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Background and Objectives: SM-1 is a new synthetic small molecule compound with antitumor activity. The metabolism of SM-1 is a key parameter that needs to be evaluated to provide further insight into drug safety and efficacy in the early phases of drug development. Methods and Results: In this study, the biotransformation process of SM-1, including the metabolic pathways and major metabolites, was investigated based on a liquid chromatography-mass spectrometry method. Upon incubation of SM-1 with human liver microsomes, five metabolites were identified, namely dihydrodiol formation (R1), hydroxylation (R2, R3, and R5), and debenzylation (R4) of SM-1, with R1 and R4 being the major metabolites. The enzyme kinetic parameters of SM-1 were determined by a liquid chromatography-tandem mass spectrometry method. The enzyme kinetics of SM-1 obeyed the Michaelis-Menten equation. The Vmax, Km, and CLint of SM-1 in HLMs were 14.5 nmol/mg protein/h, 6.32 μM, and 2.29 mL/mg protein/h, respectively. Results: The chemical inhibition studies showed that CYP450 isoenzymes were responsible for SM-1 metabolism in HLMs, and CYP3A4 was the major CYP450 isoenzyme involved in the metabolism of SM-1; these findings were confirmed by using the human recombinant CYP3A4. Conclusion: Through the identification of the biotransformation pathways and enzyme kinetics of SM-1, the metabolic enzymes for SM-1 in HLMs are characterized.

Excretion of [14C]HR011303-derived radioactivity showed significant species difference. Urine (81.50% of dose) was the main excretion route in healthy male subjects, whereas feces (87.16% of dose) was the main excretion route in rats. To further elucidate the underlying cause for excretion species differences of HR011303, studies were conducted to uncover its metabolism and excretion mechanism. M5, a glucuronide metabolite of HR011303, is the main metabolite in humans and rats. Results of rat microsomes incubation study suggested that HR011303 was metabolized to M5 in the rat liver. According to previous studies, M5 is produced in both human liver and kidney microsomes. We found M5 in human liver can be transported to the blood by multidrug resistance-associated protein (MRP) 3 and then the majority of M5 can be hydrolyzed to HR011303. HR011303 enters the human kidney or liver through passive diffusion, whereas M5 is taken up through organic anion transporter (OAT) 3, organic anion-transporting polypeptide (OATP) 1B1, and OATP1B3. When HR011303 alone was present, it can be metabolized to M5 in both sandwich-cultured rat hepatocytes (SCRH) and sandwich-cultured human hepatocytes (SCHH) and excreted into bile as M5 in SCRH. Using transporter inhibitors in sandwich-cultured model and membrane vesicles that expressing MRP2 or Mrp2, we found M5 was substance of MRP2/Mrp2 and the bile efflux of M5 mainly mediated by MRP2/Mrp2. Considering the significant role of MRP3/Mrp3 and MRP2/Mrp2 in the excretion of glucuronides, the competition between them for M5 was possibly the determinant for the different excretion routes in humans and rats. Significance Statement Animal experiments are necessary to predict dosage and safety of candidate drugs prior to clinical trials. However, extrapolation results often differ from actual situation. For HR011303, excretory pathways exhibited a complete reversal, through urine in humans and feces in rats. Such phenomena have been observed in several drugs, but no in-depth studies have been conducted to date. In the present study, the excretion species differences of HR011303 can be explained by the competition for M5 between MRP2/Mrp2 and MRP3/Mrp3.

8-methylene-tert-butylamine-3',5,7-trihydroxy-4'-methoxyflavanone (MTBH), a novel hesperidin derivative, has potential in the prevention of hepatic disease, however, its effects on cytochrome P450 isoforms (P450s) remains unexplored. The purpose was to investigate the effects of MTBH on the mRNA, protein levels and activities of six P450s (1A2, 2C11/9, 2D2/6, 3A1/4, 2C13/19 and 2E1) in vitro and in vivo.In vitro study, rat and human liver microsomes were adopted to elucidate the inhibitory effect of MTBH on six P450s using probe drugs. In vivo study, Sprague-Dawley male rats were treated with MTBH (25, 50 or 100 mg/kg for 28 consecutive days), phenobarbital (80 mg/kg for 12 consecutive days) or 0.5% CMC-Na solution (control group) by intragastric administration, then, the mRNA, protein levels and activities of liver P450s were analyzed by real-time PCR, western blotting and probe-drug incubation systems, respectively.The in vitro study indicated that MTBH inhibits the activities of CYP3A1/4 and CYP2E1 in rat and human liver microsomes. In vivo data showed that MTBH inhibits mRNA, protein levels and activities of CYP3A1 and CYP2E1 in medium- and high-dose MTBH groups.MTBH has the potential to cause drug-drug interactions when co-administered with drugs that are metabolized by CYP3A1/4 and CYP2E1.

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.

Mexedrone is a synthetic cathinone structurally related to mephedrone, which belongs to the class of N-alkyl cathinone derivatives, whose metabolic profile has not been fully clarified yet. This study considers the in vitro phase I metabolism of mexedrone, to pre-select the most appropriate marker(s) of intake. Mexedrone was incubated in the presence of either human liver microsomes or single recombinant CYP450 isoforms. The metabolic profile was outlined by ultra-high-performance liquid chromatography coupled to both high- and low-resolution mass spectrometry. In detail, the phase I metabolic profile of mexedrone was initially defined by a time-of-flight analyzer, while the chemical structures of the detected metabolites and the potential presence of minor metabolites were subsequently studied by tandem mass spectrometry, using a triple quadrupole analyzer. The main phase I metabolic reactions were hydroxylation and N- and O-dealkylation. The CYP450 isoforms most involved were CYP2C19, responsible for the formation of both hydroxylated and dealkylated metabolites, followed by CYP2D6 and CYP1A2, involved in the hydroxylation reactions only. Finally, a significant fraction of mexedrone unchanged was also detected. Based on this evidence, the most appropriate markers of intake are mexedrone unchanged and the hydroxylated metabolites.

Venlafaxine (VLF), an antidepressant agent, is widely used to combat major depressive disorders, particularly for the treatment of selective serotonin reuptake inhibitor-resistant depression. VLF has been shown to cause liver injury. The present study aimed to investigate the metabolic activation of VLF and explore the mechanisms of hepatotoxicity induced by VLF. One glutathione (GSH) conjugate and one cysteine conjugate were both detected in mouse and human liver microsomal incubations containing VLF and GSH or cysteine. The two conjugates were also detected in cultured mouse primary hepatocytes and bile of rats after exposure to VLF. The in vitro and in vivo studies demonstrated that VLF was metabolized to a quinone methide intermediate reactive to GSH and cysteine residues of hepatic protein. The observed protein covalent binding revealed dose-dependency. The metabolic activation of VLF was P450-dependent, and CYP3A4 was found as the predominant enzyme involved in the bioactivation process. These findings facilitate better understanding of the metabolic activation-hepatotoxicity relationship of VLF and provide chemists with information about new potential structural alerts during drug design process.

Zolmitriptan (ZOL), a member of triptans, has been used for the treatment of migraine with definite therapeutic effects. However, several cases of liver injury associated with ZOL have been reported and the underlying mechanisms remain unclear.The present study aimed to investigate the metabolic activation of ZOL in vitro and in vivo. ZOL-derived glutathione (GSH) and N-acetyl cysteine (NAC) conjugates were detected in rat liver microsomal incubations. In addition, the GSH and NAC conjugates were also found in bile and urine of rats given ZOL, respectively.ZOL-derived GSH conjugate M1 was also observed in ZOL-treated rat primary hepatocytes, and the formation of M1 was inhibited by pre-cultured with quinidine (a selective inhibitor of CYP2D6). Combining with recombinant P450 enzymes incubations, we found that CYP2D6 was the predominant enzyme responsible for the metabolic activation of ZOL.ZOL can be metabolized to an α,β-unsaturated imine intermediate by CYP2D6. Pre-treatment of primary hepatocytes with quinidine was able to reverse ZOL-induced cytotoxicity. The finding facilitates the understanding of the mechanisms involved in ZOL-associated liver adverse reactions.

