Lactobacillus paracasei IMC 502 ameliorates type 2 diabetes by mediating gut microbiota-SCFA-hormone/inflammation pathway in mice.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a complex and prevalent metabolic disease that seriously threatens human health. Numerous studies have shown that probiotics as dietary supplements have the potential to prevent and treat T2DM. However, the ability of various strains to improve diabetes symptoms and corresponding mechanisms are different. Thus, mechanistic investigation is required to validate the pharmacology of each probiotic strain for T2DM treatment. Lactobacillus paracasei IMC 502 was originally isolated from Italian elderly human feces and its probiotic attributes have been demonstrated. Here, the antidiabetic pharmacodynamics of L. paracasei IMC 502 on T2DM mice was explored. RESULTS: Lactobacillus paracasei IMC 502 significantly decreased blood glucose, HbA1c and lipid levels, improved insulin resistance and glucose intolerance, regulated the mRNA/protein expression of key hepatic enzymes associated with gluconeogenesis, de novo lipogenesis and PI3K/Akt pathway, and repaired pancreatic and hepatic tissue damage. This probiotic conferred beneficial outcomes in the gut microbiome of diabetic mice, which induced transformation of short-chain fatty acids (SCFAs) and further enhanced the secretion of downstream hormones, and ultimately ameliorated the inflammatory response. CONCLUSION: Lactobacillus paracasei IMC 502 prevents and alleviates T2DM by mediating the gut microbiota-SCFA-hormone/inflammation pathway. © 2022 Society of Chemical Industry.

Physiologically-Based Pharmacokinetic Modeling-Guided Dose Management of Oral Anticoagulants when Initiating Nirmatrelvir/Ritonavir (Paxlovid) for COVID-19 Treatment.

Patients with coronavirus disease 2019 (COVID-19) with cardiovascular diseases who are at higher risk of progressing to critical illness should be treated with nirmatrelvir/ritonavir (Paxlovid). Ritonavir, the booster in nirmatrelvir/ritonavir, modulates multiple drug metabolizing enzymes and transporters, complicating its use in real-world clinics. We aimed to apply physiologically-based pharmacokinetic (PBPK) modeling to simulate the complex drug-drug interactions (DDIs) of ritonavir with two anticoagulants, rivaroxaban and racemic warfarin, to address this important clinical conundrum. Simulations were implemented within Simcyp Simulator. Compound and population models were adopted from Simcyp and our previous studies. Upon verification and validation of the PBPK model of ritonavir, prospective DDI simulations with the anticoagulants were performed in both the general population (20-65 years) and geriatric subjects (65-85 years) with or without moderate renal impairment. Elevated rivaroxaban concentrations were simulated with nirmatrelvir/ritonavir treatment, where the impact was more profound among geriatric subjects with renal impairment. The overexposure of rivaroxaban was restored to normal range on day 4 post-discontinuation of nirmatrelvir/ritonavir, corroborating with the recovery of enzyme activity. A lower 10 mg daily dose of rivaroxaban could effectively maintain acceptable systemic exposure of rivaroxaban during nirmatrelvir/ritonavir treatment. Treatment of ritonavir marginally declined simulated S-warfarin concentrations, but substantially elevated that of R-warfarin, resulting in a decrease in the international normalized ratio (INR). As INR only recovered 2 weeks post-nirmatrelvir/ritonavir treatment, a longer surveillance INR for warfarin becomes important. Our PBPK-guided simulations evaluated clinically important yet untested DDIs and supports clinical studies to ensure proper anticoagulation management of patients with COVID-19 with chronic coagulative abnormalities when initiating nirmatrelvir/ritonavir therapy.

Dose-Dependent Increase in Unconjugated Cinnamic Acid Concentration in Plasma Following Acute Consumption of Polyphenol Rich Curry in the Polyspice Study.

Spices that are rich in polyphenols are metabolized to a convergent group of phenolic/aromatic acids. We conducted a dose-exposure nutrikinetic study to investigate associations between mixed spices intake and plasma concentrations of selected, unconjugated phenolic/aromatic acids. In a randomized crossover study, 17 Chinese males consumed a curry meal containing 0 g, 6 g, and 12 g of mixed spices. Postprandial blood was drawn up to 7 h at regular intervals and plasma phenolic/aromatic acids were quantified via liquid chromatography tandem mass spectrometry (LC-MS/MS). Cinnamic acid (CNA, p < 0.0001) and phenylacetic acid (PAA, p < 0.0005) concentrations were significantly increased with mixed spices consumption, although none of the other measured phenolic/aromatic acids differ significantly between treatments. CNA displayed a high dose-exposure association (R² > 0.8, p < 0.0001). The adjusted mean area under the plasma concentration-time curve until 7 h (AUC0⁻7 h) for CNA during the 3 increasing doses were 8.4 ± 3.4, 376.1 ± 104.7 and 875.7 ± 291.9 nM.h respectively. Plasma CNA concentration may be used as a biomarker of spice intake.

