Osteoprotegerin(OPG), secreted by osteoblasts, is a marker of bone turnover. OPG can inhibit osteoclastic differentiation by binding receptor activator of nuclear factor-κB ligand(RANKL). In this study, we found that rutaecarpine(RUT) had the up-regulating OPG activity, and it could significantly increase OPG protein levels in both mouse embryonic osteogenic precursor MC3 T3-E1 and human osteosarcoma U-2OS cells. Osteoblastogenic differentiation calcified nodules staining results showed that RUT significantly promoted the osteogenic differentiation of MC3 T3-E1 cells. Osteoclastic differentiation tartrate resistant acid phosphatase(TRAP) staining results showed that RUT obviously inhibited the osteoclast differentiation of mouse macrophages RAW264.7 induced by RANKL. In vivo studies showed that low-dose RUT group(5 mg·kg^-1·day^-1) and high-dose RUT group(45 mg·kg^-1·day^-1) treatments for 3 months significantly increased bone density in ovariectomized(OVX) rats; calcein double labeling experiment and toluidine blue staining results indicated that low-dose RUT group promoted bone formation and decreased bone loss in vivo; immunohistochemistry results showed that low-dose RUT group increased the expression of OPG in rat femur. All animal procedures were performed in accordance with the regulations of the Institutional Animal Care and Use Committee of Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. In summary, this study demonstrated that RUT could up-regulate OPG expression and had promoting osteoblastic differentiation and inhibiting osteoclastic differentiation effects in vitro and in vivo.

We investigated the inhibitory effect and mechanism of action of bruceantin (BCT) on the proliferation, invasion and migration of non-small cell lung cancer (NSCLC) cells. The cytotoxic activity of BCT was measured by MTT assay; a colony forming assay, wound healing assay, and a Transwell assay were used to investigate the anti-proliferative, anti-migration, and anti-invasion effects, respectively; immunoblotting and RT-qPCR were used to detect the expression of related proteins, miRNA, and mRNA, respectively, that were involved in cell proliferation, migration, and invasion. Two gene prediction websites were used to predict the downstream target gene of miRNA. Our results show that BCT has a potent cytotoxic effect on NSCLC cell lines, with a half maximal inhibitory concentration (IC₅₀) of BCT against H1299, PC-9, and A549 of 0.12 ± 0.02, 0.31 ± 0.20, and 2.07 ± 0.70 μmol·L^-1, respectively. When H1299 cells were treated with 0.03, 0.15, and 0.75 μmol·L^-1 BCT for 24 h, the proliferation, migration, and invasive ability were inhibited in a concentration-dependent manner. It is worth noting that the expression level of miRNAs related to cell migration and invasion, such as miR-29a-3p, miR-21-3p, miR-183-5p, and miR-34b-5p increased with the concentration of BCT, especially for miR-29a-3p. Using the two gene prediction websites, we predict that integrin β1 (ITGB1) may be the target gene of miR-29a-3p; immunoblot results further show that a variety of proteins related to cell proliferation, migration, and invasion, such as various proteins of the integrin family, β-catenin, p-Src, and vascular endothelial growth factor, all decreased in a concentration-dependent manner, among which the reduction of ITGB1 protein was the most obvious. RT-qPCR results showed that there was no change in ITGB1 mRNA expression. We speculate that BCT might inhibit the expression of ITGB1 protein by up-regulating miR-29a-3p independent of its mRNA level. The in-depth mechanism needs to be further explored. This study suggests that BCT has the potential for further development in the treatment of NSCLC.

The antidepressant effect of Xiaoyaosan has been demonstrated. It is of value to explore the biological mechanism of Xiaoyaosan in the treatment of depression from the perspective of functional modules by using the method of functional module division of the metabolic network. The differential metabolites and related enzymes and proteins regulated by Xiaoyaosan were identified in the database. Pathway enrichment analysis and crosstalk pathway analysis of Xiaoyaosan regulated metabolites was carried out. A network of differentially regulated metabolites and their enzymes and proteins was constructed by using the STRING tool. The CNM decomposition algorithm was used to extract the functional modules of the network and enrichment analysis of functional modules was carried out. The results show that Xiaoyaosan regulates 97 differential metabolites, 234 related enzymes and258 depression-related proteins. The pathways crosstalk analysis was divided into two sub-networks, one of which is related to the neural system and cell signal transduction, the other is related to the endocrine system and metabolic pathways. KEGG pathway enrichment analysis of the network and 9 functional modules extracted by the CNM algorithm shows that module 1 and module 3 belong to the pathways that can be enriched into more pathways with fewer proteins. The corresponding functions of these pathways include the endocrine system, amino acid metabolism, the nervous system and signal transduction. In this study, pathway crosstalk analysis and metabolic network module division strategies were used to explain the biological mechanism of Xiaoyaosan in the treatment of depression, providing ideas and methods for in-depth study of the pharmacological mechanism of this traditional Chinese medicine from the perspective of metabolic regulation.

