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Aims: Characterizing cannabinoid receptors (CBRs) expressed in Ewing sarcoma (EWS) cell lines as potential targets for anti-cancer drug development., Main Methods: CBR affinity and function were examined by competitive binding and G-protein activation, respectively. Cannabinoid-mediated cytotoxicity and cell viability were evaluated by LDH, and trypan blue assays, respectively., Key Findings: qRT-PCR detected CB1 (CB1R) and CB2 receptor (CB2R) mRNA in TC-71 cells. However, binding screens revealed that CBRs expressed exhibit atypical properties relative to canonical receptors, because specific binding in TC-71 could only be demonstrated by the established non-selective CB1/CB2R radioligand [ 3 H]WIN-55,212-2, but not CB1/CB2R radioligand [ 3 H]CP-55,940. Homologous receptor binding demonstrated that [ 3 H]WIN-55,212-2 binds to a single site with nanomolar affinity, expressed at high density. Further support for non-canonical CBRs expression is provided by subsequent binding screens, revealing that only 9 out of 28 well-characterized cannabinoids with high affinity for canonical CB1 and/or CB2Rs were able to displace [ 3 H]WIN-55,212-2, whereas two ligands enhanced [ 3 H]WIN-55,212-2 binding. Five cannabinoids producing the greatest [ 3 H]WIN-55,212-2 displacement exhibited high nanomolar affinity (K i ) for expressed receptors. G-protein modulation and adenylyl cyclase assays further indicate that these CBRs exhibit distinct signaling/functional profiles compared to canonical CBRs. Importantly, cannabinoids with the highest affinity for non-canonical CBRs reduced TC-71 viability and induced cytotoxicity in a time-dependent manner. Studies in a second EWS cell line (A-673) showed similar atypical binding properties of expressed CBRs, and cannabinoid treatment produced cytotoxicity., Significance: Cannabinoids induce cytotoxicity in EWS cell lines via non-canonical CBRs, which might be a potential therapeutic target to treat EWS., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In 2020, nearly one-third of new drugs on the global market were synthetic cannabinoids including the drug of abuse N -(1-adamantyl)-1-(5-pentyl)-1 H -indazole-3-carboxamide (5F-APINACA, 5F-AKB48). Knowledge of 5F-APINACA metabolism provides a critical mechanistic basis to interpret and predict abuser outcomes. Prior qualitative studies identified which metabolic processes occur but not the order and extent of them and often relied on problematic "semi-quantitative" mass spectroscopic (MS) approaches. We capitalized on 5F-APINACA absorbance for quantitation while leveraging MS to characterize metabolite structures for measuring 5F-APINACA steady-state kinetics. We demonstrated the reliability of absorbance and not MS for inferring metabolite levels. Human liver microsomal reactions yielded eight metabolites by MS but only five by absorbance. Subsequent kinetic studies on primary and secondary metabolites revealed highly efficient mono- and dihydroxylation of the adamantyl group and much less efficient oxidative defluorination at the N -pentyl terminus. Based on regiospecificity and kinetics, we constructed pathways for competing and intersecting steps in 5F-APINACA metabolism. Overall efficiency for adamantyl oxidation was 17-fold higher than that for oxidative defluorination, showing significant bias in metabolic flux and subsequent metabolite profile compositions. Lastly, our analytical approach provides a powerful new strategy to more accurately assess metabolic kinetics for other understudied synthetic cannabinoids possessing the indazole chromophore.

AKB48 and its fluorinated derivative 5F-AKB48 are synthetic cannabinoids (SCs) which have caused hospitalizations and deaths in human users. Abuse of SCs is dangerous because users may mistake them for natural cannabis, which is generally considered to be unlikely to elicit adverse effects. The present studies were designed to investigate the in vitro oxidative metabolism of 5F-AKB48 by human microsomal fractions from different organs and sexes as well as recombinant human cytochrome P450s (P450s). Mass spectrometry data tentatively provides evidence for the existence of mono-, di-, and trihydroxylated metabolites in a successive metabolism. Experiments utilizing P450s revealed that the most active enzymes (CYP2D6, CYP2J2, CYP3A4, and CYP3A5) effectively produced mono- and dihydroxylated metabolites, while CYP3A4/5 also produced significant amounts of the trihydroxylated metabolite. Moreover, although the affinity and potency of Phase I metabolite 4OH-5F-AKB48 is reduced when compared to that of the parent drug, this metabolite nevertheless retains similar high affinity for CB1 receptors, and greater efficacy for G protein activation, when compared to THC. Finally, 5F-AKB48 produced time- and dose-dependent cannabimimetic effects in mice which were more potent, but shorter acting, than those of Δ 9 -THC, and were attenuated by prior treatment with the CB1 antagonist rimonabant. Based on our data, we hypothesize that while many cases of toxicity result from genetic mutations, which can lead to a decrease or even absence of activity for Phase I drug-metabolizing enzymes, other P450s could potentially increase their role in the metabolism of these SCs. Because many metabolites of SCs remain biologically active, they could contribute to the deleterious effects of these substances., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Synthetic cannabinoids (SCBs), designer drugs marketed as legal alternatives to marijuana, act as ligands to cannabinoid receptors; however, they have increased binding affinity and potency, resulting in toxicity symptoms such as cardiovascular incidents, seizures, and potentially death. N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135) is a third generation SCB. When incubated with hepatocytes, it undergoes oxidation, hydrolysis, and glucuronidation, resulting in 29 metabolites, with monohydroxy STS-135 (M25) and dihydroxy STS-135 (M21) being the predominant metabolites. The enzymes responsible for this oxidative metabolism were unknown. Thus, the aim of this study was to identify the cytochrome P450 (P450s or CYPs) enzymes involved in the oxidative metabolism of STS-135. In this study, STS-135 was incubated with liver, intestinal, and brain microsomes and recombinant P450s to determine the enzymes involved in its metabolism. Metabolite quantification was carried out using ultra-performance liquid chromatography. STS-135 was extensively metabolized in HLMs and HIMs. Screening assays indicated CYP3A4 and CYP3A5 could be responsible for STS-135's oxidation. Through incubations with genotyped HLMs, CYP3A4 was identified as the primary oxidative enzyme. Interestingly, CYP2J2, a P450 isoform expressed in cardiovascular tissues, showed high activity towards the formation of M25 with a K m value of 11.4 μmol/L. Thus, it was concluded that STS-135 was primarily metabolized by CYP3A4 but may have extrahepatic metabolic pathways as well. Upon exposure to STS-135, individuals with low CYP3A4 activity could retain elevated blood concentration, resulting in toxicity. Additionally, CYP2J2 may aid in protecting against STS-135-induced cardiovascular toxicity., (© 2020 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

Recently, there has been a rise in abuse of synthetic cannabinoids (SCBs). The consumption of SCBs results in various effects and can induce toxic reactions, including paranoia, seizures, tachycardia and even death. 1-Naphthyl 1-(4-fluorobenzyl)-1H-indole-3-carboxylate (FDU-PB-22) is a third generation SCB whose metabolic pathway has not been fully characterized. In this study, we conducted in vitro pharmacokinetic analysis of FDU-PB-22 metabolism. Metabolic reactions containing FDU-PB-22 and human liver microsomes (HLMs) were independent of NADPH but not UDP-glucuronic acid (UDPGA), suggesting that UDP-glucuronosyltransferases (UGTs) are the primary enzymes involved in this metabolism. It was further determined that the metabolite extensively formed after incubating FDU-PB-22 with UDPGA in HLMs was the glucuronide of FDU-PB-22 3-carboxyindole (FBI-COOH). Various hepatic UGTs showed enzymatic activity for FBI-COOH. A series of UGT inhibitors showed moderate to strong inhibition of FBI-COOH-glucuronidation in HLMs, suggesting that multiple UGT isoforms are involved in FBI-COOH-glucuronidation in the liver. Interestingly, an extra-hepatic isoform, UGT1A10, exhibited the highest activity with a K m value of 38 µM and a V max value of 5.90 nmol/min/mg. Collectively, these results suggest that both genetic mutations of and the co-administration of inhibitors for FDU-PB-22-metabolizing UGTs will likely increase the risk of FDU-PB-22-induced toxicity.

