Recombinant Cytochrome P450 Research Articles

Ketoprofen in horses: Metabolism, pharmacokinetics, and effects on inflammatory biomarkers.

Ketoprofen is an anti-inflammatory drug that is commonly administered to racehorses for the alleviation of musculoskeletal pain and inflammation. This study represents a comprehensive examination of the metabolism (in vivo and in vitro), pharmacokinetics and ex vivo pharmacodynamics, of ketoprofen in horses. The in vitro metabolism as well as specific enzymes responsible for metabolism was determined by incubating liver microsomes and recombinant CYP450 and UGT enzymes with ketoprofen. For the in vivo portion, 15 horses were administered a single intravenous dose of 2.2-mg/kg ketoprofen. Blood and urine samples were collected prior to and up to 120 h post-drug administration. Additional blood samples were collected at select time points and were stimulated with calcium ionophore or lipopolysaccharide, ex vivo, to induce eicosanoid production. Drug, metabolite, and eicosanoid concentrations were determined using LC-MS/MS. Incubation of ketoprofen with equine liver microsomes generated 3-hydroxy ketoprofen, an unidentified hydroxylated metabolite, and ketoprofen glucuronide. Recombinant equine CYP2C23 produced the greatest amount of hydroxylated ketoprofen and recombinant equine UGT1A2 generated ketoprofen glucuronide. Dihydro, 3-hydroxy, and glucuronide metabolites were identified in blood and urine samples. The Vdss was 0.280, 0.385, and 0.319L/kg for total ketoprofen, S (+) ketoprofen, and R (-) ketoprofen, respectively. The mean half-life was 6.01 h for total ketoprofen, 2.22 h for S (+) ketoprofen, and 1.72 h for R (-) ketoprofen. Stimulation of ketoprofen-treated blood with lipopolysaccharide and calcium ionophore resulted in an inhibition of TXB2 , PGE2 , PGF2alpha , LTB4 , and 15(s)-HETE production for up to 120 h post-drug administration.

Metabolite profiling and identification of enzymes responsible for the metabolism of hirsutine, a major alkaloid from Uncaria rhynchophylla

The in vitro metabolism of hirsutine was determined using liver microsomes and human recombinant cytochrome P450 enzymes. Under the current conditions, a total of 14 phase I metabolites were tentatively identified. Ketoconazole showed significant inhibitory effect on the metabolism of hirsutine. Human recombinant cytochrome P450 enzyme analysis revealed that metabolism of hirsutine was mainly catalysed by CYP3A4. Our data revealed that hirsutine was metabolised via mono-oxygenation, di-oxygenation, N-oxygenation, dehydrogenation, demethylation and hydrolysis. In glutathione (GSH)-supplemented liver microsomes, four GSH adducts were identified. Hirsutine underwent facile P450-mediated metabolic activation, forming reactive 3-methyleneindolenine and iminoquinone intermediates. This study provided valuable information on the metabolic fates of hirsutine in liver microsomes, which would aid in understanding the hepatotoxicity caused by hirsutine or hirsutine-containing herb preparation.

Effect of Saccharolactone on CYP-mediated Metabolism of Xenobiotics.

Saccharolactone is used as a β-glucuronidase inhibitor in in vitro microsomal and recombinant uridine diphosphoglucuronosyl transferases (rUGTs) incubations to enhance glucuronide pathway and, thereby, formation of glucuronide metabolites. We investigated its effect on CYP mediated metabolism of drugs (compound-174, phenacetin and quinidine) using human liver microsomes (HLM) supplemented with Phase-1 and Phase-2 co-factors. Compounds were incubated in HLM supplemented with co-factors to assess Phase-1 (NADPH) and Phase-2 (NADPH, alamethicin, saccharolactone and UDPGA) metabolism. CYP phenotype assay for compound-174 was conducted in HLM (± 1-ABT) and human recombinant CYP isoforms. CYP inhibition profile of saccharolactone was also generated in HLM. The metabolism of compound-174, phenacetin and quinidine in HLM significantly decreased in reactions containing additional components like alamethicin, saccharolactone and UDPGA and indicated that the addition of saccharolactone inhibited the metabolism. Phenacetin and quinidine are known substrates of CYP1A2 and CYP3A4 isoforms. The metabolism of compound- 174 was significantly inhibited in the presence of 1-ABT in HLM, and CYP3A4 and CYP2C8 isoforms were found to be the predominant isoforms responsible for its metabolism. Further evaluation of CYP inhibition in HLM indicated saccharolactone to be a strong inhibitor of CYP1A2, 2D6, 3A4 and 2C8 isoforms with IC50 values of less than 4 mM. The findings indicated that saccharolactone being a strong inhibitor of CYP1A2, 2D6, 3A4 and 2C8 isoforms (IC50 < 4 mM), resulted in significant inhibition of the metabolism of compound-174, phenacetin and quinidine in HLM and caution should be exercised in using it with proper titration of the concentrations.

