Chinese Journal of Clinical Pharmacology and Therapeutics

Research and development of antiviral drugs against new coronavirus 2019-nCoV

LIU Qi, XIA Shuai, JIANG Shibo

2020, 25(3): 241-245. doi:10.12092/j.issn.1009-2501.2020.03.001

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At the end of 2019,an outbreak of 2019 new coronavirus infectious disease (COVID-19) in Wuhan spread rapidly in China and reported cases in other countries.The number of infected cases and deaths quickly exceeded Severe Acute Respiratory Syndrome (SARS) in a short period of time,resulting in incalculable losses to China.Chinese researchers have quickly identified the pathogen,2019-nCoV (or SARS-CoV-2 or HCoV-19) and carried out the research and development of antiviral drugs at different levels.This article briefly reviews the current advancement of research and development of new therapeutics against 2019-nCoV infection.Given that the progress of research and development of antiviral drugs against the emerging and reemerging pathogens is relatively backwarded,we suggest that the research and development of antivirals against pathogens with pandemic or epidemic potential should be carried out in advance before the occurrence of the outbreaks. At the national level,we should promote the research and development of broad-spectrum antivirals and clinical trials to combat the pathogens with epidemic risk.

Study on high on-aspirin platelet reactivity in diabetic mice based on metabonomics

ZHANG Haowen, ZHU Yejin, WU Xiang, CHEN Hanyu, SONG Yulei, BIAN Yaoyao, HAO Haiping, CHEN Xiaohu

2020, 25(3): 246-256. doi:10.12092/j.issn.1009-2501.2020.03.002

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AIM: To explore the underlying mechanism of aspirin-related high on-aspirin platelet reactivity (HAPR) in diabetic state from the perspective of endogenous metabolic changes in metabolomics. METHODS:Type 2 diabetes mellitus (T2DM) mice model was established by the combination treatment of high-fat diet and streptozotocin injections. Mice were randomly divided into Normal group, T2DM group, Normal+aspirin group and T2DM+aspirin group. GC/TOF-MS was used to determine the plasmatic endogenous metabolites of mice. The data were analyzed applying multivariate statistical method. RESULTS:The expression of platelet CD62P and ratio of TXB2/6-keto-PGF1α in the plasma of female T2DM mice showed no significant difference compared with that of the model group. The degree of divergence between normal and normal+aspirin groups was significantly greater than that between T2DM and T2DM+aspirin groups. Biochemical metabolic pathway analysis revealed that HAPR in diabetic mice was linked to glycolysis, gluconeogenesis, pyruvate metabolism, citric acid cycle, ascorbic acid metabolism, and arachidonic acid metabolic pathways. CONCLUSION:The different metabolites obtained in metabolome assay suggests that the HAPR in diabetic state involved several different metabolic pathways, which can better elucidate the biological mechanism of diabetes-associated HAPR.

Anti-inflammatory and analgesic effects of total saponin of Dioscorea

QI Qin, HE Xiang, WANG Gang, NA Sha, LV Lei, CHEN Guangliang

2020, 25(3): 257-264. doi:10.12092/j.issn.1009-2501.2020.03.003

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AIM: To study the anti-inflammatory and analgesic effects of total saponin of Dioscorea(TSD). METHODS: The rat acute gouty arthritis (AGA) and the mice air-pouch inflammation models were induced by monosodium urate(MSU), the pain models were adopted including the hot-plate and acetic acid-induced writhing test in mice. Then the content of IL-1β, IL-18 and PGE2 levels were determined by ELISA. RESULTS:TSD significantly inhibited the MSU-induced ankle swelling and air pouch inflammation in mice(P<0.01 or P<0.05). Meanwhile, TSD markedly reduced the levels of IL-1β and IL-18 in synovial fluid exudates and IL-1β and PGE2 in air pouch exudates (P<0.01 or P<0.05).In addition, TSD remarkably suppressed acetic acid-induced writhing response and prolonged pain threshold in hot plate assay(P<0.01 or P<0.05).CONCLUSION: TSD has anti-inflammatory and analgesic effects, which are mainly relevant to the inhibition of the synthesis and release of inflammatory factors IL-1β, IL-18 and PGE2.

Ginsenoside Rb1 protects brain through Cav-1 for mice with cerebral ischemia-reperfusion injury