In vitro enzyme kinetics and inhibition data was compared for UGT1A1 and UGT1A3 isoforms under similar assay conditions using human liver microsomes (HLM), human intestinal microsomes (HIM) and recombinant UGT (rUGT) enzyme systems.UGT1A1 catalysed ��-estradiol 3-��-D-glucuronide formation showed allosteric sigmoidal kinetics in all enzyme systems; while UGT1A3 catalysed CDCA 24-acyl-��-D-glucuronide formation exhibited Michaelis���Menten kinetics in HLM, substrate inhibition kinetics in HIM and rUGT systems. Corresponding Km or S50 concentrations of ��-estradiol and CDCA were employed in the respective UGT inhibition studies.Atazanavir inhibited the production of ��-estradiol 3-��-D-glucuronide with IC50 values of 0.54 ��M and 0.16 ��M in HLM and rUGT1A1, respectively. But its inhibition potential was not observed in HIM, indicating potential cross-talk with other high-affinity intestinal UGT isozymes. On the other hand, zafirlukast, a pan UGT inhibitor, exhibited moderate inhibition in HIM with an IC50 value of 16.70 ��M. Lithocholic acid, inhibited the production of CDCA 24-acyl-��-D-glucuronide with IC50 values of 1.68, 1.84, and 12.42 ��M in HLM, rUGT1A3, and HIM, respectively.These results indicated that HLM, HIM, and rUGTs may be used as complementary in vitro systems to evaluate hepatic and intestinal UGT mediated DDIs at the screening stage. In vitro enzyme kinetics and inhibition data was compared for UGT1A1 and UGT1A3 isoforms under similar assay conditions using human liver microsomes (HLM), human intestinal microsomes (HIM) and recombinant UGT (rUGT) enzyme systems. UGT1A1 catalysed ��-estradiol 3-��-D-glucuronide formation showed allosteric sigmoidal kinetics in all enzyme systems; while UGT1A3 catalysed CDCA 24-acyl-��-D-glucuronide formation exhibited Michaelis���Menten kinetics in HLM, substrate inhibition kinetics in HIM and rUGT systems. Corresponding Km or S50 concentrations of ��-estradiol and CDCA were employed in the respective UGT inhibition studies. Atazanavir inhibited the production of ��-estradiol 3-��-D-glucuronide with IC50 values of 0.54 ��M and 0.16 ��M in HLM and rUGT1A1, respectively. But its inhibition potential was not observed in HIM, indicating potential cross-talk with other high-affinity intestinal UGT isozymes. On the other hand, zafirlukast, a pan UGT inhibitor, exhibited moderate inhibition in HIM with an IC50 value of 16.70 ��M. Lithocholic acid, inhibited the production of CDCA 24-acyl-��-D-glucuronide with IC50 values of 1.68, 1.84, and 12.42 ��M in HLM, rUGT1A3, and HIM, respectively. These results indicated that HLM, HIM, and rUGTs may be used as complementary in vitro systems to evaluate hepatic and intestinal UGT mediated DDIs at the screening stage.

[Image: see text] BMS-813160 (compound 3) was identified as a potent and selective CCR2/5 dual antagonist. Compound 3 displayed good permeability at pH = 7.4 in PAMPA experiments and demonstrated excellent human liver microsome stability. Pharmacokinetic studies established that 3 had excellent oral bioavailability and exhibited low clearance in dog and cyno. Compound 3 was also studied in the mouse thioglycollate-induced peritonitis model, which confirmed its ability to inhibit the migration of inflammatory monocytes and macrophages. As a result of this profile, compound 3 was selected as a clinical candidate.

In this work, a series of novel substituted polycyclic pyridone derivatives were designed and synthesized as potent anti-influenza agents. The cytopathic effect (CPE) assay and cytotoxicity assay indicated that all of the compounds possessed potent anti-influenza virus activity and relatively low cytotoxicity; some of them inhibited the replication of influenza A virus (IAV) at picomolar concentrations. Further studies revealed that, at a concentration of 3 nM, three compounds (10a, 10d, and 10g) could significantly reduce the M2 RNA amounts and M2 protein expression of IAV and inhibit the activity of RNA-dependent RNA polymerase (RdRp). Among them, (R)-12-(5H-dibenzo[a,d][7]annulen-5-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (10a) was found to be a promising anti-influenza drug candidate with good human liver microsomal stability, as well as with better selectivity index and oral bioavailability than Baloxavir.

[Image: see text] An indazole/aza-indazole scaffold was developed as a novel chemotype for JNK3 inhibition. Extensive structure activity relationship (SAR) studies utilizing various in vitro and in vivo assays led to potent and highly selective JNK3 inhibitors with good oral bioavailability and high brain penetration. One lead compound, 29, was a potent and selective JNK3 inhibitor (IC(50) = 0.005 μM) that had significant inhibition (>80% at 1 μM) to only JNK3 and JNK2 in a panel profiling of 374 wild-type kinases, had high potency in functional cell-based assays, had high stability in the human liver microsome (t(1/2) = 92 min), and was orally bioavailable and brain penetrant (brain/plasma ratio: 56%). The cocrystal structure of 29 in human JNK3 at a 2.1 Å resolution showed that indazole or aza-indazole-based JNK3 inhibitors demonstrated a type I kinase inhibition/binding.

The UDP-glucuronosyltransferase (UGT) family of enzymes play a central role in the metabolism and detoxification of a wide range of endogenous and exogenous compounds. UGTs exhibit a high degree of structural similarity and display overlapping substrate specificity, often making estimations of potential drug-drug interactions difficult to fully elucidate. One such interaction yet to be examined may be occurring between UGTs and cannabinoids, as the legalization of recreational and medicinal cannabis and subsequent co-usage of cannabis and therapeutic drugs increases in the U.S. and internationally. In the present study, the inhibition potential of the major cannabinoids Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN), as well as their major metabolites, was determined in microsomes isolated from HEK293 cells over-expressing individual recombinant UGTs and in microsomes from human liver and kidney specimens. The highest inhibition was seen by CBD against the glucuronidation activity of UGTs 1A9, 2B4,1A6 and 2B7, with binding- corrected IC50,u values of 0.12 {plus minus} 0.020 µM, 0.22 {plus minus} 0.045 µM, 0.40 {plus minus} 0.10 µM and 0.82 {plus minus} 0.15 µM, respectively. Strong inhibition of UGT1A9 was also demonstrated by THC and CBN, with IC50,u values of 0.45 {plus minus} 0.12 µM and 0.51 {plus minus} 0.063 µM, respectively. Strong inhibition of UGT2B7 was also observed for THC and CBN; no or weak inhibition was observed with cannabinoid metabolites. This inhibition of UGT activity suggests that in addition to playing an important role in drug-drug interactions, cannabinoid exposure may have important implications in patients with impaired hepatic or kidney function. Significance Statement Major cannabinoids found in the plasma of cannabis users inhibit several UGT enzymes, including UGT1A6, UGT1A9, UGT2B4, and UGT2B7. This study is the first to show the potential of cannabinoids and their metabolites to inhibit all major kidney UGTs and the two most abundant UGTs present in liver. This study suggests that as all three major kidney UGTs are inhibited by cannabinoids, greater drug-drug interaction effects might be observed from co-use of cannabinoids and therapeutics that are cleared renally.

Razuprotafib, a sulphamic acid-containing phosphatase inhibitor, is shown in vivo to undergo enzymatic oxidation and methylation to form a major metabolite in monkey and human excreta with an m/z- value of 633.LC-MS/MS analysis of samples derived from incubations of razuprotafib with human liver microsomes and recombinant CYP2C8 enzyme has elucidated the metabolic pathway for formation of the thiol precursor to the S-methyl metabolite MS633 (m/z- 633).Under in vitro conditions, the major pathway of razuprotafib metabolism involves extensive oxidation of the thiophene and phenyl rings.A single oxidation takes place at one of the phenyl groups. Multiple oxidations occur at the thiophene moiety: initial oxidation results in the formation of a thiolactone followed by a second oxidation giving rise to an S-oxide of the thiolactone, which is further metabolised to the ring-opened form and ultimate formation of a thiol (m/z- 619).An additional mono-oxidation pathway involves epoxidation of the thiophene followed by hydrolysis to a diol.The thiol and diol metabolites are trapped by the addition of a nucleophilic trapping agent, 3-methoxyphenacyl bromide (MPB), giving adducts with m/z- 767.The thiol is a likely precursor to the major in vivo razuprotafib metabolite, MS633.

