In vitro and in vivo metabolic profiling of PD105, a PI3Kδ inhibitor, using UHPLC-Q-Exactive plus-MS.

PD105, a PI3Kδ inhibitor, is a candidate for the treatment of rheumatoid arthritis. This study aims to identify the metabolic profiling in vitro and in vivo by UHPLC-Q-Exactive Plus-MS.The in vitro metabolism of PD105 was studied by mouse liver microsomes and hepatocytes, while the in vivo metabolic profiling was obtained from mouse plasma, urine, and faeces. A total of 20 metabolites were tentatively identified based on accurate mass, fragment pathways, and characteristic fragment ions, including 4 in vitro and 20 in vivo.The proposed metabolic pathways of PD105 showed that there were 18 phase I metabolites and 2 phase II metabolites. The phase I metabolic pathways included oxidation, hydration, desaturation and oxidative dechlorination, while the phase II metabolic reactions were mainly methylation and arginine conjugation. Among them, oxidation was the main metabolic pathway of PD105.The comprehensive metabolic profiling contributed to further elucidation of pharmacological action and mechanism of PD105.

Metabolic profiling in liver microsomes and mice of E28, a potent FLT3 inhibitor.

The objective of this study was to clarify the species differences of metabolic stability of E28 in liver microsomes, and to study metabolic phenotypes of E28 in human liver microsomes by chemical inhibition method.The metabolites in plasma, urine, and faeces samples from mice received caudal vein intravenous were detected and identified by UHPLC-HRMS, and the tissue distribution was studied after oral administration.E28 was metabolised rapidly in liver microsomes of each species with a short half-live T1/2 and a moderate clearance, except for rats. The metabolic properties of E28 were similar in human and mouse liver microsomes. Data from metabolic phenotype studies indicated that CYP2D6, CYP3A4 and CYP2C9 were the main metabolic enzymes participating in the metabolism of E28.The main metabolic pathways implicated include oxidation, methylation, amide hydrolysis, acetylation, glucuronide conjugation.Tissue distribution studies showed that E28 could be detected in all organs and tissues after oral administration, with the highest level in the stomach and the lowest in the brain. In bone marrow cells, the concentration of E28 in all sample points were consistently higher than its half inhibitory concentration against MV4-11 tumour cells.

Ethanol extract of Piper wallichii ameliorates DSS-induced ulcerative colitis in mice: Involvement of TLR4/NF-κB/COX-2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper wallichii (family: Piperaceae), a folk herbal medicine with anti-inflammatory and anti-thrombotic properties, has been traditionally used to treat rheumatic arthralgia, lumbocrural pain, gastrointestinal flatulence, and other intestinal diseases in China, Thailand, and India. However, there is no scientific report on the efficacy and potential mechanisms of Piper wallichii for ulcerative colitis (UC). AIM OF THE STUDY: The study aims to investigate the therapeutic effect and possible molecular mechanisms of the ethanol extract of Piper wallichii (EEPW) on DSS-induced UC in BALB/c mice. MATERIALS AND METHODS: The main components in EEPW were characterized by UPLC-QE-Orbitrap-MS. Subsequently, the anti-inflammatory effect of EEPW in vitro was preliminarily evaluated in RAW264.7 cells stimulated with LPS. UC model mice were triggered by free access to 4% DSS aqueous solution for 12 consecutive days, and simultaneously, EEPW (25, 50, and 100 mg/kg) and tofacitinib (positive control, 30 mg/kg) were orally administrated, respectively. The therapeutic efficacy of EEPW on UC was assessed by body weight, DAI, colon length, and pathological morphology. Besides, we investigated the effects of EEPW on intestinal barrier function, inflammatory factors, and immune systems of UC mice through immunohistochemistry (IHC), flow cytometry, and other techniques. Moreover, the expression of related proteins in the TLR4/NF-κB/COX-2 pathway was analyzed by Western blot. RESULTS: A total of 14 components were identified in the positive and negative modes, including isofutoquinol A (11), hancinone C (12), and futoquinol (14) which characterized by references. In the RAW264.7 cells experiments, the extract significantly suppressed the levels of TNF-α and IL-6. More importantly, EEPW distinctly improved the symptoms of DSS-induced UC mice as reflected by a significant recovery from body weight, colon length, pathological injuries of the colon, and so on. Further research found that EEPW remarkably restored the levels of occludin, promoted proliferation, and inhibited apoptosis in colon to maintain the integrity of intestinal barrier. In addition, the down-regulation of TNF-α and IL-1ß in colon, Th1 and Th17 cells in spleen, as well as the up-regulation of IL-10 in colon and Th2 cells in spleen were distinctly observed in EEPW-treated groups. Furthermore, the protein expression of TLR4, p-IκB-α, p-p65, and COX-2 were significantly inhibited by EEPW. CONCLUSIONS: This study confirmed for the first time that EEPW effectively ameliorated DSS-induced UC in mice, which might be related to improving intestinal barrier function, maintaining the levels of inflammatory factors, and regulating the immune system. In addition, we found that the anti-inflammatory effect of EEPW on UC mice was involved in the TLR4/NF-κB/COX-2 signaling pathway. In conclusion, Piper wallichii can be used as a candidate for the treatment of UC.

