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Licarin A, a dihydrobenzofuranic neolignan presents in several medicinal plants and seeds of nutmeg, exhibits strong activity against protozoans responsible for Chagas disease and leishmaniasis. From biomimetic reactions by metalloporphyrin and Jacobsen catalysts, seven products were determined: four isomeric products yielded by epoxidation from licarin A, besides a new product yielded by a vicinal diol, a benzylic aldehyde, and an unsaturated aldehyde in the structure of the licarin A. The incubation with rat and human liver microsomes partially reproduced the biomimetic reactions by the production of the same epoxidized product of m/z 343 [M + H]+. In vivo acute toxicity assays of licarin A suggested liver toxicity based on biomarker enzymatic changes. However, microscopic analysis of tissues sections did not show any tissue damage as indicative of toxicity after 14 days of exposure. New metabolic pathways of the licarin A were identified after in vitro biomimetic oxidation reaction and in vitro metabolism by rat or human liver microsomes.

The cytochrome P450 is a remarkable family of enzymes with widespread occurrence in animals and plants. In humans, they catalyze phase I metabolism reactions, such as oxidations and hydroxylations, playing a significant role in xenobiotics detoxification. Although mechanistic details of the catalytic cycle of cytochrome P450 are known, and human isoforms have their crystal structure solved, mimicking cytochrome P450 reactions using in vitro systems is still challenging. For this reason, biological assays using isolated enzymes or microsomal fractions are typically required to study the oxidative metabolism of new drugs before approval by regulatory agencies. Here we demonstrate that a series of metalloporphyrin catalysts generate the same oxidative product in vitro as a liver microsomal assay. The bioactive diterpene kaurenoic acid was chosen as a substrate model because it is a component of widely used phytomedicines in Brazil, such as guaco syrups and copaiba waxes. We evaluated the influence of reaction parameters such as catalyst type, oxygen donor type, solvent, and catalyst concentration on substrate consumption. Three products had their structures proposed by mass spectrometry, and among them, a dihydroxylated oxidation product was detected as a major metabolite after liver microsomal biotransformation and using porphyrin catalysts. We envision that the use of metalloporphyrins could be a useful strategy for biomimetic oxidation of kaurenic terpenes.

Ocotea fasciculata presents yangambin (YAN) and its isomer epi-yangambin (EPI-YAN) as major lignans, which are employed as the plant markers for quality control purposes and as potential pharmacological compounds. However, a gap between the pure isomers and safety and efficacy protocols is faced by the scientific community. In this context, this work aimed to report (i) a new and advantageous purifying process in a semi-preparative scale for YAN and EPI-YAN isolation from Ocotea fasciculata, and (ii) an in vitro cytotoxicity study to estimate, for the first time, the LD50 values of the isolated epimers, as well as the influence of albumin concentration in cell culture medium. The best condition for epimers isolation was achieved in normal-phase liquid chromatography. The lignan fraction (LF), previously obtained from the plant ethanolic extract, was purified yielding 17% and 29% of YAN and EPI-YAN, respectively. The in vitro study demonstrated that YAN and EPI-YAN were safe, and only at the highest concentration studied, a decrease on cell viability was observed. The estimated LD50 value was higher than 1612 mg/kg for both epimers. The LF, on the other hand, demonstrated an estimated LD50 of 422 mg/kg. Lignan cytotoxicity studies also evidenced that the higher cell viability was related to the higher concentration of fetal bovine serum as a source of albumin in medium. This is the first time the LD50 and safety of the isolated epimers were estimated, opening up great perspectives of success in in vivo studies.

Amphotericin B (AmB) has been the gold standard to treat systemic fungal infections. The use of AmB is restricted to hospitals because it poses several risks, mainly risks related to its high nephrotoxicity. Given the importance of this drug in medicine, new therapeutics and AmB formulations with nanotechnological improvements are required and could bring many benefits to patients. A new drug formulation with gastro-resistant coated granules has been proposed. The lipid-based system containing AmB was produced and used as raw material in the granulation/coated process. The new developed formulation (AmB-NP-GR) was characterized by optical microscopy, granulometry, and atomic force microscopy (AFM) after disintegration test. AmB-NP-GR showed granular shape, with most granules measured between 250 and 500 µm (37 ± 7% w/w). The AFM images indicated that the granule formulation should disintegrate in the intestine, to release the lipid-based carriers, making them available for absorption and allowing them to reach the blood circulation. The developed formulation was administered to rats in a single dose of 4.0 or 8.0 mg kg

Made available in DSpace on 2022-04-29T08:46:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-05-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Growing human demand for food has culminated in increased use of pesticides worldwide. Prothioconazole (PTC), a profungicide, is bioactivated by metabolic PTC oxidation to prothioconazole-desthio (D-PTC). Here, the in vitro phase I metabolism of PTC to D-PTC in human liver microsomes and human CYP450 forms was studied. The kinetic parameters for the formation of (+)-D-PTC (KM = 1.2 μmol L−1, VMAX = 1.7 pmol min−1 mg−1), (−)-D-PTC (KM = 7 μmol L−1, VMAX = 5.1 pmol min−1 mg−1), and both D-PTC enantiomers (KM = 9 μmol L−1, VMAX = 7 pmol min−1 mg−1) from rac-PTC indicated an enantioselective behavior. Formation of the enantiomer (+)-D-PTC was twice more extensive than the formation of the enantiomer (−)-D-PTC. Furthermore, CLH prediction revealed the same enantioselective behavior. The phenotyping study indicated that CYP2C19 was the sole CYP450 form accounting for the metabolism of PTC. The estimated apparent distribution volume of PTC was predicted as 2 L kg−1. This study showed that D-PTC may be formed in the human organism due to hepatic metabolism of PTC, mediated by CYP2C19 and that the enantiomer (+)-D-PTC is preferentially formed. However, it was not extensively formed (~1%). Considering a risk assessment point of view, this study provided positive evidence of PTC safety. Departamento de Química Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo, SP Núcleo de Pesquisas de Produtos Naturais e Sintéticos Departamento de Ciências Biomoleculares Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo, SP National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT–DATREM) Unesp Institute of Chemistry, P.O. Box 355, SP National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT–DATREM) Unesp Institute of Chemistry, P.O. Box 355, SP FAPESP: 2014/50945-4 FAPESP: 2016/14668-7 FAPESP: 2018/07534-4 FAPESP: 2020/02139-0 FAPESP: 2021/10098-4 CNPq: 465571/2014-0