Background: Herbal medicine Angelica dahurica is widely employed for the treatment of rheumatism and pain relief in China. Oxypeucedanin is a major component in the herb. Objectives : The objectives of this study are aimed at the investigation of mechanism-based inactivation of CYP2B6 and CYP2D6 by oxypeucedanin, characterization of the reactive metabolites associated with the enzyme inactivation, and identification of the P450s participating in the bioactivation of oxypeucedanin. Methods : Oxypeucedanin was incubated with liver microsomes or recombinant CYPs2B6 and 2D6 under designed conditions, and the enzyme activities were measured by monitoring the generation of the corresponding products. The resulting reactive intermediates were trapped with GSH and analyzed by LC-MS/MS. Results : Microsomal incubation with oxypeucedanin induced a time-, concentration-, and NADPH-dependent inhibition of CYPs2B6 and 2D6 with kinetic values of KI/kinact 1.82 μM/0.07 min-1 (CYP2B6) and 8.47 μM/0.044 min-1 (CYP2D6), respectively. Ticlopidine and quinidine attenuated the observed time-dependent enzyme inhibitions. An epoxide and/or γ-ketoenal intermediate(s) derived from oxypeucedanin was/were trapped in microsomal incubations. CYP3A4 was the primary enzyme involved in the bioactivation of oxypeucedanin. Conclusion : Oxypeucedanin was a mechanism-based inactivator of CYP2B6 and CYP2D6. An epoxide and/or γ- ketoenal intermediate(s) may be responsible for the inactivation of the two enzymes.

To identify the CYP isoforms involved in the production of 2-hydroxyestradiol 17-sulfate (2-OH-ES), which we assume to be an antioxidant in vivo, the 2-hydroxylation reaction of estradiol 17-sulfate (ES) by human liver microsome was investigated. As a result, it was estimated that CYP2C8 and 2C9 were largely involved in the production of 2-OH-ES. Therefore, the 2-hydroxylation kinetic analysis of ES was performed for both CYPs, and the metabolic clearance Vmax/Km (µL/nmol CYP/min) was determined. On comparing the results of ES with those of estradiol (E2), it was found that CYP2C8 was about 2.5 times higher and CYP2C9 was about 3 times higher, and ES was more likely to be a substrate for the 2-hydroxylation reaction by both CYPs. The CYP isoforms involved in A-ring hydroxylation of E2 and ES differed. From this, it was speculated that 2-OH-ES plays a different role to 2-hydroxyestradiol (2-OH-E2), which is recognized as an antioxidant in the body.

Background Obtusofolin is the major active ingredient of Catsia tora L., which possesses the activity of improving eyesight and protecting the optic nerve. Investigation on the interaction of obtusofolin with cytochrome P450 enzymes (CYP450s) could provide a reference for the clinical application of obtusofolin. Methods The effect of obtusofolin on the activity of CYP450s was investigated in the presence of 100 μM obtusofolin in pooled human liver microsomes (HLMs) and fitted with the Lineweaver–Burk plots to characterize the specific inhibition model and kinetic parameters. Results Obtusofolin was found to significantly inhibited the activity of CYP3A4, 2C9, and 2E1. In the presence of 0, 2.5, 5, 10, 25, 50, and 100 μM obtusofolin, the inhibition of these CYP450s showed a dose-dependent manner with the IC50 values of 17.1 ± 0.25, 10.8 ± 0.13, and 15.5 ± 0.16 μM, respectively. The inhibition of CYP3A4 was best fitted with the non-competitive inhibition model with the Ki value of 8.82 μM. While the inhibition of CYP2C9 and 2E1 was competitive with the Ki values of 5.54 and 7.79 μM, respectively. After incubating for 0, 5, 10, 15, and 30 min, the inhibition of CYP3A4 was revealed to be time-dependent with the KI value of 4.87 μM− 1 and the Kinact value of 0.0515 min− 1. Conclusions The in vitro inhibitory effect of obtusofolin implying the potential drug-drug interaction between obtusofolin and corresponding substrates, which needs further in vivo validations.

Osimertinib is the only third-generation epidermal growth factor receptor tyrosine-kinase inhibitor (EGFR-TKI) approved by Food and Drug Administration (FDA). This study aimed to know the inhibitory effect of osimertinib on human UDP-glucosyltransferases (UGTs) and human liver microsomes (HLMs), as well as to identify its potential to cause drug-drug interaction (DDI) arising from the modulation of UGT activity. High inhibitory effect of osimertinib was shown towards UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A10, 2B7 and 2B15. Especially, osimertinib exhibited competitive inhibition against UGT1A1 with a Ki,u of 0.87 ± 0.12 μM. It also noncompetitively inhibited SN-38 glucuronidation in pooled HLMs with a Ki,u of 3.32 ± 0.25 μM. Results from quantitative prediction study indicated that osimertinib administered at 80 mg/day may result in a 4.83 % increase in the AUC of drugs mainly metabolized by UGT1A1, implying low risk of DDI via liver metabolism. However, the ratios of [I]gut/Ki,u are much higher than 11 in HLMs and recombinant UGT1A1, indicating a risk for interaction in intestine. The effects of osimertinib on intestinal UGT should be paid more attention on to avoid unnecessary clinical DDI risks.

Tenofovir (TFV) is the cornerstone nucleotide reverse transcriptase inhibitor (NtRTI) in many combination antiretroviral therapies prescribed to patients living with HIV/AIDS. Due to poor cell permeability and oral bioavailability, TFV is administered as one of two FDA-approved prodrugs, both of which metabolize prematurely in the liver and/or plasma. This premature prodrug processing depletes significant fractions of each oral dose and causes toxicity in kidney, bone, and liver with chronic administration. Although TFV exalidex (TXL), a phospholipid-derived prodrug of TFV, was designed to address this issue, clinical pharmacokinetic studies indicated substantial hepatic extraction, redirecting clinical development of TXL toward HBV. To circumvent this metabolic liability, we synthesized and evaluated ω-functionalized TXL analogues with dramatically improved hepatic stability. This effort led to the identification of compounds 21 and 23, which exhibited substantially longer t1/2 values than TXL in human liver microsomes, potent anti-HIV activity in vitro, and enhanced pharmacokinetic properties in vivo.

Chlorinated paraffins (CPs) are environmental pollutants of emerging concern. Long-chain CPs (LCCPs) are considered of lesser concern than other CPs in food due to their lower accumulation in most organisms. However, LCCPs have been shown to accumulate preferentially in birds. We used ultrahigh-performance liquid chromatography combined with electrospray ionization Orbitrap mass spectrometry (UPLC-ESI-Orbitrap MS) to analyze CPs (C10-26Cl4-12) in tissues of free-range hens, their feed, and local soils. Feed was found to be the main source of CP intake. The CP carbon chain length had little impact on their absorption. C18-CPs were excreted in preference to C13-CPs by laying. The metabolic elimination rates of CPs (0.2 μg/mL) estimated using chicken liver microsomes were in the order C12Cl6 (91%) > C12Cl8 (57%) > C18Cl6 (12%) > C18Cl8 (6%). CPs with longer carbon chains accumulated preferentially in muscle and adipose tissues, and the accumulation of specific carbon chain lengths was related to the content and composition of different CPs in the intake source.

Atomoxetine (ATX) is a neurological drug widely used for the treatment of attention deficit-hyperactivity disorder. Liver injury has been documented in patients administered ATX. The mechanism of ATX's toxic action is less clear. This study is aimed to characterize reactive metabolites of ATX in vitro and in vivo to assist our understanding of the mechanisms of ATX hepatotoxicity. A hydroxylated metabolite, along with an O-dealkylation metabolite, was found in ATX-supplemented rat liver microsome incubations. Additionally, two glutathione (GSH) conjugates and two N-acetylcysteine (NAC) conjugates were observed in rat liver microsome incubations containing ATX, NADPH, and GSH or NAC. The corresponding GSH conjugates and NAC conjugates were found in bile and urine of ATX-treated rats, respectively. Recombinant P450 enzyme incubation study demonstrated that CYP2D6 dominated the metabolic activation of ATX. The insights gained from this study may be of assistance to illuminate the mechanisms of ATX-induced hepatotoxicity.

Marrubiin, a furanoid compound, is a well-known diterpenoid lactone isolated from Marrubium vulgare, which displays a wide spectrum of pharmacological effects and potential hepatotoxicity. Considering that marrubiin contains a structural alert, furan ring, metabolic activation may be one of the major metabolic pathways, and the reactive metabolite may be involved in the hepatotoxicity. The present study was carried out to investigate the bioactivation mechanism of marrubiin in rats and humans. Marrubiin was initially metabolized into cis-butene-1,4-dial intermediate, which was readily trapped by glutathione (GSH) and N-acetyl-lysine (NAL) in the microsomal incubations supplemented with NADPH. A total of nine conjugates were detected and identified by high-resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. M1-M3 and M6 and M7 were characterized as mono-GSH conjugates, and M4 and M5 were identified as bis-GSH conjugates. M8 and M9 were identified as NAL conjugates. In rat bile, five GSH conjugates (M1-M3; M6 and M7) were detected. M1, M8, and M9 were chemically synthesized, and their structures were characterized by 13C NMR. Sulfaphenazole, ticlopidine, and ketoconazole displayed significant inhibitory effect on the bioactivation of marrubiin. Further phenotyping revealed that CYP2C9, CYP2C19, and CYP3A4 were the primary enzymes catalyzing the bioactivation of marrubiin. The current study provides evidence for the CYP-dominated bioactivation of marrubiin to the corresponding cis-butene-1,4-dial intermediate, which enables us to better understand the potential side effects caused by marrubiin.

