuse was evaluated.MethodsFor the drug metabolism study, fentanyl, a powerful synthetic opioid, and acetylfentanyl, an N-acetyl analog of fentanyl, were selected as model drugs. PXB-cells were cultured with the drug for 24–48 h and then the media were collected and analyzed by liquid chromatography/mass spectrometry after deproteinization with acetonitrile.ResultsThe main metabolite formed from fentanyl by PXB-cells was the desphenethylated metabolite (nor-fentanyl), and the other major metabolites formed were 4′-hydroxy-fentanyl, β-hydroxy-fentanyl and (ω-1)-hydroxy-fentanyl. ω-Hydroxy-fentanyl and 4′-hydroxy-3′-methoxy-fentanyl were the minor metabolites. Similar results were obtained for acetylfentanyl. The metabolite profile of fentanyl in PXB-cells was consistent with the in vivo metabolite profile of fentanyl reported previously. Most of the 4′-hydroxy- and 4′-hydroxy-3′-methoxy-metabolites of fentanyl and acetylfentanyl were conjugated in PXB-cells, indicating that PXB-cells had high conjugation enzyme activities. From experiments using human liver microsomes and anti-CYP antibodies, it was revealed that CYP3A4 was involved in the production of nor-fentanyl, β-hydroxy-fentanyl and (ω-1)-hydroxy-fentanyl, while CYP2D6 was partially involved in the production of 4′-hydroxy-fentanyl.ConclusionsOur results indicated that PXB-cells have high activities of phase I and phase II drug-metabolizing-enzymes, can be stably supplied, and are easy to use; thus, PXB-cells are highly useful for the prediction of the in vivo metabolism of drugs of abuse.

Microbial synthesis of mammalian metabolites of spironolactone by thermophilic fungus Thermomyces lanuginosus

Mesophilic fungi are well recognized as models of mammalian drug metabolism. Thermophilic fungi remained unexplored despite having a unique mechanism of growing at higher temperatures and performing wide diverse reactions. The present investigation is directed to isolate a promising thermophilic fungal strain capable of biotransformation using spironolactone as a model drug. Two-stage fermentation protocol was followed for the process. The transformation of spironolactone was identified by HPLC and structure elucidation of the metabolites was done with the help of LC-MS/MS analysis and previous reports. A strain of Thermomyces lanuginosus isolated from decomposed banana peel waste was found to be most promising in transforming spironolactone to 4 metabolites viz.7α-thiospironolactone (M1) canrenone (M2), 7α-thiomethylspironolactone (M3) and 6β-OH-7α-thiomethylspironolactone (M4), the major mammalian metabolites reported previously.The synthesis of metabolites of spironolactone by T. lanuginosus similar to mammals clearly states that this fungus possess enzyme system similar to mammals. Hence, this fungus has the potential to use as a model organism for studying drug metabolism.