RESUMEN
A convenient fluorometrical test method to identify skin sensitizers in chemico was developed using reactivity with glutathione (GSH), a low molecular weight endogenous substance. Following incubation of test chemicals with GSH, the remaining GSH was quantitated fluorometrically by using monobromobimane (mBBr), a thiol-detecting agent, for determining % depletion of this endogenous substance by test chemicals. The experimental conditions optimized were: (1) reactivity of thiol compounds including GSH with mBBr, (2) effects of vehicles on reactivity, (3) molar ratios of GSH to test chemicals, and (4) reactivity of endogenous substance with test substances under different incubation times. When an optimized condition with DMSO as a vehicle for test chemicals and in 1:60 ratio for 24 hr at 4°C was applied to classify 48 well-known skin sensitizers and non-sensitizers, the predictive capacity was as follows: 88.2% sensitivity, 78.6% specificity, and 85.4% accuracy with 95.8% consistency of three trials when 10.3% depletion of GSH was used as a cutoff value. Because the present method employed relatively simple GSH as an acceptor for sensitizers and/or a relatively convenient fluorometric detection system in 96-well plates for a high throughput test, it would be a useful test tool for screening skin sensitization potential of test chemicals.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Compuestos Bicíclicos con Puentes/química , Fluorometría/métodos , Glutatión/análisis , Piel/efectos de los fármacos , Piel/fisiopatologíaRESUMEN
To initiate skin sensitization, haptens react with endogenous proteins. During this process, skin sensitizers react with small endogenous molecules containing thiol or amino groups. In this study, a simple spectrophotometric method to identify skin sensitizers in chemico was developed using the reactivity of glutathione (GSH) with test chemicals in a 96-well plate. To quantitate the remaining GSH following the reaction with a skin sensitizer, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was employed. The optimized experimental conditions included the pH- and time-dependent stability of GSH, stability of derivatized products of GSH with optimal concentration and incubation time of DTNB, incubation time of GSH with the test chemicals, and molar ratios of GSH to the test chemicals. With the optimized conditions with both acetonitrile and DMSO as vehicles and incubation of GSH with test chemicals in 1:10 and 1:15 ratios for 24 h at 4 °C, 23 skin sensitizers and 23 non-sensitizers, based on the local lymph node assay, were tested to determine the predictive capacity of individual conditions. The best result showed a predictive capacity of 95.2% sensitivity, 91.3% specificity, and 93.2% accuracy, with 93.2% consistency in three trials, when 5.8% depletion was used as a cut-off value in 1:10 of GSH:test chemicals in DMSO. It would be an economic and useful screening tool for determining the skin sensitization potential of small molecules, because the present method employs simple endogenous GSH as an electron donor for sensitizers with a spectrophotometric detection system in 96-well plates, and because the method requires neither experimental animals nor cell cultures. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-023-00218-9.
RESUMEN
Oxaliplatin-induced peripheral neurotoxicity (OIPN) is a debilitating side effect that afflicts ~90% of patients that is initiated by OCT2-dependent uptake of oxaliplatin in DRG neurons. The antidepressant drug duloxetine has been used to treat OIPN, although its usefulness in preventing this side effect remains unclear. We hypothesized that duloxetine has OCT2-inhibitory properties and can be used as an adjunct to oxaliplatin-based regimens to prevent OIPN. Transport studies were performed in cells stably transfected with mouse or human OCT2 and in isolated mouse DRG neurons ex vivo. Wild-type and OCT2-deficient mice were used to assess effects of duloxetine on hallmarks of OIPN, endogenous OCT2 biomarkers, and the pharmacokinetics of oxaliplatin, and the translational feasibility of a duloxetine-oxaliplatin combination was evaluated in various models of colorectal cancer. We found that duloxetine potently inhibited the OCT2-mediated transport of several xenobiotic substrates, including oxaliplatin, in a reversible, concentration-dependent manner, and independent of species and cell context. Furthermore, duloxetine restricted access of these substrates to DRG neurons ex vivo and prevented OIPN in wild-type mice to a degree similar to the complete protection observed in OCT2-deficient mice, without affecting the plasma levels of oxaliplatin. Importantly, the uptake and cytotoxicity of oxaliplatin in tumor cell lines in vitro and in vivo were not negatively influenced by duloxetine. The observed OCT2-targeting properties of duloxetine, combined with the potential for clinical translation, provide support for its further exploration as a therapeutic candidate for studies aimed at preventing OIPN in cancer patients requiring treatment with oxaliplatin. Significance: We found that duloxetine has potent OCT2-inhibitory properties and can diminish excessive accumulation of oxaliplatin into DRG neurons. In addition, pre-treatment of mice with duloxetine prevented OIPN without significantly altering the plasma pharmacokinetics and antitumor properties of oxaliplatin. These results suggest that intentional inhibition of OCT2-mediated transport by duloxetine can be employed as a prevention strategy to ameliorate OIPN without compromising the effectiveness of oxaliplatin-based treatment.
Asunto(s)
Antineoplásicos , Síndromes de Neurotoxicidad , Enfermedades del Sistema Nervioso Periférico , Humanos , Ratones , Animales , Oxaliplatino/efectos adversos , Antineoplásicos/toxicidad , Clorhidrato de Duloxetina/farmacología , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Síndromes de Neurotoxicidad/tratamiento farmacológicoRESUMEN
In addition to the hepatic metabolism, the role of intestinal microbiota in drug metabolism has been considered important in the biotransformation of xenobiotics. Crocin and its aglycone, crocetin, isolated from many plants, including the dried stigma of Crocus sativus and the fruit of Gardenia jasminoides, have been used in treatment of inflammation, cancer, and metabolic disorders. In this study, the effect of intestinal microbiota on the pharmacokinetics of crocin was studied following single oral treatment with 600 mg/kg crocin to male rats pre-treated with a mixture of antibiotics, such as cefadroxil, oxytetracycline, and erythromycin, for three consecutive days. Following crocin treatment, blood, urine, and feces were collected at various time points for evaluating pharmacokinetic characteristics of crocin and crocetin by using LC-MS. Results showed that intestinal absorption of crocin was relatively marginal when compared with that of crocetin, and that crocin metabolism to crocetin by intestinal microbiota would be a critical step for absorption. The present results clearly suggested that the in vivo pharmacological effects of crocin might be considered as the effects by its aglycone, crocetin, mainly, and that the metabolism of glycosidic natural products by intestinal microbiota should be considered to understand their pharmacodynamic actions.
RESUMEN
Rutaecarpine, an alkaloid originally isolated from Evodia rutaecarpa, has been used for the treatment of gastrointestinal disorders in Asia. In the present study, the phase I and phase II metabolites of rutaecarpine were investigated in freshly isolated hepatocytes from male Sprague-Dawley rats. The individual metabolites were characterized via liquid chromatography-tandem mass spectrometry. The incubation of rutaecarpine with freshly isolated hepatocytes for 2 h yielded five major phase I metabolites. In addition, three glucuronide conjugates and four sulfate conjugates were observed. Because the majority of metabolites observed in vivo were identified, freshly isolated hepatocytes might be useful for the identification of certain metabolites formed from drug candidates from a reduced number of experimental animals.