RESUMO
RATIONALE: Indazole carboxamide synthetic cannabinoids, a prevalent class of recreational drugs, are a major clinical, forensic and public health challenge. One such compound, 5F-ADB, has been implicated in fatalities worldwide. Understanding its metabolism and distribution facilitates the development of laboratory assays to substantiate its consumption. Synthetic cannabinoid metabolites have been extensively studied in urine; studies identifying metabolites in blood are limited and no data on the metabolic stability (half-life, clearance and extraction ratio) of 5F-ADB have been published prior to this report. METHODS: The in vitro metabolism of 5F-ADB was elucidated via incubation with human liver microsomes for 2 h at 37°C. Samples were collected at multiple time points to determine its metabolic stability. Upon identification of metabolites, authentic forensic human blood samples underwent liquid-liquid extraction and were screened for metabolites. Extracts were analyzed via ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOFMS) operated in positive electrospray ionization mode. RESULTS: Seven metabolites were identified including oxidative defluorination (M1); carboxypentyl (M2); monohydroxylation of the fluoropentyl chain (M3.1/M3.2) and indazole ring system (M4); ester hydrolysis (M5); and ester hydrolysis with oxidative defluorination (M6). The half-life (3.1 min), intrinsic clearance (256.2 mL min-1 kg-1 ), hepatic clearance (18.6 mL min-1 kg-1 ) and extraction ratio (0.93) were determined for the first time. In blood, M1 was present in each sample as the most abundant substance; two samples contained M5; one contained 5F-ADB, M1 and M5. CONCLUSIONS: 5F-ADB is rapidly metabolized in HLM. 5F-ADB, M1 and M5 are pharmacologically active at the cannabinoid receptors (CB1 /CB2 ) and M1 and M5 may contribute to a user's impairment profile. The results demonstrate that it is imperative that synthetic cannabinoid assays screen for pharmacologically active metabolites, especially for drugs with short half-lives. The authors propose that M1 and M5 are appropriate markers to include in laboratory blood tests screening for 5F-ADB.
Assuntos
Canabinoides , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Metaboloma/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Adulto , Canabinoides/sangue , Canabinoides/metabolismo , Canabinoides/farmacologia , Feminino , Humanos , Masculino , Metabolômica , Pessoa de Meia-Idade , Adulto JovemRESUMO
FUB-AMB, an indazole carboxamide synthetic cannabinoid recreational drug, was one of the compounds most frequently reported to governmental agencies worldwide between 2016 and 2019. It has been implicated in intoxications and fatalities, posing a risk to public health. In the current study, FUB-AMB was incubated with human liver microsomes (HLM) to assess its metabolic fate and stability and to determine if its major ester hydrolysis metabolite (M1) was present in 12 authentic forensic human blood samples from driving under the influence of drug cases and postmortem investigations using UHPLC-MS/MS. FUB-AMB was rapidly metabolized in HLM, generating M1 that was stable through a 120-min incubation period, a finding that indicates a potential long detection window in human biological samples. M1 was identified in all blood samples, and no parent drug was detected. The authors propose that M1 is a reliable marker for inclusion in laboratory blood screens for FUB-AMB; this metabolite may be pharmacologically active like its precursor FUB-AMB. M1 frequently appears in samples in which the parent drug is undetectable and can point to the causative agent. The results suggest that it is imperative that synthetic cannabinoid laboratory assay panels include metabolites, especially known or potential pharmacologically active metabolites, particularly for compounds with short half-lives.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Indazóis/sangue , Indazóis/metabolismo , Microssomos Hepáticos/metabolismo , Espectrometria de Massas em Tandem/métodos , Valina/análogos & derivados , Adulto , Ésteres/metabolismo , Toxicologia Forense , Humanos , Hidrólise , Indazóis/análise , Indazóis/química , Masculino , Pessoa de Meia-Idade , Valina/análise , Valina/sangue , Valina/química , Valina/metabolismo , Adulto JovemRESUMO
Studies of the metabolic and pharmacological profiles of indole carboxamide synthetic cannabinoids (a prevalent class of new psychoactive substances) are critical in ensuring that their use can be detected through bioanalytical testing. We have determined the in vitro Phase I metabolism of one such compound, PX-1 (5F-APP-PICA), and appropriate markers to demonstrate human consumption. PX-1 was incubated with human liver microsomes, followed by analysis of the extracts via high-resolution mass spectrometry. A total of 10 metabolites were identified, with simultaneous defluorination and monohydroxylation of the pentyl side chain as the primary biotransformation product (M1). Additional metabolites formed were hydroxylation products of the indole and benzyl moieties, distal amide hydrolysis, N-desfluoropentyl, and carboxypentyl metabolites. Three monohydroxylated metabolites specific to PX-1 were identified and are reported for the first time in this study. The primary metabolite, M1, was further oxidized to M5, a carboxypentyl metabolite. M8 is PX-1 specific, possessing an intact fluoropentyl side chain. These three metabolites are the most suitable for implementation into bioanalytical assays for demonstrating PX-1 consumption. The findings of this study can be used by analytical scientists and medical professionals to determine PX-1 ingestion and predict the metabolites of synthetic cannabinoids sharing structural elements.
