Active Metabolite Research Articles

Protective Effects of Dioscin and Diosgenin on Plateau Hyperuricemia by Attenuating Renal Inflammation via EPHX2

Plateau hyperuricemia is a common disease in the plateau area, and the incidence is much higher than that in the plain area. Dioscin (DIO) and its active metabolite Diosgenin (DG) exert therapeutic effects on hyperuricemia through oxidative stress and inflammation. In this study, DIO and its active metabolite DG were taken as the research objects to explore their therapeutic effects on high-altitude hyperuricemia in rats. To evaluate the therapeutic effect of DIO on the rat model of high-altitude hyperuricemia, the evaluation indexes include blood biochemical indexes, renal histopathology, oil red O staining of the kidney, rat kidney index, and rat renal inflammatory factors. Transcriptomics was used to analyze the control group, model group, and drug-administered group to preliminarily explore the protective mechanism of DIO in rats with high-altitude hyperuricemia. An HK-2 high-altitude hyperuricemia cell injury model was established to verify the therapeutic mechanism of DIO in rats with high-altitude hyperuricemia. Western blot was used to detect the expression of related proteins in renal tissues and cell models. The results showed that DIO and its active metabolite DG regulate renal lipid metabolism through the EPHX2 gene, attenuate renal inflammatory reaction, and then promote the excretion of uric acid and reduce its reabsorption, which ultimately achieves the effect of treating plateau hyperuricemia.

Enzymatic Synthesis of Biologically Active H-Phosphinic Analogue of α-Ketoglutarate

Amino acid analogues with a phosphorus-containing moiety replacing the carboxylic group are promising sources of biologically active compounds. The H-phosphinic group, with hydrogen–phosphorus–carbon (H-P-C) bonds and a flattened tetrahedral configuration, is a bioisostere of the carboxylic group. Consequently, amino-H-phosphinic acids undergo substrate-like enzymatic transformations, leading to new biologically active metabolites. Previous studies employing NMR-based metabolomic and proteomic analyses show that in Escherichia coli, α-KG-γ-PH (the distal H-phosphinic analogue of α-ketoglutarate) can be converted into L-Glu-γ-PH. Notably, α-KG-γ-PH and L-Glu-γ-PH are antibacterial compounds, but their intracellular targets only partially overlap. L-Glu-γ-PH is known to be a substrate of aspartate transaminase and glutamate decarboxylase, but its substrate properties with NAD+-dependent glutamate dehydrogenase (GDH) have never been investigated. Compounds containing P-H bonds are strong reducing agents; therefore, enzymatic NAD+-dependent oxidation is not self-evident. Herein, we demonstrate that L-Glu-γ-PH is a substrate of eukaryotic GDH and that the pH optimum of L-Glu-γ-PH NAD+-dependent oxidative deamination is shifted to a slightly alkaline pH range compared to L-glutamate. By 31P NMR, we observe that α-KG-γ-PH exists in a pH-dependent equilibrium of keto and germinal diol forms. Furthermore, the stereospecific enzymatic synthesis of α-KG-γ-PH from L-Glu-γ-PH using GDH is a possible route for its bio-based synthesis.

Post-harvest processing methods have critical roles on the contents of active metabolites and pharmacological effects of Astragali Radix

BackgroundProcessing methods of traditional Chinese medicinal materials are critical in influencing the active metabolites and pharmacological effects. The fresh processing method effectively prevents the loss and degradation of metabolites, common in traditional drying and softening processes, while also reducing production costs. Astragali Radix (AR), a leguminous botanical drug, is widely utilized in clinical practice and functional foods. Therefore, optimizing its the post-harvest processing method is crucial for enhancing production and application.MethodsAR samples were processed using different methods with varying moisture content, including unpretreated samples and those subjected to kneading and sweating treatments. These samples were evaluated for physical appearance and active metabolite content. The entropy weight method, combined with the technique for order preference by similarity to ideal solution (TOPSIS), was employed to optimize the fresh processing method. A comparative study between freshly processed AR (AR-F) and traditionally processed AR (AR-T) assessed active metabolites, pharmacological effects and mechanisms.ResultsThe appearance and active metabolites content of AR samples were affected by moisture content, kneading, and sweating treatments. After these treatments, the content of polysaccharides and calycosin-7-O-glucoside increased compared to unpretreated samples at the same moisture level. Entropy weight-TOPSIS analysis showed that the sample with 35% moisture, 100 kneading cycles, and 12 h of sweating had the highest score. Comparative studies revealed that AR-F had significantly higher content of total polysaccharides, total flavonoids, and calycosin-7-O-glucoside compared to AR-T, along with an increased flavonoid glycoside/aglycone ratio. However, no significant differences were observed in the total saponins and their metabolites. Pharmacological studies demonstrated that total flavonoids in AR-F exhibited superior macrophage RAW264.7 activation, compared to AR-T. Furthermore, we confirmed that the enhanced immunomodulatory capacity of AR-F was linked to its ability to stimulate the release of TNF, SRC, ER-α, AKT, HSP90, and Caspase-3 in RAW264.7 cells.ConclusionOur study optimized the fresh processing method of AR, and conducted a systematic comparative analysis between fresh and traditional processing samples, providing a basis for post-harvest processing in the AR production areas.