Made available in DSpace on 2022-04-28T19:43:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-15 Tebuconazole (TEB) is a chiral triazole fungicide worldwide employed to control plant pathogens and preserve wood. People can be exposed to TEB either through diet and occupational contamination. This work investigates the in vitro inhibitory potential of rac-TEB, S-(+)-TEB, and R-(−)-TEB over the main cytochrome P450 enzymes (CYP450) using human liver microsomes to predict TEB in vivo inhibition potential. The IC50 values showed that in vitro inhibition was enantioselective for CYP2C9, CYP2C19, and CYP2D6, but not for CYP3A4/5. Despite enantioselectivity, rac-TEB and its single enantiomers were always classified in the same category. The inhibition mechanisms and constants were determined for rac-TEB and it has shown to be a mixed inhibitor of CYP3A4/5 (Ki = 1.3 ± 0.3 μM, αKi = 3.2 ± 0.5 μM; Ki = 0.6 ± 0.3 μM, αKi = 1.3 ± 0.3 μM) and CYP2C9 (Ki = 0.7 ± 0.1 μM, αKi = 2.7 ± 0.5 μM), and a competitive inhibitor of CYP2D6 (Ki = 11.9 ± 0.7 μM) and CYP2C19 (Ki = 0.23 ± 0.02 μM), respectively, suggesting that in some cases, rac-TEB has a higher or comparable inhibitory potential than well-known strong inhibitors of CYP450 enzymes, especially for CYP2C9 and CYP2C19. In vitro-in vivo extrapolations (IVIVE) were conducted based on the results and data available in the literature about TEB absorption and metabolism. R1 values were estimated based on the Food and Drug Administration guideline and suggested that in a chronic oral exposure scenario considering the acceptable daily intake dose proposed by the European Food and Safety Authority, the hypothesis of rac-TEB to inhibit the activities of CYP3A4/5, CYP2C9, and CYP2C19 in vivo and cause pesticide-drug interactions cannot be disregarded. Departamento de Química Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo Departamento de Física e Química Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT–DATREM) Unesp Institute of Chemistry, P.O. Box 355 National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT–DATREM) Unesp Institute of Chemistry, P.O. Box 355

Tebuconazole (TEB) is a chiral triazole fungicide that is globally marketed and used as a racemic mixture to control plant pathogens. Due to its use as a racemic mixture, TEB may exhibit enantioselective toxicokinetics toward nontarget organisms, including humans. Therefore, the in vitro enantioselective metabolism of TEB by cytochrome P450 enzymes (CYP450) was studied using human liver microsomes, and the in vivo toxicokinetic parameters were predicted. A new enantioselective, reversed-phase LC-MS/MS method was developed and validated to analyze the enantiomers of TEB and its main metabolite, 1-hydroxytebuconazole (TEBOH). In vitro metabolic parameters were obtained, and in vitro-in vivo extrapolations were performed. Michaelis-Menten and atypical biphasic kinetic profiles were observed with a total intrinsic clearance ranging from 53 to 19 mL min−1 mg−1. The in vitro-in vivo extrapolation results showed that TEB first passage effect by the liver seems to be negligible, with hepatic clearance and extraction ratios ranging from 0.53 to 5.0 mL min−1 kg−1 and 2.7–25%, respectively. Preferential metabolism of (+)-TEB to rac-TEB and (−)-TEB was observed, with preferential production of (+)-TEBOH. Furthermore, reaction phenotyping studies revealed that, despite the low hepatic clearance in the first pass metabolism of TEB, multiple human CYP450 isoforms were involved in TEB metabolism when TEBOH enantiomers were generated, mainly CYP3A4 and CYP2C9, which makes TEB accumulation in the human body more difficult due to multiple metabolic pathways.

The chiral pesticide fipronil is employed as a racemic mixture to control pests. Although there are no enantioselective differences in the fipronil enantiomer activities toward target organisms, fipronil enantiomers may exhibit enantioselective differences in their bioaccumulation, toxicity, and metabolism toward non-target organisms, including humans. The present work aims to provide significant reliable enantioselective information concerning fipronil risk assessment in humans. For that, the in vitro metabolism of rac-fipronil, S-fipronil, and R-fipronil by human liver microsomes was evaluated, the in vivo enantioselective toxicokinetic parameters were predicted and the main CYP450 isoforms involved in the enantioselective metabolism were determined. The obtained results demonstrated that fipronil may undergo a clearance by the liver and it is exclusively metabolized by the CYP3A4 isoform. Although no significative stereoselective differences were observed, the results provide reliable information on fipronil risk assessment for humans.

Enantioresolution processes are vital tools for investigating the enantioselectivities of chiral compounds. An analyst resolves to optimize enantioresolution conditions once they are determined. Generally, optimization is conducted by a one-factor-at-a-time (OFAT) approach. Although this approach may determine an adequate condition for the method, it does not often allow the estimation of the real optimum condition. Experimental designs are conducive for the optimization of enantioresolution methods via capillary electromigration techniques (CETs). They can efficiently extract information from the behavior of a method and enable the estimation of the real optimum condition. Furthermore, the application of the analytical quality by design (AQbD) approach to the development of CET-based enantioselective methods is a trend. This article (i) offers an overview of the application of experimental designs to the development of enantioselective methods from 2015 to mid-2020, (ii) reveals the experimental designs that are presently employed in CET-based enantioresolutions, and (iii) offers a critical point of view on how the different experimental designs can aid the optimization of enantioresolution processes by considering the method parameters.

Made available in DSpace on 2020-12-12T02:24:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Seriniquinone is a secondary metabolite isolated from a rare marine bacterium of the genus Serinicoccus. This natural quinone is highlighted for its selective cytotoxic activity toward melanoma cancer cells, in which rapid metastatic properties are still a challenge for clinical treatment of malignant melanoma. The progress of seriniquinone as a promising bioactive molecule for drug development requires the assessment of its clinical interaction potential with other drugs. This study aimed to investigate the in vitro inhibitory effects of seriniquinone on the main human CYP450 isoforms involved in drug metabolism. The results showed strong inhibition of CYP1A2, CYP2E1 and CYP3A, with IC50 values up to 1.4 μM, and moderate inhibition of CYP2C19, with IC50 value >15 μM. Detailed experiments performed with human liver microsomes showed that the inhibition of CYP450 isoforms can be explained by competitive and non-competitive inhibition mechanisms. In addition, seriniquinone demonstrated to be an irreversible and time-dependent inhibitor of CYP1A2 and CYP3A. The low inhibition constants values obtained experimentally suggest that concomitant intake of seriniquinone with drug metabolized by these isoforms should be carefully monitored for adverse effects or therapeutic failure. Núcleo de Pesquisas de Produtos Naturais e Sintéticos Departamento de Ciências BioMoleculares Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo Departamento de Química Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo Departamento de Ciências do Mar Instituto do Mar Universidade Federal de São Paulo CMBB Scripps Institution of Oceanography UC San Diego, 9500 Gilman Drive No. 0204 Departamento de Farmacologia Instituto de Ciências Biomédicas Universidade de São Paulo National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT–DATREM) Unesp Institute of Chemistry, P.O. Box 355 National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT–DATREM) Unesp Institute of Chemistry, P.O. Box 355 FAPESP: 2014/50265-3 FAPESP: 2014/50945-4 FAPESP: 2015/17177-6 FAPESP: 2016/06366-5 FAPESP: 2016/15680-5 FAPESP: 2018/07534-4 CNPq: 465571/2014-0

