Neurodegenerative diseases (ND) mainly include Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, ataxia, and other diseases. The number of patients with ND is increasing, but the proportion of patients who can be diagnosed and treated early is less than 30% and the cause of ND is still unclear. In order to intervene in the disease as early as possible, researchers are committed to finding biomarkers that facilitate the early diagnosis of ND. Among them, cerebrospinal fluid (CSF) closely reflects the composition of the extracellular space of the brain, and may be the most sensitive biomarker for evaluating ND. However, the method of taking cerebrospinal fluid is more complicated, and it is not a common method in primary care or elderly medical institutions for the treatment of ND patients. Imaging examinations are expensive and difficult to spread among the community. The peripheral blood collection is convenient and less traumatic, which is a potential early screening and follow-up method. There are many components in the blood for analysis and research. This article reviews the research progress of the changes of apolipoprotein in the blood of ND patients as markers.

Chemokines are small cytokines with chemotactic activity, they are involved in regulating immune responses and inflammatory responses. In the development of tumors, chemokines are multi-functional mediators that not only affect the infiltration of immune cells into the tumor, but also have an important impact on tumor growth, angiogenesis, invasion, and metastasis. Besides, they are important targets of tumor therapy. Here we review chemokines involved in the regulation of signaling pathways, analyze the mechanism of chemokines in the development of breast cancer, summarize the chemokines targeted drugs for breast cancer in recent years and make a prospect about the role of chemokines in anti-breast cancer therapy.

Torpor refers to a state in which the metabolic activity in the body of the living animal is greatly reduced during the period of reduced food supply, which is manifested as a substantial decrease in body temperature, metabolic level, and exercise level. Mammals have a strict body temperature regulation system to maintain a constant body temperature. When the energy supply is insufficient for a long time, some mammals will enter a hibernation state. Torpor is very similar to the hibernation state. The research on the mechanism of torpor state is of great significance in aerospace, military medicine and other fields. This review summarizes the specific mechanisms regulating the occurrence of torpor from four aspects: adenylate cyclase activating polypeptide (adcyap) neurons, leptin, pyroglutamylated RFamide peptide (QRFP) neurons, and sympathetic nervous system, aiming to provide ideas for further research on the mechanism of torpor.

Flavonoids baicalin is the main bioactive component extracted from Scutellaria baicalensis Georgi. Baicalin has high medicinal value and shows extensive pharmacological effects including antitumor, antibiosis, anti-inflammatory, antioxidation, neuro-protection, and significant potential in tumor treatment. Recent studies have shown that baicalin suppresses the growth of many kinds of human cancer. The underlying mechanisms include induction of apoptosis, induction of cell cycle arrest, inhibition of tumor metastasis, suppression of angiogenesis, and so on. This article reviewed the research progress of baicalin on its antitumor pharmacology and possible mechanisms at home and abroad, and provided the basis for its further research.

Idiosyncratic drug-induced liver injury (IDILI) is an unpredictable serious adverse drug reaction, which only occurs in a minority of special susceptible individuals. Although the mechanism of IDILI has not been fully understood, several hypotheses have been proposed to explain the action mode and specific mechanism of IDILI. Of these hypotheses, inflammatory stress hypothesis is one of the most important theories. Under the condition of inflammatory stress, drugs interact with inflammation and mediate the occurrence of IDILI through a variety of mechanisms, which can induce the production of inflammatory cytokines, activate coagulation system, affect the activity of metabolites, induce cholestasis, affect mitochondrial damage, and others. This review will summarize the main mechanisms and influencing factors of IDILI mediated by inflammatory stress, in order to provide a reference for preclinical drug development and basic research on drug-induced liver injury.