Pharmacokinetic Interaction Between Olaparib and Regorafenib in an Animal Model

Abstract: Background: Olaparib (OLA) and regorafenib (REG) are metabolized by the CYP3A4 isoenzyme of cytochrome P450. Both drugs are also substrates and inhibitors of the membrane transporters P-glycoprotein and BCRP. Therefore, the potential concomitant use of OLA and REG may result in clinically relevant drug–drug interactions. Knowledge of the influence of membrane transporters and cytochrome P450 enzymes on the pharmacokinetics of drugs makes it possible to assess their impact on the efficacy and safety of therapy. Purpose: The study aimed to evaluate the bilateral pharmacokinetic interactions of OLA and REG and its active metabolites after a single administration in healthy rats. Methods: The study was performed in male Wistar rats (n = 24) randomly divided into three groups: one study group, IREG+OLA (n = 8), received REG with OLA, and two control groups, IIREG (n = 8) and IIIOLA (n = 8), received REG and OLA, respectively. The concentrations of OLA, REG, REG-N-oxide (M-2), and N-desmethyl-REG-N-oxide (M-5) were determined by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). The values of the pharmacokinetic parameters of OLA, REG, M-2, and M-5 were determined by non-compartmental analysis with linear interpolation. Results: After OLA administration, the pharmacokinetic parameters of REG (AUC0–∞, tmax, and t0.5) increased significantly by 3.38-, 2.66-, and 1.82-fold, respectively. On the other hand, REG elimination parameters, i.e., kel and Cl/F, were significantly reduced in the study group by 1.77- and 1.70-fold, respectively. In the study group, Cmax and AUC0-t values were also 7.22- and 8.86-fold higher for M-2 and 16.32- and 17.83-fold higher for M-5, respectively. The Metabolite M-2/Parent and Metabolite M-5/Parent ratios for Cmax and AUC0-t increased by 6.52-, 10.74-, 28-, and 13-fold, respectively. After administration of OLA with REG, the Cmax, AUC0-t, and AUC0-∞ of OLA increased by 2.0-, 3.4-, and 3.4-fold, respectively, compared to the control group. Meanwhile, Cl/F and Vd/F of OLA were significantly decreased in the presence of REG. Conclusions: OLA was shown to significantly affect the pharmacokinetics of REG and its active metabolites M-2 and M-5 in rats after co-administration of both drugs. There was also a significant effect of REG on the pharmacokinetics of OLA, which may have clinical relevance. The AUC ratios (study group/control group) were 3.41 and 3.39 for REG and OLA, respectively, indicating that REG and OLA were moderate inhibitors in this preclinical study. The results obtained need to be confirmed in clinical studies. This study may provide guidance on the safety of using both drugs in clinical practice.

Abstract A007: Anti-cancer activity of (Z)-endoxifen-related compounds in ERα+ breast cancer