Made available in DSpace on 2021-06-25T11:02:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fenamiphos (FS) is a chiral organophosphate pesticide that is used to control nematodes in several crops. Enantioselective differences may be observed in FS activity, bioaccumulation, metabolism, and toxicity. Humans may be exposed to FS through occupational and chronic (food, water, and environmental) exposure. FS may cause undesirable CYP450 pesticide-drug interactions, which may impact human health. Here, the CYP450 isoforms involved in enantioselective FS metabolism were identified, and CYP450 inhibition by rac-FS, (+)-FS, and (−)-FS was evaluated to obtain reliable information on enantioselective FS risk assessment in humans. CYP3A4 and CYP2E1 metabolized FS enantiomers, and CYP2B6 may participate in rac-FS metabolism. In addition, rac-FS, (+)-FS, and (−)-FS were reversible competitive CYP1A2, CYP2C19, and CYP3A4/5 inhibitors. High stereoselective inhibition potential was verified; rac-FS and (−)-FS strongly inhibited and (+)-FS moderately inhibited CYP1A2. Stereoselective differences were also detected for CYP2C19 and CYP3A4/5, which were strongly inhibited by rac-FS, (+)-FS, and (−)-FS. Our results indicated a high potential for CYP450 drug-pesticide interactions, which may affect human health. The lack of stereoselective research on the effect of chiral pesticides on the activity of CYP450 isoforms highlights the importance of assessing the risks of such pesticides in humans. Departamento de Química Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo Departamento de Física e Química Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo Departamento de Química Instituto de Ciências Ambientais Químicas e Farmacêuticas Universidade Federal de São Paulo, Campus Diadema Departamento de Análises Clínicas Toxicológicas e Bromatológicas Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM) Unesp Institute of Chemistry, P.O. Box 355 National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM) Unesp Institute of Chemistry, P.O. Box 355 FAPESP: 2014/50945-4 FAPESP: 2015/02533-1 FAPESP: 2016/15680-5 FAPESP: 2018/07534-4 CNPq: 465571/2014-0

The potential of chiral pesticides to exhibit stereoselective behavior in regards to activity, toxicity, degradation, bioaccumulation, and/or metabolism has inspired numerous studies, e.g., several works have investigated the contamination of water, food, and soil by chiral pesticides and the consequences of non-targeted species (including human) exposure to these chemicals. As these studies typically rely on enantioselective separation methods such as liquid chromatography, supercritical fluid chromatography, gas chromatography, and capillary electromigration techniques, this review aims to provide a critical point of view on these analytical techniques used for the enantioselective analysis of pesticides. Accordingly, we selected papers from different areas and several issues (as chiral stationary phases or chiral selectors, mobile phases, and method detectability) regarding each technique were critically discussed. Finally, we outlined the future trends for each technique in the chiral analysis of pesticides.

Risperidone is an antipsychotic drug commonly used in the treatment of schizophrenia and other psychiatric illnesses. Its main metabolite, 9-hydroxyrisperidone, also called paliperidone, exhibits pharmacological activity similar to risperidone, and the active fraction is the sum of the therapeutic concentrations of both. In this study, a novel method combining dispersive liquid-liquid microextraction and liquid chromatography coupled to mass spectrometry was developed to quantify the active fraction of these antipsychotic drugs in human plasma samples. The chromatographic analysis was performed employing an Ascentis® Express C18 chromatographic column (100 × 4.6 mm × 2.7 µm) and ammonium acetate (10 mmol/L): acetonitrile in gradient elution was employed as mobile phase. The flow rate was 0.5 mL/min, and the total run time was 8 min. The best microextraction conditions were chlorobenzene (80 µL) and acetone (500 µL) as extractor and dispersing solvent, respectively. The sample pH was adjusted to 12.0 and 10% NaCl was added in the samples in order to increase the ionic strength. The method was completely validated according to Guideline on Bioanalytical Method Validation. Finally, therapeutic drug monitoring capability of the new bioanalytical method was confirmed with the analysis of schizophrenic patient plasma.

Made available in DSpace on 2020-12-12T01:22:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-08-05 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Ethofumesate (ETO) is a chiral herbicide that is marketed as a racemic mixture in the European Union and the United States. The growing consumption of pesticides in the world, along with their presence in water and food, has increased human exposure to these chemicals. Another issue concerning these compounds is that each enantiomer of a chiral pesticide may interact with biomolecules differently. For this reason, this study aimed to investigate the in vitro metabolism of ethofumesate (the racemic mixture as well as the isolated enantiomers) by human liver microsomes (HLM) and to explore the in vitro-in vivo correlation. Before the kinetics was determined, the method was fully validated by evaluating its selectivity, linearity, precision, accuracy, carryover, and stability. All the evaluated parameters agreed with the European Medicines Agency guideline. The enzyme kinetic parameters and the in vitro-in vivo correlation demonstrated that there was no enantioselective difference for the metabolism and bioavailable fraction of each enantiomer. The enzyme kinetics was biphasic; the KM1 values were 15, 5.8, and 5.6 for rac-ETO, (+)-ETO, and (–)-ETO, respectively. The total in vitro intrinsic clearance was 0.10 mg mL min−1 mg−1 for rac-ETO and its enantiomers. The enantiomer (–)-ETO was only metabolized by CYP2C19, while (+)-ETO was metabolized by both CYP2C19 and CYP3A4. CYP2C19 polymorphism and/or inhibition may represent a risk for humans exposed to this pesticide. Departamento de Química Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT–DATREM) Unesp Institute of Chemistry, P.O. Box 355 National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT–DATREM) Unesp Institute of Chemistry, P.O. Box 355 FAPESP: 2014/50945-4 FAPESP: 2018/07534-4 CNPq: 465571/2014-0

