Tissue distribution and metabolic profiling of cyclosporine (CsA) in mouse and rat investigated by DESI and MALDI mass spectrometry imaging (MSI) of whole-body and single organ cryo-sections.

Therapeutic peptides are a fast-growing class of pharmaceuticals. Like small molecules, the costs associated with their discovery and development are significant. In addition, since the preclinical data guides first-in-human studies, there is a need for analytical techniques that accelerate and improve our understanding of the absorption, distribution, metabolism, and excretion (ADME) characteristics of early drug candidates. Mass spectrometry imaging (MSI), which can be used to visualize drug distribution in intact tissue, has been extensively used to study small molecule drugs, but only applied to a limited extent to larger molecules, such as peptides, after dosing. Herein, we use MSI to obtain spatial information on the distribution and metabolism of a peptide drug. The immunosuppressant cyclosporine (CsA), a cyclic undecapeptide, was used as a-proof-of-concept peptide and investigated by desorption electrospray ionization (DESI) MSI. Calibration curves were made based on a spiked tissue homogenate model. Different washing protocols were tested to improve sensitivity, but CsA, being a quite lipophilic peptide, was found not to benefit from tissue washing. The distribution of CsA and its metabolites were mapped in whole-body mouse sections and within rat organs. Whole-body DESI-MSI studies in mice showed widespread distribution of CsA with highest abundance in organs like the pancreas and liver. After 24 h, hydroxy and dihydroxy metabolites of CsA were detected predominantly in the intestines, which were largely devoid of CsA. In addition to the DESI-MSI experiments, MALDI-MSI was also conducted on rat jejunum at higher spatial resolution, revealing the morphology of the jejenum at greater detail; however, DESI provided similar results for drug and metabolite distribution in rat jejunum at apparent slightly better sensitivity. Given its label-free nature, MSI could provide valuable ADME insight, especially for candidates in the early-stage pipeline before radiolabeling.

Ketamine analogues: Comparative toxicokinetic in vitro-in vivo extrapolation and quantification of 2-fluorodeschloroketamine in forensic blood and hair samples.

Recently, the new psychoactive substance (NPS) ketamine analogue 2-fluoro-deschloroketamine (2FDCK) was observed in driving-under-the-influence-of-drugs whole blood samples and in a forensic hair investigation case in Denmark. The molecular structure variations among the NPS subgroups may alter the metabolic fate and drug potency, thereby posing a threat for drug users. This study reports quantification of 2FDCK in whole blood samples and forensic hair and compares the following toxicokinetic parameters: unbound fraction (fu) and in vitro-in vivo-extrapolation (IVIVE) of hepatic clearance for ketamine, norketamine, 2FDCK, methoxetamine and deschloroketamine. The fu was investigated with ultrafiltration, while clearance studies were conducted at 1 µM with pooled human liver microsomes. Samples were analysed by liquid chromatography and mass spectrometry. For the first time, 2FDCK was determined in a concentration range between 0.005 and 0.48 mg/kg in blood samples. Segmental hair analysis demonstrated 2FDCK at concentrations from 0.007 to 0.034 ng/mg throughout the three investigated segments. Toxicokinetic comparison of the five compounds gave a fu between 0.54 and 0.84, with ketamine being the most bound and deschloroketamine being the least bound, in accordance with the logP of the compounds. Conversely, a negative correlation was observed between the molecular weight of the halogen in the ortho-position and IVIVE hepatic clearance. The IVIVE of hepatic clearance, CLparallel-tube, gave values from 18.1 to 5.44 mL/min/kg for ketamine and methoxetamine, respectively. The deschloroketamine IVIVE was disregarded due to low drug elimination under the experimental conditions used. This study provides a basis for toxicokinetic understanding of ketamine analogues.

Glycine-modified growth hormone secretagogues identified in seized doping material.

A number of unknown pharmaceutical preparations seized by Danish customs authorities were submitted for liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis. Comparison with reference standards unequivocally identified the content of the powders as analogs of the growth hormone secretagogues GHRP-2 (Pralmorelin), GHRP-6, Ipamorelin, and modified growth hormone releasing factor (modified GRF 1-29), which can be used as performance-enhancing substances in sports. In all cases, the detected modification involved the addition of an extra glycine amino acid at the N-terminus, and analytical methods targeting growth hormone secretagogues should hence be updated accordingly.

Bromo-dragonfly, a psychoactive benzodifuran, is resistant to hepatic metabolism and potently inhibits monoamine oxidase A.