1. Leonurine hydrochloride (LH) is derived from an ingredient of Leonurus japonicus Houtt which is widely used for diseases in women. 2. The influence of LH on the activity of cytochrome P450 (CYPs) enzymes was investigated in this study. 3. The effect of LH on CYPs enzyme activities were studied using the enzyme-selective substrates phenacetin (1A2), coumarin (2A6), diclofenac (2C9), S-mephenytoin (2C19), paclitaxel (2C8), dextromethorphan (2D6), chlorzoxazone (2E1) and testosterone (3A4). The IC50 value was calculated to express the strength of inhibition. The inhibition of CYPs was fitted with competitive or non-competitive inhibition models and corresponding parameters were also obtained. 4. LH exerted inhibitory effects on the activity of CYP1A2, 2D6, and 3A4 with the IC50 values of 18.05, 15.13, and 20.09 μM, respectively. The obtained results showed that LH inhibited the activity of CYP1A2 and CYP2D6 via competitive manners (Ki=8.667 μM and Ki=7.805 μM, respectively), while LH attenuated the CYP3A4 activity via a non-competitive manner (Ki=9.507 μM). Moreover, LH showed time-dependent inhibition on CYP3A4 with the KI/Kinact value of 4.31/0.044 min−1·μM−1. 5. The inhibition of CYP1A2, CYP2D6, and CYP3A4 by LH, demonstrated in vitro, indicated the potential herb-drug interaction. Therefore, pharmacokinetic interactions involving LH and CYP1A2 or CYP2D6 or CYP1A2 substrates are likely to occur.

Precision biotransformation is an envisioned strategy offering detailed insights into biotransformation pathways in real environmental settings using experimentally guided high-accuracy quantum chemistry. Emerging pollutants, whose metabolites are easily overlooked but may cause idiosyncratic toxicity, are important targets of such a strategy. We demonstrate here that complex metabolic reactions of tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) catalyzed by human CYP450 enzymes can be mapped via a three-step synergy strategy: (i) screening the possible metabolites via high-throughout (moderate-accuracy) computations; (ii) analyzing the proposed metabolites in vitro by human liver microsomes and recombinant human CYP450 enzymes; and (iii) rationalizing the experimental data via precise mechanisms using high-level targeted computations. Through the bilateral dialogues from qualitative to semi-quantitative to quantitative levels, we show how TDCIPP metabolism especially by CYP3A4 generates bis(1,3-dichloro-2-propyl) phosphate (BDCIPP) as an O-dealkylation metabolite and bis(1,3-dichloro-2-propyl) 3-chloro-1-hydroxy-2-propyl phosphate (alcoholβ-dehalogen) as a dehalogenation/reduction metabolite via the initial rate-determining H-abstraction from αC- and βC-positions. The relative yield ratio [dehalogenation/reduction]/[O-dealkylation] is derived from the relative barriers of H-abstraction at the βC- and αC-positions by CYP3A4, estimated as 0.002 to 0.23, viz., an in vitro measured ratio of 0.04. Importantly, alcoholβ-dehalogen formation points to a new mechanism involving successive oxidation and reduction functions of CYP450, with its precursor aldehydeβ-dehalogen being a key intermediate detected by trapping assays and rationalized by computations. We conclude that the proposed three-step synergy strategy may meet the increasing challenge of elucidating biotransformation mechanisms of substantial synthesized organic compounds in the future.

Nonclinical metabolite profiling of DS-1971a, a potent selective NaV1.7 inhibitor, was performed to predict human metabolites.After the oral administration of radiolabelled DS-1971a, the predominant metabolite in mouse plasma was M4, a monoxide at the pyrimidine ring, while the major metabolites with the first and second highest exposure in monkey plasma were M2, a monoxide at the cyclohexane ring, and M11, a demethylated pyrazole metabolite.Incubation studies with liver cytosolic and microsomal fractions in the absence or presence of NADPH indicated that the metabolising enzyme responsible for M4 formation was aldehyde oxidase (AO), while cytochrome P450s (P450s) were responsible for M2 and M11 formation. These results suggest that DS-1971a is a substrate for both AO and P450.When DS-1971a was incubated with liver S9 fractions and NADPH, the most abundant metabolites were M4 in mice, and M2 and M11 in monkeys, indicating that the results of in vitro incubation studies could provide information reflecting the in vivo plasma metabolite profiles in mice and monkeys. The results obtained from the incubation with the human liver S9 fraction and NADPH suggested that a major circulating metabolite in humans is M1, a regioisomer of M2. Nonclinical metabolite profiling of DS-1971a, a potent selective NaV1.7 inhibitor, was performed to predict human metabolites. After the oral administration of radiolabelled DS-1971a, the predominant metabolite in mouse plasma was M4, a monoxide at the pyrimidine ring, while the major metabolites with the first and second highest exposure in monkey plasma were M2, a monoxide at the cyclohexane ring, and M11, a demethylated pyrazole metabolite. Incubation studies with liver cytosolic and microsomal fractions in the absence or presence of NADPH indicated that the metabolising enzyme responsible for M4 formation was aldehyde oxidase (AO), while cytochrome P450s (P450s) were responsible for M2 and M11 formation. These results suggest that DS-1971a is a substrate for both AO and P450. When DS-1971a was incubated with liver S9 fractions and NADPH, the most abundant metabolites were M4 in mice, and M2 and M11 in monkeys, indicating that the results of in vitro incubation studies could provide information reflecting the in vivo plasma metabolite profiles in mice and monkeys. The results obtained from the incubation with the human liver S9 fraction and NADPH suggested that a major circulating metabolite in humans is M1, a regioisomer of M2.

We previously reported a potent tubulin inhibitor CH-2-77. In this study, we optimized the structure of CH-2-77 by blocking metabolically labile sites and synthesized a series of CH-2-77 analogues. Two compounds, 40a and 60c, preserved the potency while improving the metabolic stability over CH-2-77 by 3- to 4-fold (46.8 and 29.4 vs 10.8 min in human microsomes). We determined the high-resolution X-ray crystal structures of 40a (resolution 2.3 Å) and 60c (resolution 2.6 Å) in complex with tubulin and confirmed their direct binding at the colchicine-binding site. In vitro, 60c maintained its mode of action by inhibiting tubulin polymerization and was effective against P-glycoprotein-mediated multiple drug resistance and taxol resistance. In vivo, 60c exhibited a strong inhibitory effect on tumor growth and metastasis in a taxol-resistant A375/TxR xenograft model without obvious toxicity. Collectively, this work showed that 60c is a promising lead compound for further development as a potential anticancer agent.

1. Nine forms of recombinant cytochrome P450 (P450 or CYP) enzymes were used to study roles of individual P450 enzymes in the oxidation of flavone and some other flavonoids, 4´-hydroxyflavone and 4´-, 3´-, and 2´-methoxyflavones, by human liver microsomes using LC-MS/MS analysis. 2. As has been reported previously (Nagayoshi et al., Xenobiotica 50, 1158-1169, 2020), 4´-, 3´-, and 2´-methoxyflavones were preferentially O-demethylated by human liver P450 enzymes to form 4´-, 3´-, and 2´-hydroxylated flavones and also 3´,4´-dihydroxyflavone from the former two substrates. 3. In comparisons of product formation by oxidation of these methoxylated flavones, CYP2A6 was found to be a major enzyme catalyzing flavone 4´- and 3´-hydroxylations by human liver microsomes but did not play significant roles in 2´-hydroxylation of flavone, O-demethylations of three methoxylated flavones, and the oxidation of 4´-hydroxyflavone to 3´,4´-dihydroxyflavone. 4. The effects of anti-CYP2A6 IgG and chemical P450 inhibitors suggested that different P450 enzymes, as well as CYP2A6, catalyzed oxidation of these flavonoids at different positions by liver microsomes. 5. These studies suggest that CYP2A6 catalyzes flavone 4´- and 3´-hydroxylations in human liver microsomes and that other P450 enzymes have different roles in oxidizing these flavonoids.