Made available in DSpace on 2020-12-12T01:32:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-24 Capillary forces are commonly employed to transport fluids in pump-free microfluidic platforms such as paper-based microfluidics. However, since paper is a porous material consisting of nonuniform cellulose fibers, it has some limitations in performing stable flow functions like mixing. Here, we developed a pump-free microfluidic device that enables rapid mixing by combining paper and plastic. The device was fabricated by laminating transparency film and double-sided adhesive and is composed of an overlapping inlet ending in a paper-based reaction area. The mixing performance of the developed device was confirmed experimentally using aqueous dyes and pH indicators. In addition, the absolute mixing index was evaluated by numerically calculating the concentration field across the microfluidic channels. To demonstrate the utility of the new approach, the detection of an organophosphate pesticide was carried out using a colorimetric enzymatic inhibition assay. The developed device and a smartphone application were used to detect organophosphate pesticide on food samples, demonstrating the potential for onsite analysis. Department of Chemistry Colorado State University Institute of Nano Science and Technology Hanyang University Departamento de Quĺmica Faculdade de Filosofia Ciências e Letras de Ribeiraõ Preto Universidade de Saõ Paulo National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM) Unesp Institute of Chemistry, P.O. Box 355 National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM) Unesp Institute of Chemistry, P.O. Box 355

Made available in DSpace on 2022-04-29T08:45:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Casearin X (CAS X) is the major clerodane diterpene isolated from the leaves of Casearia sylvestris and has been extensively studied due to its powerful cytotoxic activity at low concentrations. Promising results for in vivo antitumor action have also been described when CAS X was administered intraperitoneally in mice. Conversely, loss of activity was observed when orally administered. Since the advancement of natural products as drug candidates requires satisfactory bioavailability for their pharmacological effect, this work aimed to characterize the CAS X metabolism by employing an in vitro microsomal model for the prediction of preclinical pharmacokinetic data. Rat and human liver microsomes were used to assess species differences. A high-performance liquid chromatography with diode-array detection (HPLC-DAD) method for the quantification of CAS X in microsomes was developed and validated according to European Medicines Agency guidelines. CAS X was demonstrated to be a substrate for carboxylesterases via hydrolysis reaction, with a Michaelis-Menten kinetic profile. The enzyme kinetic parameters were determined, and the intrinsic clearance was 1.7-fold higher in humans than in rats. The hepatic clearance was estimated by in vitro - in vivo extrapolation, resulting in more than 90% of the hepatic blood flow for both species. A qualitative study was also carried out for the metabolite identification by mass spectrometry and indicated the formation of the inactive metabolite CAS X dialdehyde. These findings demonstrate that CAS X is susceptible to first-pass metabolism and is a substrate for specific carboxylesterases expressed in liver, which may contribute to a reduction in antitumor activity when administered by the oral route. Núcleo de Pesquisa em Produtos Naturais e Sintéticos (NPPNS) Departamento de Física e Química Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo Departamento de Química Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo, Avenida Bandeirantes 3900 Núcleo de Bioensaios Biossíntese e Ecofisiologia de Produtos Naturais (NuBBE) Departamento de Química Orgânica Instituto de Química Universidade Do Estado de São Paulo Departamento de Química Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo

Social insects are frequently observed in symbiotic association with bacteria that produce antimicrobial natural products as a defense mechanism. There is a lack of studies on the microbiota associated with stingless bees and their antimicrobial compounds. To the best of our knowledge, this study is the first to report the isolation of Paenibacillus polymyxa ALLI-03-01 from the larval food of the stingless bee Melipona scutellaris. The bacterial strain was cultured under different conditions and produced (L)-(-)-3-phenyllactic acid and fusaricidins, which were active against entomopathogenic fungi and Paenibacillus larvae. Our results indicate that such natural products could be related to colony protection, suggesting a defense symbiosis between P. polymyxa ALLI-03-01 and Melipona scutellaris.

The increasing awareness and public concern with hazard exposure to endocrine-disrupting chemicals calls for methods capable to handle numerous samples in short analysis time. In this present study, a novel method combining air-assisted liquid-liquid microextraction and liquid chromatography coupled to mass spectrometry was developed and validated for the extraction, preconcentration, and determination of 7 bisphenols (bisphenol A, bisphenol S, bisphenol AP, bisphenol P, bisphenol F, bisphenol AF, bisphenol Z), 7 parabens (methyl-, ethyl-, propyl-, butyl-, benzyl-paraben, methyl-protocatechuic acid, and ethyl-protocatechuic acid), 5 benzophenones (benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-8, and 4-hydroxybenzophenone), and two antimicrobials (triclosan and triclocarban) in human urine samples. Type and volume of solvent, extraction time (cycles), pH sample, ionic strength, agitation, and needle dimensions were evaluated. The matrix-matched calibration curves of all analytes were linear with correlation coefficients higher than 0.99 in the range level of 1.0–20.0 ng mL−1. The relative standard deviation, precision, at three concentrations (1.0, 10.0 and 20.0 ng mL−1) was lower than 15% with accuracy ranging from 90% to 114%. The biomonitoring capability of the new proposed method was confirmed with the analysis of 50 human urine samples randomly collected from Brazilian children. High urinary concentrations of several EDCs associated with usage of personal care products were found.

Piperlongumine is an amide alkaloid found in Piperaceae species that shows a broad spectrum of biological properties, including antitumor and antiparasitic activities. Herein, the leishmanicidal effect of piperlongumine and its derivatives produced by a biomimetic model using metalloporphyrins was investigated. The results showed that IC50 values of piperlongumine in promastigote forms of Leishmania infantum and Leishmania amazonensis were 7.9 and 3.3 µM, respectively. The IC50 value of piperlongumine in the intracellular amastigote form of L. amazonensis was 0.4 µM, with a selectivity index of 25. The piperlongumine biomimetic derivatives, Ma and Mb, also showed leishmanicidal effects. We also carried out an in vitro metabolic degradation study showing that Ma is the most stable piperlongumine derivative in rat liver microsome incubations. The results presented here indicate that piperlongumine is a potential leishmanicidal candidate and support the biomimetic approach for development of new antileishmanial derivatives.

Topiramate (TPM) is a second-generation antiepileptic drug (AED), acting on drug-resistant epilepsy. The aim of the study was to evaluate the influence of the dose, use of other AEDs on TPM plasma concentration (Cp ), and frequency of epileptic seizures. A cross-sectional analytical study was developed with patients aged 18-60 years, for diagnosis of drug-resistant epilepsy, using TPM in monotherapy or associated with other AEDs. The following variables were analyzed: age, frequency of epileptic seizures, pharmacotherapeutic regimen with its respective doses, adherence to medication treatment, and adverse events score. Thirty-seven patients were included, 83.8% of the patients presented Cp below the therapeutic range. Multiple linear regression estimated that the increase of 1.0 mg/kg/d promoted an increase of 0.68 μg/mL in TPMCp , while the use of inducers predicted a reduction of 2.97 μg/mL (P

The consumption of pesticides worldwide has been growing in recent decades, and consequently the exposure of humans and other animals to them as well. However, even though it is known that chiral pesticides can behave stereoselectively, the knowledge about the risks to human health and the environment is scarce. Among the pesticides registered to date, approximately 30% have at least one center of asymmetry, and just 7% of them are currently marketed as a pure stereoisomer or as an enriched mixture of the active stereoisomer. There are several in vitro, in vivo, and in silico models available to evaluate the enantioselective metabolism of chiral pesticides aiming ecotoxicological and risk assessment. Therefore, this paper intends to provide a critical view of the metabolism of chiral pesticides in non-target species, including humans, and discuss their implications, as well as, conduct a review of the analytical techniques employed for in vitro and in vivo metabolism studies of chiral pesticides.

