Characterization of Casearin X Metabolism by Rat and Human Liver Microsomes.

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.

A possible solution to model nonlinearity in elimination and distributional clearances with α2 -adrenergic receptor agonists: Example of the intravenous detomidine and methadone combination in sedated horses.

The alpha(α)2 -agonist detomidine is used for equine sedation with opioids such as methadone. We retrieved the data from two randomized, crossover studies where detomidine and methadone were given intravenously alone or combined as boli (STUDY 1) (Gozalo-Marcilla et al., 2017, Veterinary Anaesthesia and Analgesia, 2017, 44, 1116) or as 2-hr constant rate infusions (STUDY 2) (Gozalo-Marcilla et al., 2019, Equine Veterinary Journal, 51, 530). Plasma drug concentrations were measured with a validated tandem Mass Spectrometry assay. We used nonlinear mixed effect modelling and took pharmacokinetic (PK) data from both studies to fit simultaneously both drugs and explore their nonlinear kinetics. Two significant improvements over the classical mammillary two-compartment model were identified. First, the inclusion of an effect of detomidine plasma concentration on the elimination clearances (Cls) of both drugs improved the fit of detomidine (Objective Function Value [OFV]: -160) and methadone (OFV: -132) submodels. Second, a detomidine concentration-dependent reduction of distributional Cls of each drug further improved detomidine (OFV: -60) and methadone (OFV: -52) submodel fits. Using the PK data from both studies (a) helped exploring hypotheses on the nonlinearity of the elimination and distributional Cls and (b) allowed inclusion of dynamic effects of detomidine plasma concentration in the model which are compatible with the pharmacology of detomidine (vasoconstriction and reduction in cardiac output).

Pharmacokinetics and Tissue Distribution of Benznidazole after Oral Administration in Mice.

Specific chemotherapy using benznidazole (BNZ) for Chagas disease during the chronic stage is controversial due to its limited efficacy and toxic effects. Although BNZ has been used to treat Chagas disease since the 1970s, few studies about the biodistribution of this drug exist. In this study, BNZ tissue biodistribution in a murine model and its pharmacokinetic profile in plasma were monitored. A bioanalytical high-performance liquid chromatography method with a UV detector (HPLC-UV) was developed and validated according to the European Medicines Agency for quantification of BNZ in organs and plasma samples prepared by liquid-liquid extraction using ethyl acetate. The developed method was linear in the BNZ concentration, which ranged from 0.1 to 100.0 µg/ml for plasma, spleen, brain, colon, heart, lung, and kidney and from 0.2 to 100.0 µg/ml for liver. Validation assays demonstrated good stability for BNZ under all conditions evaluated. Pharmacokinetic parameters confirmed rapid, but low, absorption of BNZ after oral administration. Biodistribution assays demonstrated different maximum concentrations in organs and similar times to maximum concentration and mean residence times, with means of 40 min and 2.5 h, respectively. Therefore, the biodistribution of BNZ is extensive, reaching organs such as the heart and colon, which are the most relevant organs affected by Trypanosoma cruzi infection, and also the spleen, brain, liver, lungs, and kidneys. Simultaneous analyses of tissues and plasma indicated high BNZ metabolism in the liver. Our results suggest that low bioavailability, instead of inadequate biodistribution, could be responsible for therapeutic failure during the chronic phase of Chagas disease.

Evaluation of the Intestinal Absorption Mechanism of Casearin X in Caco-2 Cells with Modified Carboxylesterase Activity.

The clerodane diterpene casearin X (1), isolated from the leaves of Casearia sylvestris, is a potential new drug candidate due to its potent in vitro cytotoxic activity. In this work, the intestinal absorption mechanism of 1 was evaluated using Caco-2 cells with and without active carboxylesterases (CES). An LC-MS method was developed and validated for the quantification of 1. The estimation of permeability coefficients was possible only under CES-inhibited conditions in which 1 is able to cross the Caco-2 cell monolayer. The mechanism is probably by active transport, with no significant efflux, but with a high retention of the compound inside the cells. The enzymatic hydrolysis assay demonstrates the susceptibility of 1 to first-pass metabolism as substrate for specific CES expressed in human intestine.

The metabolic function of pyruvate kinase M2 regulates reactive oxygen species production and microbial killing by neutrophils.