Bromo-dragonfly is a benzodifuran derivative known as one of the most potent 5-HT2A-receptor agonists within this chemical class, with long-lasting effects of up to 2-3 days. In addition to hallucinogenic effects, the drug is a potent vasoconstrictor, resulting in severe adverse effects, such as necrosis of the limbs. In some cases, intoxication has had fatal outcomes. Little is known about the metabolism of bromo-dragonfly. The aims of this study were to investigate the pharmacokinetics of bromo-dragonfly, determine the plasma protein binding, examine the human hepatic metabolism in vitro, and compare with those of its close analogue, 2C-B-fly. Additionally, we assayed the inhibition potency of both compounds on the monoamine oxidase (MAO) A- and B-mediated oxidative deamination of serotonin (5-HT) and dopamine, respectively. Liquid chromatography high-resolution mass spectrometry was used for metabolism studies in pooled human liver microsomes (HLM), pooled human liver cytosol (HLC) and recombinant enzymes. Inhibition studies of the deamination of 5-HT and dopamine were carried out using LC-MS/MS. Bromo-dragonfly was not metabolised in the tested in vitro systems. On the other hand, 2C-B-fly was metabolised in HLM by CYP2D6 and in HLC to some extent, with the main biotransformations being monohydroxylation and N-acetylation. Furthermore, MAO-A metabolised 2C-B-fly, producing the aldehyde metabolite, which was trapped in vitro with methoxyamine. Inhibition experiments revealed that bromo-dragonfly is a competitive inhibitor of MAO-A with a Ki of 0.352 µM. The IC50 value for bromo-dragonfly indicated that the inhibition of MAO-A may be clinically relevant. However, more data are needed to estimate its impact on the increase of 5-HT in vivo.

Characterization of the hepatic cytochrome P450 enzymes involved in the metabolism of 25I-NBOMe and 25I-NBOH.

The dimethoxyphenyl-N-((2-methoxyphenyl)methyl)ethanamine (NBOMe) compounds are potent serotonin 5-HT2A receptor agonists and have recently been subject to recreational use due to their hallucinogenic effects. Use of NBOMe compounds has been known since 2011, and several non-fatal and fatal intoxication cases have been reported in the scientific literature. The aim of this study was to determine the importance of the different cytochrome P450 enzymes (CYP) involved in the metabolism of 2-(4-iodo-2,5-dimethoxyphenyl)-N-(2methoxybenzyl)ethanamine (25I-NBOMe) and 2-[[2-(4-iodo-2,5dimethoxyphenyl)ethylamino]methyl]phenol (25I-NBOH) and to characterize the metabolites. The following approaches were used to identify the main enzymes involved in primary metabolism: incubation with a panel of CYP and monoamine oxidase (MAO) enzymes and incubation in pooled human liver microsomes (HLM) with and without specific CYP chemical inhibitors. The study was further substantiated by an evaluation of 25I-NBOMe and 25I-NBOH metabolism in single donor HLM. The metabolism pathways of 25I-NBOMe and 25I-NBOH were NADPHdependent with intrinsic clearance values of (CLint) of 70.1 and 118.7 mL/min/kg, respectively. The biotransformations included hydroxylation, O-demethylation, N-dealkylation, dehydrogenation, and combinations thereof. The most abundant metabolites were all identified by retention time and spectrum matching with synthesized reference standards. The major CYP enzymes involved in the metabolism of 25I-NBOMe and 25INBOH were identified as CYP3A4 and CYP2D6, respectively. The compound 25I-NBOH was also liable to direct glucuronidation, which may diminish the impact of CYP2D6 genetic polymorphism. Users of 25I-NBOMe may be subject to drug-drug interactions (DDI) if 25I-NBOMe is taken with a strong CYP3A4 inhibitor. Copyright © 2016 John Wiley & Sons, Ltd.

JWH-018 ω-OH, a shared hydroxy metabolite of the two synthetic cannabinoids JWH-018 and AM-2201, undergoes oxidation by alcohol dehydrogenase and aldehyde dehydrogenase enzymes in vitro forming the carboxylic acid metabolite.

Synthetic cannabinoids are new psychoactive substances (NPS) acting as agonists at the cannabinoid receptors. The aminoalkylindole-type synthetic cannabinoid naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) was among the first to appear on the illicit drug market and its metabolism has been extensively investigated. The N-pentyl side chain is a major site of human cytochrome P450 (CYP)-mediated oxidative metabolism, and the ω-carboxylic acid metabolite appears to be a major in vivo human urinary metabolite. This metabolite is, however, not formed to any significant extent in human liver microsomal (HLM) incubations raising the possibility that the discrepancy is due to involvement of cytosolic enzymes. Here we demonstrate in incubations with human liver cytosol (HLC), that JWH-018 ω-OH, but not the JWH-018 parent compound, is a substrate for nicotinamide adenine dinucleotide (NAD(+))-dependent alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. The sole end-product identified in HLC was the JWH-018 ω-COOH metabolite, while trapping tests with methoxyamine proved the presence of the aldehyde intermediate. ADH/ALDH and UDP-glucuronosyl-transferases (UGT) enzymes may therefore both act on the JWH-018 ω-OH substrate. Finally, we note that for [1-(5-fluoropentyl)indol-3-yl]-naphthalen-1-yl-methanone (AM-2201), the ω-fluorinated analog of JWH-018, a high amount of JWH-018 ω-OH was formed in HLM incubated without NADPH, suggesting that the oxidative defluorination is efficiently catalyzed by non-CYP enzyme(s). The pathway presented here may therefore be especially important for N-(5-fluoropentyl) substituted synthetic cannabinoids, because the oxidative defluorination can occur even if the CYP-mediated metabolism preferentially takes place on other parts of the molecule than the N-alkyl side chain. Controlled clinical studies in humans are ultimately required to demonstrate the in vivo importance of the oxidation pathway presented here.