Made available in DSpace on 2022-04-29T08:45:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-08-25 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Ethofumesate is a chiral herbicide that may display enantioselective behavior in humans. For this reason, the enantioselective potential of ethofumesate and its main metabolite ethofumesate-2-hydroxy to cause pesticide-drug interactions on cytochrome P450 forms (CYPs) has been evaluated by using human liver microsomes. Among the evaluated CYPs, CYP2C19 had its activity decreased by the ethofumesate racemic mixture (rac-ETO), (+)-ethofumesate ((+)-ETO), and (−)-ethofumesate ((−)-ETO). CYP2C19 inhibition was not time-dependent, but a strong inhibition potential was observed for rac-ETO (IC50 = 5 ± 1 μmol L−1), (+)-ETO (IC50 = 1.6 ± 0.4 μmol L−1), and (−)-ETO (IC50 = 1.8 ± 0.4 μmol L−1). The reversible inhibition mechanism was competitive, and the inhibition constant (Ki) values for rac-ETO (2.6 ± 0.4 μmol L−1), (+)-ETO (1.5 ± 0.2 μmol L−1), and (−)-ETO (0.7 ± 0.1 μmol L−1) were comparable to the Ki values of strong CYP2C19 inhibitors. Inhibition of CYP2C19 by ethofumesate was enantioselective, being almost twice higher for (−)-ETO than for (+)-ETO, which indicates that this enantiomer may be a more potent inhibitor of this CYP form. For an in vitro-in vivo correlation, the Food and Drug Administration's (FDA) guideline on the assessment of drug-drug interactions used in the early stages of drug development was used. The FDA's R1 values were estimated on the basis of the obtained ethofumesate Ki and distribution volume, metabolism, unbound plasma fraction, gastrointestinal and dermal absorption data available in the literature. The correlation revealed that ethofumesate probably inhibits CYP2C19 in vivo for both chronic (oral) and occupational (dermal) exposure scenarios. Departamento de Química Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo Núcleo de Pesquisas de Produtos Naturais e Sintéticos Departamento de Ciências Biomoleculares Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT–DATREM) Unesp Institute of Chemistry, P.O. Box 355 National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT–DATREM) Unesp Institute of Chemistry, P.O. Box 355 FAPESP: 2014/50945-4 FAPESP: 2016/14668-7 FAPESP: 2018/07534-4 FAPESP: 2019/12149-5 FAPESP: 2020/02139-0 CNPq: 465571/2014-0

Grandisin, a lignan isolated from many species of plants, such as Virola surinamensis, is a potential drug candidate due to its biological properties, highlighted by its antitumor and trypanocidal activities. In this study, the inhibitory effects of grandisin on the activities of human cytochrome P450 enzymes were investigated by using human liver microsomes. Results showed that grandisin is a competitive inhibitor of CYP2C9 and a competitive and mechanism-based inhibitor of CYP3A4/5. The apparent Ki value for CYP2C9 was 50.60 µM and those for CYP3A4/5 were 48.71 µM and 31.25 µM using two different probe substrates, nifedipine and midazolam, respectively. The apparent KI, kinact, and kinact/KI ratio for the mechanism-based inhibition of CYP3A4/5 were 6.40 µM, 0.037 min−1, and 5.78 mL · min−1 µmol−1, respectively, by examining nifedipine oxidation, and 31.53 µM, 0.049 min−1, and 1.55 mL · min−1 µmol−1, respectively, by examining midazolam 1′-hydroxylation. These apparent kinact/KI values were comparable to or even higher than those for several therapeutic drugs that act as mechanism-based inhibitors of CYP3A4/5. CYP1A2 and CYP2D6 activities, in turn, were not substantially inhibited by grandisin (IC50 > 200 µM and 100 µM, respectively). In contrast, from a concentration of 4 µM, grandisin significantly stimulated CYP2E1 activity. These results improve the prediction of grandisin-drug interactions, suggesting that the risk of interactions with drugs metabolized by CYP3A4/5 and CYP2E1 cannot be overlooked.

A simple and rapid procedure for simultaneous determination of 21 potential endocrine-disrupting chemicals (EDs) in urine samples by liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. The novelty of the present procedure is based on the use of low-density solvent air-assisted liquid-liquid microextraction (LDS–AALLME) as a sample preparation step. The target analytes were 7 bisphenols, 7 parabens, 5 benzophenones, and two antimicrobials. The parameters (type and volume of solvent, extraction time (cycles), sample pH, ionic strength, and agitation) that influence the extraction efficiency in the LDS–AALLME were investigated and optimized. Under the optimized conditions, the matrix-matched calibration curves of all analytes presented correlation coefficients higher than 0.99 (range level of 1–20 ng mL−1). Very low LODs and LOQs ranging in 0.0003–0.15 ng mL−1 and 0.001–0.2 ng mL−1, respectively, were obtained. Recoveries from spiked samples at two concentrations (2.50 ng mL−1 and 15 ng mL−1) ranged from 91% to 120% while the precision (%, RSD) was lower than 15% for all studied compounds. The proposed combination of LDS–AALLME with LC–MS/MS is an environmentally friendly, sensitive and simple analytical alternative for the determination of EDs in urine samples without the use of any special apparatus for sample preparation, which is particularly suitable for human biomonitoring studies where many samples should be processed in less time. Finally, the proposed procedure was successfully applied to the determination of the analytes in 20 urine samples from Brazilians adults.