Pharmacokinetics of ivermectin metabolites and their activity against Anopheles stephensi mosquitoes

BackgroundIvermectin (22,23-dihydroavermectin B1a: H2B1a) is an endectocide used to treat worm infections and ectoparasites including lice and scabies mites. Furthermore, survival of malaria transmitting Anopheles mosquitoes is strongly decreased after feeding on humans recently treated with ivermectin. Currently, mass drug administration of ivermectin is under investigation as a potential novel malaria vector control tool to reduce Plasmodium transmission by mosquitoes. A “post-ivermectin effect” has also been reported, in which the survival of mosquitoes remains reduced even after ivermectin is no longer detectable in blood meals. In the present study, existing material from human clinical trials was analysed to understand the pharmacokinetics of ivermectin metabolites and feeding experiments were performed in Anopheles stephensi mosquitoes to assess whether ivermectin metabolites contribute to the mosquitocidal action of ivermectin and whether they may be responsible for the post-ivermectin effect.MethodsIvermectin was incubated in the presence of recombinant human cytochrome P450 3A4/5 (CYP 3A4/5) to produce ivermectin metabolites. In total, nine metabolites were purified by semi-preparative high-pressure liquid chromatography. The pharmacokinetics of the metabolites were assessed over three days in twelve healthy volunteers who received a single oral dose of 12 mg ivermectin. Blank whole blood was spiked with the isolated metabolites at levels matching the maximal blood concentration (Cmax) observed in pharmacokinetics study samples. These samples were fed to An. stephensi mosquitoes, and their survival and vitality was recorded daily over 3 days.ResultsHuman CYP3A4 metabolised ivermectin more rapidly than CYP3A5. Ivermectin metabolites M1–M8 were predominantly formed by CYP3A4, whereas metabolite M9 (hydroxy-H2B1a) was mainly produced by CYP3A5. Both desmethyl-H2B1a (M1) and hydroxy-H2B1a (M2) killed all mosquitoes within three days post-feeding, while administration of desmethyl, hydroxy-H2B1a (M4) reduced survival to 35% over an observation period of 3 days. Ivermectin metabolites that underwent deglycosylation or hydroxylation at spiroketal moiety were not active against An. stephensi at Cmax levels. Interestingly, half-lives of M1 (54.2 ± 4.7 h) and M4 (57.5 ± 13.2 h) were considerably longer than that of the parent compound ivermectin (38.9 ± 20.8 h).ConclusionIn conclusion, the ivermectin metabolites M1 and M2 contribute to the activity of ivermectin against An. stephensi mosquitoes and could be responsible for the “post-ivermectin effect”.

Metabolism of ropinirole is mediated by several canine CYP enzymes.

Ropinirole has been shown to provoke vomiting in dogs by activating the dopamine D2-like receptors in the chemoreceptor trigger zone. In humans, ropinirole is metabolized primarily by CYP1A2. Corresponding dog CYP1A2 is known to be a polymorphic enzyme which can cause variation in pharmacokinetics of compounds metabolised via this enzyme. The aim of this study was to understand metabolic clearance of ropinirole in dogs, the enzymes involved in ropinirole metabolism and specially to estimate whether the clearance can be sensitive towards the polymorphism of canine CYP1A2. Metabolism of ropinirole was studied in dog hepatocytes and specific recombinant canine CYP isoforms. Metabolite identification and metabolite formation were evaluated using LC-mass spectrometry. Ropinirole was moderately stable in dog hepatocytes with Clint of 16.3μL/min/million cells, and the metabolites detected were 7-hydroxy ropinirole and respective glucuronide conjugate as well as despropyl ropinirole. Regarding recombinant CYPs either 7-hydroxy ropinirole, despropyl ropinirole or both were detected for each CYP isoform studied. The highest rates of metabolite formation were observed in CYP2B11, CYP2C21, CYP2D15, CYP1A2 and CYP1A1. Fluvoxamine, a fairly selective human CYP1A/CYP2C19 inhibitor inhibited ropinirole metabolism by CYP1A1, CYP1A2, CYP2B11, CYP2C21 and CYP2D15 for 65.8%-100% showing no selectivity towards canine CYP isoforms. Although human metabolism of ropinirole is mainly mediated through CYP1A2, the current study shows that several canine CYP isoforms are able to contribute to the clearance of ropinirole in dogs. This is expected to reduce a possible impact of canine CYP1A2 polymorphism on ropinirole pharmacokinetics.