Convulsant effects of abused synthetic cannabinoid (SCB) drugs have been reported in humans and laboratory animals, but the mechanism of these effects is not known. We compared convulsant effects of partial CB1R agonist ∆ 9 -tetrahydrocannabinol (THC), full CB1R agonist SCBs JWH-018 and 5F-AB-PINACA, and classic chemical convulsant pentylenetetrazol (PTZ) using an observational rating scale in mice. THC did not elicit convulsions, but both SCBs did so as effectively as and more potently than PTZ. SCB-elicited convulsions were attenuated by the CB1R antagonist rimonabant or by THC, or by dose regimens of THC and JWH-018, which downregulate and desensitize CB1Rs. None of these treatments altered the convulsant effects of PTZ, although diazepam attenuated PTZ-elicited convulsions without altering SCB-induced convulsant effects. Repeated administration of a subthreshold dose of PTZ kindled convulsant effects, but this was not observed with the SCBs, and no cross-kindling was observed. Repeated administration of the SCBs resulted in tolerance to convulsant effects, but no cross-tolerance to PTZ was observed. Inhibition on Phase I metabolism via nonselective inhibition of CYP450s with 1-aminobenzotriazole potentiated the hypothermic effects of the SCBs and protected against the convulsant effects of JWH-018, but not those of 5F-AB-PINACA or PTZ. Incubation of human liver microsomes with the SCBs showed that JWH-018 is eliminated via oxidation, whereas 5F-AB-PINACA is not. These studies suggest that SCB-elicited convulsions are mediated by high intrinsic efficacy at CB1Rs and that benzodiazepines may not be effective treatments. Finally, drug metabolism may dramatically modulate the convulsant effects of some, but not all, SCBs., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Recreational use of marijuana is associated with few adverse effects, but abuse of synthetic cannabinoids (SCBs) can result in anxiety, psychosis, chest pain, seizures and death. To potentially explain higher toxicity associated with SCB use, we hypothesized that AB-PINACA, a common second generation SCB, exhibits atypical pharmacodynamic properties at CB1 cannabinoid receptors (CB1Rs) and/or a distinct metabolic profile when compared to Δ 9 -tetrahydrocannabinol (Δ 9 -THC), the principal psychoactive cannabinoid present in marijuana. Liquid chromatography tandem mass spectrometry (LC/MS) identified AB-PINACA and monohydroxy metabolite(s) as primary phase I metabolites (4OH-AB-PINACA and/or 5OH-AB-PINACA) in human urine and serum obtained from forensic samples. In vitro experiments demonstrated that when compared to Δ 9 -THC, AB-PINACA exhibits similar affinity for CB1Rs, but greater efficacy for G-protein activation and higher potency for adenylyl cyclase inhibition. Chronic treatment with AB-PINACA also results in greater desensitization of CB1Rs (e.g., tolerance) than Δ 9 -THC. Importantly, monohydroxy metabolites of AB-PINACA retain affinity and full agonist activity at CB1Rs. Incubation of 4OH-AB-PINACA and 5OH-AB-PINACA with human liver microsomes (HLMs) results in limited glucuronide formation when compared to that of JWH-018-M2, a major monohydroxylated metabolite of the first generation SCB JWH-018. Finally, AB-PINACA and 4OH-AB-PINACA are active in vivo , producing CB1R-mediated hypothermia in mice. Taken collectively, the atypical pharmacodynamic properties of AB-PINACA at CB1Rs relative to Δ 9 -THC (e.g., higher potency/efficacy and greater production of desensitization), coupled with an unusual metabolic profile (e.g., production of metabolically stable active phase I metabolites) may contribute to the pronounced adverse effects observed with abuse of this SCB compared to marijuana.

Synthetic cannabinoids (SCBs), synonymous with 'K2', 'Spice' or 'synthetic marijuana', are psychoactive drugs of abuse that frequently result in clinical effects and toxicity more severe than those classically associated with Δ 9 -tetrahydrocannabinol such as extreme agitation, hallucinations, supraventricular tachycardia, syncope, and seizures. JWH-018 is one of the earliest compounds identified in various SCB products, and our laboratory previously demonstrated that JWH-018 undergoes extensive metabolism by cytochromes P450 (P450), binds to, and activates cannabinoid receptors (CBRs). The major enzyme involved in the metabolism of JWH-018 is CYP2C9, a highly polymorphic enzyme found largely in the intestines and liver, with *1 being designated as the wild type, and *2 and *3 as the two most common variants. Three different major products have been identified in human urine and plasma: JWH-018 (ω)-OH, JWH-018 (ω-1)-OH(R), and JWH-018 (ω-1)-OH(S). The (ω-1)-OH metabolite of JWH-018 is a chiral molecule, and is thus designated as either (ω-1)-OH(R) or (ω-1)-OH(S). Here, in vitro enzyme kinetic assays performed with human recombinant CYP2C9 variants (*1, *2, and *3) revealed that oxidative metabolism by CYP2C9*3 resulted in significantly less formation of (ω)-OH and (ω-1)-OH metabolites. Surprisingly, CYP2C9*2 was roughly 3.6-fold more efficient as the CYP2C9*1 enzyme based on V max /K m , increasing the rate of JWH-018 metabolism and allowed for a much more rapid elimination. These results suggest that genetic polymorphisms of P450 enzymes result in the production of varying levels of biologically active JWH-018 metabolites in some individuals, offering a mechanistic explanation for the diverse clinical toxicity often observed following JWH-018 abuse., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Tamoxifen (Tam) is a selective estrogen receptor (ER) modulator (SERM) that is an essential drug to treat ER-positive breast cancer. Aside from known actions at ERs, recent studies have suggested that some SERMs like Tam also exhibit novel activity at cannabinoid subtype 1 and 2 receptors (CB1R and CB2Rs). Interestingly, cis- (E-Tam) and trans- (Z-Tam) isomers of Tam exhibit over a 100-fold difference in affinity for ERs. Therefore, the current study assessed individual isomers of Tam and subsequent cytochrome P450 metabolic products, 4-hydroxytamoxifen (4OHT) and 4-hydroxy-N-desmethyl tamoxifen (End) for affinity and activity at CBRs. Results showed that Z-4OHT, but not Z-Tam or Z-End, exhibits higher affinity for both CB1 and CB2Rs relative to the E-isomer. Furthermore, Z- and E-isomers of Tam and 4OHT show slightly higher affinity for CB2Rs, while both End isomers are relatively CB1R-selective. When functional activity was assessed by G-protein activation and regulation of the downstream effector adenylyl cyclase, all isomers examined act as full CB1 and CB2R inverse agonists. Interestingly, Z-Tam appears to be more efficacious than the full inverse agonist AM630 at CB2Rs, while both Z-Tam and Z-End exhibit characteristics of insurmountable antagonism at CB1 and CB2Rs, respectively. Collectively, these results suggest that the SERMs Tam, 4OHT and End elicit ER-independent actions via CBRs in an isomer-specific manner. As such, this novel structural scaffold might be used to develop therapeutically useful drugs for treatment of a variety of diseases mediated via CBRs., Competing Interests: The authors have declared that no competing interests exist.

Increased aerobic glycolysis and de novo lipid biosynthesis are common characteristics of invasive cancers. UDP-glucuronosyltransferases (UGTs) are phase II drug metabolizing enzymes that in normal cells possess the ability to glucuronidate these lipids and speed their excretion; however, de-regulation of these enzymes in cancer cells can lead to an accumulation of bioactive lipids, which further fuels cancer progression. We hypothesize that UGT2B isoform expression is down-regulated in cancer cells and that exogenous re-introduction of these enzymes will reduce lipid content, change the cellular phenotype, and inhibit cancer cell proliferation. In this study, steady-state mRNA levels of UGT isoforms from the 2B family were measured using qPCR in 4 breast cancer and 5 pancreatic cancer cell lines. Expression plasmids for UGT2B isoforms known to glucuronidate cellular lipids, UGT2B4, 2B7, and 2B15 were transfected into MCF-7 and Panc-1 cells, and the cytotoxic effects of these enzymes were analyzed using trypan blue exclusion, annexin V/PI staining, TUNEL assays, and caspase-3 immunohistochemistry. There was a significant decrease in cell proliferation and a significant increase in the number of dead cells after transfection with each of the 3 UGT isoforms in both cell lines. Cellular lipids were also found to be significantly decreased after transfection. The results presented here support our hypothesis and emphasize the important role UGTs can play in cellular proliferation and lipid homeostasis. Evaluating the effect of UGT expression on the lipid levels in cancer cell lines can be relevant to understanding the complex regulation of cancer cells, identifying the roles of UGTs as "lipid-controllers" in cellular homeostasis, and illustrating their suitability as targets for future clinical therapy development.

Graphene and single-walled carbon nanotubes were used to deliver the natural low-toxicity drug gambogic acid (GA) to breast and pancreatic cancer cells in vitro, and the effectiveness of this complex in suppressing cellular integrity was assessed. Cytotoxicity was assessed by measuring lactate dehydrogenase release, mitochondria dehydrogenase activity, mitochondrial membrane depolarization, DNA fragmentation, intracellular lipid content, and membrane permeability/caspase activity. The nanomaterials showed no toxicity at the concentrations used, and the antiproliferative effects of GA were significantly enhanced by nanodelivery. The results suggest that these complexes inhibit human breast and pancreatic cancer cells grown in vitro. This analysis represents a first step toward assessing their effectiveness in more complex, targeted, nanodelivery systems., (Copyright © 2014 John Wiley & Sons, Ltd.)