Assuntos
Agonistas de Receptores de Canabinoides/farmacocinética , Cromatografia Líquida de Alta Pressão/métodos , Indóis/farmacocinética , Espectrometria de Massas/métodos , Microssomos Hepáticos/metabolismo , Agonistas de Receptores de Canabinoides/análise , Agonistas de Receptores de Canabinoides/química , Humanos , Indóis/análise , Indóis/química , Modelos MolecularesRESUMO
Indazole carboxamide synthetic cannabinoids remain the most prevalent subclass of new psychoactive substances (NPS) reported internationally. However, the metabolic and pharmacological properties of many of these compounds remain unknown. Elucidating these characteristics allows members of the clinical and forensic communities to identify causative agents in patient samples, as well as render conclusions regarding their toxic effects. This work presents a detailed report on the in vitro phase I metabolism of indazole carboxamide synthetic cannabinoid APP-CHMINACA (PX-3). Incubation of APP-CHMINACA with human liver microsomes, followed by analysis of extracts via high-resolution mass spectrometry, yielded 12 metabolites, encompassing 7 different metabolite classes. Characterization of the metabolites was achieved by evaluating the product ion spectra, accurate mass and chemical formula generated for each metabolite. The predominant biotransformations observed were hydrolysis of the distal amide group and hydroxylation of the cyclohexylmethyl (CHM) substituent. Nine metabolites were amide hydrolysis products, of which five were monohydroxylated, one dihydroxylated and two were ketone products. The metabolites in greatest abundance in the study were products of amide hydrolysis with no further biotransformation (M1), followed by amide hydrolysis with monohydroxylation (M2.1). Three APP-CHMINACA-specific metabolites were generated, all of which were hydroxylated on the CHM group; one mono-, di- and tri-hydroxylated metabolite each was produced, with dihydroxylation (M6) present in the greatest abundance. The authors propose that metabolites M1, M2.1 and M6 are the most appropriate markers to determine consumption of APP-CHMINACA. The methods used in the current study have broad applicability and have been used to determine the in vitro metabolic profiles of multiple synthetic cannabinoids and other classes of NPS. This research can be used to guide analytical scientists in method development, synthesis of reference material, pharmacological testing of proposed metabolites and prediction of metabolic processes of compounds yet to be studied.
Assuntos
Canabinoides/metabolismo , Medicamentos Sintéticos/metabolismo , Biotransformação , Cromatografia Líquida , Humanos , Hidrólise , Hidroxilação , Indazóis , Metaboloma , Microssomos Hepáticos , Espectrometria de Massas em TandemRESUMO
Synthetic cannabinoids have proliferated over the last decade and have become a major public health and analytical challenge, critically impacting the clinical and forensic communities. Indazole carboxamide class synthetic cannabinoids have been particularly rampant, and exhibit severe toxic effects upon consumption due to their high binding affinity and potency at the cannabinoid receptors (CB1 and CB2 ). MDMB-CHMINACA, methyl 2-[1-(cyclohexylmethyl)-1H-indazole-3-carboxamido]-3,3-dimethylbutanoate, a compound of this chemical class, has been identified in forensic casework and is structurally related to several other synthetic cannabinoids. This study presents the first extensive report on the Phase I metabolic profile of MDMB-CHMINACA, a potent synthetic cannabinoid. The in vitro metabolism of MDMB-CHMINACA was determined via incubation with human liver microsomes and high-resolution mass spectrometry. The accurate masses of precursor and fragments, mass error (ppm), and chemical formula were obtained for each metabolite. Twenty-seven metabolites were identified, encompassing twelve metabolite types. The major biotransformations observed were hydroxylation and ester hydrolysis. Hydroxylations were located predominantly on the cyclohexylmethyl (CHM) moiety. Ester hydrolysis was followed by additional biotransformations, including dehydrogenation; mono- and dihydroxylation and ketone formation, each with dehydrogenation. Minor metabolites were identified and reported. The authors propose that CHM-monohydroxylated metabolites specific to MDMB-CHMINACA are the most suitable candidates for implementation into bioanalytical assays to demonstrate consumption of this synthetic cannabinoid. Due to the structural similarity of MDMB-CHMINACA and currently trending synthetic cannabinoids whose metabolic profiles have not been reported, the results of this study can be used as a guide to predict their metabolic pathways.