Traditional antitumor drugs are cytotoxic chemotherapeutic drugs that can directly kill tumor cells and inhibit the growth and proliferation of tumor cells. Modern chemotherapy for tumors was initiated by use of nitrogen mustard to treat lymphomas in 1946, which was derived from mustard gas. Starting with nitrogen mustard, many kinds of anti-tumor drugs, including alkylating agents, anti-metabolism drugs, anti-tumor antibiotics, and anti-tumor plant drugs, have been successively developed for clinical treatment. Traditional antitumor drugs are the cornerstone of tumor chemotherapy and play important roles in the comprehensive treatment and neoadjuvant therapy of malignant tumors. In recent years, the combination of traditional antitumor drugs with molecular targeted therapy, immunotherapy, and radiotherapy has greatly improved the survival rate of tumor patients. With the deepening understanding of tumor genome as well as tumor initiation and promotion, the concepts of precision medicine and individualized treatment have been proposed and achieved success in clinical practice. In this context, the strategies leading to personalized therapy with traditional anti-tumor drugs also need to be further studied and optimized. This review summarized the recent clinical application and research progress of traditional antitumor drugs.

The RAS (rat sarcoma) gene is one of the important oncogenes, and its mutation is present in about 30% human tumors. KRAS (kirsten rat sarcoma viral oncogene) is one of the three RAS subtypes, and KRAS mutations are more common than the mutations in other two RAS subtypes. In recent years, with the continuous research, new ideas have been provided for the treatment of cancers via targeting-KRAS. Efforts have been made to develop various KRAS inhibitors. Here, based on the mechanism of action, we classified KRAS inhibitors into two categories: inhibitors that directly target KRAS and inhibitors that indirectly act on KRAS. The representative KRAS inhibitors were summarized and introduced in this paper.

Local focal adhesion kinase (FAK) is a non-receptor intracellular tyrosine kinase that plays an important role in tumor initiation, development, metastasis and invasion, and is considered to be an important target for the development of antineoplastic drugs. It has both kinase-dependent and non-kinase-dependent scaffolding functions. However, traditional small molecular inhibitors can only inhibit its kinase-dependent activity, so it is difficult to target the kinase-independent scaffolding function. Therefore, there is an urgent need for novel strategies to enhance FAK targeting to lay the foundation for determining the druggability and discovery of FAK inhibitors. Proteolysis targeting chimera (PROTAC) is a new drug development strategy that can recruit E3 ligase to specifically ubiquitinylate target proteins for degradation through the proteasome system. The unique mechanism of action of the PROTAC system could be used to target and degrade the FAK protein, thus eliminating the scaffolding function of FAK. In this review, FAK protein, the signaling pathway, and small molecule inhibitors are briefly described, and the latest research progress in targeting the degradation of FAK using PROTAC technology is summarized.

The tumor microenvironment (TME), a dynamic and complex local environment, interacts with the tumor cells and is closely related to tumor growth, metastasis, immune escape and drug resistance. Thus, targeting the TME has been a worldwide focus in cancer therapy. Many natural products possess the advantages of multiple targets, multiple pathways and wide pharmacological functions, and are the main source of antitumor drugs. In recent years studies have found that some natural products had advantageous effects on the TME. In this review, we summarize the components and functions of the TME and some natural products that target the TME, with references to the drug therapy of cancer.

The efficient and safe delivery of drugs to the therapeutic site through the biofilm has traditionally been a difficult and hot topic in the field of drug delivery. In recent years, alkyl polyglycoside (APG) have become ideal penetration enhancers for drug delivery systems because of their high permeability, good safety and biodegradability, which has attracted wide attention of domestic and foreign researchers. In this paper, the physical and chemical properties, characteristics, action mechanism and application of APG in drug delivery system are reviewed, and its application prospect in drug delivery system is prospected.

In this study, the regulatory effects of chlorogenic acid (CGA) on the expression of programmed cell death ligand 1 (PD-L1) in esophageal squamous cell carcinoma (ESCC), as well as the role of interferon γ (IFN-γ), has been discussed using both in vitro and in vivo animal models. ESCC murine model was established according to the standard operating procedures (SOP) of Animal Experiment Center of Institute of Materia Medica, Chinese Academy of Medical Sciences. The expression of PD-L1 in esophageal tissues of murine models was analyzed using the microarray assay. Then, the results were verified by qRT-PCR, Western blot and immunohistochemistry (IHC) staining, the molecular mechanism was explored in KYSE180 and KYSE510 ESCC cells in vitro. The results showed that CGA could suppress the expression of PD-L1 in tumor tissues in murine models significantly, rather than the expression in KYSE180 and KYSE510 ESCC cells in vitro. However, after the pretreatment of IFN-γ, the expression of PD-L1 was significantly increased, then it was down-regulated by CGA in both dose- and time-dependent manner. Meanwhile, the expression of interferon regulatory factor 1 (IRF1), an upstream regulatory factor of PD-L1, was suppressed by CGA in both KYSE180 and KYSE510 pretreated with IFN-γ, which was consistent with the expression of PD-L1. These results indicate that CGA down-regulates the expression of PD-L1 in ESCC via IFN-γ-IRF1 signaling pathway, providing the molecular theoretical basis for exploration of new treatment of ESCC.