Tamoxifen (Tam) is a selective estrogen receptor modulator used to inhibit breast tumor growth. Tam can be directly N-glucuronidated via the tertiary amine group or O-glucuronidated after cytochrome P450-mediated hydroxylation. In this study, the glucuronidation of Tam and its hydroxylated and/or chlorinated derivatives [4-hydroxytamoxifen (4OHTam), toremifene (Tor), and 4-hydroxytoremifene (4OHTor)] was examined using recombinant human UDP-glucuronosyltransferases (UGTs) from the 1A subfamily and human hepatic microsomes. Recombinant UGT1A4 catalyzed the formation of N-glucuronides of Tam and its derivatives and was the most active UGT enzyme toward these compounds. Therefore, it was hypothesized that single nucleotide polymorphisms (SNPs) in the promoter region of UGT1A4 have the ability to significantly decrease the glucuronidation rates of Tam metabolites in the human liver. In vitro activity of 64 genotyped human liver microsomes was used to determine the association between the UGT1A4 promoter and coding region SNPs and the glucuronidation rates of Tam, 4OHTam, Tor, and 4OHTor. Significant decreases in enzymatic activity were observed in microsomes for individuals heterozygous for -163G/A and -217T/G. These alterations in glucuronidation may lead to prolonged circulating half-lives and may potentially modify the effectiveness of these drugs in the treatment of breast cancer., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Trans-Resveratrol (tRes) has been shown to have powerful antioxidant, anti-inflammatory, anticarcinogenic, and antiaging properties; however, its use as a therapeutic agent is limited by its rapid metabolism into its conjugated forms by UDP-glucuronosyltransferases (UGTs). The aim of the current study was to test the hypothesis that the limited bioavailability of tRes can be improved by modifying its structure to create analogs which would be glucuronidated at a lower rate than tRes itself. In this work, three synthetic stilbenoids, (E)-3-(3-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid (NI-12a), (E)-2,4-dimethoxy-6-(4-methoxystyryl)benzaldehyde oxime (NI-ST-05), and (E)-4-(3,5-dimethoxystyryl)-2,6-dinitrophenol (DNR-1), have been designed based on the structure of tRes and synthesized in our laboratory. UGTs recognize and glucuronidate tRes at each of the 3 hydroxyl groups attached to its aromatic rings. Therefore, each of the above compounds was designed with the majority of the hydroxyl groups blocked by methylation and the addition of other novel functional groups as part of a drug optimization program. The activities of recombinant human UGTs from the 1A and 2B families were examined for their capacity to metabolize these compounds. Glucuronide formation was identified using HPLC and verified by β-glucuronidase hydrolysis and LC-MS/MS analysis. NI-12a was glucuronidated at both the -COOH and -OH functions, NI-ST-05 formed a novel N-O-glucuronide, and no product was observed for DNR-1. NI-12a is primarily metabolized by the hepatic and renal enzyme UGT1A9, whereas NI-ST-05 is primarily metabolized by an extrahepatic enzyme, UGT1A10, with apparent Km values of 240 and 6.2 μM, respectively. The involvement of hepatic and intestinal UGTs in the metabolism of both compounds was further confirmed using a panel of human liver and intestinal microsomes, and high individual variation in activity was demonstrated between donors. In summary, these studies clearly establish that modified, tRes-based stilbenoids may be preferable alternatives to tRes itself due to increased bioavailability via altered conjugation.

K2 or Spice products are emerging drugs of abuse that contain synthetic cannabinoids (SCBs). Although assumed by many teens and first time drug users to be a "safe" and "legal" alternative to marijuana, many recent reports indicate that SCBs present in K2 produce toxicity not associated with the primary psychoactive component of marijuana, ∆(9)-tetrahydrocannabinol (Δ(9)-THC). This mini-review will summarize recent evidence that use of K2 products poses greater health risks relative to marijuana, and suggest that distinct pharmacological properties and metabolism of SCBs relative to Δ(9)-THC may contribute to the observed toxicity. Studies reviewed will indicate that in contrast to partial agonist properties of Δ(9)-THC typically observed in vitro, SCBs in K2 products act as full cannabinoid receptor type 1 (CB1R) and type 2 (CB2R) agonists in both cellular assays and animal studies. Furthermore, unlike Δ(9)-THC metabolism, several SCB metabolites retain high affinity for, and exhibit a range of intrinsic activities at, CB1 and CB2Rs. Finally, several reports indicate that although quasi-legal SCBs initially evaded detection and legal consequences, these presumed "advantages" have been limited by new legislation and development of product and human testing capabilities. Collectively, evidence reported in this mini-review suggests that K2 products are neither safe nor legal alternatives to marijuana. Instead, enhanced toxicity of K2 products relative to marijuana, perhaps resulting from the combined actions of a complex mixture of different SCBs present and their active metabolites that retain high affinity for CB1 and CB2Rs, highlights the inherent danger that may accompany use of these substances., (© 2013.)

New designer drugs such as K2, Spice, and "bath salts" present a formidable challenge for law enforcement and public health officials. The following report summarizes a three-year study of 1320 law enforcement cases involving over 3000 products described as vegetable material, powders, capsules, tablets, blotter paper, or drug paraphernalia. All items were seized in Arkansas from January 2010 through December 2012 and submitted to the Arkansas State Crime Laboratory for analysis. The geographical distribution of these seizures co-localized in areas with higher population, colleges, and universities. Validated forensic testing procedures confirmed the presence of 26 synthetic cannabinoids, 12 designer stimulants, and 5 hallucinogenic-like drugs regulated by the Synthetic Drug Prevention Act of 2012 and other state statutes. Analysis of paraphernalia suggests that these drugs are commonly used concomitantly with other drugs of abuse including marijuana, MDMA, and methamphetamine. Exact designer drug compositions were unpredictable and often formulated with multiple agents, but overall, the synthetic cannabinoids were significantly more prevalent than all the other designer drugs detected. The synthetic cannabinoids JWH-018, AM2201, JWH-122, JWH-210, and XLR11 were most commonly detected in green vegetable material and powder products. The designer stimulants methylenedioxypyrovalerone (MDPV), 3,4-methylenedioxy-N-methylcathinone (methylone), and α-methylamino-valerophenone (pentedrone) were commonly detected in tablets, capsules, and powders. Hallucinogenic drugs were rarely detected, but generally found on blotter paper products. Emerging designer drug products remain a significant problem and continued surveillance is needed to protect public health., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Tamoxifen (Tam) is classified as a selective estrogen receptor modulator (SERM) and is used for treatment of patients with ER-positive breast cancer. However, it has been shown that Tam and its cytochrome P450-generated metabolite 4-hydroxy-Tam (4OH-Tam) also exhibit cytotoxic effects in ER-negative breast cancer cells. These observations suggest that Tam and 4OH-Tam can produce cytotoxicity via estrogen receptor (ER)-independent mechanism(s) of action. The molecular targets responsible for the ER-independent effects of Tam and its derivatives are poorly understood. Interestingly, similar to Tam and 4OH-Tam, cannabinoids have also been shown to exhibit anti-proliferative and apoptotic effects in ER-negative breast cancer cells, and estrogen can regulate expression levels of cannabinoid receptors (CBRs). Therefore, this study investigated whether CBRs might serve as novel molecular targets for Tam and 4OH-Tam. We report that both compounds bind to CB1 and CB2Rs with moderate affinity (0.9-3 μM). Furthermore, Tam and 4OH-Tam exhibit inverse activity at CB1 and CB2Rs in membrane preparations, reducing basal G-protein activity. Tam and 4OH-Tam also act as CB1/CB2R-inverse agonists to regulate the downstream intracellular effector adenylyl cyclase in intact cells, producing concentration-dependent increases in intracellular cAMP. These results suggest that CBRs are molecular targets for Tam and 4OH-Tam and may contribute to the ER-independent cytotoxic effects reported for these drugs. Importantly, these findings also indicate that Tam and 4OH-Tam might be used as structural scaffolds for development of novel, efficacious, non-toxic cancer drugs acting via CB1 and/or CB2Rs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Limited forensic and clinical experience and the lack of confirmatory testing strategies for synthetic cannabinoids (SC) prevent adequate characterization of SC toxicity and the potential impact on public health. A statewide surveillance system identified a fatality involving a 23-year-old man found with a large stab wound to the neck following use of a SC product suspected of containing AM2201. Analytical testing for common SCs, SC metabolites, routine drugs of abuse, and over-the-counter medications was performed on heart blood obtained at autopsy. Additionally, assays were performed on the SC raw material and drug paraphernalia found on the decedent. High concentrations of AM2201 were detected in all samples. AM2201 metabolites were detected in postmortem blood. Other than a trace amount of JWH-073 found in smoke residue, no other substances were detected. Psychiatric complications including self-induced, lethal trauma can occur after the use of SC products., (© 2013 American Academy of Forensic Sciences.)