Intake of synthetic cannabinoids (SC), one of the largest classes of new psychoactive substances, was reported to be associated with acute liver damage but information about their hepatotoxic potential is limited. The current study aimed to analyze the hepatotoxicity including the metabolism-related impact of JWH-200, A-796260, and 5F-EMB-PINACA in HepG2 cells allowing a tentative assessment of different SC subclasses. A formerly adopted high-content screening assay (HCSA) was optimized using a fully automated epifluorescence microscope. Metabolism-mediated effects in the HCSA were additionally investigated using the broad CYP inhibitor 1-aminobenzotriazole. Furthermore, phase I metabolites and isozymes involved were identified by in vitro assays and liquid chromatography–high-resolution tandem mass spectrometry. A strong cytotoxic potential was observed for the naphthoylindole SC JWH-200 and the tetramethylcyclopropanoylindole compound A-796260, whereas the indazole carboxamide SC 5F-EMB-PINACA showed moderate effects. Numerous metabolites, which can serve as analytical targets in urine screening procedures, were identified in pooled human liver microsomes. Most abundant metabolites of JWH-200 were formed by N-dealkylation, oxidative morpholine cleavage, and oxidative morpholine opening. In case of A-796260, most abundant metabolites included an oxidative morpholine cleavage, oxidative morpholine opening, hydroxylation, and dihydroxylation followed by dehydrogenation. Most abundant 5F-EMB-PINACA metabolites were generated by ester hydrolysis plus additional steps such as oxidative defluorination and hydroxylation. To conclude, the data showed that a hepatotoxicity of the investigated SC cannot be excluded, that metabolism seems to play a minor role in the observed effects, and that the extensive phase I metabolism is mediated by several isozymes making interaction unlikely. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03148-3.

Numerous studies have been reported in the past 50-plus years regarding the stimulatory role of cytochrome b5 (b5) in some but not all microsomal cytochrome P450 (P450, CYP) reactions with drugs and steroids. A missing element in most of these studies has been a sensitive and accurate measure of binding affinities of b5 with P450s. In the course of work with P450 17A1, we developed a fluorescent derivative of a human b5 site-directed mutant, Alexa 488-T70C-b5, that could be utilized in binding assays at sub-µM concentrations. Alexa 488-T70C-b5 bound to human P450s 1A2, 2B6, 2C8, 2C9, 2E1, 2S1, 4A11, 3A4, and 17A1 with estimated Kd values ranging from 2.5 to 61 nM. Only weak binding was detected with P450 2D6, and no fluorescence attenuation was observed with P450 2A6. All of the P450s that bound b5have some reported activity stimulation except for P450 2S1. The affinity of P450 3A4 for b5 was decreased somewhat by the presence of a substrate or inhibitor. The fluorescence of a P450 3A4•Alexa 488-T70C-b5 complex was partially restored by titration with NADPH-P450 reductase (POR) (Kd,apparent 89 nM), suggesting the existence of a ternary P450 3A4-b5-POR complex, as observed previously with P450 17A1. Gel filtration evidence was also obtained for this ternary complex with P450 3A4. Overall, the results indicated that the affinity of b5 for many P450s is very high and that ternary P450-b5-POR complexes are relevant in P450 3A4 reactions, as opposed to a shuttle mechanism. Significance Statement High affinity binding of cytochrome b5 (b5) (Kd

Cytochrome P450 3A (CYP3A) is a frequent target for time-dependent inhibition (TDI) that can give rise to drug-drug interactions (DDI). Yet many drugs that exhibit in vitro TDI for CYP3A do not result in DDI. There were 23 drugs with published clinical DDI evaluated for CYP3A TDI in human liver microsomes (HLM) and hepatocytes (HHEP), and these data were used in static and dynamic models for projecting DDI caused by inactivation of CYP3A in both liver and intestine. TDI parameters measured in HHEP, particularly the maximal rate of enzyme inactivation, were generally lower than those measured in HLM. In static models, the use of estimated average unbound organ exit concentrations offered the most accurate projections of DDI with geometric mean fold errors of 2.0 and 1.7 for HLM and HHEP, respectively. Use of maximum organ entry concentrations yielded marked overestimates of DDI. When evaluated in a binary fashion (i.e., projection of DDI of 1.25-fold or greater), data from HLM offered the greatest sensitivity (100%) and specificity (67%) and yielded no missed DDI when average unbound organ exit concentrations were used. In dynamic physiologically based pharmacokinetic modeling, accurate projections of DDI were obtained with geometric mean fold errors of 1.7 and 1.6 for HLM and HHEP, respectively. Sensitivity and specificity were 100% and 67% when using TDI data generated in HLM and Simcyp modeling. Overall, DDI caused by CYP3A-mediated TDI can be reliably projected using dynamic or static models. For static models, average organ unbound exit concentrations should be used as input values otherwise DDI will be markedly overestimated. SIGNIFICANCE STATEMENT CYP3A time-dependent inhibitors (TDI) are important in the design and development of new drugs. The prevalence of CYP3A TDI is high among newly synthesized drug candidates, and understanding the potential need for running clinical drug-drug interaction (DDI) studies is essential during drug development. Ability to reliably predict DDI caused by CYP3A TDI has been difficult to achieve. We report a thorough evaluation of CYP3A TDI and demonstrate that DDI can be predicted when using appropriate models and input parameters generated in human liver microsomes or hepatocytes.

Ciguatoxins (CTX) are potent marine neurotoxins, which can bioaccumulate in seafood, causing a severe and prevalent human illness known as ciguatera poisoning (CP). Despite the worldwide impact of ciguatera, effective disease management is hindered by a lack of knowledge regarding the movement and biotransformation of CTX congeners in marine food webs, particularly in the Caribbean and Western Atlantic. In this study we investigated the hepatic biotransformation of C-CTX across several fish and mammalian species through a series of in vitro metabolism assays focused on phase I (CYP P450; functionalization) and phase II (UGT; conjugation) reactions. Using liquid chromatography high-resolution mass spectrometry to explore potential C-CTX metabolites, we observed two glucuronide products of C-CTX-1/-2 and provided additional evidence from high-resolution tandem mass spectrometry to support their identification. Chemical reduction experiments confirmed that the metabolites were comprised of four distinct glucuronide products with the sugar attached at two separate sites on C-CTX-1/-2 and excluded the C-56 hydroxyl group as the conjugation site. Glucuronidation is a novel biotransformation pathway not yet reported for CTX or other related polyether phycotoxins, yet its occurrence across all fish species tested suggests that it could be a prevalent and important detoxification mechanism in marine organisms. The absence of glucuronidation observed in this study for both rat and human microsomes suggests that alternate biotransformation pathways may be dominant in higher vertebrates.

Background: Dermorphin is a heptapeptide with an analgesic potential higher than morphine that does not present the same risk for the development of tolerance. These pharmacological features make dermorphin a potential doping agent in competitive sports and is already prohibited for racehorses. For athletes, the development of an efficient strategy to monitor for its abuse necessitates an investigation of the metabolism of dermorphin in humans. Methods: Here, human liver microsomes and zebrafish were utilized as model systems of human metabolism to evaluate the presence and kinetics of metabolites derived from dermorphin. Five hours after its administration, the presence of dermorphin metabolites could be detected in both models by liquid chromatography coupled to high resolution mass spectrometry. Results: Although the two models showed common results, marked differences were also observed in relation to the formed metabolites. Six putative metabolites, based on their exact masses of m/z 479.1915, m/z 501.1733, m/z 495.1657, m/z 223.1073, m/z 180.1017 and m/z 457.2085, are proposed to represent the metabolic pattern of dermorphin. The major metabolite generated from the administration of dermorphin in both models was YAFG-OH (m/z 457.2085), which is the N-terminal tetrapeptide previously identified from studies with rats. Conclusion: Its extensive characterization and commercial availability suggests that it could serve as a primary target analyte for the detection of dermorphin misuse. The metabolomics approach also allowed the assignment of other confirmatory metabolites.

