Phase I-metabolism studies of the synthetic cannabinoids PX-1 and PX-2 using three different in vitro models

Purpose Synthetic cannabinoids (SCs), highly metabolized substances, are rarely found unmodified in urine samples. Urine screening relies on SC metabolite detection, requiring metabolism knowledge. Metabolism data can be acquired via in vitro assays, e.g., human hepatocytes, pooled human liver microsomes (pHLM), cytochrome P450 isoforms and a fungal model; or in vivo by screening, e.g., authentic human samples or rat urine. This work describes the comprehensive study of PX-1 and PX-2 in vitro metabolism using three in vitro models. 5F-APP-PICA (PX-1) and 5F-APP-PINACA (PX-2) were studied as they share structural similarity with AM-2201, THJ-2201 and 5F-AB-PINACA, the metabolism of which was described in the literature. Methods For SC incubation, pHLM, cytochrome P450 isoenzymes and the fungal model Cunninghamella elegans LENDNER (C. elegans) were used. PX-1 and PX-2 in vitro metabolites were revealed comprehensively by liquid chromatography–high-resolution mass spectrometry measurements. Results In total, 30 metabolites for PX 1 and 15 for PX-2 were detected. The main metabolites for PX-1 and PX-2 were the amide hydrolyzed metabolites, along with an indole monohydroxylated (for PX-1) and a defluorinated pentyl-monohydroxylated metabolite (for PX-2). Conclusions CYP isoforms along with fungal incubation results were in good agreement to those obtained with pHLM incubation. CYP2E1 was responsible for many of the metabolic pathways; particularly for PX-1. This study shows that all three in vitro assays are suitable for predicting metabolic pathways of synthetic cannabinoids. To establish completeness of the PX-1 and PX-2 metabolic pathways, it is not only recommended but also necessary to use different assays.

Oligochitosan Synthesized by Cunninghamella elegans, a Fungus from Caatinga (The Brazilian Savanna) Is a Better Antioxidant than Animal Chitosan

Animal chitosan (Chit-A) is gaining more acceptance in daily activities. It is used in a range of products from food supplements for weight loss to even raw materials for producing nanoparticles and hydrogel drug carriers; however, it has low antioxidant activity. Fungal oligochitosan (OChit-F) was identified as a potential substitute for Chit-A. Cunninghamella elegans is a fungus found in the Brazilian savanna (Caatinga) that produces OligoChit-F, which is a relatively poorly studied compound. In this study, 4 kDa OChit-F with a 76% deacetylation degree was extracted from C. elegans. OChit-F showed antioxidant activity similar to that of Chit-A in only one in vitro test (copper chelation) but exhibited higher activity than that of Chit-A in three other tests (reducing power, hydroxyl radical scavenging, and iron chelation). These results indicate that OChit-F is a better antioxidant than Chit-A. In addition, Chit-A significantly increased the formation of calcium oxalate crystals in vitro, particularly those of the monohydrate (COM) type; however, OChit-F had no effect on this process in vitro. In summary, OChit-F had higher antioxidant activity than Chit-A and did not induce the formation of CaOx crystals. Thus, OChit-F can be used as a Chit-A substitute in applications affected by oxidative stress.

USO DE RESÍDUOS AGROINDUSTRIAS COMO ALTERNATIVA SUSTENTÁVEL NA PRODUÇÃO FÚNGICA DE BIOPOLÍMEROS

Introdução: O uso de resíduos agroindustriais na biotecnologia de fungos tem se tornado alvo de diversos estudos sendo empregados como substrato alternativo na produção de biomassa em bioprocessos. Entre os produtos de maior destaque, estão os biopolímeros como a quitina e a quitosana, seu co-polímero, cujas aplicações vão desde as áreas biotecnológicas, agricultura, têxtil, insumos farmacêuticos, bioengenharia e alimentícia. Objetivo: Foi analisado o uso de resíduos agroindustriais como alternativa sustentável na produção fúngica de biopolímeros. Material e Métodos: Através de um planejamento fatorial 23 foi investigado o uso dos resíduos milhocina e manipueira como fontes de carbono e nitrogênio na produção de biomassa, quitina e quitosana pela espécie Cunninghamella elegans (UCP 1306), tendo como variáveis a temperatura (25 ºC, 30 ºC e 35 ºC), concentração de milhocina (2%, 4% e 6%) e manipueira (5%, 7,5% e 10%). Resultados: Os maiores rendimentos de biomassa (9,86 g/L) de Cunninghamella elegans (UCP 1306) foram obtidos nas condições de ensaio 4 (6% milhocina, 10% manipueira, 25 ºC), em cultivo submerso sob agitação constante durante 96 h. Isso sugere que um meio de cultura com maior concentração de milhocina em relação às concentrações de manipueira pode favorecer o crescimento da espécie. Comportamento similar foi observado na produção de quitina, onde o melhor rendimento foi encontrado no ensaio 6 (5% manipueira, 6% milhocina, 35 ºC), que apresentou 257,57 mg/g de quitina, indicando que maiores concentrações de milhocina favorecem a produção do biopolímero. Já os ensaios com menores concentrações de milhocina e maiores concentrações de manipueira, apresentaram maior rendimento de quitosana (132,42 mg/g). Na análise estatística, a maior concentração de milhocina contribuiu significativamente para o crescimento da espécie (p= 0,0316). As bandas da FT-IR confirmaram o grau de desacetilação de 80,7% da quitosana produzida por CCunninghamella elegans (UCP 1306). Conclusão: Os dados experimentais mostraram que o uso dos resíduos milhocina e manipueira como fontes sustentáveis de carbono e nitrogênio é uma associação promissora para a produção de biomassa e de biopolímeros como quitina e quitosana.