Abstarct Introduction: (Z)-endoxifen (ENDX) is a potent orally bioavailable selective estrogen receptor modulator (SERM) and the most active metabolite of tamoxifen. Given its effective anti-estrogen activity and favorable safety profile, ENDX is being developed as a therapeutic solution in breast health. During its synthesis, two chemical biproducts (compound AT416 and AT402) with Z- and E- isomeric forms are produced. This study aimed to assess the anti-estrogenic activity of these four ENDX-related compounds with ENDX in ERα+ breast cancer. Methods: Compounds AT416E/Z are formed by acetylation of the final API with ethyl acetate during Step 3 of the manufacturing process of ENDX. Compounds AT402 E/Z are formed by coupling of impurity AT300 with propiophenone as a side reaction during Step 2 of the ENDX manufacturing process. The anti-cancer activities of the four ENDX-related compounds were tested across multiple ERα+ breast cancer cell lines (MCF7, T47D, UCD12), including those with ESR1 mutations (Y537S and D538G) known to confer endocrine resistance. Studies included cell proliferation, migration and invasion assays, cell cycle progression assessments, induction of apoptosis, activity of an estrogen response element (ERE) luciferase reporter construct and RT-qPCR analyses of ERα target gene expression. Results: In estrogen-containing media, ENDX exhibited strong anti-proliferative activity in MCF7 cells, regardless of ESR1 mutations. In MCF7 cells cultured in estrogen-deficient media, the four compounds demonstrated more pronounced anti-proliferative effects than ENDX. In T47D cells with the ESR1 Y537S mutation, AT416E showed superior anti-proliferative activity compared to ENDX, while in T47D cells with the D538G mutation, these two compounds exhibited comparable effects. In UCD12 cells, ENDX displayed the strongest anti-proliferative effects. Regarding the inhibition of migration and invasion, AT416E consistently outperformed ENDX in both T47D and MCF7 cells. ENDX was the most effective at inducing G1 phase cell cycle arrest followed. Both AT402E and AT402Z elicited pro-apoptotic effects comparable to ENDX in MCF7 cells, while in T47D cells, ENDX was the most effective at inducing apoptosis, followed by AT402E and AT402Z. In T47D and MCF7 cells, luciferase-based transactivation assays showed that ENDX was the most potent anti-estrogen followed by AT402Z. Finally, based on RT-qPCR analyses, ENDX was a more effective inhibitor of estrogen-induced ERα target gene expression, compared to the biproducts. Summary: These studies have revealed that four novel compounds derived from the manufacturing process of ENDX confer notable anti-tumor activity, with some performing comparably to or better than ENDX in several cancer-relevant cellular readouts. Future studies will validate these findings in additional in vitro/in vivo breast cancer models with the goal of identifying the basis for their varied anti-estrogenic and anti-cancer effects. Such discoveries should allow for the rational design of novel molecules with more potent therapeutic efficacy. Citation Format: Natalie Farris, Lena Batoon, Rajeev S. Muthyala, H. Lawrence Remmel, Sandra S. Hammer, John R. Hawse, Steven C. Quay. Anti-cancer activity of (Z)-endoxifen-related compounds in ERα+ breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A007.

Evaluation of dimethandrolone undecanoate in non-human primates as a candidate for long-acting injectable male contraceptive.

Dimethandrolone undecanoate (DMAU) is under development as a single agent hormonal male contraceptive. DMAU is a prodrug hydrolyzed by esterase(s) to the active metabolite dimethandrolone (DMA) which has dual androgenic and progestogenic actions. Phase 1 clinical trial results show DMAU to be well-tolerated as an oral contraceptive in healthy men; however, delivery of DMAU as a long-acting injectable rather than a daily oral formulation would provide user compliance benefits and address oral bioavailability concerns. To assess the safety, pharmacokinetics (PK), and long-acting contraceptive potential of DMAU in male non-human primates (NHP) when delivered as an injectable or oral formulation. DMAU was administered to cynomolgus macaques orally for 9 months or as five weekly intramuscular (IM) injections and to rhesus macaques as a single IM injection. Evaluations of safety, fertility indicators, and serum levels of DMAU and DMA were followed>2 years post-dose. Repeat dose oral and IM administrations were well-tolerated with no significant toxicological findings. Dramatic reductions in serum testosterone concentration occurred within days of administration followed by sustained suppression of additional fertility indicators (e.g., serum inhibin B, sperm count, and testicular spermatogenesis). Slight body weight increases and reductions in testes weight also occurred. Repeat DMAU injections resulted in DMA serum concentrations above the lower limit of quantification (1ng/mL) for>500 days. DMAU PK parameters increased with increasing IM dose, while dose dependence was not seen for serum DMA concentrations suggesting a depot effect with a reservoir of non-circulating prodrug remaining at the injection site which gets slowly hydrolyzed to DMA and absorbed into circulation. Testosterone levels and spermatogenesis returned by the end of the recovery period. Nonclinical safety, PK, and pharmacodynamic data in male NHP demonstrate the safe, well-tolerated, long-acting, and reversible contraceptive potential of injectable DMAU.