Made available in DSpace on 2019-10-06T15:49:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-10-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fipronil is a chiral insecticide employed worldwide in crops, control of public hygiene and control of veterinary pests. Humans can be exposed to fipronil through occupational, food, and environmental contamination. Therefore, the risk assessment of fipronil in humans is important to protect human health. Fipronil sulfone is the major metabolite formed during fipronil metabolism by humans. Since the CYP450 enzymes are the main ones involved in drug metabolism, the evaluation of their inhibition by fipronil and its main metabolite is important to predict drug-pesticide interactions. The aim of this work was to investigate the inhibition effects of rac-fipronil, S-fipronil, R-fipronil and fipronil sulfone on the main human CYP450 isoforms. The results showed that CYP2D6 is the only CYP450 isoform inhibited by these xenobiotics. In addition, no enantioselective differences were observed in the inhibition of CYP450 isoforms by fipronil and its individuals’ enantiomers. Rac-fipronil, S-fipronil and R-fipronil are moderate CYP2D6 inhibitors showing a competitive inhibition profile. On the other hand, the metabolite fipronil sulfone showed to be a strong inhibitor of CYP2D6 also by competitive inhibition. These results highlight the importance of metabolite evaluation on pesticide safety since the metabolism of fipronil into fipronil sulfone increases the risk of pesticide-drug interactions for drugs metabolized by CYP2D6. Departamento de Química Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo Departamento de Física e Química Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM) Unesp Institute of Chemistry, P.O. Box 355 National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM) Unesp Institute of Chemistry, P.O. Box 355 FAPESP: 2014/50945-4 FAPESP: 2016/07597-0 FAPESP: 2016/15680-5 FAPESP: 2018/07534-4 FAPESP: 2018/14668-7

Myclobutanil is a chiral triazole fungicide that is employed worldwide. Although enantiomers have the same physical-chemical properties, they may differ in terms of activity, metabolism, and toxicity. This investigation consisted of in vitro enantioselective metabolism studies that employed a human model to assess the risks of myclobutanil in humans. A LC-MS/MS enantioselective method was developed and validated. The enzymatic kinetic parameters (VMAX, KMapp, and CLINT) determined for in vitro rac-myclobutanil and S-(+)-myclobutanil metabolism revealed enantioselective differences. Furthermore, human CYP450 enzymes did not metabolize R-(-)-myclobutanil. The predicted in vivo toxicokinetic parameters indicated that S-(+)-myclobutanil may be preferentially eliminated by the liver and suffer the first-pass metabolism effect. However, because CYP450 did not metabolize R-(-)-myclobutanil, this enantiomer could reach the systemic circulation and stay longer in the human body, potentially causing toxic effects. The CYP450 isoforms CYP2C19 and CYP3A4 were involved in rac-myclobutanil and S-(+)-myclobutanil metabolism. Although there were differences in the metabolism of the myclobutanil enantiomers, in vitro inhibition studies did not show significant enantioselective differences. Overall, the present investigation suggested that myclobutanil moderately inhibits CYP2D6 and CYP2C9 in vitro and strongly inhibits CYP3A and CYP2C19 in vitro. These results provide useful scientific information for myclobutanil risk assessment in humans.

Finding new drugs is imperative especially in the case of diseases such as serious infections and cancer, for which a gap in drug development exists. Plant-derived compounds display large structural diversity and constitute a source of novel medicinal agents. However, some drug candidates have failed in advanced stages of clinical and commercial use. In vitro tools have been effectively used to anticipate and to explore undesirable drug properties during early drug development. In this review, we address the purpose and advantages of conducting metabolic studies on natural products, and we describe how in vitro metabolism methods have contributed to delineating plant-based drug candidates. We also deal with the applications of in vitro metabolism studies on natural product-based drug development, which include characterization of the metabolites, investigation into enzymatic kinetics, prediction of drug clearance, phenotyping studies, and evaluation of natural product–drug interactions.

A three phase hollow fiber liquid-phase microextraction technique combined with capillary electrophoresis was developed to quantify lamotrigine (LTG) in plasma samples. The analyte was extracted from 4.0 mL of a basic donor phase (composed of 0.5 mL of plasma and 3.5 mL of sodium phosphate solution pH 9.0) through a supported liquid membrane composed of 1-octanol immobilized in the pores of the hollow fiber, and to an acidic acceptor phase (hydrochloric acid solution pH 4.0) placed in the lumen of the fiber. The extraction was carried out for 30 min at 500 rpm. The eletrophoretic analysis was carried out in 130 mmol/L MES buffer, pH 5.0 with a constant voltage of +15 kV and 20°C. Sample injections were performed for 10 s, at a pressure of 0.5 psi. The detection was performed at 214 nm for both LTG and the internal standard lidocaine. Under the optimized conditions, the method showed a limit of quantification of 1.0 μg/mL and was linear over the plasmatic concentration range of 1.0-20.0 μg/mL. Finally, the validated method was applied for the quantification of LTG in plasma samples of epileptic patients.

Dihydroergotamine is a semisynthetic natural product derived from ergotamine, an ergot alkaloid. It is used to treat migraines, a neurological disease characterized by recurrent moderate to severe headaches. In this work, the in vitro metabolism of dihydroergotamine was evaluated in a biomimetic phase I reaction, aiming to verify all possible formed metabolites. Dihydroergotamine was submitted to an in vitro metabolism assay using rat liver microsomes, and the metabolites were analyzed by HPLC-MS/MS. The biomimetic reactions were performed with Jacobsen catalyst for scaling up production of oxidized metabolites. Two hydroxylated metabolites were isolated and characterized by MS/MS and 1H NMR analysis.

Rice, a staple food for over half of the world’s population, represents a significant dietary source of inorganic arsenic. Thus, it is of great importance to reduce the levels of inorganic arsenic in rice with minimal cost and efficiency. Based on this, the present study focused on the isolation and identification of fungi present in soil rhizosphere to be used as a low-cost and environmentally-friendly procedure for inorganic arsenic remediation. Soil samples, from rice production areas, were evaluated in matter of toxic and essential elements (Pb, Cd, Ni, Mn, Se, Co, Cr, Ba, Rb, U, Cs, V, Cu, Fe, Mg, Zn) by inductively coupled mass spectrometry (ICP-MS). The isolated and identified filamentous fungi were Penicillium sp., Aspergillus sp., Trichoderma sp., Cladosporium sp., Rhizopus sp. and Westerdykella sp. Fungi were submitted to As3+ exposure in Czapeck growth media, and the biomass and broth of each fungus were analyzed by high performance liquid chromatography coupled to ICP-MS. The results have shown capacity of arsenic biotransformation and accumulation. Arsenic-tolerance test (0–50 mg l−1 As3+) has shown that the most arsenic-tolerant genera were Penicillium sp. and Aspergillus sp. Our data led us to believe that filamentous fungi may influence on arsenic biogeochemistry in rice paddy soils and further these microorganisms might be potentially used for bioremediation.