Identification of Cytochrome P450 Enzymes Responsible for Oxidative Metabolism of Synthetic Cannabinoid (1-Hexyl-1H-Indol-3-yl)-1-naphthalenyl-methanone (JWH-019)

(1-Hexyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-019) is one of the second-generation synthetic cannabinoids which as a group have been associated with severe adverse reactions in humans. Although metabolic activation can be involved in the mechanism of action, the metabolic pathway of JWH-019 has not been fully investigated. In the present study, we aimed to identify the enzymes involved in the metabolism of JWH-019. JWH-019 was incubated with human liver microsomes (HLMs) and recombinant cytochrome P450s (P450s or CYPs). An animal study was also conducted to determine the contribution of the metabolic reaction to the onset of action. Using an ultra-performance liquid chromatography system connected to a single-quadrupole mass detector, we identified 6-OH JWH-019 as the main oxidative metabolite in HLMs supplemented with NADPH. JWH-019 was extensively metabolized to 6-OH JWH-019 in HLMs with the KM and Vmax values of 31.5 µM and 432.0 pmol/min/mg. The relative activity factor method estimated that CYP1A2 is the primary contributor to the metabolic reaction in the human liver. The animal study revealed that JWH-019 had a slower onset of action compared to natural and other synthetic cannabinoids. CYP1A2 mediates the metabolic activation of JWH-019, contributing to the slower onset of its pharmacological action.

Metabolism and toxicity of usnic acid and barbatic acid based on microsomes, S9 fraction, and 3T3 fibroblasts in vitro combined with a UPLC-Q-TOF-MS method.

Introduction: Usnic acid (UA) and barbatic acid (BA), two typical dibenzofurans and depsides in lichen, have a wide range of pharmacological activities and hepatotoxicity concerns. This study aimed to clarify the metabolic pathway of UA and BA and illuminate the relationship between metabolism and toxicity. Methods: An UPLC-Q-TOF-MS method was developed for metabolite identification of UA and BA in human liver microsomes (HLMs), rat liver microsomes (RLMs), and S9 fraction (RS9). The key metabolic enzymes responsible for UA and BA were identified by enzyme inhibitors combined with recombinant human cytochrome P450 (CYP450) enzymes. The cytotoxicity and metabolic toxicity mechanism of UA and BA were determined by the combination model of human primary hepatocytes and mouse 3T3 fibroblasts. Results: The hydroxylation, methylation, and glucuronidation reactions were involved in the metabolic profiles of UA and BA in RLMs, HLMs, and RS9. CYP2C9, CYP3A4, CYP2C8, and UGT1A1 are key metabolic enzymes responsible for metabolites of UA and CYP2C8, CYP2C9, CYP2C19, CYP1A1, UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 for metabolites of BA. UA and BA did not display evident cytotoxicity in human primary hepatocytes at concentrations of 0.01-25 and 0.01-100 µM, respectively, but showed potential cytotoxicity to mouse 3T3 fibroblasts with 50% inhibitory concentration values of 7.40 and 60.2 µM. Discussion: In conclusion, the attenuated cytotoxicity of BA is associated with metabolism, and UGTs may be the key metabolic detoxification enzymes. The cytotoxicity of UA may be associated with chronic toxicity. The present results provide important insights into the understanding of the biotransformation behavior and metabolic detoxification of UA and BA.

