Search Results (30)

Background/Objectives: The opioid crisis is an ongoing problem in the United States, and fentanyl analogs play a major role in the issue, as novel fentanyl analogs are constantly being developed. Substitutions and additions to the fentanyl scaffold impact the potency of the substances and can sometimes influence the biotransformation of the drugs. This study aimed to determine whether one or more common metabolites could be detected among a group of five methoxylated fentanyl analogs, for the purpose of eventually providing a more rapid detection method for new and unknown fentanyl-related compounds in toxicological samples. Methods: In vitro metabolism via pooled human liver microsomes (HLMs) was performed for five methoxylated fentanyl analogs (para-methoxyfentanyl, ortho- and para-methoxy butyryl fentanyl, and ortho- and para-methoxy furanyl fentanyl) to generate metabolites. Assays were analyzed via liquid chromatography–tandem mass spectrometry. Results: Nine metabolites were detected. A common metabolite was observed between fentanyl analogs that were methoxylated at the para-position. Conclusions: Similarities between metabolites of five methoxylated fentanyl analogs were noted. It appeared that the major pathway of metabolism for methoxylated fentanyl analogs is largely consistent, regardless of the location of the methoxy substituent so long as the piperidine nitrogen is unobstructed and is available for N-dealkylation. This research provides valuable insight to potentially find new ways for toxicologists to identify novel methoxylated fentanyl analogs in biological matrices. Full article

Synthetic cannabinoids (SCs) are a rapidly developing kind of novel psychoactive substance, frequently associated with acute intoxication and public health concerns. This study aimed to elucidate and compare the phase I metabolic pathways of two structurally related SCs, 5F-ADB-PINACA and 5F-ADBICA, using in vitro and in vivo models. Temporal metabolic profiling was performed to identify potential signature metabolites. Temporal abundance patterns and correlation cluster analysis of metabolites were analyzed to determine metabolite biomarkers. The two SCs were incubated with pooled human liver microsomes for 24 h and were also evaluated in vivo in zebrafish. Metabolite profiles were characterized using UHPLC-QE Orbitrap-MS. HLM analysis identified 21 5F-ADB-PINACA metabolites and 28 5F-ADBICA metabolites. Metabolites of 5F-ADBICA were detected for the first time in vitro and in a zebrafish model. Zebrafish studies confirmed the presence of all key metabolites observed in HLM. Comparative analysis of their metabolic pathways revealed differences in metabolism driven by structural differences between the indazole and indole cores. This is the first time that correlation analysis has been used in the temporal metabolic profiling of SCs. This study comprehensively characterized the metabolism of 5F-ADB-PINACA and 5F-ADBICA, identifying M13 (hydrolytic defluorination) as a potential metabolite biomarker for 5F-ADB-PINACA and M19 (hydrolytic defluorination) as a potential metabolite biomarker for 5F-ADBICA. The metabolic reactions of the main metabolites of the two synthetic cannabinoids are consistent. However, their metabolic processes (i.e., the overall metabolic pathways and temporal progression of these reactions) are different, which illustrates the metabolic similarity of structurally similar synthetic cannabinoids and the impact of different structures on the metabolic processes. Full article

Theaflavins, benzotropolone compounds formed during black tea processing via catechin condensation, have drawn attention for their potential health benefits and diverse biological effects. This study evaluated the inhibitory effects of four theaflavin monomers—theaflavin-3′-gallate, theaflavin-3,3′-digallate, theaflavin-3-gallate, and theaflavin—on eight CYP450 enzymes using pooled human liver microsomes and specific probe substrates, and seven UGT enzymes using human recombinant UGT enzymes and specific probe substrates. Theaflavin-3′-gallate moderately inhibited CYP1A2-catalyzed phenacetin metabolism and CYP2C8-mediated amodiaquine metabolism, with IC₅₀ values of 8.67 μM and 10–20 μM, respectively. Theaflavin-3,3′-digallate exhibited similar effects. Both compounds showed negligible inhibition with other CYP enzymes. In UGT assays, theaflavin-3′-gallate and theaflavin-3,3′-digallate moderately inhibited UGT1A1- and UGT1A3-mediated beta-estradiol glucuronidation (IC₅₀: 1.40–5.22 μM), with weak or no effects on other UGT enzymes. Molecular docking revealed that CYP1A2-theaflavin-3′-gallate and CYP2C8-theaflavin-3,3′-digallate interactions were non-competitive, primarily mediated by hydrogen bonding and π-interactions. UGT1A1-theaflavin interactions suggested non-competitive inhibition, while UGT1A3-theaflavin interactions indicated competitive inhibition. Other enzyme-theaflavin interactions exhibited minimal binding energy differences, implying mixed-type inhibition. These findings highlight the selective inhibitory effects of theaflavins on specific hepatic enzymes, with potential implications for nutrient interactions, particularly for nutrients metabolized by CYP1A2, CYP2C8, UGT1A1, and UGT1A3. Further research is needed to explore the in vivo relevance and assess the dietary implications of theaflavin-rich black tea in nutrition and metabolism. Full article