Attenuated Salmonella VNP20009 specifically colonizes and proliferates within tumor tissues and inhibits tumor growth. It has been used as drug delivery vehicle or in combination with other therapies (such as chemotherapy), which shows a good application potential in tumor therapy. In this paper, study was conducted to determine the physiological changes of growth curve and formation of bacterial biofilm of VNP20009 under various environmental stresses, such as temperature, pH, and H₂O₂. The results showed that VNP20009 could grow normally under the conditions of 42 ℃, pH 6.5, and 1 mmol·L^-1 H₂O₂. Furthermore, the weak acid environment was beneficial to the biofilm formation of VNP20009. This study provides a basis for in-depth study of the survival mechanism and application of attenuated Salmonella.

Hyperglycemic kidney injury (HKI) is a common complication of diabetic patients. We examined the relationship between HKI and the abnormal expression of 5-hydroxytryptamine (5-HT) system induced by hyperglycemia in type 2 diabetes mellitus (T2DM). In animal experiments, a T2DM model was established in mice by feeding a high-fat diet with intraperitoneal injection of streptozotocin. The mice were treated with the 5-HT_2A receptor (5-HT_2AR) antagonist sarpogrelate hydrochloride (SH) and 5-HT synthesis inhibitor carbidopa (CDP) (respectively or in combination). In cell culture experiments, human glomerular mesangial cells (HMC) were stimulated with D-glucose (D-Glu), and 5-HT_2AR, 5-HT synthesis, and 5-HT degradation were inhibited by SH, CDP, or monoamine oxidase A (MAO-A) inhibitor clorgyline. Periodic acid-Schiff (PAS) staining and Masson staining, immunohistochemistry and Western blot, fluorescent probe, and enzyme linked immunosorbent assay (ELISA) and enzyme reagent were respectively used to detect histopathology, protein expression, intracellular reactive oxygen species (ROS), and biochemical indexes. The animal experiments were in accordance with the regulations of the Animal Ethics Committee of China Pharmaceutical University. The results showed that 5-HT_2AR, 5-HT synthases, and MAO-A were expressed in glomerular basement membrane and kidney tubular epithelial cells of mouse kidney and HMC. The expression of these proteins was significantly up-regulated in T2DM mice or when HMC cells were exposed to high concentration of D-Glu. HKI, characterized by abnormal renal function, glomerular swelling, and glomerular basement membrane thickening and fibrosis, is closely associated with an increase in kidney 5-HT_2AR, 5-HT synthesis, and 5-HT degradation. Among them, 5-HT_2AR can mediate the expression of 5-HT synthases and MAO-A; MAO-A can catalyze the degradation of 5-HT to increase the production of mitochondrial ROS, leading to the phosphorylation of nuclear factor kappa B (NF-κB) with the production of inflammatory cytokines, and the up-regulation of matrix metalloproteinase-2 (MMP-2) and α-smooth muscle actin (α-SMA) with the production of collagens. SH and CDP can effectively treat HKI, and the combination of SH and CDP has a clear synergistic effect.