Introduction: Alcohol abuse is a global health problem. The liver maintains high muscle damage by over drinking because it is a major source of ethanol metabolism. Among substance abusers, about 35 % develop advanced liver disease because the number of viral mutations increases, slows down, or inhibits the progression of liver disease. Glutathione-S-transferase is a family of Phase II enzyme-releasing toxins that cause the synthesis of glutathione in a variety of chronic and external electrophilic types. GSTs are divided into two very different family members: family members bound by microsomal membrane and cytosolic. Aim: To study the Glutathione S Transferase π and Malondialdehyde in Alcoholic Patients. Materials and Methods: Present study comprises 100 Subjects were included in the study and distributed in two groups. Patients from group one was alcoholic patients, enrolled from medicine ward and 50 non-alcoholic healthy individuals from group two were from non-alcoholic population as well as medicine ward. Results: Rise of GGT, AST and ALT in Alcoholic patients (54.54 ± 3.72, 19.21 ± 0.68 and 24.32 ± 1.27 respectively) as compare to healthy individuals (24.40 ± 3.16, 10.36±0.35 and 17.06±0.84 respectively). The level of GST-π was decreased in alcoholic patients (62.44±26.30) as compare to control group (83.26±32.71). Similarly, the level of MDA was raised in alcoholic patients (5.36 ± 0.51) as compare to healthy individuals (4.73 ± 0.21). Conclusion: Present study suggests that it would be vital to contain SGOT, SGPT, GGT, MDA and GST-π calculation in the prognostic assessment of alcoholic patients.

Cytochrome P450 (CYP) 1A1 and CYP1B1 enzymes play a significant role in the pathogenesis of cancer and cardiovascular diseases (CVD) such as cardiac hypertrophy and heart failure. Previously, we have demonstrated that R- and S-enantiomers of 19-hydroxyeicosatetraenoic acid (19-HETE), an arachidonic acid endogenous metabolite, enantioselectively inhibit CYP1B1. The current study was conducted to test the possible inhibitory effect of novel synthetic analogues of R- and S-enantiomers of 19-HETE on the activity of CYP1A1, CYP1A2, and CYP1B1. The O-dealkylation rate of 7-ethoxyresorufin (EROD) by recombinant human CYP1A1 and CYP1B1, in addition to the O-dealkylation rate of 7-methoxyresorufin (MROD) by recombinant human CYP1A2, were measured in the absence and presence of varying concentrations (0–40 nM) of the synthetic analogues of 19(R)- and 19(S)-HETE. Also, the possible inhibitory effect of both analogues on the catalytic activity of EROD and MROD, using RL-14 cells and human liver microsomes, was assessed. The results showed that both synthetic analogues of 19(R)- and 19(S)-HETE exhibited direct inhibitory effects on the activity of CYP1A1 and CYP1B1, while they had no significant effect on CYP1A2 activity. Nonlinear regression analysis and comparisons showed that the mode of inhibition for both analogues is noncompetitive inhibition of CYP1A1 and CYP1B1 enzymes. Also, nonlinear regression analysis and Dixon plots showed that the R- and S-analogues have KI values of 15.7 ± 4.4 and 6.1 ± 1.5 nM for CYP1A1 and 26.1 ± 2.9 and 9.1 ± 1.8 nM for CYP1B1, respectively. Moreover, both analogues were able to inhibit EROD and MROD activities in a cell-based assay and human liver microsomes. Therefore, the synthetic analogues of 19-HETE could be considered as a novel therapeutic approach in the treatment of cancer and CVD.

A challenge in the development of novel 18F-labelled positron emission tomography (PET) imaging probes is identification of metabolically stable sites to incorporate the 18F radioisotope. Metabolic loss of 18F from PET probes in vivo can lead to misleading biodistribution data as displaced 18F can accumulate in various tissues.In this study we report on in vitro hepatic microsomal metabolism of novel caffeine containing bifunctional compounds (C8-6-I, C8-6-N, C8-6-C8) that can prevent in vitro aggregation of α-synuclein, which is associated with the pathophysiology of Parkinson’s disease. The metabolic profile obtained guided us to synthesize stable isotope 19F-labelled analogues in which the fluorine was introduced at the metabolically stable N7 of the caffeine moiety.An in vitro hepatic microsomal metabolism study of the 19F-labelled analogues resulted in similar metabolites to the unlabelled compounds and demonstrated that the fluorine was metabolically stable, suggesting that these analogues are appropriate PET imaging probes. This straightforward in vitro strategy is valuable for avoiding costly stability failures when designing radiolabelled compounds for PET imaging. A challenge in the development of novel 18F-labelled positron emission tomography (PET) imaging probes is identification of metabolically stable sites to incorporate the 18F radioisotope. Metabolic loss of 18F from PET probes in vivo can lead to misleading biodistribution data as displaced 18F can accumulate in various tissues. In this study we report on in vitro hepatic microsomal metabolism of novel caffeine containing bifunctional compounds (C8-6-I, C8-6-N, C8-6-C8) that can prevent in vitro aggregation of α-synuclein, which is associated with the pathophysiology of Parkinson’s disease. The metabolic profile obtained guided us to synthesize stable isotope 19F-labelled analogues in which the fluorine was introduced at the metabolically stable N7 of the caffeine moiety. An in vitro hepatic microsomal metabolism study of the 19F-labelled analogues resulted in similar metabolites to the unlabelled compounds and demonstrated that the fluorine was metabolically stable, suggesting that these analogues are appropriate PET imaging probes. This straightforward in vitro strategy is valuable for avoiding costly stability failures when designing radiolabelled compounds for PET imaging.

Dendrobine is the main sesquiterpene alkaloid of Dendrobium nobile Lindl, which exhibits potent neuroprotective activity. However, its metabolism and disposition are little known. In this study, we investigated the metabolic characteristics of dendrobine in vitro and in rats. The metabolic stability and temporal profile of metabolites formation of dendrobine were assayed in human/rat liver microsomal and S9 fractions. Dendrobine metabolites were separated and identified mainly by UPLC-Q/Orbitrap MS. After oral administration of dendrobine (50 mg/kg) to rats, the accumulative excretion rate of dendrobine in feces, urine, and bile was 0.27%, 0.52%, and 0.031%, respectively, and low systematic exposure of dendrobine (AUC(0–∞) = 629.2 ± 56.4 ng·h/mL) was observed. We demonstrated that the elimination of dendrobine was very rapid in liver microsomal incubation (the in vitro elimination t(1/2) in rat and human liver microsomes was 1.35 and 5.61 min, respectively). Dendrobine underwent rapid and extensive metabolism; cytochrome P450, especially CYP3A4, CYP2B6, and CYP2C19, were mainly responsible for its metabolism. Aldehyde dehydrogenase, alcohol dehydrogenase and aldehyde oxidase were involved in the formation of carboxylic acid metabolites. By the aid of in-source fragmentation screening, hydrogen/deuterium exchange experiment, post-acquisition processing software, and available reference standards, 50 metabolites were identified and characterized in liver microsomal incubation and in rats. The major metabolic pathways of dendrobine were N-demethylation, N-oxidation, and dehydrogenation, followed by hydroxylation and glucuronidation. Collectively, the metabolic fate of dendrobine elucidated in this study not only yields benefits for its subsequent metabolism study but also facilitates to better understanding the mode of action of dendrobine and evaluating the pharmacologic efficiency of the high exposure metabolites.

The aim of this study was to investigate the effect of the water-soluble form of quercetin on the indicators of oxidative-nitrosative stress in the tissues of the submandibular salivary glands (SG) under conditions of lipopolysaccharide-induced systemic inflammatory response (SIR). The study was performed on 30 white Wistar male rats weighing 180-220 g, divided into 3 groups: 1st group included intact animals, 2nd group included animals, subjected to the systemic administration of lipopolysaccharide of Salmonella typhi, 3rd group involved animals, which received water-soluble quercetin complex with polyvinylpyrrolidone (corvitin) in a dosage of 100 mg/kg (10 mg/ g in terms of quercetin) intraperitoneally every 3 days, starting on the 30 day of the experiment with using S. typhi lipopolysaccharide. The latter was administered in a dose of 0.4 μg/kg body weight 3 times during the 1st week, and then once a week for the next 7 weeks. The study has demonstrated that applying quercetin under SIR restrains the production of reactive oxygen species in SG tissues: it reduces unstimulated production of superoxide anion radical and its induced generation with the administration of NADPH (by microsomes and NO-synthase), NADH (by mitochondria), S. typhi lipopolysaccharide (by leukocyte NADPH-oxidase). The administration of quercetin under SIR condition diminishes the signs of nitrosative stress in SG tissues, as evidenced by decrease in inducible NO-synthase activity without significant changes in ornithine decarboxylase activity and the level of coupling of constitutive isoform of NO-synthase, decreased concentration of highly active peroxynitrite, but, however, without significant shift in S-nitrosothiols content.