Antioxidant capacities and non-volatile metabolites changes after solid-state fermentation of soybean using oyster mushroom (Pleurotus ostreatus) mycelium

Given the abundance of beneficial properties and enzymes secreted by edible oyster mushrooms, their mycelium could serve as a starter for fermented foods to enhance their nutritional and bioactive quality. This study aimed to investigate the effects on the nutritional ingredients, antioxidant activity, and non-volatile metabolites during solid-state fermentation (SSF) of soybeans by Pleurotus ostreatus mycelium. The results indicated that the contents of dietary fiber and starch in fermented soybeans decreased, while the amounts of protein and lipid increased after SSF (P < 0.05). Analysis of the total phenolic content (TPC) and antioxidant activities of the fermented soybeans revealed that the methanolic extracts significantly increased TPC and antioxidant activities against intracellular reactive oxygen species (ROS) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, as well as against DPPH and ABTS radicals in vitro. A total 154 differential metabolites were identified after SSF, and a Spearman correlation study revealed a direct relationship between antioxidant activities and certain metabolites including phenolic compounds, oligopeptides, and free fatty acids etc. Among these metabolites, phenolic compounds produced by the shikimic acid pathway were diverse in variety and had the greatest multiple differences. The study discovered that a potential mechanism involving SSF with P. ostreatus mycelium increased the antioxidant activity of soybeans.

Tailored culture strategies to promote antimicrobial secondary metabolite production in Diaporthe caliensis: a metabolomic approach

BackgroundIn the search for new antimicrobial secondary metabolites of fungi, optimizing culture conditions remains a critical challenge, as standard laboratory approaches often result in low yields. While non-selective methods, such as modifying culture media, have been effective in expanding the chemical diversity of fungal metabolites, they have not yet established a direct link to key process parameters crucial for further optimization. This study investigates the capacity of Diaporthe caliensis as a biofactory for biologically active secondary metabolites, employing tailored culture media to explore the relationship between chemical diversity and critical process variables.ResultsThe metabolomic profiles, antibacterial activities, and production yields of the extracts were analyzed to progressively adjust the culture conditions. This study was conducted in five steps, evaluating carbon and nitrogen source concentration, nitrogen source type, salt supplementation, and pH adjustment. Altering the rice starch concentration affected biomass yield per unit of oxygen consumed, while modifications to the nitrogen source concentration influenced both the bioactivity and chemical space by Diaporthe caliensis. Despite changes at the metabolome level, the extracts consistently exhibited potent antibacterial activities, influenced by the nitrogen source, added salts and pH adjustments. For instance, when using corn steep liquor and rice starch, supplemented with micronutrients, different metabolites were produced depending on whether buffer or water was used, though both conditions showed similar antibacterial activities (IC50 ≈ 0.10 mg mL− 1 against Staphylococcus aureus and ≈ 0.14 mg mL− 1 against Escherichia coli). In the treatment where buffer was used to stabilize pH change, there was an increase in the production of phomol-like compounds which are associated with known antibiotic properties. In contrast, in the treatments using water, the drop in pH stimulated the production of previously unidentified metabolites with potential antimicrobial activity.ConclusionsThis study proposes a strategic methodology for the tailored formulation of culture media aiming to promote the biosynthesis of diverse secondary metabolites. This approach revealed the critical role of nutrient limitation and pH regulation in stimulating the production of polyketide-lactone derivatives, including the antibiotic phomol. Ultimately, the systematic, custom-designed culture conditions developed in this work offer a promising strategy for expanding the chemical diversity of Diaporthe caliensis, while providing valuable insights into the key parameters needed for optimizing this fungal biofactory.Graphical

Immunomodulatory properties of polysaccharide extract samples from Cyanobacterium sp. Rippka B-1200

Cyanobacteria are most abundant in aquatic systems and can grow in freshwater, saline or brackish water, and cold/hot springs. Cyanobacteria have attracted considerable research attention in the last decade as a potential source of numerous biological products in large quantities, such as biofuels, pigments, polyunsaturated fatty acids, nutraceuticals, enzymes, and polysaccharides. Unlike most plant and fungal polysaccharides, the chemical composition, immunomodulatory activity, and molecular mechanisms of action of Cyanobacterium sp. Rippka B-1200 polysaccharides have been studied much less. The complexity of their primary structure due to the high variability of monosaccharides, their diverse bonds, the presence of substituents and high viscosity made detailed structural studies of cyanobacterial polysaccharides rare, which determines the need for analysis of cyanobacteria biomass components to identify active metabolites with promising biological activity. The aim of this study was to investigate the immunomodulatory properties of polysaccharides from Cyanobacterium sp. Rippka B-1200. Pharmacological and nutraceutical value of Cyanobacterium sp. Rippka B-1200 has been set, defining our study’s scientific novelty. As a result, the molecular weight of immunoactive polysaccharide (6.0–8.0 kDa) was determined. The analysis shows that endopolysaccharide samples at a concentration of 300 mg/kg showed no significant immunomodulatory effect (1.60 ± 0.15 mg/g), and the thymus mass index of animals in the experimental group was comparable to that of the control group in which animals were immunosuppressed with cyclophosphamide (1.15 ± 0.24 mg/g). When exopolysaccharide samples were used at a concentration of 600 mg/kg, the thymus mass index of animals in the experimental group (3.60 ± 0.32 mg/g) was statistically comparable to that of the control group (without polysaccharide) in which immunosuppression was not induced (thymus index was 3.70 ± 0.25 mg/g). It was found that endopolysaccharide samples at a concentration of 600 mg/kg also exhibited high immunomodulatory activity. When Cyanobacterium sp. Rippka B-1200 endopolysaccharide samples were used, no internal organ changes were observed in experimental animals after immunosuppression. The empirical results presented in the study may find application in the development of both pharmaceutical and cosmetic products.