CONVERSÃO METABÓLICA VERDE DE MILHOCINA E MANIPUEIRA NA PRODUÇÃO DE QUITOSANA COM ELEVADO GRAU DE DESACETILAÇÃO

Introdução: O uso de resíduos agroindustriais em processos fermentativos tem despertado grande interesse no âmbito da sustentabilidade ambiental e econômica, por se tratar de fontes naturais e renováveis com grande potencial biotecnológico praticamente inexplorado, sendo descartados na maioria das vezes. Diversos estudos têm discutido as inúmeras vantagens do uso desses resíduos como substrato alternativo para produção de biomassa empregada em bioprocessos. Objetivo: O presente estudo apresenta um método que utiliza uma conversão metabólica verde de dois resíduos agroindustrais, milhocina e manipueira, na produção de quitosana, um biopolímero de alta massa molecular e grande aplicabilidade biotecnológica. Material e métodos: Um Planejamento Fatorial completo 22 foi desenhado para avaliar os principais efeitos e interações das variáveis independentes, milhocina (licor de maceração de milho) e manipueira (água residual da prensa da mandioca), no rendimento de biomassa e quitosana, como variáveis respostas, produzidas pela espécie Cunninghamella elegans (UCP 1306). Também foi realizada a análise das alterações morfológicas observadas na espécie cultivada em cada ensaio do Planejamento Fatorial, e a quitosana obtida foi analisada em Espectroscopia de Infravermelho com transformada de Fourier (FT-IR). Resultados: O maior rendimento de biomassa (6,375 g/L) e quitosana (101,7 mg/g) foi alcançado no meio composto de milhocina 4% e manipueira 4%, no qual C. elegans (UCP 1306) apresentou predominância de hifas frouxas e ramificadas com presença de septações e formou pellets de 0,4 a 0,5 mm e clumps de até 0,1 mm. Na análise estatística, a maior concentração de milhocina contribuiu significativamente para o crescimento da espécie (p=0,00124). As bandas da FT-IR confirmaram o grau de desacetilação de 84,61% da quitosana produzida por C. elegans (UCP 1306). Conclusão: Os dados experimentais sugerem que o uso de milhocina e manipueira como fontes sustentáveis de carbono e nitrogênio é uma associação promissora para a produção de quitosana com elevado grau de desacetilação.

2C-B-Fly-NBOMe Metabolites in Rat Urine, Human Liver Microsomes and C. elegans: Confirmation with Synthesized Analytical Standards

Compounds from the N-benzylphenethylamine (NBPEA) class of novel psychoactive substances are being increasingly utilized in neurobiological and clinical research, as diagnostic tools, or for recreational purposes. To understand the pharmacology, safety, or potential toxicity of these substances, elucidating their metabolic fate is therefore of the utmost interest. Several studies on NBPEA metabolism have emerged, but scarce information about substances with a tetrahydrobenzodifuran (“Fly”) moiety is available. Here, we investigated the metabolism of 2-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b’]difuran-4-yl)-N-(2-methoxybenzyl)ethan-1-amine (2C-B-Fly-NBOMe) in three different systems: isolated human liver microsomes, Cunninghamella elegans mycelium, and in rats in vivo. Phase I and II metabolites of 2C-B-Fly-NBOMe were first detected in an untargeted screening and identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Several hypothesized metabolites were then synthesized as reference standards; knowledge of their fragmentation patterns was utilized for confirmation or tentative identification of isomers. Altogether, thirty-five phase I and nine phase II 2C-B-Fly-NBOMe metabolites were detected. Major detected metabolic pathways were mono- and poly-hydroxylation, O-demethylation, oxidative debromination, and to a lesser extent also N-demethoxybenzylation, followed by glucuronidation and/or N-acetylation. Differences were observed for the three used media. The highest number of metabolites and at highest concentration were found in human liver microsomes. In vivo metabolites detected from rat urine included two poly-hydroxylated metabolites found only in this media. Mycelium matrix contained several dehydrogenated, N-oxygenated, and dibrominated metabolites.