Levetiracetam (LEV) is an antiepileptic drug that is clinically effective in generalized and partial epilepsy syndromes. The use of this drug has been increasing in clinical practice and intra- or -interindividual variability has been exhibited for special population. For this reason, bioanalytical methods are required for drug monitoring in biological matrices. So this work presents a dispersive liquid-liquid microextraction method followed by gas chromatography-mass spectrometry (DLLME-GC-MS) for LEV quantification in human plasma. However, due to the matrix complexity a previous purification step is required. Unlike other pretreatment techniques presented in the literature, for the first time, a procedure employing ultrafiltration tubes Amicon® (10 kDa porous size) without organic solvent consumption was developed. GC-MS analyses were carried out using a linear temperature program, capillary fused silica column, and helium as the carrier gas. DLLME optimized parameters were type and volume of extraction and dispersing solvents, salt addition, and vortex agitation time. Under chosen parameters (extraction solvent: chloroform, 130 μL; dispersing solvent: isopropyl alcohol, 400 μL; no salt addition and no vortex agitation time), the method was completely validated and all parameters were in agreement with the literature recommendations. LEV was quantified in patient’s plasma sample using less than 550 μL of organic solvent.

Made available in DSpace on 2020-12-12T02:25:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-10-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Simplicity, speed, and reduced cost are essential demands for routine analysis in human biomonitoring studies. Moreover, the availability of higher volumes of human specimens is becoming more restrictive due to ethical controls and to the costs associated with sample transportation and storage. Thus, analytical methods requiring much lower sample volumes associated with simultaneous detection capability (multiclass analysis) are with a very high claim. In this sense, the present approach aimed at the development of a method for preconcentration and simultaneous determination of four classes of endocrine disruptors (seven bisphenols, seven parabens, five benzophenones, and two antimicrobials) in the urine. The approach is based on vortex-assisted dispersive liquid-liquid microextraction (VADLLME) and high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). After optimization of the significant parameters of VADLLME extraction, the proposed procedure showed to be simple, fast, sensitive, requiring only 1.0 mL of urine, 400 μL of organic solvents with a total stirring time of 20 s. Moreover, a variation of inter-day and between-day runs were lower than 10.0% and 11.0%, respectively. Finally, the proposed method was successfully applied to the analysis of 50 urine samples of Brazilian pregnant women to establish reference ranges. Departamento de Análises Clínicas Toxicológicas e Bromatológicas Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo Faculdade de Ciências Farmacêuticas Universidade de Ribeirão Preto Departamento de Química Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM) Unesp Institute of Chemistry Department of Chemistry University of Central Florida, P.O.Box 25000 National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM) Unesp Institute of Chemistry FAPESP: 14/50867–3 CNPq: 154866/2018–0 FAPESP: 2014/19914–5 FAPESP: 2014/50945–4 FAPESP: 2015/20725–5 FAPESP: 2018/07534–4 FAPESP: 2018/24069–3 CNPq: 309822/2017–3 CNPq: 422588/2016–4 CNPq: 465389/2014–7 CNPq: 465571/2014–0 CAPES: 88887.370750/2019–00

This work presents the synthesis and characterization of two novel binuclear ruthenium compounds of general formula [Ru2O(carb)2(py)6](PF6)2, where py=pyridine and carb are the non-steroidal anti-inflammatory drugs ibuprofen (1) and ketoprofen (2). Both complexes were characterized by ESI-MS/MS spectrometry. The fragmentation patterns, which confirm the proposed structures, are presented. Besides that, compounds 1 and 2 present the charge transfer transitions within 325-330nm; and the intra-core transitions around 585nm, which is the typical spectra profile for [Ru2O] analogues. This suggests the carboxylate bridge has little influence in their electronic structure. The effects of the diruthenium complexes on Ig-E mediated mast cell activation were evaluated by measuring the enzyme β-hexosaminidase released by mast cells stimulated by antigen. The inhibitory potential of the ketoprofen complex against mast cell stimulation suggests its promising application as a therapeutic agent for treating or preventing IgE-mediated allergic diseases. In addition, in vitro metabolism assays had shown that the ibuprofen complex is metabolized by the cytochrome P450 enzymes.

This paper reports the biomimetic oxidation of hydroxydihydrocarvone by iodosylbenzene using tetraphenyl-porphine iron(III) chloride as the catalyst in ethyl acetate. Mass spectrometry fragmentation maps of hydroxydihydrocarvone (obtained by gas chromatography-mass spectrometry analyses) allowed for the identification of the major product as 4-hydroxy-5-(2-hydroxypropan-2-yl)-2-methylcyclohex-2-en-1-one (4-hydroxy-hydroxydihydrocarvone). This compound was also observed in an in vitro metabolism assay that employed isolated rat liver microsomes, thereby validating the biomimetic procedure.

The genus Erythrina, Fabaceae, is widely distributed in tropical and subtropical regions. Their flowers, fruits, seeds and bark are frequently used in folk medicine for its effects on the central nervous system such as anticonvulsant, antidepressant, analgesic, sedative, and hypnotic effects. Erythraline has been reported as one of the active compounds from Erythrina, but until now there are no pharmacokinetics data about this compound and only few results showing a putative metabolism were reported. To improve the information about erythraline metabolism, this article reports and discusses, for the first time, the in vitro metabolism biotransformation of erythraline by cytochrome P450 enzymes. Keywords: Fabaceae, Erythraline, CYP450, Drug metabolism, Microsomal model

Albendazole (ABZ) is an anthelmintic drug used for the treatment of infectious diseases in veterinary and human medicine. This drug is a prochiral drug that after administration, is rapidly oxidized in the pharmacologically active sulfoxide metabolite, which is also known as ricobendazole (ABZSOX). ABZSOX has a stereogenic center and possibly two enantiomers, (+)-ABZSOX and (-)-ABZSOX. In the present work, we investigate the pH effect on the asymmetric stereoselective sulfoxidation of ABZ into ABZSOX by employing the fungi Nigrospora sphaerica, Papulaspora immera Hotson, and Mucor rouxii. The results show a possibility of obtaining the pure enantiomers of the ricobendazole drug using fungi as biocatalytic agents. The three fungi showed a high degree of enantioselectivity expressed by enantiomeric excess. In addition, M. rouxii can be used as an alternative to obtain the (+)-ABZSOX enantiomer (ee 89.8%).