Identification and kinetics of microsomal and recombinant equine liver cytochrome P450 enzymes responsible for in vitro metabolism of omeprazole

In humans, omeprazole is metabolised by cytochrome P450 (CYP450) CYP2C19 and CYP3A4 with differences in CYP2C19 genotypes leading to variable response to therapy. Despite a wide use of omeprazole in horses with evidence of variable therapeutic efficiency, information regarding enzymatic metabolism is not currently available. This study aims to describe the in vitro kinetics of omeprazole metabolism and determine which enzyme(s) are responsible for omeprazole metabolism in horses. Omeprazole (0–800 uM) was incubated with liver microsomes and a panel of equine recombinant CYP450s (eq-rCYP). Metabolite concentrations were quantified by LC-MS and the kinetics of metabolites’ formation were calculated by non-linear regression analysis. The in vitro liver microsomes formed three metabolites (5-hydroxy-omeprazole, 5-O-desmethyl-omeprazole and omeprazole-sulfone). The 5-O-desmesthyl-omeprazole formation was best fitted to a two enzyme Michaelis-Menten (MM) model with the high affinity site Clint double that of the low affinity site. For 5-hydroxy-omeprazole the best fit was to a 1 enzyme MM model with a Clint higher than for 5-O-desmesthyl-omeprazole (0.12 vs 0.09 pmol/min/pmol P450). The formation of omeprazole-sulfone was negligible. Recombinant CYP3A89 and CYP3A97 produced substantial amounts of 5-hydroxy-omeprazole (1551.72 ng/mL and 1665.33 ng/mL, respectively), while 5-O-desmethyl-omeprazole and omeprazole-sulfone were formed to a much lesser extent by multiple eq-rCYP from the CYP2C and CYP3A family. In vitro metabolism of omeprazole in horses is different to that in humans, with major metabolites produced by the CYP3A family. The current study provides the basis for further investigations of CYP450 single nucleotide polymorphisms that could affect omeprazole metabolism and therapeutic efficacy.

Structural modeling of cytochrome P450 51 from a deep-sea fish points to a novel structural feature in other CYP51s

Cytochromes P450 (CYP), enzymes involved in the metabolism of endogenous and xenobiotic substrates, provide an excellent model system to study how membrane proteins with unique functions have catalytically adapted through evolution. Molecular adaptation of deep-sea proteins to high hydrostatic pressure remains poorly understood. Herein, we have characterized recombinant cytochrome P450 sterol 14α-demethylase (CYP51), an essential enzyme of cholesterol biosynthesis, from an abyssal fish species, Coryphaenoides armatus. C. armatus CYP51 was heterologously expressed in Escherichia coli following N-terminal truncation and purified to homogeneity. Recombinant C. armatus CYP51 bound its sterol substrate lanosterol giving a Type I binding spectra (KD 15 μM) and catalyzed lanosterol 14α-demethylation turnover at 5.8 nmol/min/nmol P450. C. armatus CYP51 also bound the azole antifungals ketoconazole (KD 0.12 μM) and propiconazole (KD 0.54 μM) as determined by Type II absorbance spectra. Comparison of C. armatus CYP51 primary sequence and modeled structures with other CYP51s identified amino acid substitutions that may confer an ability to function under pressures of the deep sea and revealed heretofore undescribed internal cavities in human and other non-deep sea CYP51s. The functional significance of these cavities is not known. PrologueThis paper is dedicated in memory of Michael Waterman and Tsuneo Omura, who as good friends and colleagues enriched our lives. They continue to inspire us.

Estimation of contribution of CYP2D6 to tipepidine metabolism in humans and prolongation of the half-life of tipepidine by combination use with a CYP2D6 inhibitor in chimeric mice with humanised liver

Recently, it has been reported that tipepidine has various central pharmacological effects and can be expected to be safely repositioned as a treatment for psychiatric disorders. Since tipepidine has a very short half-life and requires three doses per day, the development of a once-daily medication would be highly beneficial to improve adherence and quality of life in patients with chronic psychiatric disorders. The aim of this study was to identify the enzymes involved in tipepidine metabolism and to verify that combination use with an enzyme inhibitor prolongs the half-life of tipepidine. Metabolism studies using recombinant human cytochrome P450 (P450, CYP) isoforms and inhibition studies using various selective P450 inhibitors and human liver microsomes revealed that CYP2D6 is the main enzyme catalysing tipepidine metabolism, with a metabolic contribution ratio of 85.4%. Furthermore, a pharmacokinetic study using chimeric mice with humanised liver showed that oral coadministration of a CYP2D6 inhibitor, quinidine, increased the C max, AUC 0-t, and t 1/2 of tipepidine by 1.5-, 3.2-, and 3.0-fold, respectively. These results indicated that coadministration of a CYP2D6 inhibitor is effective in increasing plasma exposure and prolonging the half-life of tipepidine and is useful for repositioning tipepidine as a treatment for psychiatric disorders.