Designer synthetic cannabinoids like JWH-018 and AM2201 have unique clinical toxicity. Cytochrome-P450-mediated metabolism of each leads to the generation of pharmacologically active (ω)- and (ω-1)-monohydroxyl metabolites that retain high affinity for cannabinoid type-1 receptors, exhibit Δ(9)-THC-like effects in rodents, and are conjugated with glucuronic acid prior to excretion in human urine. Previous studies have not measured the contribution of the specific (ω-1)-monohydroxyl enantiomers in human metabolism and toxicity. This study uses a chiral liquid chromatography-tandem mass spectroscopy approach (LC-MS/MS) to quantify each specific enantiomer and other nonchiral, human metabolites of JWH-018 and AM2201 in human urine. The accuracy (average % RE = 18.6) and reproducibility (average CV = 15.8%) of the method resulted in low-level quantification (average LLQ = 0.99 ng/mL) of each metabolite. Comparisons with a previously validated nonchiral method showed strong correlation between the two approaches (average r(2) = 0.89). Pilot data from human urine samples demonstrate enantiospecific excretion patterns. The (S)-isomer of the JWH-018-(ω-1)-monohydroxyl metabolite was predominantly excreted (>87%) in human urine as the glucuronic acid conjugate, whereas the relative abundance of the corresponding AM2201-(ω-1)-metabolite was low (<5%) and did not demonstrate enantiospecificity (approximate 50:50 ratio of each enantiomer). The new chiral method provides a comprehensive, targeted metabolomic approach for studying the human metabolism of JWH-018 and AM2201. Preliminary evaluations of specific enantiomeric contributions support the use of this approach in future studies designed to understand the pharmacokinetic properties of JWH-018 and/or AM2201.

"K2" or "Spice" is an emerging drug of abuse that is laced with psychoactive synthetic cannabinoids JWH-018 and AM2201. Previous studies have identified hydroxylated (OH) and carboxylated (COOH) species as primary human metabolites, and kinetic studies have implicated CYP2C9 and -1A2 as major hepatic P450s involved in JWH-018 and AM2201 oxidation. The present study extends these findings by testing the hypothesis that CYP2C9- and 1A2-selective chemical inhibitors, sulfaphenazole (SFZ) and α-naphthoflavone (ANF), block oxidation of JWH-018 and AM2201 in human liver microsomes (HLM). A concentration-dependent inhibition of JWH-018 and AM2201 oxidation was observed in the presence of increasing concentration of SFZ (0.5 - 50 μM) and ANF (0.1 - 5.0 μM). No metabolic inhibition was observed with omeprazole, quinidine, and ketoconazole. The results presented herein further demonstrate the importance of CYP2C9- and 1A2-mediated oxidation of JWH-018 and AM2201 and the likelihood of adverse toxicity in populations with polymorphic alleles of these enzymes.

The acridinone derivates 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) and 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) are promising antitumor agents with high activity against several experimental cellular and tumor models and are under evaluation in preclinical and early phase clinical trials. Recent evidence from our laboratories has indicated that both compounds were conjugated by several uridine diphosphate-glucuronyltransferase (UGT) isoforms, the most active being extrahepatic UGT1A10. The present studies were designed to test the ability and selectivity of UGT1A10 in the glucuronidation of acridinone antitumor agents in a cellular context. We show that in KB-3 cells, a HeLa subline lacking expression of any UGT isoforms, both C-1305 and C-1311 undergo metabolic transformation to the glucuronidated forms on overexpression of UGT1A10. Furthermore, UGT1A10 overexpression significantly increased the cytotoxicity of C-1305, but not C-1311, suggesting that the glucuronide was more potent than the C-1305 parent compound. These responses were selective for UGT1A10 because documented overexpression of UGT2B4 failed to produce glucuronide products and failed to alter the cytotoxicity for both compounds. These findings contribute to our understanding of the mechanisms of action of these agents and are of particular significance because data for C-1305 contradict the dogma that glucuronidation typically plays a role in detoxification or deactivation. In summary, these studies suggest that extrahepatic UGT1A10 plays an important role in the metabolism and the bioactivation of C-1305 and constitutes the basis for further mechanistic studies on the mode of action of this drug, as well as translational studies on the role of this enzyme in regulation of C-1305 toxicity in cancer.

Abuse of synthetic cannabinoids (SCs), such as [1-naphthalenyl-(1-pentyl-1H-indol-3-yl]-methanone (JWH-018) and [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone (AM2201), is increasing at an alarming rate. Although very little is known about the metabolism and toxicology of these popular designer drugs, mass spectrometric analysis of human urine specimens after JWH-018 and AM2201 exposure identified monohydroxylated and carboxylated derivatives as major metabolites. The present study extends these initial findings by testing the hypothesis that JWH-018 and its fluorinated counterpart AM2201 are subject to cytochrome P450 (P450)-mediated oxidation, forming potent hydroxylated metabolites that retain significant affinity and activity at the cannabinoid 1 (CB(1)) receptor. Kinetic analysis using human liver microsomes and recombinant human protein identified CYP2C9 and CYP1A2 as major P450s involved in the oxidation of the JWH-018 and AM2201. In vitro metabolite formation mirrored human urinary metabolic profiles, and each of the primary enzymes exhibited high affinity (K(m) = 0.81-7.3 μM) and low to high reaction velocities (V(max) = 0.0053-2.7 nmol of product · min(-1) · nmol protein(-1)). The contribution of CYP2C19, 2D6, 2E1, and 3A4 in the hepatic metabolic clearance of these synthetic cannabinoids was minimal (f(m) = <0.2). In vitro studies demonstrated that the primary metabolites produced in humans display high affinity and intrinsic activity at the CB(1) receptor, which was attenuated by the CB(1) receptor antagonist (6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (O-2050). Results from the present study provide critical, missing data related to potential toxicological properties of "K2" parent compounds and their human metabolites, including mechanism(s) of action at cannabinoid receptors.

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone, C-1311 (NSC-645809), is an antitumor agent shown to be effective against breast cancer in phase II clinical trials. A similar compound, 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, shows high activity against experimental tumors and is expected to have even more beneficial pharmacological properties than C-1311. Previously published studies showed that these compounds are not substrates for cytochrome P450s; however, they do contain functional groups that are common targets for glucuronidation. Therefore, the aim of this work was to identify the human UDP-glucuronosyltransferases (UGTs) able to glucuronidate these two compounds. High-performance liquid chromatography analysis was used to examine the activities of human recombinant UGT1A and UGT2B isoforms and microsomes from human liver [human liver microsomes (HLM)], whole human intestinal mucosa [human intestinal microsomes (HIM)], and seven isolated segments of human gastrointestinal tract. Recombinant extrahepatic UGT1A10 glucuronidated 8-hydroxyl groups with the highest catalytic efficiency compared with other recombinant UGTs, V(max)/K(m) = 27.2 and 8.8 μl · min⁻¹ · mg protein⁻¹, for C-1305 and C-1311, respectively. In human hepatic and intestinal microsomes (HLM and HIM, respectively), high variability in UGT activities was observed among donors and for different regions of intestinal tract. However, both compounds underwent UGT-mediated metabolism to 8-O-glucuronides by microsomes from both sources with comparable efficiency; V(max)/K(m) values were from 4.0 to 5.5 μl · min⁻¹ · mg protein⁻¹. In summary, these studies suggest that imid azoacridinone and triazoloacridinone drugs are glucuronidated in human liver and intestine in vivo and may form the basis for future translational studies of the potential role of UGTs in resistance to these drugs.

Recently, hydroxylated metabolites of JWH-018, a synthetic cannabinoid found in many K2/Spice preparations, have been shown to retain affinity and activity for cannabinoid type 1 receptors (CB1Rs). The activity of glucuronidated metabolites of JWH-018 is not known; hence, this study investigated the affinity and activity of a major metabolite, JWH-018-N-(5-hydroxypentyl) β-D-glucuronide (018-gluc), for CB1Rs. The 018-gluc binds CB1Rs (K(i) = 922 nM), has no effect on G-protein activity, but antagonizes JWH-018 activity at CB1Rs. The data suggests that hydroxylation by cytochrome P450s and subsequent glucuronidation by UDP-glucuronosyltransferases produces a metabolite, 018-gluc, which possesses antagonistic activity at CB1Rs.