Can aged Camellia oleifera Abel oil truly be used to treat atopic dermatitis?

Atopic dermatitis is an inflammatory skin condition characterized by erythema, eruption, lichenification, and pruritus. Aged Camellia oleifera Abel oil, an effective empirical plant oil utilized by the Gannan Hakka people in China to alleviate the symptoms of atopic dermatitis. However, no scientific studies have been reported to prove whether this oil is truly effective. We conducted this study to confirm whether aged C. oleifera oil could alleviate the symptoms of 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis in mice. Differences in the thickness and weight of the right and left ears were measured. ELISA was used to determine the serum levels of the inflammatory factors IL-4, IgE, IFN-γ, and TNF-α. HE staining was performed to observe inflammatory cell infiltration in the mouse skin lesions. In addition, the metabolites of aged C. oleifera oils were analyzed, and molecular docking was used to assess the binding affinity of the major metabolites to filaggrin, a protein involved in skin barrier function. Animal studies showed that aged C. oleifera oil significantly improved the symptoms of atopic dermatitis. HE staining and measurement of inflammatory factor levels revealed similar results. A total of 41 metabolites were tentatively identified in the oil, with fatty acids emerging as the major metabolites. Molecular docking confirmed that the three most abundant fatty acids, i.e., oleic acid, n-hexadecanoic acid, and octadecanoic acid, bind well to filaggrin. Our results suggest that aged C. oleifera oils can be used to ameliorate the symptoms of atopic dermatitis. Fatty acids may be the major active metabolites responsible for the observed therapeutic effects by reducing transdermal water loss, increasing skin hydration, alleviating DNCB-induced skin barrier alterations, and eliminating itchy scratching caused by dry skin.

Identification and characterization of the cytochrome p450 complement in <i>Streptomyces cavourensis</i> YBQ59

Cytochrome P450 enzymes (CYPs) are regarded as some of the most versatile biocatalysts. They are attractive candidates for natural product development because of their ability to selectively oxidize a broad range of substrates. Streptomyces spp. are not only producers of biologically active secondary metabolites but also a rich source of P450 enzymes. However, only a limited number of studies have explored the function and potential of P450 enzymes encoded in the Streptomyces genomes. In this study, the endophytic Streptomyces cavourensis YBQ59 isolated from Cinnamomum cassia J. Presl was sequenced using the Illumina sequencing platform to identify its P450 enzymes. The genome of YBQ59 was approximately 8,126,002 bp in size, with a G + C content of 72.1% and contained 7,020 genes. Genome annotation identified 21 CYP genes, distributed across 10 CYP families and 17 subfamilies. The possible role of these P450 enzymes in the synthesis of secondary metabolites was discussed. Since CYPs often require electron transport proteins to function, we analyzed the physical map of the genes encoding ferredoxins and ferredoxin reductases found in the genome of S. cavourensis YBQ59. Additionally, a phylogenetic tree was constructed to compare the P450 enzyme system from S. cavourensis YBQ59 with those of closely related and well-studied Streptomyces species, including Streptomyces sp. CFMR7, S. fulvissimus DSM 40593, S. griseus IFO 13350, and S. globisporus 1912. These results provide a basis for exploiting potential P450 enzymes from S. cavourensis YBQ59 for agricultural and medicinal applications.

Buprenorphine Salivary Gland Accumulation Sustaining High Oral Fluid Exposure and Increasing the Risk of Streptococcus mutans Biofilm Formation.