Background: Sirolimus (SRL, rapamycin) is a clinically important mTOR inhibitor used in immunosuppression, oncology, and cardiovascular drug-eluting devices. Despite its long-standing FDA approval, the human metabolic profile of SRL remains incompletely characterized. SRL is primarily metabolized by CYP3A enzymes in the liver and intestine, but the diversity, pharmacokinetics, and biological activity of its metabolites have been poorly explored due to the lack of structurally identified standards. Methods: To investigate SRL metabolism, we incubated SRL with pooled human liver microsomes (HLM) and isolated the resulting metabolites. Structural characterization was performed using high-resolution mass spectrometry (HRMS) and ion trap MSⁿ. We also applied Density Functional Theory (DFT) calculations to assess the energetic favorability of metabolic transformations and conducted molecular dynamics (MD) simulations to model metabolite interactions within the CYP3A4 active site. Results: We identified 21 unique SRL metabolites, classified into five major structural groups: O-demethylated, hydroxylated, didemethylated, di-hydroxylated, and mixed hydroxylated/demethylated derivatives. DFT analyses indicated that certain demethylation and hydroxylation reactions were energetically preferred, correlating with metabolite abundance. MD simulations further validated these findings by demonstrating the favorable orientation and accessibility of key sites within the CYP3A4 binding pocket. Conclusions: This study provides a comprehensive structural map of SRL metabolism, offering mechanistic insights into the formation of its metabolites. Our integrated approach of experimental and computational analyses lays the groundwork for future investigations into the pharmacodynamic and toxicodynamic effects of SRL metabolites on the mTOR pathway. Full article

In this study, to better understand the mechanisms of the profound impact of alcohol consumption on drug pharmacokinetics, efficacy, and toxicity, we characterized the alcohol-induced changes in the ensemble of drug-metabolizing enzymes and transporters (DMETs) in the human liver by performing global proteomic analysis of human liver microsomes from 94 donors. DMET protein levels were analyzed concerning alcohol consumption, smoking history, and sex using non-parametric tests, which were further strengthened by correlational analysis. To this end, we used a provisional index of alcohol exposure formulated based on the relative abundances of four marker proteins best correlating with the level of alcohol consumption. Alcohol-induced changes in the cytochrome P450 pool include significant increases in CYP2E1, CYP2B6, CYP2J2, and NADPH-cytochrome P450 reductase levels and the lowering of CYP1A2, CYP2C8, CYP2C9, CYP4A11, and cytochrome b₅. Changes in UDP-glucuronosyltransferase (UGT) abundances comprise elevated UGT1A6, UGT1A9, and UGT2A1, and reduced UGT1A3, UGT1A4, UGT2B7, UGT2B10, and UGT2B15 levels. Tobacco smokers showed elevated CYP1A2, UGT1A6, and UGT2B4 and reduced FMO3, FMO4, and FMO5 levels, while in females, CYP1A2, UGT2B17, and UGT2B15 levels were lower, and UGT2A3 and STS were higher compared to males. The alcohol-induced changes in the DMET ensemble at the protein level reported herein provide deep insights into how alcohol impacts drug and xenobiotic metabolism. Full article