K2 and several similar purported "incense products" spiked with synthetic cannabinoids are abused as cannabis substitutes. We hypothesized that metabolism of JWH-073, a prevalent cannabinoid found in K2, contributes to toxicity associated with K2 use. Competition receptor binding studies and G-protein activation assays, both performed by employing mouse brain homogenates, were used to determine the affinity and intrinsic activity, respectively, of potential monohydroxylated (M1, M3-M5) and monocarboxylated (M6) metabolites at cannabinoid 1 receptors (CB1Rs). Surprisingly, M1, M4 and M5 retain nanomolar affinity for CB1Rs, while M3 displays micromolar affinity and M6 does not bind to CB1Rs. JWH-073 displays equivalent efficacy to that of the CB1R full agonist CP-55,940, while M1, M3, and M5 act as CB1R partial agonists, and M4 shows little or no intrinsic activity. Further in vitro investigation by Schild analysis revealed that M4 acts as a competitive neutral CB1R antagonist (K(b)∼40nM). In agreement with in vitro studies, M4 also demonstrates CB1R antagonism in vivo by blunting cannabinoid-induced hypothermia in mice. Interestingly, M4 does not block agonist-mediated responses of other measures in the cannabinoid tetrad (e.g., locomotor suppression, catalepsy or analgesia). Finally, also as predicted by in vitro results, M1 exhibits agonist activity in vivo by inducing significant hypothermia and suppression of locomotor activity in mice. In conclusion, the present study indicates that further work examining the physiological effects of synthetic cannabinoid metabolism is warranted. Such a complex mix of metabolically produced CB1R ligands may contribute to the adverse effect profile of JWH-073-containing products., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Rationale: The therapeutic promise of trans-resveratrol (tRes) is limited by poor bioavailability following rapid metabolism. We hypothesise that trans-arachidin-1 (tA1) and trans-arachidin-3 (tA3), peanut hairy root-derived isoprenylated analogs of tRes, will exhibit slower metabolism/enhanced bioavailability and retain biological activity via cannabinoid receptor (CBR) binding relative to their non-prenylated parent compounds trans-piceatannol (tPice) and tRes, respectively., Results: The activities of eight human UDP-glucuronosyltransferases (UGTs) toward these compounds were evaluated. The greatest activity was observed for extrahepatic UGTs 1A10 and 1A7, followed by hepatic UGTs 1A1 and 1A9. Importantly, an additional isoprenyl and/or hydroxyl group in tA1 and tA3 slowed overall glucuronidation. CBR binding studies demonstrated that all analogs bound to CB1Rs with similar affinities (5-18 µM); however, only tA1 and tA3 bound appreciably to CB2Rs. Molecular modelling studies confirmed that the isoprenyl moiety of tA1 and tA3 improved binding affinity to CB2Rs. Finally, although tA3 acted as a competitive CB1R antagonist, tA1 antagonised CB1R agonists by both competitive and non-competitive mechanisms., Conclusions: Prenylated stilbenoids may be preferable alternatives to tRes due to increased bioavailability via slowed metabolism. Similar structural analogs might be developed as novel CB therapeutics for obesity and/or drug dependency.

Coumadin (R-, S-warfarin) is a challenging drug to accurately dose, both initially and for maintenance, because of its narrow therapeutic range and wide interpatient variability and is typically administered as a racemic (Rac) mixture, which complicates the biotransformation pathways. The goal of the current work was to identify the human UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of the separated R- and S-enantiomers of 6-, 7-, and 8-hydroxywarfarin and the possible interactions between these enantiomers. The kinetic and inhibition constants for human recombinant 1A family UGTs toward these separated enantiomers have been assessed using high-performance liquid chromatography (HPLC)-UV-visible analysis, and product confirmations have been made using HPLC-mass spectrometry/mass spectrometry. We found that separated R- and S-enantiomers of 6-, 7-, and 8-hydroxywarfarin demonstrate significantly different glucuronidation kinetics and can be mutually inhibitory. In some cases significant substrate inhibition was observed, as shown by K(m), V(max), and K(i), comparisons. In particular, UGT1A1 and extrahepatic UGT1A10 have significantly higher capacities than other isoforms for S-7-hydroxywarfarin and R-7-hydroxywarfarin glucuronidation, respectively. Activity data generated using a set of well characterized human liver microsomes supported the recombinant enzyme data, suggesting an important (although not exclusive) role for UGT1A1 in glucuronidation of the main warfarin metabolites, including Rac-6- and 7-hydroxywarfarin and their R- and S-enantiomers in the liver. This is the first demonstration that the R- and S-enantiomers of hydroxywarfarins are glucuronidated, with significantly different enzymatic affinity and capacity, and supports the importance of UGT1A1 as the major hepatic isoform involved.

Little is currently known about the substrate binding site of the human UDP-glucuronosyltransferases (UGTs) and the structural elements that affect their complex substrate selectivity. In order to further understand and extend our earlier findings with phenylalanines 90 and 93 of UGT1A10, we have replaced each of them with Gly, Ala, Val, Leu, Ile or Tyr, and tested the activity of the resulting 12 mutants toward eight different substrates. Apart from scopoletin glucuronidation, the F90 mutants other than F90L were nearly inactive, while the F93 mutants' activity was strongly substrate dependent. Hence, F93L displayed high entacapone and 1-naphthol glucuronidation rates, whereas F93G, which was nearly inactive in entacapone glucuronidation, was highly active toward estradiol, estriol and even ethinylestradiol, a synthetic estrogen that is a poor substrate for the wild-type UGT1A10. Kinetic analyses of 4-nitrophenol, estradiol and ethinylestradiol glucuronidation by the mutants that catalyzed the respective reactions at considerable rates, revealed increased K(m) values for 4-nitrophenol and estradiol in all the mutants, whilst the K(m) values of F93G and F93A for ethinylestradiol were lower than in control UGT1A10. Based on the activity results and a new molecular model of UGT1A10, it is suggested that both F90 and F93 are located in a surface helix at the far end of the substrate binding site. Nevertheless, only F93 directly affects the selectivity of UGT1A10 toward large and rigid estrogens, particularly those with substitutions at the D ring. The effects of F93 mutations on the glucuronidation of smaller or less rigid substrates are indirect, however., (Copyright © 2011 Elsevier Inc. All rights reserved.)

5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, being the close structural analogue of the clinically tested imidazoacridinone anti-tumour agent, C-1311, expressed high activity against experimental tumours and is expected to have more advantageous pharmacological properties than C-1311. The aim of this study was to elucidate the role of selected liver enzymes in the metabolism of C-1305. We demonstrated that the studied triazoloacridinone was transformed with rat and human liver microsomes, HepG2 hepatoma cells and with human recombinant flavin-containing monooxygenases FMO1, FMO3 but not with CYPs. Furthermore, this compound was an effective inhibitor of CYP1A2 and CYP3A4. The product of FMO catalysed metabolism was shown to be identical to the main metabolite from liver microsomes and HepG2 cells. It was identified as an N-oxide derivative and, under hypoxia, it underwent retroreduction back to C-1305, what was extremely effective with participation of CYP3A4. In summary, this work revealed that the involvement of the P450 enzymatic system in microsomal and cellular metabolism of C-1305 was negligible, whereas this agent was an inhibitor of CYP1A2 and CYP3A4. In contrast, FMO1 and FMO3 were crucial for metabolism of C-1305 by liver microsomes and in HepG2 cells, which makes C-1305 an attractive potent anti-tumour agent.

K2, a synthetic cannabinoid (SC), is an emerging drug of abuse touted as "legal marijuana" and marketed to young teens and first-time drug users. Symptoms associated with K2 use include extreme agitation, syncope, tachycardia, and visual and auditory hallucinations. One major challenge to clinicians is the lack of clinical, pharmacological, and metabolic information for the detection and characterization of K2 and its metabolites in human samples. Information on the metabolic pathway of SCs is very limited. However, previous reports have shown the metabolites of these compounds are excreted primarily as glucuronic acid conjugates. Based on this information, this study evaluates nine human recombinant uridine diphosphate-glucuronosyltransferase (UGT) isoforms and human liver and intestinal microsomes for their ability to glucuronidate hydroxylated metabolites of 1-naphthalenyl-1(1-pentyl-1H-indol-3-yl)-methanone (JWH-018) and (1-butyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-073), the two most common SCs found in K2 products. Conjugates were identified and characterized using liquid chromatography/tandem mass spectrometry, whereas kinetic parameters were quantified using high-performance liquid chromatography-UV-visible methods. UGT1A1, UGT1A3, UGT1A9, UGT1A10, and UGT2B7 were shown to be the major enzymes involved, showing relatively high affinity with K(m) ranging from 12 to 18 μM for some hydroxylated K2s. These UGTs also exhibited a high metabolic capacity for these compounds, which indicates that K2 metabolites may be rapidly glucuronidated and eliminated from the body. Studies of K2 metabolites will help future development and validation of a specific assay for K2 and its metabolites and will allow researchers to fully explore their pharmacological actions.