The US Food and Drug Administration (FDA) issued a warning about buprenorphine-induced dental caries of unknown mechanism in 2022. To investigate the potential mechanism, the association between local buprenorphine exposure and dental biofilm formation will be explored in this study. Female F344 rats were dosed with sublingual buprenorphine film or intravenous injection to explore the oral cavity exposure of the buprenorphine. The buprenorphine distribution in salivary glands after the sublingual and intravenous administration was also evaluated. To investigate the effects of buprenorphine exposure on dental caries formation, buprenorphine's impact on the biofilm formation of S. mutans in vitro was measured. The absolute sublingual bioavailability of buprenorphine in rats was 17.8% with a high ratio of oral fluid exposure to blood concentration in the pharmacokinetic study. Salivary gland concentrations of buprenorphine and its active metabolite norbuprenorphine were significantly higher than their blood concentrations after both sublingual (s.l.) and intravenous (i.v.) administration. Correlation analysis showed that the oral fluid concentration of buprenorphine and norbuprenorphine was highly correlated to salivary gland concentration rather than blood concentration. These data indicate that the salivary gland serves as an accumulation organ for buprenorphine, allowing prolonged oral fluid exposure to buprenorphine. Lastly, buprenorphine and its metabolites contributed to the biofilm formation of S. mutans in high concentration. Sublingual administration substantially increased the salivary gland distribution of buprenorphine and norbuprenorphine. Depot effects following sublingual dosing and salivary gland accumulation likely sustained high oral fluid exposure to buprenorphine and stimulated the biofilm formation of S. mutans.

Highly sensitive ultra-high-performance liquid chromatography coupled with tandem mass spectrometry method for the multiplex analysis of levosimendan and its metabolites OR-1855 and OR-1896 in human plasma

Levosimendan is a positive inotrope and vasodilator used in patients with acute and chronic decompensated heart failure. It is metabolized into OR-1855 (inactive metabolite), which is further acetylated into OR-1896 (active metabolite having a prolonged half-life, hence a sustained effect). Levosimendan represents a valuable alternative to traditional inotropes with broad clinical applications in critically ill patients with cardiogenic shock, advanced heart failure and post-cardiac surgery. However, while levosimendan demonstrates dose-dependent hemodynamic effects, its pharmacokinetics has not yet been investigated in adult critically ill patients, a vulnerable population characterized by complex and unstable conditions that may significantly alter drug disposition. Therefore, pharmacokinetics studies of levosimendan and metabolites in critically ill patients require a reliable and sensitive quantification method.We developed and validated a highly sensitive method using ultra-high-performance liquid-chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) for the quantification of levosimendan, OR-1855 and OR-1896 in human plasma. To achieve the required analytical sensitivity, plasma sample preparation included protein precipitation with acetonitrile, subsequent supernatant’s evaporation to dryness under nitrogen, and reconstitution of the solid residues with a solution of H2O:MeOH 4:1, followed by a 40 µL-aliquot injection into the LC column. Chromatographic separation of the three analytes was achieved in a 6-minute run in gradient mode, using an Acquity UPLC BEH C18 1.7µm, 2.1 ×150mm column. The method was extensively validated according to international bioanalytical assay guidelines, on a clinically relevant concentration range of 0.1 to 100ng/mL, for each analyte. Considering these very low concentrations, the assay showed excellent performances in terms of trueness (94.3-105.3%), repeatability (1.9-7.2%) and intermediate fidelity (2.3-9.7%).Of note, during our ex vivo studies on whole blood samples stability, acetylation of the metabolite OR-1855 into the active OR-1896 metabolite was observed in the presence of red blood cells.The UHPLC method is being applied for a prospective observational pharmacokinetics study of levosimendan in patients undergoing extracorporeal membrane oxygenation support. The benefit of levosimendan therapeutic drug monitoring in intensive care patients remains to be assessed.

Analysis of Concentrations of Monomethyl Fumarate in Patients with Multiple Sclerosis: Result from Routine Health Care