Piper nigrum Extract Inhibits the Growth of Human Colorectal Cancer HT-29 Cells by Inducing p53-Mediated Apoptosis.

Colorectal cancer (CRC) is a prevalent malignancy of the digestive tract with the second highest mortality rate globally. Piper nigrum is a widely used traditional medicinal plant, exhibiting antitumor activity against various tumor cells. At present, research on the effect of Piper nigrum on CRC is limited to in vitro cytotoxicity, lacking comprehensive mechanism investigations. This study aimed to explore the inhibitory effect and mechanism of Piper nigrum extract (PNE) on HT-29 cells. Firstly, we identified the chemical components of PNE. Then, MTT assay, colony formation assay, JC-1 staining, and flow cytometry were used to analyze the effect of PNE on HT-29 cells in vitro. A xenograft model, histopathological examination, immunohistochemistry, and western blot were used to evaluate the tumor growth inhibitory activity and mechanism of PNE in vivo. The results indicated that PNE could inhibit cell proliferation and colony formation, reduce mitochondrial membrane potential, induce cell apoptosis in vitro, and inhibit tumor growth in vivo. Furthermore, PNE could regulate p53 and its downstream proteins, and subsequently activate the caspase-3 pathway. In summary, PNE probably induced apoptosis of HT-29 cells through the mitochondrial pathway mediated by p53. All these results suggested that PNE might be a potential natural-origin anti-CRC drug candidate.

UPLC-MS/MS method development and application to pharmacokinetic study in rats and dogs of Flonoltinib Maleat.

Flonoltinib Maleate (FM) is a novel selective inhibitor of Janus kinase 2/FMS-like tyrosine kinase 3 (JAK2/FLT3). In this study, we developed an ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to measure the plasma concentrations of FM in rats and dogs for pharmacokinetic studies. For chromatographic separation, we used a BEH C18 column (2.1 × 50 mm, 1.7 µm particle size) in HPLC. The mobile phase A consisted of a water solution containing 0.1% formic acid (FA) and 2 mM NH4OAc, mixed with acetonitrile (ACN) (V:V = 95:5). The mobile phase B was a water solution containing 0.1% FA and 2 mM NH4OAc, mixed with ACN (V:V = 5:95), which was used for gradient elution. We used multiple reactive ion detection (MRM) mode and electrospray ionization positive (ESI+) mode for quantitative analysis. The standard curve was linear in the concentration range of 0.5 to 500 ng/ml in rat and dog plasma. The intra-batch and inter-batch precision (RSD%) of FM in rat and dog plasma was less than 15%. The intra-batch and inter-batch accuracy was 88.3-106.5% and 92.0-100.6% in rats, and 94.7-106.6% and 95.3-103.8% in dogs, respectively. The RSD (%) of matrix factors (MF) normalized to the internal standard (IS) of FM in rat and dog plasma was ≤5.6% and ≤3.0%, respectively. The extraction recovery and carryover were considered acceptable. When the sample concentration was higher than the upper limit of quantitation (ULOQ), the 10-fold dilution was reliable within the limits of acceptability. The UPLC-MS/MS method developed in this study was successfully applied in measuring the pharmacokinetic parameters of FM in rats and dogs after intravenous and oral administration, laying a foundation for the preclinical pharmacokinetic study of FM and providing a reference for clinical pharmacokinetic studies.