M701 is a bispecific CD3/EpCAM T-cell engager antibody for the treatment of malignant ascites. We developed a population pharmacokinetic/pharmacodynamic (PK/PD) model to quantitatively describe and predict the antitumor effect of M701 in human colorectal cancer xenograft mice. We developed the M701 PK model based on plasma concentration data after i.v. administration. A tumor growth model for human colorectal cancer xenograft was developed to evaluate the antitumor effect of M701. We additionally simulated the inhibitory effect of M701 on tumor volume under different dose regimens based on a PK/PD model. A two-compartment model was developed to predict the PK in human colorectal cancer xenograft mice. The relationship between the M701 concentration and tumor growth inhibition was characterized by a combined Simeoni tumor growth/transit compartment model. The estimated pharmacodynamic parameters were related to the tumor growth characteristics λ₀ (0.212 d^-1) and λ₁ (0.044 7 cm³·d^-1), to the drug potency k₂ (0.071 5 mL·ng^-1·d^-1), and to the kinetics of tumor cell death k₁ (2×10^-5 d^-1). A model visual predictive check showed that both the PK model and the tumor growth model closely fit the observed data. Simulated tumor growth after administration of M701 (0.5 mg·kg^-1 every 6 days and 0.25 mg·kg^-1 every 3 days) could be effectively inhibited. This population PK/PD model of M701 provides insight into the antitumor effect of M701 and supports the further therapeutic development of M701.

The purpose of this study was to discover novel inhibitors of sirtuin-1(SIRT1) that could be used in the treatment of acute myeloid leukemia(AML).Eight potential SIRT1 inhibitors were identified from 231 511 natural drug-like molecules by virtual screening-based molecular docking and molecular mechanics-generalized Born surface area(MM-GBSA) calculation of binding free energies.Using existing SIRT1 inhibitor molecules as training and test sets, a series of quantitative structure-activity relationship models were established, and the best quantitative structure-activity relationship(QSAR) model was used to predict the IC₅₀ of these 8 potential inhibitor molecules for SIRT1.Subsequently, molecular dynamics simulations were performed to verify the binding mode and stability of these complexes of potential inhibitors and SIRT1 protein.Finally, the activity of these potential SIRT1 inhibitors was verified by cell proliferation assays of OCI-AML2, OCI-AML3 and MV4-11 cells and SIRT1 enzyme activity assays, and it was found that 5 compounds could inhibit AML cell proliferation.Among them, the most active compound, ZINC000001774455, had an IC₅₀ of 2.29 ± 0.09 μmol·L^-1 with OCI-AML2 cells, and at a concentration of 1 μmol·L^-1, the inhibitory ratio of this compound on SIRT1 protein activity was 65.33%.ZINC000001774455 can be used as a lead compound for the development of new AML treatments.

(±)-Bicoryanhunine B(1), a new dimeric benzylisoquinoline alkaloid was isolated from the dried tubers of Corydalis yanhusuo by various chromatographic methods, including silica gel, Sephadex LH-20, reverse phase C18, and semi-preparative HPLC.Its structure was determined by spectroscopic methods, including UV, IR, ESI-MS, HR-ESI-MS and 1D/2D NMR.(±)-Bicoryanhunine B(1) was a moderate PD-1/PD-L1 interaction inhibitor with an IC₅₀ value of 7.80±0.49 μmol·L^-1.In addition, 1 exhibited potent inhibitory activities against LPS-induced NO production in RAW 264.7 macrophages with an IC₅₀ value of 4.83 ± 2.21 μmol·L^-1.