Dimethyl fumarate is used to treat patients with relapsing-remitting multiple sclerosis. After ingestion, it is rapidly hydrolyzed to the active primary metabolite monomethyl fumarate. The main objective of our study was to analyze serum concentrations of monomethyl fumarate during routine health care in patients with multiple sclerosis treated with a fixed dose of dimethyl fumarate. In the pilot cross-sectional study, data from 42 patients treated with dimethyl fumarate at a dose of 240 mg twice daily were collected. Concentrations of the active metabolite monomethyl fumarate were determined at 1-8 h (median, 3 h) or 10-14 h (median, 13 h) after taking the dose. The relationship between monomethyl fumarate concentrations and absolute lymphocyte count was evaluated. Concentrations of monomethyl fumarate ranged from 2.5-3177.9 μg/L, with most concentrations being undetectable approximately 10 hours after administration. In the 1-8 h (median, 3 h) post-dose subgroup, the concentration/dose ratio ranged widely from 0.04-6.62. The median concentration of monomethyl fumarate in the group with the absolute lymphocyte count <0.8 x 10^9/l was more than four times higher than in the group with the absolute lymphocyte count ≥0.8 x 10^9/l (median 440.1 μg/L versus 98.4 μg/L). The wide interindividual variability in monomethyl fumarate pharmacokinetics could contribute to the differential response to dimethyl fumarate in multiple sclerosis patients. A nonsignificant but noticeable trend was observed in the relationship of higher serum monomethyl fumarate concentrations to absolute lymphocyte counts.

Development and Validation of a Sensitive LC-MS/MS Method for Determination of Lenvatinib and Its Major Metabolites in Human Plasma and Its Application in Hepatocellular Carcinoma Patients.

Lenvatinib has been demonstrated effective in advanced hepatocellular carcinoma (HCC), but the pharmacokinetic-pharmacodynamics behavior of lenvatinib and its metabolites remains unclear. To investigate the pharmacokinetic-pharmacodynamics behavior of lenvatinib and its active metabolites in advanced HCC patients, it is important to develop a simple and rapid method to analyze the exposures of lenvatinib and its metabolites in human samples. Here, we established and validated a simple and rapid method for determining lenvatinib and its three major metabolites, descyclopropyl lenvatinib (M1), O-demethyl lenvatinib hydrochloride (M2), and lenvatinib N-Oxide (M3) by liquid chromatography-tandem mass spectrometry method. Lenvatinib and its main metabolites were separated on an X-Terra RP18 column (50 × 2.1mm, 3.5 µm) at 35°C within 3min, and the analytes were isocratically eluted with the mobile phase of methanol-water (10:90, v/v) containing 0.1% of formic acid at a flow rate of 0.15mL/min. The calibration range was 1-1000ng/mL for lenvatinib, while 0.1-100ng/mL for M1-M3 under positive electrospray ionization mode. The inter- and intra-batch precisions and accuracy were acceptable for lenvatinib and its metabolites. This method was successfully applied to measure lenvatinib and its metabolites in plasma samples from HCC patients, which provides a robust tool for pharmacokinetic-pharmacodynamics studies of lenvatinib.

Microbiota responses to mutations affecting NO homeostasis in Arabidopsis thaliana.

Interactions between plants and microorganisms are pivotal for plant growth and productivity. Several plant molecular mechanisms that shape these microbial communities have been identified. However, the importance of nitric oxide (NO) produced by plants for the associated microbiota remains elusive. Using Arabidopsis thaliana isogenic mutants overproducing NO (nox1, NO overexpression) or down-producing NO (i.e. nia1nia2 impaired in the expression of both nitrate reductases NR1/NIA1 and NR2/NIA2; the 35s::GSNOR1 line overexpressing nitrosoglutathione reductase (GSNOR) and 35s::AHB1 line overexpressing haemoglobin 1 (AHB1)), we investigated how altered NO homeostasis affects microbial communities in the rhizosphere and in the roots, soil microbial activity and soil metabolites. We show that the rhizosphere microbiome was affected by the mutant genotypes, with the nox1 and nia1nia2 mutants causing opposite shifts in bacterial and fungal communities compared with the wild-type (WT) Col-0 in the rhizosphere and roots, respectively. These mutants also exhibited distinctive soil metabolite profiles than those from the other genotypes while soil microbial activity did not differ between the mutants and the WT Col-0. Our findings support our hypothesis that changes in NO production by plants can influence the plant microbiome composition with differential effects between fungal and bacterial communities.

Antimicrobial activity of herbal biomass derived metabolites fabricated wound dressing material against microbial strains causing skin infection

In the present study, anti-microbial activity of wound dressing material coated with Aloe vera and Azadirachta indica metabolites was investigated on pathogenic microbial strains associated with skin infection. Anti-microbial active metabolites extracted from the respective medicinal plant were fabricated on the wound dressing material by simple, in situ green science principles. Confirmation of phytochemicals fabrication on the fibre surface of the material was studied by Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) Additionally, the impact of phyto-constituents fabrication on mechanical properties such as thickness and tensile strength was assessed. The anti-microbial efficacy of the fabricated dressing against pathogenic microorganisms (Candida albicans, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 25923) was evaluated using agar diffusion assay. The release profile of phytochemical constituents from the fabricated wound dressing was investigated using ethanol and distilled water as solvents. The results showed that the phytochemical constituents were fabricated effectively on the wound dressing material and exhibiting notable growth inhibition potential against tested pathogenic strains. The release profile study revealed an initial burst release of phytochemical constituents followed by a controlled or sustained pattern. In conclusion, wound dressings infused with plant extracts present a promising approach for wound care applications and show potential for various healing-related purpose.