Dronedarone-Induced Cardiac Mitochondrial Dysfunction and Its Mitigation by Epoxyeicosatrienoic Acids.

Dronedarone and amiodarone are structurally similar antiarrhythmic drugs. Dronedarone worsens cardiac adverse effects with unknown causes while amiodarone has no cardiac adversity. Dronedarone induces preclinical mitochondrial toxicity in rat liver and exhibits clinical hepatotoxicity. Here, we further investigated the relative potential of the antiarrhythmic drugs in causing mitochondrial injury in cardiomyocytes. Differentiated rat H9c2 cardiomyocytes were treated with dronedarone, amiodarone, and their respective metabolites namely N-desbutyldronedarone (NDBD) and N-desethylamiodarone (NDEA). Intracellular ATP content, mitochondrial membrane potential (Δψm), and inhibition of carnitine palmitoyltransferase I (CPT1) activity and arachidonic acid (AA) metabolism were measured in H9c2 cells. Inhibition of electron transport chain (ETC) activities and uncoupling of ETC were further studied in isolated rat heart mitochondria. Dronedarone, amiodarone, NDBD and NDEA decreased intracellular ATP content significantly (IC50 = 0.49, 1.84, 1.07, and 0.63 µM, respectively) and dissipated Δψm potently (IC50 = 0.5, 2.94, 12.8, and 7.38 µM, respectively). Dronedarone, NDBD, and NDEA weakly inhibited CPT1 activity while amiodarone (IC50 > 100 µM) yielded negligible inhibition. Only dronedarone inhibited AA metabolism to its regioisomeric epoxyeicosatrienoic acids (EETs) consistently and potently. NADH-supplemented ETC activity was inhibited by dronedarone, amiodarone, NDBD and NDEA (IC50 = 3.07, 5.24, 11.94, and 16.16 µM, respectively). Cytotoxicity, ATP decrease and Δψm disruption were ameliorated via exogenous pre-treatment of H9c2 cells with 11, 12-EET and 14, 15-EET. Our study confirmed that dronedarone causes mitochondrial injury in cardiomyocytes by perturbing Δψm, inhibiting mitochondrial complex I, uncoupling ETC and dysregulating AA-EET metabolism. We postulate that cardiac mitochondrial injury is one potential contributing factor to dronedarone-induced cardiac failure exacerbation.

An investigation of the bioactivation potential and metabolism profile of Zebrafish versus human.

The zebrafish model has been increasingly explored as an alternative model for toxicity screening of pharmaceutical drugs. However, little is understood about the bioactivation of drug to reactive metabolite and phase I and II metabolism of chemical in zebrafish as compared with human. The primary aim of our study was to establish the bioactivation potential of zebrafish using acetaminophen as a probe substrate. Our secondary aim was to perform metabolite profiling experiments on testosterone, a CYP3A probe substrate, in zebrafish and compare the metabolite profiles with that of human. The glutathione trapping assay of N-acetyl-p-benzoquinone imine demonstrated that zebrafish generates the same reactive metabolite as humans from the bioactivation of acetaminophen. Zebrafish possesses functional CYP3A4/5-like and UDP-glucuronosyltransferase metabolic activities on testosterone. Differential testosterone metabolism was observed among the two species. In silico docking studies suggested that the zebrafish CYP3A65 was responsible for the bioactivation of acetaminophen and phase I hydroxylation of testosterone. Our findings reinforce the need to further characterize the drug metabolism phenotype of zebrafish before the model can fully achieve its potential as an alternative toxicity screening model in drug research.

Ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOFMS) for time-dependent profiling of raw and steamed Panax notoginseng.

The metabolic profiles of Panax notoginseng and its associated therapeutic values are critically affected by the duration of steaming. The time-dependent steaming effect of P. notoginseng is not well-characterized and there is also no official guideline on its duration of steaming. In this paper, a UHPLC/TOFMS-based metabolomic platform was developed for the qualitative profiling of multiparametric metabolic changes of raw P. notoginseng during the steaming process. Our method was successful in discriminating the differentially processed herbs. Both the unsupervised principal component analysis (PCA) score plot (R(2)X=0.664, Q(2) (cum)=0.622, and PCs=2) and the supervised partial least square-data analysis (PLS-DA) model (R(2)X=0.708, R(2)Y=0.461, and Q(2)Y=0.271) demonstrated strong classification and clear trajectory patterns with regard to the duration of steaming. The PLS-DA model was validated for its robustness via a prediction set, confirming that the UHPLC/TOFMS metabolic profiles of the raw and differentially steamed P. notoginseng samples were highly reproducible. Based on our method, the minimum durations of steaming for the maximum production of bioactive ginsenosides such as Rg3 and Rh2 were also predicted. Our novel time-dependent metabolic profiling approach represents the paradigm shift in the quality control of P. notoginseng products.