Background/Objectives: Perillyl alcohol (POH), a monoterpene natural product derived from the essential oils of plants such as perilla (Perilla frutescens), is currently in phase I and II clinical trials as a chemotherapeutic agent. In this study, we investigated the effect of POH on cytochrome P450 (CYP) activity for evaluating POH–drug interaction potential. Methods: The investigation was conducted using pooled human liver microsomes (HLMs), recombinant CYP3A4 (rCYP3A4) enzymes, and human pluripotent stem cell-derived hepatic organoids (hHOs) employing liquid chromatography-tandem mass spectrometry. Results: POH inhibited the activities of CYP2A6 and CYP2B6 with K_i of 6.35 and 3.78 μM, respectively, whereas it stimulated CYP3A4 activity in pooled HLMs incubated with midazolam (MDZ). In a direct CYP inhibition assay using HLMs, activities of CYP2C9, CYP2C19, and CYP2E1 were also inhibited by POH, with IC₅₀ values greater than 50 μM, but those of CYP1A2, CYP2C8, CYP2D6, and CYP3A4 (testosterone) were not significantly inhibited. In pooled HLMs, the V_max/K_m value of 1′-hydroxy MDZ, but not that of 4-hydroxy MDZ, was increased 2.7-fold by 100 μM POH compared with that in the absence of POH. Moreover, stimulation of MDZ 1′-hydroxylation by CYP3A4 was observed in hHOs and rCYP3A4 with cytochrome b₅ but not rCYP3A4 without cytochrome b₅. Furthermore, activation of CYP3A4-mediated metabolism by POH was observed in HLMs incubated with fimasartan but not atorvastatin, buspirone, donepezil, nifedipine, or tadalafil, suggesting a substrate-dependent activation of CYP3A4 by POH. Conclusions: POH inhibits CYP2A6 and CYP2B6, but it activates CYP3A4. These findings underscore the need for further evaluation of the interactions of clinical drugs with POH. Full article

Ketamine derivatives such as deschloroketamine and deschloro-N-ethyl-ketamine show dissociative and psychoactive properties and their abuse as new psychoactive substances (NPSs) has been reported. Though some information is available on the biotransformation of dissociative NPSs, data on deschloro-N-cyclopropyl-ketamine deschloro-N-isopropyl-ketamine and deschloro-N-propyl-ketamine concerning their biotransformation and, thus, urinary detectability are not available. The aims of the presented work were to study the in vivo phase I and II metabolism; in vitro phase I metabolism, using pooled human liver microsomes (pHLMs); and detectability, within a standard urine screening approach (SUSA), of five deschloroketamine derivatives. Metabolism studies were conducted by collecting urine samples from male Wistar rats over a period of 24 h after their administration at 2 mg/kg body weight. The samples were analyzed using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) and gas chromatography–mass spectrometry (GC-MS). The compounds were mainly metabolized by N-dealkylation, hydroxylation, multiple oxidations, and combinations of these metabolic reactions, as well as glucuronidation and N-acetylation. In total, 29 phase I and 10 phase II metabolites were detected. For the LC-HRMS/MS SUSA, compound-specific metabolites were identified, and suitable screening targets could be recommended and confirmed in pHLMs for all derivatives except for deschloro-N-cyclopropyl-ketamine. Using the GC-MS-based SUSA approach, only non-specific acetylated N-dealkylation metabolites could be detected. Full article

(−)-trans-Δ⁹-tetrahydrocannabinol (Δ⁹-THC) is the primary psychoactive compound in the Cannabis sativa plant. Δ⁹-THC undergoes extensive metabolism, with the main human phase I metabolites being 11-hydroxy-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH). Early animal studies have indicated that the 9-10 double bond may be reduced in vivo to yield 11-hydroxy-hexahydrocannabinol (11-OH-HHC) and 11-nor-9-carboxy-hexahydrocannabinol (HHC-COOH). These metabolites have not been confirmed in humans. In this study, we aimed to investigate whether this metabolic transformation occurs in humans. A range of cannabinoids and metabolites, including 11-OH-HHC and HHC-COOH, were measured in whole blood from 308 authentic forensic traffic cases, of which 222 were positive for Δ⁹-THC. HHC-COOH and 11-OH-HHC were detected in 84% and 15% of the Δ⁹-THC positive cases, respectively, and the estimated median concentration of HHC-COOH was 7%, relative to that of THC-COOH. To corroborate the in vivo findings, Δ⁹-THC and its metabolites 11-OH-THC and THC-COOH were incubated with pooled human liver microsomes. HHC-COOH was detected in both the Δ⁹-THC and 11-OH-THC incubations, while 11-OH-HHC was only detectable in the 11-OH-THC incubation. Hexahydrocannabinol was not detected in any of the incubations, indicating that it is 11-OH-THC or the corresponding aldehyde that undergoes double bond reduction with subsequent oxidation of the aliphatic alcohol to HHC-COOH. In summary, the presented data provide the first evidence of HHC-COOH and 11-OH-HHC being human phase I metabolites of Δ⁹-THC. These findings have implications for interpretation of analytical results from subjects exposed to Δ⁹-THC or HHC. Full article