Hyperuricemia is not only the biochemical basis of gout, but also closely related to the development of metabolic syndrome, cardiovascular diseases, chronic kidney disease, etc. Xanthine oxidase (XOD) is the key catalytic enzyme for uric acid biosynthesis, therefore the vital target for anti-hyperuricemic drugs. In this study, compound CC18022 was designed and synthesized specifically targeting to XOD. Molecular docking analysis indicated a fairly tight binding between CC18022 and XOD. In the in vitro study, CC18022 significantly inhibited XOD activity with a half maximal inhibitory concentration (IC₅₀) value in the order of nmol·L^-1, which is relative to the XOD inhibitor febuxostat. By using both acute and chronic hyperuricemic mice model, compound CC18022 was found to have serum uric acid-lowering effect in a dose-dependent manner in vivo. The animal welfare and experimental processes were in accordance with the provisions of the Animal Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences. In the acute hyperuricemic mice, CC18022 significantly inhibited serum XOD activity, and also the XOD activity in intestine and liver, which were related to purine absorption and metabolism. Therefore, the novel compound CC18022 exhibited significant inhibition on XOD activity and anti-hyperuricemic effects, making it a favorable candidate for further research.

Strict regulation of human immunodeficiency virus type 1 (HIV-1) protease function is critical for efficient production of mature viral particles. During viral protein expression and viral assembly, HIV-1 protease (PR) located within Gag-Pol precursor must be inactive to prevent premature cytoplasmic processing of the viral Gag and Gag-Pol precursors. Premature activation of HIV-1 precursors leads to major defects in viral assembly and production of viral particles. Specifically activating the protease in the precursor protein can directly inhibit the replication of the virus. In addition, HIV-1 PR is able to induce cell apoptosis. In this study, we identified 6 small molecule compounds using a cell-based assay for screening compounds that activate HIV-1 PR and induce premature of HIV-1 precursors. Results showed the active compounds are able to activate HIV-1 PR, inhibit HIV-1 replication, and induce cell apoptosis. This study provides ideas for the research and development of antiviral drugs.

This study was designed to explore the protective effect and underlying mechanism of catalpol on hepatocyte apoptosis in nonalcoholic fatty liver disease (NAFLD). High fat diet (HFD) was used to establish NAFLD model in the in vivo experiment, and the procedures of the experiments and animal care protocol were approved by the Animal Care and Use Committee of Jianghan University. Human liver cancer cell line HepG2 was treated with palmitate (PA) to establish a lipid toxicity model in the in vitro experiments. The results showed that catalpol significantly decreased the contents of serum total glyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), and aspartate transaminase (AST) in HFD-fed mice. Results of TUNEL staining and flow cytometry analyses revealed that catalpol significantly inhibited hepatocytes apoptosis in HFD-fed mice and PA-treated HepG2 cells. Moreover, catalpol treatment significantly reduced the endoplasmic reticulum stress-related protein expression levels of binding immunoglobulin protein (BiP), phosphorylated PKR-like endoplasmic reticulum kinase (p-PERK), inositol-requiring kinase 1α (IRE1α), and transcriptional factor activating transcription factor 6 (ATF6), and apoptosis-related protein expression levels of C/EBP homology protein (CHOP), phosphorylated c-Jun N-terminal kinase (p-JNK), and cleaved cysteinyl aspartate specific proteinases (caspases)-12, -9, and -3 in HFD-fed mice and PA-treated HepG2 cells. Furthermore, endoplasmic reticulum stress agonist tunicamycin (TM) significantly reversed the inhibitory effect of catalpol on protein expression levels of BiP, p-PERK, IRE1α, and ATF6, subsequently the inhibitory effect of catalpol on expression levels of CHOP, p-JNK, Bcl-2, Bax, and cleaved caspases (-12, -9, and -3) was also attenuated in PA-treated HepG2 cells. Taken together, these findings demonstrated that catalpol could inhibit hepatocytes apoptosis and had a significant protective effect on liver injury, and its mechanism might be related to the relief of endoplasmic reticulum stress.