The aminoalkylindole agonists JWH-018 and JWH-073 are contained in "K2/SPICE" products sold as "legal marijuana". Previous human metabolic studies have identified (ω)-hydroxyl and (ω)-carboxyl metabolites as biomarkers that are indicative of product use. However, other primary metabolites exhibiting similar chromatographic properties and mass spectra are also excreted in human urine. Analytical standards were used in this study to identify new primary metabolites as (ω-1)-hydroxyl derivatives of JWH-018 and JWH-073. The liquid chromatography tandem mass spectrometry (LC-MS/MS) procedure, coupled with an automated solid-phase extraction procedure incorporating deuterium-labeled internal standards, provides rapid resolution of the (ω)- and (ω-1) metabolites with adequate sensitivity, precision, and accuracy for trace analysis in human urine. Results from four urine specimens collected after individuals reportedly self-administered either JWH-018 or a mixture of JWH-018 and JWH-073 showed the following: (1) all tested metabolites were excreted in high concentrations, (2) (ω)- and (ω-1)-hydroxyl metabolites were exclusively excreted as glucuronic acid conjugates, and (3) ∼5%-80% of the (ω)-carboxyl metabolites was excreted as glucuronic acid conjugates. This is the first report to identify and quantify (ω-1)-hydroxyl metabolites of JWH-018 and JWH-073 and the first to incorporate automated extraction procedures using deuterium-labeled internal standards. Full clinical validation awaits further testing.

"K2/SPICE" products are commonly laced with aminoalkylindole synthetic cannabinoids (i.e., JWH-018 and JWH-073) and are touted as "legal" marijuana substitutes. Here we validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring urinary concentrations of JWH-018, JWH-073, and several potential metabolites of each. The analytical procedure has high capacity for sample throughput and does not require solid phase or liquid extraction. Evaluation of human urine specimens collected after the subjects reportedly administered JWH-018 or a mixture of JWH-018 and JWH-073 provides preliminary evidence of clinical utility. Two subjects that consumed JWH-018 primarily excreted glucuronidated conjugates of 5-(3-(1-naphthoyl)-1H-indol-1-yl)-pentanoic acid (>30 ng/mL) and (1-(5-hydroxypentyl)-1H-indol-3-yl)(naphthalene-1-yl)-methanone (>50 ng/mL). Interestingly, oxidized metabolites of both JWH-018 and JWH-073 were detected in these specimens, suggesting either metabolic demethylation of JWH-018 to JWH-073 or a nonreported, previous JWH-073 exposure. Metabolic profiles generated from a subject who consumed a mixture of JWH-018 and JWH-073 were similar to profiles generated from subjects who presumably consumed JWH-018 exclusively. Oxidized metabolites of JWH-018 and JWH-073 were of the same pattern, but JWH-018 metabolites were excreted at lower concentrations. These results begin clinically validating the LC-MS/MS assay for detecting and quantifying aminoalkylindole metabolites. Full validation awaits further testing.

A gene delivery concept based on ethylenediamine-functionalized single-walled carbon nanotubes (f-SWCNTs) using the oncogene suppressor p53 gene as a model gene was successfully tested in vitro in MCF-7 breast cancer cells. The f-SWCNTs-p53 complexes were introduced into the cell medium at a concentration of 20 μg mL(-1) and cells were exposed for 24, 48, and 72 hours. Standard ethidium bromide and acridine orange assays were used to detect apoptotic cells and indicated that a significantly larger percentage of the cells (approx 40%) were dead after 72 hours of exposure to f-SWCNTs-p53 as compared to the control cells, which were exposed to only p53 or f-SWCNTs, respectively. To further support the uptake and expression of the genes within the cells, green fluorescent protein-tagged p53, attached to the f-SWCNTs was added to the medium and the complex was observed to be strongly expressed in the cells. Moreover, caspase 3 activity was found to be highly enhanced in cells incubated with the f-SWCNTs-p53 complex, indicating strongly induced apoptosis. This system could be the foundation for novel gene delivery platforms based on the unique structural and morphological properties of multi-functional nanomaterials.

Human UDP-glucuronosyltransferases (EC 2.4.1.17) (UGTs) are major phase II metabolism enzymes that detoxify a multitude of endo- and xenobiotics through the covalent addition of a glucuronic acid moiety. UGTs are promiscuous enzymes that regulate the levels of numerous important endobiotics in a range of tissues, and inactivate most therapeutic compounds in concert with phase I enzymes. In spite of the importance of these enzymes, we have only a limited understanding of the molecular mechanisms governing their substrate specificity and catalytic activity. Until recently, no three-dimensional structural information was available for any mammalian UGT. The 1.8-å resolution apo crystal structure of the UDP-glucuronic acid binding domain of human UGT2B7 (2B7CT) is the only structure of a mammalian UGT target determined to date. In this review, we summarize what has been learned about human UGT function from the analysis of this and other related glycosyltransferase (GT) crystal structures.

As a step toward exploring a targeted metabolomics approach to personalized warfarin (Coumadin) therapy, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of quantifying specific enantiomeric (R and S) contributions of warfarin (WAR) and the corresponding hydroxywarfarins (OH-WAR) and glucuronides (-GLUC) generated by cytochrome P450s (CYP) and UDP-glucuronosyltransferases (UGTs), respectively. Evaluation of quality control samples and three commercially available human samples showed that our analytical approach has the ability to measure 24 unique WAR metabolites in human urine. Evaluation of the human data also provides new insights for evaluating WAR toxicity and begins characterizing important UGT metabolic pathways responsible for WAR detoxification. Data revealed the significance of specific metabolites among patients and the corresponding enzymatic capacity to generate these compounds, including the first report of direct WAR glucuronidation. On the basis of total OH-WAR levels, (S)-7-OH-WAR was the predominant metabolite indicating the significance of CYP2C9 in WAR metabolism, although other CYP2C enzymes also contributed to clearance of this isomer. (R)-WAR hydroxylation to OH-WARs was more diverse among the patients as reflected in varying contributions of CYP1A2 and multiple CYP2C enzymes. There was wide variation in the glucuronidation of WAR and the OH-WARs with respect to the compounds and patients. 6- and 7-OH-WAR were primarily (>70%) excreted as glucuronides unlike 4'-OH-WAR and 8-OH-WAR. For all patients, UGT1A1 is likely responsible for 6-O-GLUC production, although UGT1A10 may also contribute in one patient. 7-O-GLUC levels reflected contributions from potentially five different UGT1A enzymes. In all cases, WAR, 4'-OH-WAR, 8-OH-WAR, and the corresponding glucuronides were minor metabolites with respect to the others. Taken together, these data suggest that both P450 and UGT reactions contribute to the generation of excretable products in human urine, thereby generating complex metabolic networks.

The purpose of this work was to identify human UDP-glucuronosyltransferases (UGTs) capable of glucuronidating dopamine. Using a sensitive liquid chromatography-tandem mass spectrometry method, we screened all 19 known human UGTs and found that only one enzyme, UGT1A10, catalyzed dopamine glucuronidation at substantial rates, yielding both dopamine-4-O-glucuronide (37.1 pmol/min/mg) and dopamine-3-O-glucuronide (32.7 pmol/min/mg). Much lower (<2 pmol/min/mg) or no dopamine glucuronidation activity was found for all other UGTs tested at 1 mM dopamine. Evaluation of the UGT1A10 expression pattern in human tissues by quantitative reverse transcription-polymerase chain reaction confirmed that it is mainly expressed in small intestine, colon, and adipose tissue, whereas only low levels were found in trachea, stomach, liver, testis, and prostate but not in brain. Dopamine glucuronidation assays using microsomes from human liver and intestine corroborated these findings because activity in intestinal microsomes was markedly higher than that in liver microsomes. Moreover, the glucuronidation regioselectivity in intestinal microsomes was similar to that of recombinant UGT1A10, and both enzyme sources exhibited sigmoidal kinetics with substrate affinity (K(A)) values in the range of 2 to 3 mM. Examination of four UGT1A10 mutants, F90A, F90L, F93A, and F93L, revealed lower dopamine glucuronidation in all of them, particularly in F90A and F93A. Nonetheless, the substrate affinities of the four mutants were similar to that of UGT1A10. It is interesting to note that mutant F93L exhibited regioselectivity, conjugating dopamine at the 4-hydroxyl (OH) position approximately 3 times more efficiently than at the 3-OH position. These results shed new light on the structure and function of UGT1A10 and indicate that dopamine may be a useful probe substrate for this enzyme.

Recent studies show that the extrahepatic human UDP-glucuronosyltransferase (UGT)1A10 is capable of phase II glucuronidation of several major cytochrome P450 metabolites of warfarin (i.e., 6-, 7-, and 8-hydroxywarfarin). This study expands on this finding by testing the hypothesis that the UGT1A10 F(90)-M(91)-V(92)-F(93) amino acid motif is important for proper recognition and conjugation of hydroxywarfarin derivatives. Site-directed mutagenesis studies demonstrate that F(90) is critical for 6- and 7-hydroxywarfarin glucuronidation based on the complete loss of enzymatic activity toward these substrates. In contrast, V92A and F93A mutants lead to higher rates of substrate turnover, have minimum changes in K(m) values, and demonstrate substrate inhibition kinetics. A completely different activity profile is observed in the presence of 8-hydroxywarfarin. No change in either activity or affinity is observed with F90A when compared with wild type, whereas F93A and V92A mutants show increases in V(max) (3- and 10-fold, respectively) and minimum changes in K(m). Liquid chromatographytandem mass spectrometry studies show that enzymatic products produced by mutants are identical to wild-type products produced in the presence of 6-, 7-, and 8-hydroxywarfarin. Because F(90) is not critical for the glucuronidation of 8-hydroxywarfarin, there is likely another, different amino acid responsible for binding this compound. In addition, an inhibitory binding site may be formed in the presence of 6- and 7-hydroxywarfarin. This new knowledge and continued characterization of the hydroxywarfarin binding site(s) for UGT1A10 will help elucidate the molecular mechanism of hydroxywarfarin glucuronidation and potentially result in more effective anticoagulant therapies.