Population pharmacokinetic analysis of quizartinib in patients with newly diagnosed FLT3-internal-tandem-duplication-positive acute myeloid leukemia.

The population pharmacokinetics (PK) of quizartinib and its pharmacologically active metabolite AC886 have been previously described in healthy volunteers (HV) and relapsed/refractory (R/R) FLT3-internal-tandem-duplication-positive (FLT3-IDT-positive) acute myeloid leukemia (AML) patients receiving quizartinib monotherapy. In this analysis, we characterized the population PK of quizartinib and AC886 in newly diagnosed FLT3-ITD-positive AML patients receiving standard induction and consolidation chemotherapy as background treatment, using data from the Phase 3 QuANTUM-First trial and 12 earlier studies. Quizartinib PK were best described by a three-compartment model with sequential zero- and first-order absorption and first-order elimination. A two-compartment model with first-order metabolite formation and first-order elimination best fitted AC886 data. The PK of both moieties showed large interindividual variability (approximately 70% coefficient of variation for systemic clearances). The use of strong cytochrome P450 3A (CYP3A) inhibitors had the largest impact on exposure, increasing the steady-state area under the curve during the dosing interval (AUCss) by 1.8-fold. This is consistent with observations in HV and R/R AML patients and confirms the need for dose adjustments during coadministration. A novel finding in newly diagnosed AML patients was the phase-dependent change in steady-state quizartinib exposure: dose-normalized AUCss values were 0.6-fold during induction, similar during consolidation, and 1.4-fold during continuation compared to R/R AML patients receiving quizartinib monotherapy. The present analysis highlighted the comparison of quizartinib and AC886 PK between newly diagnosed AML patients and previously studied populations, informed dose modifications needed with strong CYP3A inhibitors, and supported the use of derived individual exposure metrics in separate exposure-response analyses.

Synchronization of extraction and chromatographic conditions of diverse physicochemical behavior of pseudoephedrine, loratadine and desloratadine for their simultaneous determination in human plasma: Application to pharmacokinetic study

A selective, sensitive, and rapid LC-MS/MS method has been developed, optimized, and validated for the simultaneous determination of pseudoephedrine (PSE), loratadine (LOR), and LOR active metabolite, desloratadine (DES) in K3EDTA human plasma. A simple liquid–liquid extraction method was adopted for extraction of the 3 analytes and their internal standards; chlorpheniramine (CLP), loratadine D4 (LOR-D4), and desloratadine D4 (DES-D4), respectively, giving consistent recovery results (78.38––88.23 %) using diethyl ether: dichloromethane (80:20, v/v) as the extraction solvent. The chromatographic separation was challenging due to the polarity differences between PES, which is more polar, and LOR and DES, which are less polar that affected their physicochemical properties including the retention of the drugs on C18 column. The separation was achieved using of isocratic elution of 20 mM ammonium formate and 2 mM trifluoracetic acid ammonium salt: acetonitrile containing 0.15 % formic acid (12:88, v/v) as a mobile phase pumped onto a Supelco Discovery® C18 (4.6 mm x 100 mm, 5 µm) at 35 °C with a flow rate of 0.8 mL/min. The use of trifluoracetic acid ammonium salt as an ion pair reagent in the selected mobile phase allowed good retention of the polar PSE on the used C18 column with a reasonable retention time. Positive Electrospray ionization was employed followed by detection using a tandem mass spectrometer in multiple reactions monitoring (MRM) mode. The sample analysis of this study was conducted in compliance with ICH guideline M10 on bioanalytical method validation and study sample analysis. The method was linear over the concentration range of (4–1200) ng/mL for PSE, (50–15000) pg/mL for both LOR, and DES. The developed method was accurate (91.14–110.30 %), precise (0.53–7.86 %), and was applied to study the pharmacokinetic profile of the cited drugs in the plasma of healthy human volunteers after single oral administration of the fixed-dose combination Claritin D® tablet, (240/10 mg) of PSE, and LOR, respectively.

Brain distribution and muscarinic receptor occupancy of an active metabolite of fesoterodine in rats

Brain distribution and muscarinic receptor occupancy of an active metabolite of fesoterodine in rats