In this study, in vitro experiments were conducted to investigate that sinomenine inhibits the macrophage classic activation by up-regulating the expression of paired immunoglobulin-like receptor B (PIR-B). A macrophage model with classic activation was established by lipopolysaccharide and interferon-gamma co-stimulation. Real-time fluorescence reverse transcription-polymerase chain reaction was executed for evaluating the PIR-B gene expression, and Western blot for PIR-B protein expression, in macrophages, respectively. The tumor necrosis factor α and interleukin 8 in cell culture supernatant were measured by enzyme-linked immunosorbent assay. The flow cytometry was utilized to detect M1 macrophages. The PIR-B expression in situ was observed by laser scanning confocal microscope. The results showed that sinomenine significantly increased the expression of PIR-B, markedly reduced the percentage of M1 macrophages, and decreased the levels of tumor necrosis factor α and interleukin 8 in the culture supernatant. The above results indicated that sinomenine can significantly inhibit the macrophage classic activation, and its mechanism may be related to the increase of PIR-B expression in macrophages. This pharmacological effect helps explain the pharmacodynamic mechanism of sinomenine in treating rheumatoid arthritis.

We explored the pharmacodynamic material basis and network regulatory mechanism of Fufang Yuxingcao Mixture (FYM) for the treatment of fever and inflammation. Targets of the 25 compounds in FYM were predicted according to the reverse pharmacophore method and TCMSP, UniProt database. Gene ontology (GO) function enrichment and pathway analysis of the targets was analyzed by Omicsbean software and the Kyoto Gene and Genome Encyclopedia (KEGG) database. A "compound-target-pathway-pharmacological action-effect" network was established with Cytoscape 3.6.1 software. The lipopolysaccharide (LPS)-induced RAW264.7 cell inflammation model was used to verify the anti-inflammatory effects of FYM and its 10 important components. The network pharmacology experiment showed that 25 compounds affected 97 pathways through 211 targets, of which 15 key targets [including RAC-alpha serine/threonine-protein kinase (AKT1), insulin (INS), vascular endothelial growth factor A (VEGFA), interleukin-6 (IL-6), cellular tumor antigen p53 (TP53), tumor necrosis factor (TNF), transcription factor AP-1 (JUN), caspase-3 (CASP3), matrix metalloproteinase-9 (MMP9), interleukin-8 (IL-8), prostaglandin G/H synthase 2 (PTGS2), proto-oncogene c-Fos (FOS), tyrosine-protein kinase SRC (SRC), c-Jun N-terminal kinase 1 (MAPK8), estrogen receptor 1 (ESR1)] and 46 pathways (including NF-kappa B signaling pathway, Toll-like receptor signaling pathway, MAPK signaling pathway, IL-17 signaling pathway, arachidonic acid metabolism, cAMP signaling pathway, T cell receptor signaling pathway, calcium signaling pathway, inflammatory mediator regulation of TRP channels, chemokine signaling pathway, Th1 and Th2 cell differentiation, natural killer cell mediated cytotoxicity, etc.) were related to anti-inflammatory, antipyretic, immune regulation, and analgesia. In vitro cell experiments showed that FYM and the 10 components (including isoquercitrin, luteoloside, baicalein, wogonin, wogonoside, phillyrin, forsythoside A, chlorogenic acid, isochlorogenic acid A, and sweroside) could significantly reduce the expression of nitric oxide (NO), TNF-α and IL-6 in cell supernatants, indicating that the above 10 components may be the key pharmacodynamic material basis of FYM.

There is no specific drug against COVID-19, but berberine (BBR) has moderate anti-SARS-CoV-2 pseudovirus activity. Taking BBR as the lead, 18 novel N-cycloberberine derivatives were synthesized and evaluated for their anti-SARS-CoV-2 pseudovirus activities in vitro. Structure-activity relationship analysis revealed that introducing an appropriate heterocyclic group at position 9 might be beneficial for potency. Among the tested compounds, compound 3m showed the most potent activity against SARS-CoV-2, with EC₅₀ value of 1.61 μmol·L^-1 and SI value of 22.2, much better than that of BBR. Additional experiment indicated that 3m had inhibitory activity on multiple processes in viral invasion, including adsorption and membrane fusion, suggesting a multi-target synergistic mechanism of action. These results provide a novel family of lead compounds for the discovery of anti-SARS-CoV-2 candidates.