Investigation on the in vitro metabolism of bicyclol using liver microsomes, hepatocytes and human recombinant cytochrome P450 enzymes

In vitro metabolism of bicyclol was studied using liver microsomes, hepatocytes and human recombinant cytochrome P450 enzymes. Liquid chromatography-benchtop orbitrap mass spectrometry technique was utilised to identify the metabolites. A total of 19 metabolites, including 5 new metabolites (M2, M3, M4, M5 and M16) were tentatively identified. Among these metabolites, M6&M8 (demethylenation), M9&M10 (demethylation) and M19 (glucuronidation) were the major metabolites. In glutathione (GSH)-supplemented liver microsomes, 5 new GSH conjugates were found and tentatively identified. The formation was assumed to be through demethylenation of methylenedioxyphenyl to form catechol derivatives, which further underwent oxidation to form ortho-quinone intermediates, reacting with GSH to form stable adducts. CYP3A4 and 2C19 were demonstrated to be the major enzymes responsible for the bioactivation of bicyclol. This study provided valuable information on the metabolic fate of bicyclol in liver microsomes and hepatocytes, and the bioactivation pathways were reported for the first time, which would be helpful for us to understand the potential drug-drug interactions and the possible side effect of this drug.

Combined contributions of cytochrome P450s (CYPs) and non-enzymatic metabolism in the in vitro biotransformation of anaprazole, a novel proton pump inhibitor.

Anaprazole, a new proton pump inhibitor (PPI), is designed for the treatment of acid-related diseases, such as gastric ulcers and gastroesophageal reflux. This study explored the in vitro metabolic transformation of anaprazole. The metabolic stabilities of anaprazole in human plasma and human liver microsomes (HLM) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Then, the contribution (%) of non-enzymatic and cytochrome P450s (CYPs) enzyme-mediated anaprazole metabolism was assessed. To obtain the metabolic pathways of anaprazole, the metabolites generated in HLM, thermal deactivated HLM, and cDNA-expressed recombinant CYPs incubation systems were identified by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS). Results showed thatanaprazole was very stable in human plasma and unstable in HLM. The contribution (%) of non-enzymatic vs. CYPs enzyme-mediated metabolism was 49% vs. 51%. CYP3A4 was the major enzyme (48.3%), followed by CYP2C9 (17.7%) and CYP2C8 (12.3%), in responsible for the metabolism of anaprazole. Specific chemical inhibitors targeting CYP enzymes notably blocked the metabolic transformation of anaprazole. Six metabolites of anaprazole were identified in the non-enzymatic system, whereas 17 metabolites were generated in HLM. The biotransformation reactions mainly included sulfoxide reduction to thioether, sulfoxide oxidation to sulfone, deoxidation, dehydrogenation, O-dealkylation or O-demethylation of thioether, O-demethylation and dehydrogenation of thioether, O-dealkylation and dehydrogenation of thioether, thioether O-dealkylation and dehydrogenation of thioether, and O-dealkylation of sulfone. Both enzymatic and non-enzymatic metabolisms contribute to the clearance of anaprazole in human. Anaprazole is less likely to develop drug-drug interactions in clinical use compared to other PPIs.

Heterologous Expression of Recombinant Human Cytochrome P450 (CYP) in Escherichia coli: N-Terminal Modification, Expression, Isolation, Purification, and Reconstitution.

