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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 Km 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. [ABSTRACT FROM AUTHOR]

ABSTRACT 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cellular retinoic acid-binding proteins (CRABPs) are carrier proteins thought to play a crucial role in the transport and metabolism of all- trans-retinoic acid (atRA) and its derivatives within the cell. This report describes a novel photoaffinity-based binding assay involving competition between potential ligands of CRABP and [3H]atRA or [3H]-9- cis-RA for binding to the atRA-binding sites of CRABP I and II. Photoaffinity labeling of purified CRABPs with [3H]atRA was light- and concentration-dependent, saturable, and protected by several retinoids in a concentration-dependent manner, indicating that binding occurred in the CRABP atRA-binding site. Structure-function relationship studies demonstrated that oxidative changes to the atRA β-ionone ring did not affect ligand potency. However, derivatives lacking a terminal carboxyl group and some cis isomers did not bind to CRABPs. These studies also identified two novel ligands for CRABPs: 5,6-epoxy-RA and retinoyl-β-D-glucuronide (RAG). The labeling of both CRABPs with 9- cis-RA occurred with much lower affinity. Experimental evidence excluded nonspecific binding of RAG to CRABPs and UDP-glucuronosyltransferases, the enzymes responsible for RAG synthesis. These results established that RAG is an effective ligand of CRABPs. Therefore, photoaffinity labeling with [3H]atRA can be used to identify new ligands for CRABP and retinoid nuclear receptors and also provide information concerning the identity of amino acid(s) localized in the atRA-binding site of these proteins. [ABSTRACT FROM AUTHOR]

Warfarin is an anticoagulant used for the prevention of thrombosis and embolism. Hydroxylation of warfarin via CYP450s is well documented; however, its biotransformation by UGTs has not been investigated. The activities of 8 human recombinant UGTs, towards warfarin and its hydroxylated metabolites have been assessed with R-warfarin and racemic warfarin, 4′-OH-, 6-OH-, 7-OH-, and 10-OH-warfarin. HPLC-UV-Vis assays and HPLC-MS/MS product confirmations established that UGT1A10, -1A1, -1A3, and -1A8 (highest to lowest activity) conjugated 7-OH-warfarin. Kinetic assays measured Km and Vmax values for 7-OH-warfarin glucuronidation at 165.5 ± 42.7 µM and 1.14 ± 0.1 nmol/mg protein/min, respectively by UGT1A10. 6-OH-warfarin is glucuronidated by 1A1 and 1A10, and 4′-OH-warfarin is glucuronidated by 1A10, but at low levels. Warfarin, 10-OH-warfarin, or R-warfarin were not glucuronidated. This is the first demonstration that major CYP450 metabolites of warfarin are glucuronidated by human recombinant UGTs, with UGT1A10 being the major isoform involved in human phase II biotransformation. [ABSTRACT FROM AUTHOR]

UDP-Glucuronic Acid (UDP-GlcUA) is the common cofactor of all UDP-Glucuronosyltransferase (UGT) isoforms, and it is postulated that its binding site is located within the C-terminal half of the UGT molecule. Amino acid sequence alignment of UDP-glycosyl- and glucuronosyl-transferases as well as other proteins that bind sugars and nucleotides, showed that the DxxD motif in UGTs is a good candidate for the site of UDP binding. Using site-directed mutagenesis and kinetic analysis, we have demonstrated that D393 and D396 of UGT1A10 are involved in the binding of UDP-GlcUA. Mutation of D393 to alanine totally abolished the activity of UGT1A10 toward all substrates analyzed. D396 was also mutated and produced a ∼2 fold decrease in the glucuronidation activity. This mutation did not affect the affinity of this mutant for 2-nitrophenol (pNP); however, its affinity for UDP-GlcUA was decreased by more than half. This is the first direct demonstration that the DxxD motif of UGTs is involved in the binding of UDP-GlcUA. [ABSTRACT FROM AUTHOR]