Dammarane-type triterpenoid saponins are regarded as the main active constituents of Gynostemma longipes C.Y.Wu. By using MCI and silica gel column chromatography, as well as preparative HPLC, we isolated four new dammarane-type triterpenoid saponins from the polar saponin fraction of G. longipes C.Y.Wu. Their structures were determined by comprehensive analyses of NMR and MS data and identified as (20S)-3β, 20, 21-trihydroxydammar-19-oxo-24-ene-3-O-{[α-L-rhamnopyranosyl(1→2)]-[β-D-xylopyranosyl(1→3)]-α-L-arabinopyranosyl}-21-O-β-D-glucopyranosyl(1→6)-β-D-glucopyranoside (1), (20S)-3β, 20, 21-trihydroxydammar-24-ene-19-oxo-3-O-[α-L-rhamnopyranosyl(1→2)]-[β-D-xylopyranosyl(1→3)]-α-L-arabinopyranosyl-21-O-α-L-rhamnopyranosyl(1→6)-β-D-glucopyranoside (2), (20S)-3β, 19, 20, 21-terahydroxydammar-24-ene-3-O-{[α-L-rhamnopyranosyl(1→2)]-[β-D-xylopyranosyl(1→3)]-α-L-arabinopyranosyl}-21-O-[β-D-glucopyranosyl(1→6)]-β-D-glucopyranoside (3), (20S)-3β, 20, 21-trihydroxydammar-24-ene-3-O-{[α-L-rhamnopyranosyl(1→2)]-[β-D-glucopyranosyl(1→3)]-β-D-glucopyranosyl}-21-O-[β-D-glucopyranosyl(1→6)]-β-D-glucopyranoside (4). Compounds 1-4 are new dammarane-type triterpenoid saponins which contain five glycosyl residues.

Research on polymer impurities has always been important in the quality control of cephalosporins. Research on polymers in cephalosporins that lack active amino groups on the C-7 side chain has not been reported. Therefore, our study used cefazolin sodium, which is widely used in the clinic, as an example. The polymer in cefazolin sodium and its product "cefazolin sodium pentahydrate for injection" was analyzed by column switching liquid chromatography-high resolution mass spectrometry. Two polymer impurity peaks were detected and the possible structures of these polymers were suggested. Through two-dimensional liquid chromatography, the chromatographic peaks following Sephadex gel chromatography and high-performance gel chromatography were compared to those obtained by reverse high-performance liquid chromatography (HPLC) for cefazolin sodium as reported in the Chinese Pharmacopoeia. The HPLC method proves more suitable for polymer detection than Sephadex gel chromatography and high-performance gel chromatography. The method of polymer detection for cefazolin sodium was established using the method of related substances HPLC as described in the Chinese Pharmacopoeia.

The compatibility of kanamycin with sodium citrate for the formulation of kanamycin sulfate injection was determined, including optimization of the amount of sodium citrate in the injection and the sterilization process. An HPLC coupled with an evaporative light scattering detector (ELSD) was used to measure the amount of sodium citrate and the impurity profiles. A validated post-column derivatization HPLC coupled with a fluorescence detector (FLD) was used to determine the correlation between specific impurities in a domestic factory and sodium citrate, and then the formulation was evaluated by HPLC coupled with mass detector (MS) characterization of degradation products. The results show that the amount of sodium citrate in kanamycin sulfate injection from a domestic factory is about 40 times higher than that of the Meiji formulation. Several specific impurities can be detected in solutions heated under simulated sterilization conditions (121 ℃), which were correlated with the amount of sodium citrate. Impurities were characterized by HPLC-MS/MS, and data showed that the identified impurities were interaction products of kanamycin and sodium citrate. These results indicate that greater attention should be directed at formula optimization in domestic factories, as it is crucial to the safety and efficacy of the preparations. Drug-excipient chemical compatibility should also be evaluated in the development of pharmaceutical dosages forms especially when the active pharmaceutical ingredients have a primary amine group.