For drug development, species differences in drug-metabolism reactions present obstacles for predicting pharmacokinetics in humans. We characterized the species differences in hydrolases among humans and mice, rats, dogs, and cynomolgus monkeys. In this study, to expand the series of such studies, we attempted to characterize marmoset hydrolases. We measured hydrolase activities for 24 compounds using marmoset liver and intestinal microsomes, as well as recombinant marmoset carboxylesterase (CES) 1, CES2, and arylacetamide deacetylase (AADAC). The contributions of CES1, CES2, and AADAC to hydrolysis in marmoset liver microsomes were estimated by correcting the activities by using the ratios of hydrolase protein levels in the liver microsomes and those in recombinant systems. For six out of eight human CES1 substrates, the activities in marmoset liver microsomes were lower than those in human liver microsomes. For two human CES2 substrates and three out of seven human AADAC substrates, the activities in marmoset liver microsomes were higher than those in human liver microsomes. Notably, among the three rifamycins, only rifabutin was hydrolyzed by marmoset tissue microsomes and recombinant AADAC. The activities for all substrates in marmoset intestinal microsomes tended to be lower than those in liver microsomes, which suggests that the first-pass effects of the CES and AADAC substrates are due to hepatic hydrolysis. In most cases, the sums of the values of the contributions of CES1, CES2, and AADAC were below 100%, which indicated the involvement of other hydrolases in marmosets. In conclusion, we clarified the substrate preferences of hydrolases in marmosets. SIGNIFICANCE STATEMENT This study confirmed that there are large differences in hydrolase activities between humans and marmosets by characterizing marmoset hydrolase activities for compounds that are substrates of human CES1, CES2, or arylacetamide deacetylase. The data obtained in this study may be useful for considering whether marmosets are appropriate for examining the pharmacokinetics and efficacies of new chemical entities in preclinical studies.

OZ439 is a potent synthetic ozonide evaluated for the treatment of uncomplicated malaria. The metabolite profile of OZ439 was characterized in vitro using human liver microsomes combined with LC/MS-MS, chemical derivatization, and metabolite synthesis. The primary biotransformations were monohydroxylation at the three distal carbon atoms of the spiroadamantane substructure, with minor contributions from N-oxidation of the morpholine nitrogen and deethylation cleavage of the morpholine ring. Secondary transformations resulted in the formation of dihydroxylation metabolites and metabolites containing both monohydroxylation and morpholine N-oxidation. With the exception of two minor metabolites, none of the other metabolites had appreciable antimalarial activity. Reaction phenotyping indicated that CYP3A4 is the enzyme responsible for the metabolism of OZ439, and it was found to inhibit CYP3A via both direct and mechanism-based inhibition. Elucidation of the metabolic pathways and kinetics will assist with efforts to predict potential metabolic drug–drug interactions and support physiologically based pharmacokinetic (PBPK) modeling.

Carfentanil is an ultra-potent opioid with an analgesic potency 10,000 times that of morphine but has received little scientific investigation. In the present study, the human cytochrome P450 (CYP) isozymes catalyzing the oxidative metabolism of carfentanil were investigated. Using UHPLC-HRMS, Michaelis-Menten kinetics of formation for three major metabolites norcarfentanil (M1), pharmaceutical active metabolite 4-[(1-oxopropyl)phenylamino]-1-(2-hydroxyl-2-phenylethyl)-4-piperidinecarboxylic acid methyl ester (M11), and 4-[(1-oxopropyl)phenylamino]-1-(2-oxo-2-phenylethyl)-4-piperidinecarboxylic acid methyl ester (M15) were determined. Isozymes catalyzing the formation of the low abundant, highly active metabolite 1-[2-(2-hydroxylphenyl)ethyl]-4-[(1-oxopropyl)phenylamino]-4-piperidinecarboxylic acid methyl ester (M13) were also identified. Selective P450 inhibition studies with pooled human liver microsomes (HLMs) and recombinant CYP isozymes suggested that metabolites M1, M11, and M15 were predominantly formed by isozyme CYP3A5, followed by CYP3A4. Isozymes CYP2C8 and CYP2C9 also made contributions but to a much lesser extent. Highly potent metabolite M13 was predominantly formed by isozyme CYP2C9, followed by CYP2C8. These findings indicate that CYP3A5, CYP3A4, CYP2C8 and CYP2C9 play a major role in the transformation of carfentanil to M1 (norcarfentanil), M11, M13 and M15 through N-dealkylation of piperidine ring, hydroxylation of phenethyl group and ketone formation on phenethyl linker by human liver micrsomes.

Rapid, higher throughput, and predictive toxicological methods are needed to assess vast numbers of chemicals with unknown safety profiles. A current effort towards this goal is Toxicology in the 21st Century (Tox21), a United States government consortium using a battery of in vitro assays to screen a library of 10,000 compounds relevant to food, drug, and environmental safety. Recently, we implemented in vitro assays for measuring acetylcholinesterase (AChE) inhibition, a mechanism of toxicity, into Tox21’s high-throughput screening campaign (Li S., et al. Environ Health Persp 2021;129:047008, doi:10.1289/EHP6993). In this Commentary, we provide detailed insights on two topics related to our article: (1) prioritizing recently discovered AChE inhibitors from our screening based upon physiological relevance and (2) incorporating human liver microsomes into the AChE inhibition assay to identify metabolically active AChE inhibitors.

We explore the mechanism of electron transfers mediated by cytochrome c, a soluble protein involved in mitochondrial oxidative phosphorylation and cytochrome b5, a microsomal membrane protein acting as a redox aide in xenobiotic metabolism. We found minimal conservation in the sequence and surface amino acid residues of cytochrome c/b5 proteins among divergent species. Therefore, we question the evolutionary logic for electron transfer (ET) occurring through affinity binding via recognition of specific surface residues/topography. Also, analysis of putative protein-protein interactions in the crystal structures of these proteins and their redox partners did not point to any specific interaction logic. A comparison of the kinetic and thermodynamic constants of wildtype vs. mutants did not provide strong evidence to support the binding-based ET paradigm, but indicated support for diffusible reactive species (DRS)-mediated process. Topographically divergent cytochromes from one species have been substituted for reaction with proteins from other species, implying the involvement of non-specific interactions. We provide a viable alternative (murburn concept) to classical protein-protein binding-based long range ET mechanism. To account for the promiscuity of interactions and solvent-accessible hemes, we propose that the two proteins act as non- specific redox capacitors, mediating one-electron redox equilibriums involving DRS and unbound ions. Communicated by Ramaswamy H. Sarma

We developed an assay system to evaluate the cytochrome P450 (CYP) 3A4-inhibitory activity of compounds, taking account of their cellular permeability, using intestine-derived cell lines pre-treate...

Photoacoustic imaging (PAI) in the second near-infrared window (NIR-II) is conducive to deep-tissue imaging due to small scattering coefficients, but often requires exogenous imaging agents. At present, nanoparticle-based NIR-II imaging agents are mainly used in non-clinical studies, some basic components of which are resistant to metabolism in vivo. The aim of this study was to examine the ∼600 Da croconaine absorber A1094, absorbing lights around 1094 nm, as a rare, small-molecule NIR-II imaging agent in vivo. The clinical translational potential of A1094 injection were systematically revealed, including sufficient solubility and freeness in blood, good anti-interference ability, and favourable pH/oxidation-reduction/metabolic stabilities. After intravenous administration of A1094 injection, PAI of murine ears exhibited comparable capillaries visibility to that of PAI with popular Au nanorods. The contrasts achieved with A1094 and Au nanorods were 1.78 and 1.29 times higher than before administration, in the healthy group, and 3.25 and 1.58 times higher in the inflammation group. Notably, A1094 demonstrated a desired faster liver clearance than Au nanorods. The PAI signal of A1094 was cleared by 74.2 % after 3 h, whereas Au nanorods were only cleared by 43.1 %. The main metabolic mechanisms of A1094 were identified as N-methylation and lipid hydrolysis by murine liver microsomes in vitro. Therefore, A1094 may have promising clinical potential as a stable and fast-clearing NIR-II imaging agent.