UGT1A10 was recently identified as the major isoform that conjugates estrogens. In this study, real-time PCR revealed high levels of UGT1A10 and UGT2B7 mRNA in human breast tissues. The expression of UGT1A10 in breast was a novel finding. UGT1A10 and UGT2B7 mRNAs were differentially expressed among normal and malignant specimens. Their overall expression was significantly decreased in breast carcinomas as compared to normal breast specimens (UGT1A10: 68+/-26 vs. 252+/-86, respectively; p<0.05) and (UGT2B7: 1.4+/-0.7 vs. 12+/-4, respectively; p<0.05). Interestingly, in African American women, UGT1A10 expression was significantly decreased in breast carcinomas in comparison to normals (57+/-35 vs. 397+/-152, respectively; p<0.05). Among Caucasian women, UGT2B7 was significantly decreased in breast carcinomas in comparison to normals (1.1+/-0.5 vs. 13.5+/-6, respectively; p<0.05). Glucuronidation of 4-hydroxylated estrone (4-OHE(1)) was significantly reduced in breast carcinomas compared to normals (30+/-15 vs. 106+/-31, respectively; p<0.05). Differential down-regulation of UGT1A10 and UGT2B7 mRNAs, protein, and activity in breast carcinomas compared to the adjacent normal breast specimens from the same donor were also found. These data illustrate the novel finding of UGT1A10 in human breast and confirm the expression of UGT2B7. Significant individual variation and down-regulation of expression in breast carcinomas of both isoforms were also demonstrated. These findings provide evidence that decreased UGT expression and activity could result in the promotion of carcinogenesis.

All UDP-glucuronosyltransferase enzymes (UGTs) share a common cofactor, UDP-glucuronic acid (UDP-GlcUA). The binding site for UDP-GlcUA is localized to the C-terminal domain of UGTs on the basis of amino acid sequence homology analysis and crystal structures of glycosyltransferases, including the C-terminal domain of human UGT2B7. We hypothesized that the (393)DQMDNAK(399) region of human UGT1A10 interacts with the glucuronic acid moiety of UDP-GlcUA. Using site-directed mutagenesis and enzymatic analysis, we demonstrated that the D393A mutation abolished the glucuronidation activity of UGT1A10 toward all substrates. The effects of the alanine mutation at Q(394),D(396), and K(399) on glucuronidation activities were substrate-dependent. Previously, we examined the importance of these residues in UGT2B7. Although D(393) (D(398) in UGT2B7) is similarly critical for UDP-GlcUA binding in both enzymes, the effects of Q(394) (Q(399) in UGT2B7) to Ala mutation on activity were significant but different between UGT1A10 and UGT2B7. A model of the UDP-GlcUA binding site suggests that the contribution of other residues to cosubstrate binding may explain these differences between UGT1A10 and UGT2B7. We thus postulate that D(393) is critical for the binding of glucuronic acid and that proximal residues, e.g., Q(394) (Q(399) in UGT2B7), play a subtle role in cosubstrate binding in UGT1A10 and UGT2B7. Hence, this study provides important new information needed for the identification and understanding of the binding sites of UGTs, a major step forward in elucidating their molecular mechanism.

Our understanding of human phase II metabolic pathways which facilitate detoxification and excretion of warfarin (Coumadin) is limited. The goal of this study was to test the hypothesis that there are specific human hepatic and extrahepatic UDP-glucuronosyltransferase (UGT) isozymes, which are responsible for conjugating warfarin and hydroxylated metabolites of warfarin. Glucuronidation activity of human liver microsomes (HLMs) and eight human recombinant UGTs toward (R)- and (S)-warfarin, racemic warfarin, and major cytochrome P450 metabolites of warfarin (4'-, 6-, 7-, 8-, and 10-hydroxywarfarin) has been assessed. HLMs, UGT1A1, 1A8, 1A9, and 1A10 showed glucuronidation activity toward 4'-, 6-, 7-, and/or 8-hydroxywarfarin with K(m) values ranging from 59 to 480 microM and V(max) values ranging from 0.03 to 0.78 microM/min/mg protein. Tandem mass spectrometry studies and structure comparisons suggested glucuronidation was occurring at the C4'-, C6-, C7-, and C8-positions. Of the hepatic UGT isozymes tested, UGT1A9 exclusively metabolized 8-hydroxywarfarin, whereas UGT1A1 metabolized 6-, 7-, and 8-hydroxywarfarin. Studies with extrahepatic UGT isoforms showed that UGT1A8 metabolized 7- and 8-hydroxywarfarin and that UGT1A10 glucuronidated 4'-, 6-, 7-, and 8-hydroxywarfarin. UGT1A4, 1A6, 1A7, and 2B7 did not have activity with any substrate, and none of the UGT isozymes evaluated catalyzed reactions with (R)- and (S)-warfarin, racemic warfarin, or 10-hydroxywarfarin. This is the first study identifying and characterizing specific human UGT isozymes, which glucuronidate major cytochrome P450 metabolites of warfarin with similar metabolic rates known to be associated with warfarin metabolism. Continued characterization of these pathways may enhance our ability to reduce life-threatening and costly complications associated with warfarin therapy.

Human UDP-glucuronosyltransferase 2B7 (UGT2B7) is one of the major isoforms involved in the glucuronidation of endogenous compounds and xenobiotics. This isoform is the only human UGT shown to glucuronidate retinoids and their oxidized derivatives. In this study, the effects of all-trans retinoic acid (atRA), 9-cis RA, and the RAR agonist TTNPB, on UGT2B7 and UGT2B15 mRNA expression in Caco-2 cells have been examined. Each of these retinoids significantly suppressed UGT2B7 mRNA expression in a concentration-dependent manner with IC50 values of 3.5, 0.3, and 0.2 microM, respectively. However, no inhibition was observed when two other UGTs, UGT2B15 or -1A6, were exposed to atRA, 9-cis RA, or TTNPB, demonstrating that the inhibitory effect of retinoids might be specific for the UGT2B7 isoform. Further, experiments with oxidized atRA derivatives, 4-OH-atRA, 4-oxo-atRA, and 5,6-epoxy-atRA showed that these RA degradation products have no inhibitory effect on UGT2B7 mRNA expression. These data lead us to hypothesize that biologically active forms of RA suppress the expression of UGT2B7 in intestinal cells. This information provides a new pathway by which retinoids may enhance their own toxicity when accumulated in the body at pharmacological concentrations by down-regulating the enzymes involved in their biotransformation into soluble derivatives.

Recently, we have shown that UGT1A10 is actively involved in the inactivation of E(1), E(2), and their 2- and 4-hydroxylated derivatives. In the present study, we show for the first time that treatment of the MCF-7 ER-positive breast cancer cell line with E(2) produces a dose-dependent up-regulation of UGT1A10 mRNA levels, followed by a steady down-regulation. In contrast, E(2) did not stimulate mRNA expression in the MDA-MB-231 (ER)-negative breast cancer cell line. Expression of UGT1A10 mRNA was blocked by the antiestrogen, ICI 182,780, but not by the transcriptional inhibitor, actinomycin-d. These findings suggest that regulation of UGT1A10 mRNA might be a primary transcriptional response mediated through the ER. Expression of UGT1A10 mRNA was also stimulated by other estrogenic compounds including propylpyrazoletriol (PPT) and genistein (Gen). Exposure of MCF-7 cells to 0.1nM E(2) up-regulated, and then down-regulated, UGT1A protein and enzymatic activity toward E(2) at 10nM E(2) as determined by Western blot and glucuronidation activity assays. Collectively, these results suggest that induction of UGT1A10 mRNA expression by E(2) might be mediated through ER, and that this isoform is a novel, estrogen-regulated target gene in MCF-7, ER-positive human breast cancer cells. The finding of E(2)-induced expression of UGT1A10 mRNA, followed by the down-regulation of UGT1A10 at pharmacological concentrations of E(2), might have a significant moderating effect on E(2) availability for ER and estrogen clearance, thereby promoting the signaling of E(2) in breast cancer cells.