Cunninghamella spp. produce mammalian-equivalent metabolites from fluorinated pyrethroid pesticides

Cunninghamella spp. are fungi that are routinely used to model the metabolism of drugs. In this paper we demonstrate that they can be employed to generate mammalian-equivalent metabolites of the pyrethroid pesticides transfluthrin and β-cyfluthrin, both of which are fluorinated. The pesticides were incubated with grown cultures of Cunninghamella elegans, C. blakesleeana and C. echinulata and the biotransformation monitored using fluorine-19 nuclear magnetic resonance spectroscopy. Transfluthrin was initially absorbed in the biomass, but after 72 h a new fluorometabolite appeared in the supernatant; although all three species yielded this compound, it was most prominent in C. blakesleeana. In contrast β-cyfluthrin mostly remained in the fungal biomasss and only minor biotransformation was observed. Gas chromatography-mass spectrometry (GC–MS) analysis of culture supernatant extracts revealed the identity of the fluorinated metabolite of transfluthrin to be tetrafluorobenzyl alcohol, which arose from the cytochrome P450-catalysed cleavage of the ester bond in the pesticide. The other product of this hydrolysis, dichlorovinyl-2,2-dimethylcyclopropane carboxylic acid, was also detected by GC–MS and was a product of β-cyfluthrin metabolism too. Upon incubation with rat liver microsomes the same products were detected, demonstrating that the fungi can be used as models of mammalian metabolism of fluorinated pesticides.

Biodegradation of Chloroxylenol by Cunninghamella elegans IM 1785/21GP and Trametes versicolor IM 373: Insight into Ecotoxicity and Metabolic Pathways

Chloroxylenol (PCMX) is applied as a preservative and disinfectant in personal care products, currently recommended for use to inactivate the SARS-CoV-2 virus. Its intensive application leads to the release of PCMX into the environment, which can have a harmful impact on aquatic and soil biotas. The aim of this study was to assess the mechanism of chloroxylenol biodegradation by the fungal strains Cunninghamella elegans IM 1785/21GP and Trametes versicolor IM 373, and investigate the ecotoxicity of emerging by-products. The residues of PCMX and formed metabolites were analysed using GC-MS. The elimination of PCMX in the cultures of tested microorganisms was above 70%. Five fungal by-products were detected for the first time. Identified intermediates were performed by dechlorination, hydroxylation, and oxidation reactions catalysed by cytochrome P450 enzymes and laccase. A real-time quantitative PCR analysis confirmed an increase in CYP450 genes expression in C. elegans cells. In the case of T. versicolor, spectrophotometric measurement of the oxidation of 2,20-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) showed a significant rise in laccase activity during PCMX elimination. Furthermore, with the use of bioindicators from different ecosystems (Daphtoxkit F and Phytotoxkit), it was revealed that the biodegradation process of PCMX had a detoxifying nature.

25CN-NBOMe Metabolites in Rat Urine, Human Liver Microsomes and C.elegans—Structure Determination and Synthesis of the Most Abundant Metabolites

N-Benzylphenethylamines are novel psychedelic substances increasingly used for research, diagnostic, or recreational purposes. To date, only a few metabolism studies have been conducted for N-2-methoxybenzylated compounds (NBOMes). Thus, the available 2,5-dimethoxy-4-(2-((2-methoxybenzyl)amino)ethyl)benzonitrile (25CN-NBOMe) metabolism data are limited. Herein, we investigated the metabolic profile of 25CN-NBOMe in vivo in rats and in vitro in Cunninghamella elegans (C. elegans) mycelium and human liver microsomes. Phase I and phase II metabolites were first detected in an untargeted screening, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of the most abundant metabolites by comparison with in-house synthesized reference materials. The major metabolic pathways described within this study (mono- and bis-O-demethylation, hydroxylation at different positions, and combinations thereof, followed by the glucuronidation, sulfation, and/or N-acetylation of primary metabolites) generally correspond to the results of previously reported metabolism of several other NBOMes. The cyano functional group was either hydrolyzed to the respective amide or carboxylic acid or remained untouched. Differences between species should be taken into account in studies of the metabolism of novel substances.