The intestinal absorption properties of the main effective components (glycyrrhizic acid, isoliquiritigenin, 6-gingerol, ginsenoside Rb1, atractylode-Ⅰ) in Lizhong decoction (LZD) extracts were investigated with an in situ single-pass intestinal perfusion model in rats. UPLC-TQ-MS was used to determine the concentration of the five components in the intestinal perfusion. Animal welfare and experimental procedures were in accordance with the regulations of the Animal Ethics Committee of Nanjing University of Chinese Medicine. As evaluation indexes for the intestinal absorption characteristics, the absorption rate constant (K_a) and the apparent permeability coefficient (P_eff) of the five main ingredients were analyzed. Results showed that the best absorption sites for glycyrrhizic acid, isoliquiritin and 6-gingerol were the ileum, colon and duodenum, respectively, and the differences between different intestinal segments were statistically significant (P < 0.05). There was no notable difference in K_a and P_eff between ginsenoside Rb1 and atractylode-Ⅰ in the different intestinal segments (P > 0.05), suggesting that they were absorbed throughout. The five components were well-absorbed in the whole intestine (P_eff > 1.0×10^-3 cm·min^-1), indicating that LZD is suitable for preparing sustained, controlled release and enteric-coated preparations.

The study evaluates the lipolysis rate and extent of type Ⅲ lipid formulations using testosterone undecanoate as a model drug after digestion with in vitro lipolysis model, and studies the digestive regularity with optical microscope and electrical conductivity. The results showed that for testosterone undecanoate type Ⅲ lipid formulations with castor oil as oil phase and Transcutol HP as latent solvent, the lipolysis rate and extent were increased with the increase of oil phase proportion and were decreased with excessive proportion of surfactant, in which can see liquid crystal phase during lipolysis process. The lipolysis rate of type ⅢB lipid preparations with different surfactant were ordered as Labrasol > Tween 80 > Cremophor EL, but the rate of type ⅢA is different in quick digestion phase and slow digestion phase. The lipolysis extent of type Ⅲ lipid formulations with different surfactant were ordered as Cremophor EL > Tween 80 > Labrasol. These may be related to the digestive effect of pancreatic lipase on different surfactants. This study implied that the lipolysis rate and extent of type Ⅲ lipid formulations are greatly influenced by the proportion of oil phase and surfactant, and the surfactant structure. These factors will affect the in vivo digestion and should be taken into account when screening type Ⅲ lipid formulations.

In this study, artemether (ARM)-loaded mixed micelles (MM) composed of the sodium glycocholate (SGC) and soybean lecithin (SL) were prepared by film dispersion method. The effects of hydration medium, SL mass ratio and total concentration of excipients on the solubilization of ARM were investigated and the stability of MM was evaluated. Results showed that the particle size distribution of SGC-SL-MM prepared by phosphate buffer solution (PBS, pH 7.4, 0.05 mol·L^-1) was uniform, with an average size of 3.58 ±0.14 nm and the polydispersity index (PDI) value was 0.16 ±0.04. The solubility of ARM increased significantly from 0.64 ±0.04 mg·mL^-1 to 13.7 ±0.13 mg·mL^-1 along with the concentration of total excipient increasing from 1.0% to 30.0% (w/w). The calculated results of Arrhenius parameter and storage stability showed that the degradation rate constant of ARM in MM was smaller than that in acetonitrile-PBS (pH 7.4) at either 37 ℃ or 60 ℃. The experimental ARM-MM was clear after storing for two months at 25 ℃ and the degradation of ARM was less than 7.0%. In conclusion, the SGC-SL-MM can not only improve the solubility of ARM in aqueous solution, but also improve its chemical stability in aqueous solution at low temperature.

Methotrexate (MTX) injection has a short half-life and significant toxic side effects. In order to overcome the demerits of MTX injection, MTX@COF was prepared for subcutaneous injection by loading MTX in crosslinked cyclodextrin metal-organic framework (COF) in this study. The cationic lipid material (2, 3-dioleoyl-propyl)-trimethylamine (DOTAP) was then coated on the MTX@COF surface by solvent evaporation. Different surface charge characteristics were observed in the coated MTX@COF@DOTAP with no significant change in particle morphology. The in vitro release behaviors of sustained-release particles were investigated in water and phosphate buffer (pH 7.4), and the in vivo release characteristics were evaluated for pharmacokinetics in rats. The in vitro release results showed that the cumulative release of MTX, MTX@COF and MTX@COF@DOTAP within 6 h was 92.70%, 36.31% and 18.19% in water, respectively; the cumulative release of MTX, MTX@COF and MTX@COF@DOTAP within 4 h was 90.82%, 79.37% and 58.30% in phosphate buffer, respectively; the results showed that MTX@COF can significantly delay the release of MTX, the modification to MTX@COF by DOTAP can further delay the release of MTX. Pharmacokinetic studies in rats showed that the mean retention time [MRT_(0-t)] and the time to peak (T_max) of the subcutaneous injection of MTX@COF@DOTAP group were significantly prolonged compared with the MTX@COF group and the MTX group. The area under the concentration-time curve [AUC_(0-t)] of the MTX@COF@DOTAP subcutaneous injection group was 1.8 times high as that of the MTX group. In this study, MTX@COF@DOTAP particles had a certain sustained-release effect, and could prolong the bioavailability of MTX by subcutaneous injection, which provided a new idea for the development of new MTX dosage forms.