(−)-grandisin is a tetrahydrofuran lignan that displays important biological properties, such as trypanocidal, anti-inflammatory, cytotoxic, and antitumor activities, suggesting its utility as a potential drug candidate. One important step in drug development is metabolic characterization and metabolite identification. To perform a biotransformation study of (−)-grandisin and to determine its kinetic properties in humans, a high performance liquid chromatography (HPLC) method was developed and validated. After HPLC method validation, the kinetic properties of (−)-grandisin were determined. (−)-grandisin metabolism obeyed Michaelis-Menten kinetics. The maximal reaction rate (Vmax) was 3.96 ± 0.18 µmol/mg protein/h, and the Michaelis-Menten constant (Km) was 8.23 ± 0.99 μM. In addition, the structures of the metabolites derived from (−)-grandisin were characterized via gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analysis. Four metabolites, 4-O-demethylgrandisin, 3-O-demethylgrandisin, 4,4′-di-O-demethylgrandisin, and a metabolite that may correspond to either 3,4-di-O-demethylgrandisin or 3,5-di-O-demethylgrandisin, were detected. CYP2C9 isoform was the main responsible for the formation of the metabolites. These metabolites have not been previously described, demonstrating the necessity of assessing (−)-grandisin metabolism using human-derived materials. Copyright © 2015 John Wiley & Sons, Ltd.

Drug Delivery Systems (DDS) of known drugs are prominent candidates towards new and more-effective treatments of various infectious diseases as they may increase drug bioavailability, control drug delivery and target the site of action. In this sense, the encapsulation of Amphotericin B (AmB) in Nanostructured Lipid Carriers (NLCs) designed with pH-sensible phospholipids to target infectious tissues was proposed and suitable analytical methods were validated, as well as, proper nanoparticle characterization were conducted. Characterization assays by Dinamic Light Scattering (DLS) and Atomic Force Microscopy demonstrated spherical particles with nanometric size 268.0±11.8nm and Zeta Potential -42.5±1.5mV suggestive of important stability. DSC/TGA and FT-IR assessments suggested mechanical encapsulation of AmB. The AmB aggregation study indicated that the encapsulation provided AmB at the lowest cytotoxic form, polyaggregate. Analytical methods were developed and validated according to regulatory agencies in order to fast and assertively determine AmB in nanoparticle suspension and, in Drug Encapsulation Efficiency (EE%), release and stability studies. The quantification method for AmB in NLC suspension presented linearity in 5.05-60.60μgmL-1 range (y=0.07659x+0.05344) and for AmB in receptor solution presented linearity in 0.15-10.00μgmL-1 range (y=54609x+263.1), both with r≥0.999. EE% was approximately 100% and according to the release results, at pH 7.4, a sustained controlled profile was observed for up 46h. In the meantime, a micellar AmB solution demonstrated an instability pattern after 7h of contact with the medium. Degradation and release studies under acid conditions (infectious condition) firstly depicted a prominent degradation of AmB (raw-material), with 20.3±3.5% at the first hour, reaching 43.3±7.0% after 7h of study. Next, particles faster disruption in acid environment was evidenced by measuring the NLC size variation by DLS and by the loss of the bluish sheen, characteristic of the nanostructured system macroscopically observed. Finally, safety studies depicted that NLC-AmB presented reduced toxicity in fibroblast cells, corroborating with AmB aggregated form study. Therefore, an innovative AmB formulation was fully characterized and it is a new proposal for in vivo investigations.

Artepillin C, a natural product present in the Brazilian green propolis, has several biological properties. Among these properties, the antitumor action of this product is noteworthy and makes it a promising drug candidate for the treatment of several types of cancer. This paper describes the in vitro metabolism of Artepillin C in rat and human liver microsomes. The rat model suggested a sigmoidal profile for the metabolism, adapted to the Hillʼs kinetic model. The enzymatic kinetic parameters were as follows: maximal velocity = 0.757 ± 0.021 µmol/mg protein/min, Hill coefficient = 10.90 ± 2.80, and substrate concentration at which half-maximal velocity of a Hill enzyme is achieved = 33.35 ± 0.55 µM. Based on these results, the calculated in vitro intrinsic clearance for Artepillin C was 16.63 ± 1.52 µL/min/mg protein. The in vitro metabolism assay conducted on the human model did not fit any enzymatic kinetic model. Two novel metabolites were formed in both mammal microsomal models and their chemical structures were elucidated for the first time. The main human cytochrome P450 isoforms involved in Artepillin C metabolism had been identified, and the results suggest a majority contribution of CYP2E1 and CYP2C9 in the formation of the two metabolites.

The present work describes, for the first time, the simultaneous separation of oxcarbazepine (OXC) and its active metabolite 10-hydroxy-10,11-dihydrocarbamazepine (licarbazepine, Lic) by chiral CE. The developed method was employed to monitor the enantioselective biotransformation of OXC into its active metabolite by fungi. The electrophoretic separations were performed using 10 mmol/L of a Tris-phosphate buffer solution (pH 2.5) containing 1% w/v of β-CD phosphate sodium salt (P-β-CD) as running electrolyte, -20 kV of applied voltage and a 15°C capillary temperature. The method was linear over the concentration range of 1000-30 000 ng/mL for OXC and 75-900 ng/mL for each Lic enantiomer (r ≥ 0.9952). Within-day precision and accuracy evaluated by RSD and relative errors, respectively, were lower than 15% for all analytes. The validated method was used to evaluate the enantioselective biotransformation of OXC, mediated by fungi, into its active metabolite Lic. This study showed that the fungi Glomerella cingulata (VA1) and Beuveria bassiana were able to enantioselectively metabolize the OXC into Lic after 360 h of incubation. Biotransformation by the fungus Beuveria bassiana showed 79% enantiomeric excess for (S)-(+)-Lic, while VA1 gave an enantiomeric excess of 100% for (S)-(+)-Lic. This study opens a new route to the drug (S)-(+)-licarbazepine.

Monensin A is a commercially important natural product isolated fromStreptomyces cinnamonensinsthat is primarily employed to treat coccidiosis. Monensin A selectively complexes and transports sodium cations across lipid membranes and displays a variety of biological properties. In this study, we evaluated the Jacobsen catalyst as a cytochrome P450 biomimetic model to investigate the oxidation of monensin A. Mass spectrometry analysis of the products from these model systems revealed the formation of two products: 3-O-demethyl monensin A and 12-hydroxy monensin A, which are the same ones found inin vivomodels. Monensin A and products obtained in biomimetic model were tested in a mitochondrial toxicity model assessment and an antimicrobial bioassay againstStaphylococcus aureus, S. aureusmethicillin-resistant,Staphylococcus epidermidis, Pseudomonas aeruginosa,andEscherichia coli.Our results demonstrated the toxicological effects of monensin A in isolated rat liver mitochondria but not its products, showing that the metabolism of monensin A is a detoxification metabolism. In addition, the antimicrobial bioassay showed that monensin A and its products possessed activity against Gram-positive microorganisms but not for Gram-negative microorganisms. The results revealed the potential of application of this biomimetic chemical model in the synthesis of drug metabolites, providing metabolites for biological tests and other purposes.