Cytochrome P450 (CYP) enzymes play important roles in metabolising endogenous and xenobiotic substances. Characterisations of human CYP proteins have been advanced with the rapid development of molecular technology that allows heterologous expression of human CYPs. Among several hosts, bacteria systems such as Escherichia coli (E. coli) have been widely used thanks to their ease of use, high level of protein yields, and affordable maintenance costs. However, the levels of expression in E. coli reported in the literature sometimes differ significantly. This paper aims to review several contributing factors, including N-terminal modifications, co-expression with a chaperon, selections of vectors and E. coli strains, bacteria culture and protein expression conditions, bacteria membrane preparations, CYP protein solubilizations, CYP protein purifications, and reconstitution of CYP catalytic systems. The common factors that would most likely lead to high expression of CYPs were identified and summarised. Nevertheless, each factor may still require careful evaluation for individual CYP isoforms to achieve a maximal expression level and catalytic activity. Recombinant E. coli systems have been evidenced as a useful tool in obtaining the ideal level of human CYP proteins, which ultimately allows for subsequent characterisations of structures and functions.

Metabolism and pharmacokinetic study of deuterated osimertinib.

Osimertinib is a highly selective third-generation irreversible inhibitor of epidermal growth factor receptor mutant, which can be utilized to treat non-small cell lung cancer. As the substrate of cytochrome P450 enzyme, it is mainly metabolized by the CYP3A enzyme in humans. Among the metabolites produced by osimertinib, AZ5104, and AZ7550, which are demethylated that is most vital. Nowadays, deuteration is a new design approach for several drugs. This popular strategy is deemed to improve the pharmacokinetic characteristics of the original drugs. Therefore, in this study the metabolism profiles of osimertinib and its deuterated compound (osimertinib-d3) in liver microsomes and human recombinant cytochrome P450 isoenzymes and the pharmacokinetics in rats and humans were compared. After deuteration, its kinetic isotope effect greatly inhibited the metabolic pathway that produces AZ5104. The plasma concentration of the key metabolite AZ5104 of osimertinib-d3 in rats and humans decreased significantly compared with that of the osimertinib. This phenomenon was consistent with the results of the metabolism studies in vitro. In addition, the in vivo results indicated that osimertinib-d3 had higher systemic exposure (AUC) and peak concentration (Cmax ) compared with the osimertinib in rats and human body.

Primary alcohols as substrates or products in whole-cell biocatalysis: Toxicity for Escherichia coli expression strains

Whole-cell biotransformations, either with resting or growing cells, are a method of choice for reactions that involve complex settings, such as multi-enzyme systems, or require complex and costly cofactors. Alcohols may play a central role as reagents, reaction intermediates or products in such a setting but are, as many other organic solvents, toxic to microorganisms. Albeit much is known on the effect of short- and long-chain alcohols on e.g. membrane fluidity, a systematic study on the influence of alcohols with different chain lengths on viability under biotechnological conditions is missing. We close this gap and report on the effects that alcohols exert in typical microbial expression strains. Towards this aim, we tested four different expression strains, Pseudomonas putida KT2440, Escherichia coli (E. coli) BL21(DE3) and Top 10 as well as Bacillus subtilis str. 168, for their tolerance towards short to middle-length aliphatic alcohols. For growing cells in complex media, the toxicity of primary alcohols rose with the chain length with tolerated concentrations of less than 1 vol% for compounds with 5 or more carbon atoms, in good accordance with reported logPO/W-correlated toxicity development. Interestingly, higher alcohol concentrations could be tolerated by growing cells at higher cell density and in resting-cell setups. In an exemplary whole-cell biotransformation to produce 1-hexanol from n-hexane by a recombinant cytochrome P450 monooxygenase system in E. coli Top10, this knowledge is then applied. Our study thus emphasizes the need of careful substrate and product dosing in whole-cell setups to avoid lethal and inactivating effects caused by the substances.

Molecular docking and in vitro evaluations reveal the role of human cytochrome P450 3A4 in the cross-coupling metabolism of phenolic xenobiotics

Metabolism generally transforms xenobiotics into more polar and hydrophilic products, facilitating their elimination from the body. Recently, a new metabolic pathway that transforms phenolic xenobiotics into more lipophilic and bioactive dimer products was discovered. To elucidate the role of cytochrome P450 (CYP) enzymes in mediating this cross-coupling metabolism, we used high-throughput screening to identify the metabolites generated from the coupling of 20 xenobiotics with four endogenous metabolites in liver microsomes. Endogenous vitamin E (VE) was the most reactive metabolite, as VE reacted with seven phenolic xenobiotics containing various structures (e.g., an imidazoline ring or a diphenol group) to generate novel lipophilic ethers such as bakuchiol-O-VE, phentolamine-O-VE, phenylethyl resorcinol-O-VE, 2-propanol-O-VE, and resveratrol-O-VE. Seven recombinant CYP enzymes were successfully expressed and purified in Escherichia coli. Integration of the results of recombinant human CYP incubation and molecular docking identified the central role of CYP3A4 in the cross-coupling metabolic pathway. Structural analysis revealed the π–π interactions, hydrogen bonds, and hydrophobic interactions between reactive xenobiotics and VE in the malleable active sites of CYP3A4. The consistency between the molecular docking results and the in vitro human cytochrome P450 evaluation shows that docking calculations can be used to screen molecules participating in cross-coupling metabolism. The results of this study provide supporting evidence for the overlooked toxicological effects induced by direct reactions between xenobiotics and endogenous metabolites during metabolic processes.