A quantitative analytical method based on HPLC coupled with the charged aerosol detector (CAD) for quantitative analysis of multi-components with a single marker (QAMS) was established for simultaneous determinations of astragaloside Ⅰ, astragaloside Ⅱ, astragaloside Ⅳ, calycosin-7-O-β-D-glucoside, formononetin and 7, 2'-dihydroxy-3', 4'-dimethoxyisoflavan in Astragalus membranaceus. The separation was performed on an Agilent SB-C18 (150 mm×4.6 mm, 3.5 μm), with gradient elution using the mobile phase consisting of 0.05% formic acid solution and 0.05% formic acid acetonitrile at the flow rate of 1.0 mL·min^-1. The column temperature was 35 ℃, and the injection volume was 20 μL. For CAD, the drift tube temperature was at 50 ℃. The contents of six components in A. membranaceus were determined by both external standard method (ESM) and QAMS, and then were compared. The results showed that chromatographic peaks were separated well and the linear ranges of astragaloside Ⅰ, astragaloside Ⅱ, astragaloside Ⅳ, calycosin-7-glucoside, formononetin and 7, 2'-dihydroxy-3', 4'-dimethoxyisoflavan were 0.113-2.250 mg·mL^-1, 0.012-0.240 mg·mL^-1, 0.004-0.080 mg·mL^-1, 0.065-1.300 mg·mL^-1, 0.005-0.100 mg·mL^-1 and 0.007-0.150 mg·mL^-1, respectively. The content ranges of astragaloside Ⅰ, astragaloside Ⅱ, astragaloside Ⅳ, calycosin-7-glucoside, formononetin and 7, 2'-dihydroxy-3', 4'-dimethoxyisoflavan were 0.306-0.922 mg·g^-1, 0.053-0.183 mg·g^-1, 0.015-0.092 mg·g^-1, 0.069-0.823 mg·g^-1, 0-0.098 mg·g^-1 and 0.020-0.107 mg·g^-1 in 20 batches of A. membranaceus, respectively. Using astragaloside Ⅱ as an internal reference, the relative correlation factors of astragaloside Ⅰ, astragaloside Ⅳ, calycosin-7-O-β-D-glucoside, formononetin, and 7, 2'-dihydroxy-3', 4'-dimethoxyisoflavan were calculated as 0.561, 0.835, 0.299, 0.796, and 0.799, respectively. The results were compared with those obtained by the external standard method to verify the feasibility, rationality and repeatability of QAMS method, and there was no significant difference in assay results between the two methods. In conclusion, the QAMS method is accurate and feasible, and could be used to determine the contents such as astragaloside Ⅰ, astragaloside Ⅱ, astragaloside Ⅳ, calycosin-7-glucoside, formononetin and 7, 2'-dihydroxy-3', 4'-dimethoxyisoflavan, and it can be used for quality control of A. membranaceus.

The bioactivity of a working reference standard was determined by replicate bioassays with calibration against a primary reference standard. In this study the number of bioassay replicates needed for calibration first was calculated theoretically, and if the mean value of the experimental bioassay replicates fell within the predefined bioactivity level the bioactivity of the working reference was defined as 100%. Our results showed that when the total intermediate precision of the bioassay method was at 11.66% and the predefined bioactivity level was set at 95%-105% with a confidence level of 95%, 21 bioassay replicates should be carried out for calibration.The average value of the 22 experimental bioassay replicates was 101.96%, so the bioactivity of the working reference standard was consistent with that of the primary reference standard at 100%. The results suggest that a strategy of first calculating the number of bioassay replicates needed for calibration and then determining whether the resulting experimental mean value is within the predefined bioactivity level will be of value to the biopharmaceutical industry.

Four salts of ticagrelor, ticagrelor-3, 5-dinitrobenzoic acid, ticagrelor-pyrazinamide, ticagrelor-Dproline and ticagrelor-L-proline were prepared by solvent suspension and liquid-assisted grinding to improve the solubility of ticagrelor. The compounds were characterized by powder X-ray diffraction, Fourier transform infrared spectroscopy, differential scanning calorimetry, nuclear magnetic resonance spectroscopy, elemental analysis, and the intermolecular salt-bonding forces were analyzed. The equilibrium solubility of salts and pure drug in hydrochloride buffer pH 1.2 and phosphate buffer pH 6.8 were measured by high-performance liquid chromatography.Ticagrelor was salted with 3, 5-dinitrobenzoic acid, pyrazinamide, D-proline, L-proline all in a stoichiometric ratio of 1∶1; with the exception of ticagrelor-D-proline, the solubility of the other three salts provided significantly improved solubility in hydrochloride buffer pH 1.2, and the equilibrium solubility of ticagrelor-3, 5-dinitrobenzoic acid was increased by approximately 1.7 folds as compared to pure drug. Salt-forming technology is convenient and can improve the solubility of ticagrelor.

Sacubitril valsartan sodium(LCZ696) is an ionic cocrystal drug. The purpose of this study was to explore the cocrystal features of LC696 by establishing a variety of characterization methods, and thus provide basic research data for effective quality control. The cocrystal characteristics of LCZ696 and its tablets were identified by applying analytical means including powder X-ray diffraction(PXRD), fourier transform infrared spectroscopy(FTIR), Raman spectra(RM), differential scanning calorimetry(DSC) and solid-state nuclear magnetic resonance spectroscopy(ssNMR). The crystalline water and hygroscopicity of LCZ696 were analyzed by thermogravimetric analysis(TGA), dynamic vapor sorption(DVS), hygroscopicity test and Karl Fischer reaction method.The results show that PXRD, FTIR, DSC and ssNMR can effectively distinguish the features of LCZ696 cocrystal, sacubitril monomer, valsartan monomer, and sacubitril-valsartan(1∶1) mixture. RM can be used as a supplementary approach. Combined with the analysis by TGA, DVS, hygroscopicity test and Karl Fischer reaction method results, LCZ696 contains 2.5 crystalline water molecules and is very hygroscopic; we recommend that LCZ696 be stored in an environment with a relative humidity below 60%. By characterizing the crystal features we can establish quality control measure and evaluate the stability of the drug tablets. This study provides data in support for the establishment of the LCZ696 quality standard.