Fluorescence-based liver microsomal assay for screening of pharmaceutical reactive metabolites using a glutathione conjugated 96-well plate.

The purpose of our paper is to develop and validate a fluorescence-based mouse liver microsomal (MLM) assay in screening pharmaceutical reactive metabolites (RMs) using a glutathione (GSH)-conjugated 96-well plate. Poly(2-hydroxyethylmethacrylate) (pHEMA) polymeric membrane was coated on 96-well plates to provide a functional support for GSH conjugation. Oxidized GSH (GSSG) was conjugated on a cyanogen bromide (CNBr)-activated pHEMA surface. The conjugated GSH was regenerated after the reduction of GSSG using d,l-dithiothreitol (DTT). X-ray photoelectron spectroscopy, Ellman's, and fluorescence assays were applied to validate the chemistry and optimize the processes of GSH conjugation. The performance of the 96-well assay was further cross-validated using N-acetyl-p-benzo-quinone imine (NAPQI), a RM of acetaminophen (APAP), and the in vitro MLM assay of APAP. Finally, the developed method was applied to screen a batch of marketed drugs and chemicals on the formation of RMs. Our results indicated that optimum conditions were obtained for pHEMA loading, CNBr activation of pHEMA, and GSSG coupling and reduction. The detection limit of the assay for NAPQI was 500 nM with good specificity. In vitro MLM assay of APAP demonstrated a positive trapping index (TI) of 19.3%. The subsequent RM screening of a series of marketed drugs and chemical compounds resulted in a range of TI values (1.0-25.7%) that corroborated with their capacity in generating RMs. The differences of TI values are statistically significant between the compounds which are known to produce RMs and those that do not generate reactive intermediates. In conclusion, we successfully developed a fluorescence-based GSH-conjugated 96-well plate platform for the screening of RMs using MLM.

Metabonomics investigation of human urine after ingestion of green tea with gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry and (1)H NMR spectroscopy.

A method using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and (1)H NMR with pattern recognition tools such as principle components analysis (PCA) was used to study the human urinary metabolic profiles after the intake of green tea. From the normalized peak areas obtained from GC/MS and LC/MS and peak heights from (1)H NMR, statistical analyses were used in the identification of potential biomarkers. Metabolic profiling by GC/MS provided a different set of quantitative signatures of metabolites that can be used to characterize the molecular changes in human urine samples. A comparison of normalized metabonomics data for selected metabolites in human urine samples in the presence of potential overlapping peaks after tea ingestion from LC/MS and (1)H NMR showed the reliability of the current approach and method of normalization. The close agreements of LC/MS with (1)H NMR data showed that the effects of ion suppression in LC/MS for early eluting metabolites were not significant. Concurrently, the specificity of detecting the stated metabolites by (1)H NMR and LC/MS was demonstrated. Our data showed that a number of metabolites involved in glucose metabolism, citric acid cycle and amino acid metabolism were affected immediately after the intake of green tea. The proposed approach provided a more comprehensive picture of the metabolic changes after intake of green tea in human urine. The multiple analytical approach together with pattern recognition tools is a useful platform to study metabolic profiles after ingestion of botanicals and medicinal plants.

Hydrophilic interaction liquid chromatography with tandem mass spectrometry for the determination of underivatized dencichine (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid) in Panax medicinal plant species.

Dencichine (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid) is a haemostatic agent present in important Chinese medicinal herbs such as Panax notoginseng, as well as other Panax species. It is also a reported neurotoxic agent found in Lathyrus sativus (grass pea seed). A selective analytical method incorporating hydrophilic interaction chromatography with positive electrospray ionization tandem mass spectrometry (HILIC/ESI-MS/MS), for the analysis of dencichine in Panax plant species, was developed. Using multiple reaction monitoring (MRM) mode, underivatized dencichine, a small and highly polar compound, was selectively detected and quantified. The contents of dencichine in raw and steamed Panax notoginseng roots, 11 pairs of raw and steamed P. notoginseng herbal products, Panax ginseng roots, and Panax quinquefolium roots, were analyzed and compared. Optimal sensitivity of 0.3 ppm (detection limit) and 1.5 ppm (quantification limit) was achieved. The method was rapid (< or =5 min), with the HILIC peak eluting at about 1 min. Steamed P. notoginseng samples were found to contain less dencichine than the corresponding raw samples, and there were also differences among the three Panax species; raw P. ginseng and P. quinquefolium contained less dencichine than the raw P. notoginseng species. This rapid and specific method may be applied to the quantification of dencichine in complex medicinal plants and their products.