Synthetic cannabinoids (SCs) represent a large group of new psychoactive substances (NPS), sustaining a high prevalence on the drug market since their first detection in 2008. Cumyl-CBMICA and Cumyl-CBMINACA, the first representatives of a new subclass of SCs characterized by a cyclobutyl methyl (CBM) moiety, were identified in July 2019 and February 2020. This work aimed at evaluating basic pharmacological characteristics and human Phase I metabolism of these compounds. Human Phase I metabolites were tentatively identified by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QToF-MS) of urine samples and confirmed by a pooled human liver microsome (pHLM) assay. The basic pharmacological evaluation was performed by applying a competitive ligand binding assay and a functional activation assay (GTPγS) using cell membranes carrying the human cannabinoid receptor 1 (hCB1 ). Investigation of the human Phase I metabolism resulted in the identification of specific urinary markers built by monohydroxylation or dihydroxylation. Although Cumyl-CBMICA was primarily hydroxylated at the indole ring, hydroxylation of Cumyl-CBMINACA mainly occurred at the CBM moiety. Both substances acted as agonists at the hCB1 receptor, although substantial differences could be observed. Cumyl-CBMINACA showed higher binding affinity (Ki = 1.32 vs. 29.3 nM), potency (EC50 = 55.4 vs. 497 nM), and efficacy (Emax = 207% vs. 168%) than its indole counterpart Cumyl-CBMICA. This study confirms that substitution of an indole by an indazole core tends to increase in vitro potency, which is potentially reflected by higher in vivo potency. The emergence and disappearance of SCs distributed via online shops carrying a CBM moiety once more demonstrate the "cat-and-mouse" game between manufacturers and legislation.

Dairy cattle are predisposed to disease around the time of calving due to dysfunctional inflammatory responses. Oxylipids are lipid-derived mediators that regulate all aspects of the inflammatory response, and shifts in oxylipid profiles are correlated with disease risk. For example, 20-hydroxyeicosatetraenoic acid (HETE) is an oxylipid derived from cytochrome P450 enzymes (CYP450) found at significantly greater concentrations around calving and during clinical disease. Biosynthesis of 20-HETE occurs almost exclusively from two specific CYP450 of which CYP450 family four sub-family F member two (CYP4F2) is the major contributor to 20-HETE production in humans. To further study the activities of 20-HETE and potentially reduce its production in vivo, mitigation methods must be explored. Additional substrates of CYP4F2, such as vitamin E, are known to both increase and decrease the metabolism of other CYP4F2 substrates. This study aimed to determine whether vitamin E analogs may reduce the production of 20-HETE through competition for CYP4F2 activity in human CYP4F2, bovine-kidney and bovine-mammary microsomes. Gamma-tocopherol reduced 20-HETE production from human and bovine-kidney microsomes (35.3% and 27.5%, respectively) whereas γ-tocotrienol only reduced 20-HETE production from human microsomes (40.1%). Finally, bovine-mammary microsomes did not produce a quantifiable amount of 20-HETE, suggesting basal mammary CYP4F2 activity may not be a significant contributor to 20-HETE found in milk. Together, these data show that analogs of vitamin E can reduce the production of 20-HETE, potentially through competition with arachidonic acid for metabolism by CYP4F2, posing a potential means for limiting 20-HETE production during clinical diseases of dairy cattle.

Methylophiopogonanone A (MOA), an abundant homoisoflavonoid bearing a methylenedioxyphenyl moiety, is one of the major constituents in the Chinese herb Ophiopogon japonicas. This work aims to assess the inhibitory potentials of MOA against cytochrome P450 enzymes and to decipher the molecular mechanisms for P450 inhibition by MOA. The results showed that MOA concentration-dependently inhibited CYP1A, 2C8, 2C9, 2C19, and 3A in human liver microsomes (HLMs) in a reversible way, with IC50 values varying from 1.06 to 3.43 μM. By contrast, MOA time-, concentration-, and NADPH-dependently inhibited CYP2D6 and CYP2E1, along with KI and kinact values of 207 µM and 0.07 minute−1 for CYP2D6, as well as 20.9 µM and 0.03 minutes−1 for CYP2E1. Further investigations demonstrated that a quinone metabolite of MOA could be trapped by glutathione in an HLM incubation system, and CYP2D6, 1A2, and 2E1 were the major contributors to catalyze the metabolic activation of MOA to the corresponding O-quinone intermediate. Additionally, the potential risks of herb-drug interactions triggered by MOA or MOA-related products were also predicted. Collectively, our findings verify that MOA is a reversible inhibitor of CYP1A, 2C8, 2C9, 2C19, and 3A but acts as an inactivator of CYP2D6 and CYP2E1. Significance Statement Methylophiopogonanone A (MOA), an abundant homoisoflavonoid isolated from the Chinese herb Ophiopogon japonicas, is a reversible inhibitor of CYP1A, 2C8, 2C9, 2C19, and 3A but acts as an inactivator of CYP2D6 and CYP2E1. Further investigations demonstrated that a quinone metabolite of MOA could be trapped by glutathione in a human liver microsome incubation system, and CYP2D6, 1A2, and 2E1 were the major contributors to catalyze the metabolic activation of MOA to the corresponding O-quinone intermediate.

Endoplasmic reticulum (ER) maintains within, an oxidative redox state suitable for disulfide bond formation. We monitored the ER redox dynamics subsequent to proteasome inhibition using an ER redox probe ERroGFP S4. Proteasomal inhibition initially led to oxidation of the ER, but gradually the normal redox state was recovered that further led to a reductive state. These events were found to be concomitant with the increase in the both oxidized and reduced glutathione in the microsomal fraction, with a decrease of total intracellular glutathione. The ER reduction was suppressed by pretreatment of a glutathione synthesis inhibitor or by knockdown of ATF4, which induces glutathione-related genes. These results suggested cellular adaptation of ER redox homeostasis: (1) inhibition of proteasome led to accumulation of misfolded proteins and oxidative state in the ER, and (2) the oxidative ER was then reduced by ATF4 activation, followed by influx of glutathione into the ER.

Background: Timosaponin A-III is one of the most promising active saponins from Anemarrhena asphodeloides Bge. As an oral chemotherapeutic agent, there is an urgent need to clarify its biopharmaceutics and pharmacokinetics to improve its development potential. Objective: This research explores the bioavailability of timosaponin A-III and clarifies its absorption and metabolism mechanisms by a sensitive and specific HPLC-MS/MS method. Methods: Pharmacokinetics and bioavailability studies of timosaponin A-III were performed in Sprague- Dawley rats by oral (20 mg/kg) and intravenous administration (2 mg/kg). Control group was given the same volume of normal saline. The absorption of timosaponin A-III was investigated in a rat intestinal perfusion model in situ and a Caco-2 cell transport model in vitro. The metabolic rate of timosaponin A-III was determined in a rat liver microsome incubation system. Results: After the oral administration, timosaponin A-III reached Cmax of 120.90 ± 24.97 ng/mL at 8 h, and the t1/2 was 9.94 h. The absolute oral bioavailability of timosaponin A-III was 9.18%. The permeability coefficients of timosaponin A-III in four intestinal segments ranged from 4.98 to 5.42 × 10-7 cm/s, indicating a difficult absorption. A strikingly high efflux transport of timosaponin A-III was found, PappBA 3.27 ± 0.64 × 10−6 cm/s, which was abolished by a P-gp inhibitor. Rat liver microsome incubation studies showed that timosaponin A-III could hardly be metabolized, with a t1/2 of over 12 h. In addition, the solubility test showed a low solubility in PBS solution, i.e. 30.58 μg/mL. Conclusion: Timosaponin A-III exhibited low oral bioavailability by oral and intravenous administration, which was probably caused by its low permeability and solubility. This study may provide a reference for its rational clinical use and further study on the pharmacology or toxicology of timosaponin A-III.

Summary. The pathogenesis of fluoride intoxication at the molecular, cellular and functional levels has not been sufficiently studied. There are very few modern data on these issues, so they are contradictory, since the effects of this trace element are multifaceted and cannot be characterized unambiguously. The aim of the study – to learn the state of the monooxygenase system of rat hepatocytes under conditions of the formation of fluoride intoxication. Materials and Methods. In the experiment, we used 30 sexually mature rats (N=30) of the Wistar population weighing 200–210 g for 1.5 months. Sodium fluoride solution was administered orally at doses of 1/10 DL50, which was 20 mg/kg of animal body weight. Results. The results of experiments on the study of oxygen consumption by rat liver microsomes under fluoride intoxication indicated that the rate of endogenous respiration of microsomes, the rate of NADPH oxidation, the rate of NADH oxidation in the presence of EDTA, and the rate of lipid peroxidation increase under the influence of fluorides. Sodium fluoride stimulated an increase in all parameters of microsomal oxidation, except for cytochrome b5. It should be assumed that in this case there is an increase in the generation of reactive oxygen species, free radicals, which stimulate the development of free radical processes in the body and are, most likely, the leading link in oxidative stress. Conclusions. These changes indicate a violation of the bioenergetics of hepatocytes associated with the mitochondrial apparatus and the development of hypoxic processes, which lead to a decrease in the activity of redox reactions occurring at the level of intracellular membranes and organelles.