Unregulated core structures, “isatin acyl hydrazones” (OXIZIDs), have quietly appeared on the market since China legislated to ban seven general core scaffolds of synthetic cannabinoids (SCs). The fast evolution of SCs presents clinical and forensic toxicologists with challenges. Due to extensive metabolism, the parent compounds are barely detectable in urine. Therefore, studies on the metabolism of SCs are essential to facilitate their detection in biological matrices. The aim of the present study was to elucidate the metabolism of two cores, “indazole-3-carboxamide” (e.g., ADB-BUTINACA) and “isatin acyl hydrazone” (e.g., BZO-HEXOXIZID). The in vitro phase I and phase II metabolism of these six SCs was investigated by incubating 10 mg/mL pooled human liver microsomes with co-substrates for 3 h at 37 °C, and then analyzing the reaction mixture using ultrahigh-performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry. In total, 9 to 34 metabolites were detected for each SC, and the major biotransformations were hydroxylation, dihydrodiol formation (MDMB-4en-PINACA and BZO-4en-POXIZID), oxidative defluorination (5-fluoro BZO-POXIZID), hydrogenation, hydrolysis, dehydrogenation, oxidate transformation to ketone and carboxylate, N-dealkylation, and glucuronidation. Comparing our results with previous studies, the parent drugs and SC metabolites formed via hydrogenation, carboxylation, ketone formation, and oxidative defluorination were identified as suitable biomarkers. Full article

Microsomes are commonly used to perform in vitro drug metabolism, predominantly to form phase I drug metabolites. Pooled microsomes from multiple donors can contain microorganisms from underlying microbial diseases. Exposure to microbes can also occur during extraction if aseptic processing is compromised. Although microbial presence does not affect the metabolic activity of microsomes, presence of unwanted microorganisms can cause interference if the downstream application of microsomal drug metabolites is screening for antibacterial activity. In this work, traditional biochemical tests and advanced proteomics-based identification techniques were used to identify two gram-negative bacteria in pooled human liver microsomes. Several decontamination procedures were assessed to eradicate these two bacteria from the microsomes without affecting its metabolic capacity, and organic extraction was found to be the most convenient and efficient approach to decontaminate microsomes and screen drug metabolites for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Full article

Synthetic cathinones are one important group amongst new psychoactive substances (NPS) and limited information is available regarding their toxicokinetics and -dynamics. Over the past few years, nontargeted toxicometabolomics has been increasingly used to study compound-related effects of NPS to identify important exogenous and endogenous biomarkers. In this study, the effects of the synthetic cathinone PCYP (2-cyclohexyl-1-phenyl-2-(1-pyrrolidinyl)-ethanone) on in vitro and in vivo metabolomes were investigated. Pooled human-liver microsomes and blood and urine of male Wistar rats were used to generate in vitro and in vivo data, respectively. Samples were analyzed by liquid chromatography and high-resolution mass spectrometry using an untargeted metabolomics workflow. Statistical evaluation was performed using univariate and multivariate statistics. In total, sixteen phase I and one phase II metabolite of PCYP could be identified as exogenous biomarkers. Five endogenous biomarkers (e.g., adenosine and metabolites of tryptophan metabolism) related to PCYP intake could be identified in rat samples. The present data on the exogenous biomarker of PCYP are crucial for setting up analytical screening procedures. The data on the endogenous biomarker are important for further studies to better understand the physiological changes associated with cathinone abuse but may also serve in the future as additional markers for an intake. Full article

Tectorigenin and irigenin are biologically active isoflavones of Belamcanda chinensis (L.) DC. Previous studies indicated that both compounds could be metabolized in vivo; however, the kinetic parameters of enzymes involved in the metabolization of tectorigenin and irigenin have not been identified. The aim of this study was to investigate UGTs involved in the glucuronidation of tectorigenin and irigenin and determine enzyme kinetic parameters using pooled human liver microsomes (HLMs) and recombinant UGTs. Glucuronides of tectorigenin and irigenin were identified using high-performance liquid chromatography (HPLC) coupled with mass spectrometry and quantified by HPLC using a response factor method. The results showed that tectorigenin and irigenin were modified by glucuronidation in HLMs. One metabolite of tectorigenin (M) and two metabolites of irigenin (M1 and M2) were detected. Chemical inhibition and recombinant enzyme experiments revealed that several enzymes could catalyze tectorigenin and irigenin glucuronidation. Among them, UGT1A1 and UGT1A9 were the primary enzymes for both tectorigenin and irigenin; however, the former mostly produced irigenin glucuronide M1, while the latter mostly produced irigenin glucuronide M2. These findings suggest that UGT1A1 and UGT1A9 were the primary isoforms metabolizing tectorigenin and irigenin in HLMs, which could be involved in drug–drug interactions and, therefore, should be monitored in clinical practice. Full article