Characteristics of cytochrome P450-dependent metabolism against acetamiprid in the musk shrew (Suncus murinus)

Soricidae spp. (shrews) play an essential role in soil ecosystems and, due to their habitat and behavior, are exposed to soil pollutants, such as pesticides. Still, toxicity risk in Soricidae spp. has not been appropriately assessed. In this study, the musk shrew (Suncus murinus) was used as a model organism for toxicity assessment in Soricidae. Considering their carnivorous diet, it is reasonable to assume that the musk shrew has unique metabolic traits that are different from those of other common experimental models. This study describes the cytochrome P450 (CYP)-dependent metabolism affected by acetamiprid (ACP), a neonicotinoid insecticide. Pharmacokinetics analysis, an in vitro metabolic assay, and genetic analysis of CYP were performed and compared with data from mice and rats. Through phylogenetic and syntenic analyses, three families of CYP were identified in the musk shrew. Pharmacokinetic analysis showed that the blood concentration of ACP decreased more quickly in musk shrews than in mice. Moreover, the in vitro metabolic assay suggested more efficient metabolic responses toward ACP in musk shrews than in mice or rats. One of the CYP2A isoforms in musk shrews might be linked to a better ACP metabolism. From the results above, we describe novel metabolic traits of the musk shrew. Future research on recombinant CYP enzymes is necessary to fully understand CYP-dependent metabolism of xenobiotics in musk shrews.

1716. In Vitro Drug-Drug Interaction Evaluation of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor

Abstract Background Epetraborole (EBO) is a boron-containing oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. EBO is being developed for the treatment of Mycobacterium avium complex lung disease patients and will be used in combination with other drugs. Therefore, EBO and its major circulating metabolite M3 were evaluated in a comprehensive drug-drug interactions (DDI) risk assessment. Methods Stability of EBO was evaluated in human liver microsomes, hepatocytes and recombinant CYP enzymes. The inhibitory potential of EBO (0.03-100 μM) and M3 (1-1000 μM) on cytochrome P450 (CYP) activities was assessed using human hepatic microsomes. The CYP induction potential of EBO (25-200 μM), M3 was evaluated and compared to prototypical inducers in human hepatocytes (three donors) and mRNA. Fold increase in mRNA expression was utilized to investigate the CYP induction potential of EBO (0.3-100 µM) and M3 (1-250 µM). Stably transfected cell lines that expressed individual transporters were used to determine whether EBO or M3 were substrates or inhibitors for these proteins. Results In vitro studies with microsomes, hepatocytes and recombinant cytochrome P450 (CYP) enzymes indicated that EBO was a poor substrate for major CYP enzymes; drug interactions with epetraborole as victim are considered unlikely. Neither EBO nor its major metabolite M3 was a potent reversible or time-dependent inhibitor of major CYP enzymes. Half maximal inhibitory concentration (IC50) values for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 were &amp;gt;100 µM. EBO was not an inducer of CYP1A2 mRNA in human hepatocytes from three donors, while it was a weak inducer of CYP2B6 and CYP3A4. No induction of mRNA was observed in human hepatocytes at concentrations relevant to planned clinical doses. EBO is unlikely to be a substrate for P-gp, BCRP, OATP1B1, OATP1B3, OAT1, OAT3, MATE1 and MATE2K. EBO was an in vitro substrate for OCT2, a transporter involved in active renal secretion. At clinically relevant concentrations, neither EBO nor M3 inhibited major human efflux or uptake transporters. Conclusion At clinically relevant concentrations of EBO and its major circulating metabolite M3, there is a low risk of victim or perpetrator DDI. Disclosures Afshin Shafiee, PhD, AN2 Therapeutics: Stocks/Bonds Bradley K. Wong, PhD, AN2 Therapeutics: Advisor/Consultant Sanjay Chanda, PhD, AN2 Therapeutics: Stocks/Bonds.