Chemical analysis of Chrysosplenium from different species by UPLC-Q exactive orbitrap HRMS and HPLC-DAD.

Chrysosplenium is the main component of a variety of Tibetan prescription preparations. Nevertheless, there are few chemical reports for different species of Chrysosplenium, which should be further explored. To this end, ultra-performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry (UPLC-Q Exactive Orbitrap HRMS) and high-performance liquid chromatography-diode array detection (HPLC-DAD) were first integrated to qualitatively analyse the chemical characteristics of Chrysosplenium nudicaule, Chrysosplenium carnosum, Chrysosplenium sikangense, Chrysosplenium griffithii, Chrysosplenium absconditicapsulum, Chrysosplenium forrestii and Chrysosplenium axillare. As a result, a total of 40 compounds were identified or tentatively identified from these 7 species of Chrysosplenium, including 21 flavonoids, 3 triterpenoids and a variety of alkaloids, organic acids and anthraquinones, etc. Among them, 6 compounds were detected for the first time, and 8 compounds are common components in all 7 species of Chrysosplenium. In the specific chromatogram, 4 characteristic peaks, namely Riboflavin, 5,4'-dihydroxy-3,6,3'-trimethoxyflavin-7-O-ß-D-glucoside, 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone and Chrysosplenetin, were selected to evaluate the similarities of 17 batches of Chrysosplenium samples, which ranged from 0.770 to 0.994. The established method is simple, feasible and accurate, and was proven to be suitable for characterizing the chemical compositions of Chrysosplenium from different species and evaluating their similarities by specific chromatogram analysis to clarify the rationality of using Chrysosplenium from different species in clinical medication, which provides experimental data for further quality evaluation of Chrysosplenium.

A review of the genus Chrysosplenium as a traditional Tibetan medicine and its preparations.

ETHNOPHARMACOLOGICAL RELEVANCE: Plants of genus Chrysosplenium have a long history of application and are distributed in many countries, especially in Tibetan regions of China. The genus has been used locally in the treatment of various hepatobiliary diseases such as "Chiba disease" (related to cholecystitis, cholelithiasis, acute icteric hepatitis, and acute liver necrosis in modern medicine). AIM OF THE REVIEW: This review summarizes and critically analyzes the aspects of the botanical morphology and distribution, traditional uses, phytochemistry, pharmacological activities, quality control, and development status of preparations of the genus Chrysosplenium. Moreover, the future research direction and focus of the genus are also discussed. We hope to provide a valuable reference for researchers who are interested in the genus Chrysosplenium. MATERIALS AND METHODS: The relevant information of the genus Chrysosplenium was gathered through electronic databases from 1968 to 2021, including PubMed, Web of Science, ScienceDirect, Google Scholar, Springer, CNKI, and Wan Fang, as well as PhD, MSc thesis, Chinese Pharmacopoeia (2020 edition), Tibetan medicine monographs. In addition, plant names were verified by "The Plant List" (The Plant List Database, http://www.theplantlist.org). RESULTS: Based on existing studies of chemical compositions, more than 90 compounds have been identified from Chrysosplenium species, including flavonoids, triterpenoids, volatile oils, steroids, alkaloids, and other compounds. The highly hydroxylated and methoxylated flavonoids and triterpenoids are the main active components. In addition, many studies have shown that the extracts and some components isolated from the genus Chrysosplenium have a variety of pharmacological activities, such as anti-tumor, antibacterial, anti-viral, hepatoprotective, and insecticidal properties. Furthermore, there are only 9 preparations with Chrysosplenium species as one of the medicinal materials. Among these preparations, C. nudicaule is used more and other Chrysosplenium species are rarely involved. CONCLUSIONS: Most medicinal species of Chrysosplenium have not only good therapeutic effects in traditional uses, but also a great potential for development in modern pharmaceutical studies. However, the material basis and mechanism of action of this genus have not been well explained. Therefore, further systematic and comprehensive research on the genus Chrysosplenium is still required to provide a scientific basis for its clinical applications.