Schizandrol A (SZA) and schizandrol B (SZB) are two active ingredients of Wuzhi capsule (WZC), a Chinese proprietary medicine commonly prescribed to alleviate tacrolimus (FK-506)-induced hepatoxicity in China. Due to their inhibitory effects on cytochrome P450 (CYP) 3A enzymes, SZA/SZB may display drug–drug interaction (DDI) with tacrolimus. To identify the extent of this DDI, the enzymes’ inhibitory profiles, including a 50% inhibitory concentration (IC₅₀) shift, reversible inhibition (RI) and time-dependent inhibition (TDI) were examined with pooled human-liver microsomes (HLMs) and CYP3A5-genotyped HLMs. Subsequently, the acquired parameters were integrated into a physiologically based pharmacokinetic (PBPK) model to quantify the interactions between the SZA/SZB and the tacrolimus. The metabolic studies indicated that the SZB displayed both RI and TDI on CYP3A4 and CYP3A5, while the SZA only exhibited TDI on CYP3A4 to a limited extent. Moreover, our PBPK model predicted that multiple doses of SZB would increase tacrolimus exposure by 26% and 57% in CYP3A5 expressers and non-expressers, respectively. Clearly, PBPK modeling has emerged as a powerful approach to examine herb-involved DDI, and special attention should be paid to the combined use of WZC and tacrolimus in clinical practice. Full article

Quinolin-8-yl 4-methyl-3-(piperidine-1-sulfonyl)benzoate (QMPSB) and quinolin-8-yl 4-methyl-3-(piperidine-1-carbonyl)benzoate (QMPCB, SGT-11) are synthetic cannabinoid receptor agonists (SCRAs). Knowing their metabolic fate is crucial for the identification of toxicological screening targets and to predict possible drug interactions. The presented study aimed to identify the in vitro phase I/II metabolites of QMPSB and QMPCB and to study the contribution of different monooxygenases and human carboxylesterases by using pooled human liver S9 fraction (pHLS9), recombinant human monooxygenases, three recombinant human carboxylesterases, and pooled human liver microsomes. Analyses were carried out by liquid chromatography high-resolution tandem mass spectrometry. QMPSB and QMPCB showed ester hydrolysis, and hydroxy and carboxylic acid products were detected in both cases. Mono/dihydroxy metabolites were formed, as were corresponding glucuronides and sulfates. Most of the metabolites could be detected in positive ionization mode with the exception of some QMPSB metabolites, which could only be found in negative mode. Monooxygenase activity screening revealed that CYP2B6/CYP2C8/CYP2C9/CYP2C19/CYP3A4/CYP3A5 were involved in hydroxylations. Esterase screening showed the involvement of all investigated isoforms. Additionally, extensive non-enzymatic ester hydrolysis was observed. Considering the results of the in vitro experiments, inclusion of the ester hydrolysis products and their glucuronides and monohydroxy metabolites into toxicological screening procedures is recommended. Full article

Synthetic cannabinoid receptor agonists (SCRAs) remain popular drugs of abuse. As many SCRAs are known to be mostly metabolized, in vitro phase I metabolic profiling was conducted of the two indazole-3-carboxamide SCRAs: CUMYL-THPINACA and ADAMANTYL-THPINACA. Both compounds were incubated using pooled human liver microsomes. The sample clean-up consisted of solid phase extraction, followed by analysis using liquid chromatography coupled to a high resolution mass spectrometer. In silico-assisted metabolite identification and structure elucidation with the data-mining software Compound Discoverer was applied. Overall, 28 metabolites were detected for CUMYL-THPINACA and 13 metabolites for ADAMATYL-THPINACA. Various mono-, di-, and tri-hydroxylated metabolites were detected. For each SCRA, an abundant and characteristic di-hydroxylated metabolite was identified as a possible in vivo biomarker for screening methods. Metabolizing cytochrome P450 isoenzymes were investigated via incubation of relevant recombinant liver enzymes. The involvement of mainly CYP3A4 and CYP3A5 in the metabolism of both substances were noted, and for CUMYL-THPINACA the additional involvement (to a lesser extent) of CYP2C8, CYP2C9, and CYP2C19 was observed. The results suggest that ADAMANTYL-THPINACA might be more prone to metabolic drug−drug interactions than CUMYL-THPINACA, when co-administrated with strong CYP3A4 inhibitors. Full article