Neutrophils rely predominantly on glycolytic metabolism for their biological functions, including reactive oxygen species (ROS) production. Although pyruvate kinase M2 (PKM2) is a glycolytic enzyme known to be involved in metabolic reprogramming and gene transcription in many immune cell types, its role in neutrophils remains poorly understood. Here, we report that PKM2 regulates ROS production and microbial killing by neutrophils. Zymosan-activated neutrophils showed increased cytoplasmic expression of PKM2. Pharmacological inhibition or genetic deficiency of PKM2 in neutrophils reduced ROS production and Staphylococcus aureus killing in vitro. In addition, this also resulted in phosphoenolpyruvate (PEP) accumulation and decreased dihydroxyacetone phosphate (DHAP) production, which is required for de novo synthesis of diacylglycerol (DAG) from glycolysis. In vivo, PKM2 deficiency in myeloid cells impaired the control of infection with Staphylococcus aureus. Our results fill the gap in the current knowledge of the importance of lower glycolysis for ROS production in neutrophils, highlighting the role of PKM2 in regulating the DHAP and DAG synthesis to promote ROS production in neutrophils.

Preclinical monitoring of drug association in experimental chemotherapy of Chagas' disease by a new HPLC-UV method.

A combination of drugs in experimental chemotherapy of Chagas' disease may increase the effectiveness of treatment. To evaluate the possible mechanisms that influence the improvement of therapy, we investigated the pharmacokinetic interaction between benznidazole and itraconazole in a murine model treated orally with single doses of 5 mg of each compound separately or together. Blood samples from treated mice were collected at different intervals for 48 h, and a high-performance liquid chromatography (HPLC)-UV method was used to quantify both drugs in the plasma. A decrease of 1.5-fold in the maximum drug concentration in the plasma (C(max)) and an increase of 2.66-fold in the volume of distribution (V) and 7.5-fold in the elimination half-life (t(1/2ß)) of benznidazole when coadministered with itraconazole were observed. The parameters area under the curve (AUC(0-t)), area under the curve extrapolated to infinity (AUC(0-∞)), time to maximum concentration of drug in serum (T(max)), and clearance (CL) for benznidazole were not significantly different in this therapeutic regime. None of the evaluated parameters for ITC demonstrated a significant difference between isolated and associated administration. These results suggest that the main effect of this interaction leads to accumulation of benznidazole in the biological system. This effect may contribute to the improved therapeutic efficacy of this combination of drugs, in addition to synergism of the different mechanisms of action of benznidazole and itraconazole against Trypanosoma cruzi in vivo.

In vitro-in vivo correlation of the chiral pesticide prothioconazole after interaction with human CYP450 enzymes.

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.

Chemometric optimization of salting-out assisted liquid-liquid extraction (SALLE) combined with LC-MS/MS for the analysis of carvedilol enantiomers in human plasma: Application to clinical pharmacokinetics.

Carvedilol is a commonly used antihypertensive whose oral absorption is limited by low solubility and significant first-pass metabolism. This work aimed to apply chemometrics for the optimization of a salting-out assisted liquid-liquid extraction (SALLE) combined with LC-MS/MS to analyze carvedilol enantiomers in plasma samples. Method development and validation were driven for application in pharmacokinetic studies. Parameters that influence the efficiency of SALLE were evaluated using a fractional factorial 24-1 design with 4 factors and a central composite design was used to evaluate the optimal extraction condition. Carvedilol enantiomers and the internal standard lidocaine were separated on an Astec® Chirobiotic® V column and a mixture of methanol:ethanol (90:10, v/v) with 0.02% diethylamine and 0.18% acetic acid as mobile phase. The positive ion mode on electrospray ionization was used to monitor the transitions of m/z 407 > 100 and 235 > 86 for carvedilol enantiomers and lidocaine, respectively. Acetonitrile and ammonium acetate solution were selected for sample preparation by SALLE. Surface graphs and the desirability test were used to define the optimized SALLE conditions which resulted in 93% recovery for both carvedilol enantiomers. The method was linear in the range of 0.5 to 100 ng/mL in plasma, with a lower limit of quantification of 0.5 ng/mL. Within-run and between-run precision (as the relative standard deviation) were all < 9.74% and accuracy (as relative error) did not exceed ± 10.30%. Residual effect and matrix effect were not observed. Carvedilol enantiomers were stable in plasma under the storage, preparation, and analysis conditions. The validated method was successfully applied to analyze carvedilol in plasma samples from patients previously submitted to a Roux-en-Y gastric bypass surgery treated with a single oral dose of 25 mg racemic-carvedilol. Higher plasma concentrations were observed for (R)-(+)-carvedilol when compared to (S)-(-)-carvedilol in two patients post-bariatric surgery.

Enantioselective inhibition of human CYP2C19 by the chiral pesticide ethofumesate: Prediction of pesticide-drug interactions in humans.

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.

In vitro enantioselective inhibition of the main human CYP450 enzymes involved in drug metabolism by the chiral pesticide tebuconazole.

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.

Risk assessment of the chiral pesticide fenamiphos in a human model: Cytochrome P450 phenotyping and inhibition studies.

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.

Prediction of seriniquinone-drug interactions by in vitro inhibition of human cytochrome P450 enzymes.

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.