It is widely believed that the UGT1A isoforms, UGT1A8 and -1A10, are expressed exclusively in extrahepatic tissues. In this work, human primary hepatocytes from six donors were analyzed for UGT1A8 and -1A10 mRNA expression by semi-quantitative RT-PCR. New primers to amplify UGT1A8 mRNA were designed and found to differ from those previously published. We demonstrated that UGT1A8 and -1A10 mRNA are expressed in hepatocytes. Although basal UGT mRNA levels were detected in untreated hepatocytes, significant up-regulation of the levels of mRNA for these isoforms were seen after treatment with 3-methylcholanthrene (3-MC) and rifampicin (Rif). RT-PCR products for all UGTs were sequenced and unambiguously identified as matching the corresponding cDNA. The discovery of these isoforms in hepatocytes is a novel discovery and will stimulate studies on the potential role for these isoforms in hepatic detoxification.

Human UDP-glucuronosyltransferases (UGT) are the dominant phase II conjugative drug metabolism enzymes that also play a central role in processing a range of endobiotic compounds. UGTs catalyze the covalent addition of glucuronic acid sugar moieties to a host of therapeutics and environmental toxins, as well as to a variety of endogenous steroids and other signaling molecules. We report the 1.8-A resolution apo crystal structure of the UDP-glucuronic acid binding domain of human UGT isoform 2B7 (UGT2B7), which catalyzes the conjugative elimination of opioid, antiviral, and anticancer drugs. This is the first crystal structure of any region of a mammalian UGT drug metabolism enzyme. Designated UGT2B7 mutants at residues predicted to interact with the UDP-glucuronic acid cofactor exhibited significantly impaired catalytic activity, with maximum effects observed for amino acids closest to the glucuronic acid sugar transferred to the acceptor molecule. Homology modeling of UGT2B7 with related plant flavonoid glucosyltransferases indicates human UGTs share a common catalytic mechanism. Point mutations at predicted catalytic residues in UGT2B7 abrogated activity, strongly suggesting human UGTs also utilize a serine hydrolase-like catalytic mechanism to facilitate glucuronic acid transfer.

Despite its biological importance, our knowledge of active site structure and relevance of critical amino acids in CYP2E1 catalytic processes remain limited. In this study, we identified CYP2E1 active site residues using photoaffinity labeling with 7-azido-4-methylcoumarin (AzMC) coupled with a CYP2E1 homology model. In the absence of light, AzMC was an effective competitor against substrate p-nitrophenol oxidation by CYP2E1. Photoactivation of AzMC led to a concentration-dependent loss in CYP2E1 activity and structural integrity resulting from the modification of both heme and protein. The photo-labeling reaction degraded heme and produced a possible heme adduct. Probe incorporation into the protein occurred at multiple sites within substrate recognition sequence 5 (SRS-5). Based on a CYP2E1 homology model, we hypothesize AzMC labels SRS-5 residues, Leu363, Val364, and Leu368, in the active site. In addition, we propose a series of phenylalanines, especially Phe106, mediate contacts with the coumarin.

Human UDP-glucuronosyltransferase 1A10 has been identified as the major isoform involved in the biotransformation of a wide range of phenolic substrates, including native estrogens and their oxidized metabolites. Our recent studies point to the F(90)-M(91)-V(92)-F(93) amino acid motif of UGT1A10, which was identified using photoaffinity labeling followed by LC-MS/MS analysis, as a key determinant of the binding of phenolic substrates. In this report, we have evaluated the role of F(90), V(92), and F(93) in the recognition of estrogens by UGT1A10 using site-directed mutagenesis. Kinetic studies using five mutants revealed that F(90) and F(93) are critical residues for the recognition of all estrogen substrates. The substitution of F(90) with alanine totally abolished the activity of this enzyme toward all the estrogens investigated. Overall, sequential removal for the aromatic ring (F to L) and of the hydrophobic chain (F to A and V to A) from amino acids 90, 92, and 93 effectively alters estrogen recognition. This demonstrates that individual features of the native and hydroxylated estrogens determine the specific binding properties of the compound within the binding site of the human UGT1A10 and the mutants. The resulting activities are completely abolished, unchanged, increased, or decreased depending on the structures of both the mutant and the substrate. The novel identification of UGT1A10 as the major isoform involved in the glucuronidation of all estrogens and the discovery of the importance of the FMVF motif in the binding of steroids will help to elucidate the molecular mechanism of glucuronidation, resulting in the design of more effective estrogen-based therapies.

Phytoestrogens, in particular the isoflavone aglycones genistein and daidzein, are thought to be the bioactive components of soy. Like estrogens, isoflavones can be sulfur-conjugated. However, although isoflavones in the serum are found largely in the form of glucuronide and sulfur conjugates following soy consumption, little is known regarding the relative contributions of sulfotransferases in the liver and small intestine to isoflavone sulfation. Since the sulfates may be deconjugated in target tissues, circulating isoflavone sulfates may act as a source of tissue aglycones. In the current study genistein and daidzein sulfotransferase activities were measured in cytosol from human and rat liver and gastrointestinal tract. Isoflavone sulfation in the human gastrointestinal (GI) tract was correlated with activities towards substrates for previously characterized human sulfotransferases. Western blots of human cytosols were also conducted using antisera towards human sulfotransferases SULT1E1 and SULT2A1. Whereas rat liver was almost fourfold more active than small intestine in sulfation of genistein, in the human, activities in the two tissues were comparable. In contrast, intestinal sulfation of daidzein was comparable to hepatic sulfation in the rat and significantly greater in the human. Genistein and daidzein sulfation occurred throughout the human GI tract, but with a different distribution and different interindividual variability. Whereas genistein sulfation in the human GI tract correlated significantly with sulfation of the prototypical human phenolic sulfotransferase SULT1A family substrate 2-naphthol (r2 = 0.71), daidzein sulfotransferase activity did not correlate with activities towards any prototypical sulfotransferase substrate or with genistein sulfation. Our results suggest that metabolism in the human GI tract has an important role in the generation of potentially bioactive isoflavone sulfates and a major role for the human phenolic sulfotransferase SULT1A family in metabolism of genistein in the gut. However, human intestinal daidzein sulfation appears to be catalyzed by a separate enzyme.

Resveratrol (3, 5, 4'-trihydroxy-trans-stilbene), a natural polyphenol present in grapes and peanuts, has been reported to exert a variety of potentially therapeutic effects. The aim of this study was to determine the contribution of the gastrointestinal (GI) tract to the glucuronidation of this compound and its cis-isomer, which also occurs naturally. For this purpose, glucuronidation of the two resveratrol isomers was investigated in human microsomes prepared from: stomach, duodenum, four segments of the remaining small intestine (S-1 to S-4) and colon, and from the human intestinal cell lines Caco-2 and PD-7. cis- and trans-Resveratrol were efficiently glucuronidated in the GI tract with the formation of both 3-O- and 4'-O-glucuronides, however, the two stereoisomers were glucuronidated at different rates depending on the donor and the segment considered. Microsomes prepared from Caco-2 and PD-7 cells also efficiently glucuronidated cis-resveratrol and, to a lesser extent, the trans-isomer, however, only the 3-O-glucuronide was formed. Among the UDP-glucuronosyltransferases (UGT) that are known to be expressed in the GI tract, the isoforms UGT1A1, 1A6, 1A8, 1A9 and 1A10 were active in glucuronidating trans- and/or cis-resveratrol. The results demonstrate that the GI tract may contribute significantly to the first pass metabolism of these naturally occurring polyphenols., (Copyright 2006 John Wiley & Sons, Ltd.)

4-Azido-2-hydroxybenzoic acid (4-AzHBA), a novel photoactive benzoic acid derivative, has been synthesized and used as a photoprobe to identify the phenol binding site of UDP-glucuronosyltransferases (UGTs). Analysis of recombinant His-tag UGTs from the 1A family for their ability to glucuronidate p-nitrophenol (pNP) and 4-methylumbelliferone (4-MU) revealed that UGT1A10 shows high activity toward phenols and phenol derivatives. Purified UGT1A10 was photolabeled with 4-AzHBA, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometry. A single modified peak corresponding to amino acid residues 89-98 (EFMVFHAQWK) of UGT1A10 was identified. The attachment site of the 4-AzHBA probe was localized to the quadruplet Phe(90)-Met(91)-Val(92)-Phe(93) using ESI LC-MS/MS. Sequence alignment revealed that the Phe(90) and Phe(93) are conserved in UGT1A7-10. Site-directed mutagenesis of these two amino acids was then followed by kinetic analysis of the mutants with two phenolic substrates, pNP and 4-MU, containing one and two planar rings, respectively. Using the combination of photoaffinity labeling, enzymatic digestion, MALDI-TOF and LC-MS mass spectrometry, and site-directed mutagenesis, we have determined for the first time that Phe(90) and Phe(93) are directly involved in the catalytic activity of UGT1A10 toward 4-MU and pNP.