Ferulate 5-hydroxylase (F5H) is a key enzyme involved in the phenylpropane metabolism pathway. Based on our previous transcriptome sequencing study, F5H played a negative regulatory role in glycyrrhizic acid (GA) biosynthesis. Therefore, in this study we cloned the F5H gene and investigated its regulatory effect on GA accumulation through gene overexpression and knockout. F5H was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MK882511). A plant binary expression vector pCA-F5H was constructed by inserting F5H into pCAMBIA1305.1 at Spe Ⅰ and Bgl Ⅱ sites. The sgRNA sequences were designed based on the first exon of F5H. The CRISPR/Cas9 gene editing vector pHSE-F5H was constructed by inserting F5H sgRNA into pHSE401 at two Bsa Ⅰ sites. PCA-F5H and pHSE-F5H were transfected into Agrobacterium tumefaciens ATCC15834, which was used to induce hairy root overexpressing or knocking out F5H with licorice hypocotyl as explants. At the same time, wild type and negative control hairy roots were also generated. UPLC was used to assay the GA content in different hairy root lines, and results showed that the GA content in hairy root lines knocking out F5H was significantly higher, whereas in hairy root lines overexpressing F5H GA content was lower than that in the wild-type and negative control. In this work, through a reverse genetics strategy, the negative regulatory effect of F5H on GA biosynthesis was confirmed through gene overexpression and knockout. This work will lay a foundation for further elucidation of the molecular regulatory network of GA biosynthesis.

In the research and development of new drugs, it is very important to investigate the in vitro metabolism of candidate drugs. Traditional models such as liver microsomes have many limitations, while the in vitro model of recombinant human drug metabolizing enzymes is considered as an important and useful approach because of its convenient access, stable activity and low cost. In this study, six major human UDP-glucuronosyltransferases (UGTs) genes (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) were cloned from human liver cDNA and heterologously expressed in Saccharomyces cerevisiae and baculovirus-infected insect cell. UGT1A1, 1A3, 1A6 and 1A9 were successfully expressed in yeast and showed glucuronidation activity against a variety of different structural types of substrates, but their activities were low. All six UGTs were successfully expressed and exhibited significantly improved glucuronidation activity when Trichopolusia ni cells BTI-TN5B1-4 (High Five) were used as the host. The recombinant human UGTs expressed in insect cells can catalyze the glucuronidation of their specific substrates, and the glucuronidation products were synthesized at milligram-scale with yields of 13%-66% for the first time, of which the structures were identified via MS, ¹H NMR, and ¹³C NMR spectroscopic analysis. Above all, the recombinant human UGTs yeast and insect cell expression systems constructed in this study can be used for in vitro metabolism evaluation in the early stage of new drugs research and development, and also provide a new tool for the synthesis of glucuronide metabolites.

Based on the "requirements on the submitted documents for consistency evaluation of generic oral solid dosage forms of chemical drugs" and relevant guidance, this article summarized and formulated the decision tree of in vitro consistency evaluation of oral solid generic drugs, discussed the differences and common problems of in vitro evaluation research projects under different conditions, selective analyzed the technical requirements and concern problems of unconventional research projects, and proposed corresponding recommendations for concern problems, in order to provide more references for the follow-up study on consistency evaluation of oral solid generic drugs.