Assuntos
Canabinoides/metabolismo , Drogas Ilícitas/metabolismo , Indazóis/metabolismo , Microssomos Hepáticos/metabolismo , Toxicologia Forense/métodos , Humanos , Hidrólise , Hidroxilação , Espectrometria de Massas em Tandem/métodosRESUMO
In this research, electron paramagnetic resonance (EPR) spin-trapping was utilized to determine if surface radical chemistries occur for gamma (gamma)-sterilized orthopedic materials-ultra-high molecular weight polyethylene (UHMWPE) and the novel, hybrid, diurethane dimethacrylate (DUDMA)-based RHAKOSS. The materials' ability to competitively chelate catalytic ferrous ions (Fe(2+)) or readily reduce ferric ions (Fe(3+)), and hydrogen peroxide (H(2)O(2)) directly, in facilitating the Fenton reaction (FR), is indicative of cytotoxicity. Validations with a radical scavenger aids to confirm a radical mechanism. In conjunction, materials were thermally annealed and characterized by attenuated total reflectance-Fourier-transform infrared (ATR-FTIR) spectroscopy in order to explore accelerated oxidative degradation induced by residual radicals evolving from gamma-sterilization. Particularly, there was a significant decrease in spin-adduct peak areas obtained from the reduction of H(2)O(2) in the presence of RHAKOSS or UHMWPE, evaluated against their respective controls. Additionally, chelated Fe(2+) accelerated the rate of FR. This phenomenon suggests that the materials are not better chelators than the Fe-activating chelator, edta. Neither material had the propensity to readily reduce Fe(3+) to the relevant Fe(2+), as certified by a nonradical mechanism. Alternatively, the false spin-adduct signal acquired when chelated Fe(3+) is employed arises via the nucleophilic addition of water onto the DMPO spin trap. Residual radicals in UHMWPE did not recombine/terminate following thermal annealing in an inert atmosphere. The radicals in RHAKOSS, however, did recombine under mild heating in an oxidizing or inert atmosphere. Both materials displayed quenching of ( )OH; however, for UHMWPE, this mechanism was jointly accountable for its accelerated degradation, evidenced by ATR-FTIR. Quenching of ( )OH by the silica found in RHAKOSS manifested in a competing effect that counterbalanced the observed FR. Implanted RHAKOSS is not likely to promote cytotoxicity and should not degrade, but the damaging effect of gamma sterilization on UHMWPE is a serious dilemma confronting its long-term durability and biocompatibility.
Assuntos
Inflamação/etiologia , Teste de Materiais , Equipamentos Ortopédicos/efeitos adversos , Polietilenos/química , Polietilenos/toxicidade , Esterilização , Materiais Biocompatíveis/análise , Materiais Biocompatíveis/química , Espectroscopia de Ressonância de Spin Eletrônica , Raios gama , Inflamação/induzido quimicamente , Estrutura Molecular , Polietilenos/efeitos da radiação , Propriedades de Superfície/efeitos da radiaçãoRESUMO
Hydroxyl radical (*OH)-induced inflammation is a primary mode for in vivo cytotoxicity. A legitimate concern is whether particulate wear debris from orthopedic composites can stimulate inflammation via ferrous ion (Fe2+)-mediated production of *OH. The purpose of this research was to utilize electron paramagnetic resonance (EPR) spin trapping in investigating and comparing the potential for postsurgical cytotoxicity induced specifically by *OH in the presence of two composites: Simplex P and the novel, hybrid, CORTOSS. Cytotoxicity is evaluated based on the composites competitively chelating catalytic Fe2+ or readily reducing ferric ions (Fe3+), in facilitating the Fenton reaction (FR). *OH that are produced were then validated by a radical scavenger to confirm a genuine radical signal and mechanism. Spin adduct peak areas decreased in the presence of CORTOSS as opposed to increasing in the presence of Simplex P, evaluated against their respective controls. A plausible theory elucidating this finding is that CORTOSS may sequester the Fe form, by virtue of its monomers. Principally, direct comparison of composites indicated that Simplex P had greater tendency to produce *OH, yielding 25.6 and 48.7% greater spin adduct peak areas when chelated and non-chelated Fe2+ are used, respectively. Moreover, the rate of FR accelerated when chelated Fe2+ was used, leading to the formation of a ternary complex with the composites. This was more prominent in Simplex P, as coordination of chelated Fe2+ occurs on its surface via an electrostatic attraction to allow a seventh coordination site for ligand exchange in the ternary complex, stabilized by Ba2+. Conversely, the silica found in CORTOSS possesses radical quenching abilities that deactivate generated *OH in impeding the efficiency of FR. Neither composite demonstrated a capacity to readily reduce Fe3+ to the relevant Fe2+, as validated by a non-radical pathway. Instead, the artificial spin adduct signal attained when employing chelated Fe3+ was due to the nucleophilic addition of water onto DMPO. Simplex P may also serve as a template for surface catalysis of the nucleophilic addition of water onto DMPO involving chelated Fe3+. CORTOSS is thought not to induce cytotoxicity, whereas the propensity of Simplex P in promoting Fenton chemistry is a serious issue that must be addressed.