Cytochrome P450-mediated metabolism of the synthetic cannabinoids UR-144 and XLR-11.

In recent years, synthetic cannabinoids have emerged in the illicit drug market, in particular via the Internet, leading to abuse of these drugs. There is currently limited knowledge about the specific enzymes involved in the metabolism of these drugs. In this study, we investigated the cytochrome P450 (CYP) enzymes involved in the metabolism of the two synthetic cannabinoids (1-pentyl-1H-indol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone (UR-144) and [1-(5-fluoropentyl)-1H-indol-3-yl)](2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11). This study extends previous studies by identifying the specific CYP enzymes involved in the metabolism of UR-144 and XLR-11 utilizing a panel of nine recombinant enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 3A4, and 2E1). This is followed by an investigation of the effect of specific inhibitors targeted against CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4 in human liver microsomes (HLM). Incubations of UR-144 and XLR-11 with recombinant CYP enzymes revealed that UR-144 and XLR-11 are extensively metabolized by CYP3A4 at the tetramethylcyclopropyl (TMCP) moiety, but also CYP1A2 and CYP2C19 showed activity. Inhibition of CYP3A4 in HLM attenuated the metabolism of UR-144 and XLR-11, while inhibition of the other CYP enzymes in HLM had only minor effects. Thus, CYP3A4 is the major contributor to the CYP mediated metabolism of UR-144 and XLR-11 with minor contributions from CYP1A2. Users of UR-144 and XLR-11 are thus subject to the influence of potential drug-drug interactions, if they are concomitantly medicated with CYP3A4 inducers (e.g. some antiepileptics) or inhibitors (e.g. some antifungal drugs). Copyright © 2015 John Wiley & Sons, Ltd.

CYP3A4 Mediates Oxidative Metabolism of the Synthetic Cannabinoid AKB-48.

Synthetic cannabinoid designer drugs have emerged as drugs of abuse during the last decade, and acute intoxication cases are documented in the scientific literature. Synthetic cannabinoids are extensively metabolized, but our knowledge of the involved enzymes is limited. Here, we investigated the metabolism of N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (AKB-48), a compound identified in herbal blends from 2012 and onwards. We screened for metabolite formation using a panel of nine recombinant cytochrome P450 (CYP) enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) and compared the formed metabolites to human liver microsomal (HLM) incubations with specific inhibitors against CYP2D6, 2C19, and 3A4, respectively. The data reported here demonstrate CYP3A4 to be the major CYP enzyme responsible for the oxidative metabolism of AKB-48, preferentially performing the oxidation on the adamantyl moiety. Genetic polymorphisms are likely not important with regard to toxicity given the major involvement of CYP3A4. Adverse drug-drug interactions (DDIs) could potentially occur in cases with co-intake of strong CYP3A4 inhibitors, e.g., HIV antivirals and azole antifungal agents.

Metabolites of 5F-AKB-48, a synthetic cannabinoid receptor agonist, identified in human urine and liver microsomal preparations using liquid chromatography high-resolution mass spectrometry.

New types of synthetic cannabinoid designer drugs are constantly introduced to the illicit drug market to circumvent legislation. Recently, N-​(1-Adamant​yl)-​1-​(5-​fluoropentyl)-​1H-​indazole-​3-​carboxamide (5F-AKB-48), also known as 5F-APINACA, was identified as an adulterant in herbal products. This compound deviates from earlier JHW-type synthetic cannabinoids by having an indazole ring connected to an adamantyl group via a carboxamide linkage. Synthetic cannabinoids are completely metabolized, and identification of the metabolites is thus crucial when using urine as the sample matrix. Using an authentic urine sample and high-resolution accurate-mass Fourier transform Orbitrap mass spectrometry, we identified 16 phase-I metabolites of 5F-AKB-48. The modifications included mono-, di-, and trihydroxylation on the adamantyl ring alone or in combination with hydroxylation on the N-fluoropentylindazole moiety, dealkylation of the N-fluoropentyl side chain, and oxidative loss of fluorine as well as combinations thereof. The results were compared to human liver microsomal (HLM) incubations, which predominantly showed time-dependent formation of mono-, di-, and trihydroxylated metabolites having the hydroxyl groups on the adamantyl ring. The results presented here may be used to select metabolites specific of 5F-AKB-48 for use in clinical and forensic screening.

Functions of volume-sensitive and calcium-activated chloride channels.

The review describes molecular and functional properties of the volume regulated anion channel and Ca(2+)-dependent Cl(-) channels belonging to the anoctamin family with emphasis on physiological importance of these channels in regulation of cell volume, cell migration, cell proliferation, and programmed cell death. Finally, we discuss the role of Cl(-) channels in various diseases.