Hydroxychloroquine is Metabolized by Cytochrome P450 2D6, 3A4, and 2C8, and Inhibits Cytochrome P450 2D6, while its Metabolites also Inhibit Cytochrome P450 3A in vitro.

This study aimed to explore the cytochrome P450 (CYP) metabolic and inhibitory profile of hydroxychloroquine (HCQ). Hydroxychloroquine metabolism was studied using human liver microsomes (HLMs) and recombinant CYP enzymes. The inhibitory effects of HCQ and its metabolites on nine CYPs were also determined in HLMs, using an automated substrate cocktail method. Our metabolism data indicated that CYP3A4, CYP2D6, and CYP2C8 are the key enzymes involved in HCQ metabolism. All three CYPs formed the primary metabolites desethylchloroquine (DCQ) and desethylhydroxychloroquine (DHCQ) to various degrees. Although the intrinsic clearance (CLint) value of HCQ depletion by recombinant CYP2D6 was > 10-fold higher than that by CYP3A4 (0.87 versus 0.075 µl/min/pmol), scaling of recombinant CYP CLint to HLM level resulted in almost equal HLM CLint values for CYP2D6 and CYP3A4 (11 and 14 µl/min/mg, respectively). The scaled HLM CLint of CYP2C8 was 5.7 µl/min/mg. Data from HLM experiments with CYP-selective inhibitors also suggested relatively equal roles for CYP2D6 and CYP3A4 in HCQ metabolism, with a smaller contribution by CYP2C8. In CYP inhibition experiments, HCQ, DCQ, DHCQ, and the secondary metabolite didesethylchloroquine were direct CYP2D6 inhibitors, with 50% inhibitory concentration (IC50) values between 18 and 135 µM. HCQ did not inhibit other CYPs. Furthermore, all metabolites were time-dependent CYP3A inhibitors (IC50 shift 2.2-3.4). To conclude, HCQ is metabolized by CYP3A4, CYP2D6, and CYP2C8 in vitro. HCQ and its metabolites are reversible CYP2D6 inhibitors, and HCQ metabolites are time-dependent CYP3A inhibitors. These data can be used to improve physiologically-based pharmacokinetic models and update drug-drug interaction risk estimations for HCQ. SIGNIFICANCE STATEMENT: While CYP2D6, CYP3A4, and CYP2C8 have been shown to mediate chloroquine biotransformation, it appears that the role of CYP enzymes in hydroxychloroquine (HCQ) metabolism has not been studied. In addition, little is known about the CYP inhibitory effects of HCQ. Here, we demonstrate that CYP2D6, CYP3A4, and CYP2C8 are the key enzymes involved in HCQ metabolism. Furthermore, our findings show that HCQ and its metabolites are inhibitors of CYP2D6, which likely explains the previously observed interaction between HCQ and metoprolol.

The in vitro/in vivo metabolic pathways analysis of lobetyol, lobetyolin, and lobetyolinin, three polyacetylenes from Codonopsis Radix, by UHPLC-Q/TOF-MS and UHPLC-MS/MS

Polyacetylenes, lobetyol, lobetyolin and lobetyolinin, are responsible for antitumor, antioxidant, anti-inflammatory, immunomodulatory activities of Codonopsis Radix. However, their metabolic pathways are still unknown. The study was purposed to investigate the metabolism of three polyacetylenes in vitro and in vivo by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Moreover, a rapid, sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous quantitative and semi-quantitative determination of lobetyol and its 12 metabolites to investigate the metabolic stability and metabolic phenotypes. A total of 47, 30 and 34 metabolites of lobetyol, lobetyolin and lobetyolinin were found in all samples. These metabolites are produced through extensive pathways, mainly involving oxidation, glucuronidation and glutathione conjugation. Lobetyol showed good metabolic stability in liver microsomes. The results of both recombinant human CYP enzymes and chemical inhibition experiments confirmed that CYP2C19, 1A1, 2C9, and 1A2 are the major isozymes mediating lobetyol metabolism.