ZHANG Gaojiao, WANG Xinxin, ZHOU Jia, WANG Leilei, DAI Qinxue

2020, 25(3): 265-270. doi:10.12092/j.issn.1009-2501.2020.03.004

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AIM: To investigate the protective effect of ginsenoside Rb1 on brain through Cav-1 in mice with cerebral ischemia-reperfusion injury. METHODS: One hundred and twenty C57/B6 mice were randomly divided into sham operation group, model group, model + ginsenoside Rb1 group, ginsenoside Rb1+ Cav-1 siRNA group, ginsenoside Rb1+siNC group, 24 in each group. The model of cerebral ischemia-reperfusion injury in mice was established by middle cerebral artery occlusion (MCAO). The ginsenoside Rb1 group received intraperitoneally injection of ginsenoside Rb1 (40 mg/kg); the sham operation group and model group were intraperitoneally injected with an equal amount of physiological saline immediately after modeling. For the ginsenoside Rb1+ cav-1 siRNA group and the ginsenoside Rb1+siNC group, cav-1 siRNA and siNC were injected into the lateral ventricle 24 h before molding, respectively, and the other operations were the same as the ginsenoside Rb1 group. The neurobehavioral scores of the mice in each group were measured at 24 h after reperfusion, and the water content of brain tissue, cerebral infarction volume, Cav-1 mRNA and Cav-1, Bcl-2 and Bax protein expressions in the cerebral cortex penumbra were measured in each group. RESULTS:Compared with the sham operation group, the neurobehavioral scores, cerebral infarction volume and brain tissue water content in the model group were significantly increased (P<0.05), and the expressions of Cav-1 mRNA and Cav-1 protein, and the Bcl-2 /Bax ratio were significantly decreased (P<0.05). Compared with the model group, the neurobehavioral scores, cerebral infarction volume and brain tissue water content in the ginsenoside Rb1 group were significantly decreased, and the expressions of Cav-1 mRNA and Cav-1 protein, and the Bcl-2 /Bax ratio were significantly increased (P<0.05). Compared with the ginsenoside Rb1 group, the neurobehavioral scores, cerebral infarction volume and brain tissue water content in the ginsenoside Rb1 + cav-1 siRNA group were significantly increased, and the expressions of Cav-1 mRNA and Cav-1 protein, and the Bcl-2 /Bax ratio were significantly decreased (P<0.05). CONCLUSION: Ginsenoside Rb1 can protects brain for mice with cerebral ischemia-reperfusion injury. After Cav-1 siRNA decreased the expression of Cav-1 protein in the brain tissue of mice, it significantly reverses the cerebral protective effect of ginsenoside Rb1, indicating that Cav-1 protein mediated the cerebral protective effect of ginsenoside Rb1 on cerebral ischemia reperfusion injury mice.

Apoptosis of Tca8113 cell induced by antitumor component-I from Agkistrodon acutus venom through CHOP/Caspase-12 pathway

CHAI Lin, WANG Zhenjie, XU Ran

2020, 25(3): 271-277. doi:10.12092/j.issn.1009-2501.2020.03.005

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AIM: To investigate the effect of endoplasmic reticulum stress pathway on the apoptosis of tongue squamous cancer Tca8113 cells induced by antitumor component-I from Agkistrodon acutus venom (AAVC-I). METHODS: The in vitro experiments were performed on subculture tongue squamous cancer Tca8113 cells in their growth period. A normal control group, a DL-dithiothreitol (DTT) positive control group and different AAVC-I concentrations were set according to the experiment objective. MTT assay was used to detect the proliferation inhibition of Tca8113 cells after been treated with different concentrations of DTT and AAVC-I for 24 h. The results were used to choose appropriate concentrations of DTT and AAVC-I in DTT positive control group and AAVC-I treated group, respectively. HE staining and Annexin V-FITC/PI double fluorescence staining were used to monitor the apoptosis of Tca8113 cells. Western blot was used to identify the expression levels of apoptosis-related proteins including endoplasmic reticulum stress glucose-regulatory protein 78 (GRP78), enhance-binding protein-homologousprotein (CHOP), cysteine-containing aspartate specific protease-12 (Caspase-12), cysteine-containing aspartate specific protease-9 (Caspase-9) and cysteine-containing aspartate specific protease-3 (Caspase-3).RESULTS:The proliferation inhibition of Tca8113 cells increased with an increased concentration of AAVC-I concentration (P<0.05), causing cell shrinkage, increased cell gaps, cytonuclear condensation, cell fragmentation, the appearance of apoptotic bodies, and increased rate of apoptosis (P<0.05). In addition, the expression level of GRP78 protein, CHOP protein, proteins of Caspase-12, Caspase-9 and Caspase-3 were increased (P<0.05).CONCLUSION: Endoplasmic reticulum stress CHOP/Caspase-12 pathway plays an important role in AAVC-I induced Tca8113 cells apoptosis.

Synergistic effects of Cortex Mori alkaloids combined with Simvastatin in rats with hyperlipidaemia

LI Jiadan，LU Chen

2020, 25(3): 278-284. doi:10.12092/j.issn.1009-2501.2020.03.006

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AIM: To explore the lipid lowering effect of Cortex Mori alkaloids combined with simvastatin on hyperlipidemia rats. METHODS: Hyperlipidemia model was established by feeding golden hamsters with high-fat diet for 6 weeks, and 40 golden hamsters were randomly divided into normal group, model group, Cortex Mori alkaloids group (CMA, 25 mg·kg-1·d-1),simvastatin group (15 mg·kg-1·d-1) and combination group (Cortex Mori alkaloids, 25 mg·kg-1·d-1, simvastatin 15 mg·kg-1·d-1). 2 weeks after the intragastric administration, cardiac blood sampling was used to determine the blood biochemical indexes such as TG，TC，LDL-C，HDL-C, as well as the levels of ALT，AST，CRP， NO，Lp-PLA2.Liver HE staining was used to observe pathological changes, and liver cell lipid deposition was observed by oil red O staining. RESULTS:Compared with the model group, TC levels in combination group were significantly reduced (P<0.05), LDL-C levels in simvastatin group and the combination group were significantly reduced (P<0.05).The level of NO in the combined group was significantly increased (P<0.05), and the difference was statistically significant compared with that in Cortex Mori alkaloids group and simvastatin group (P<0.05) .The levels of CRP were significantly decreased in Cortex Mori alkaloids group, simvastatin group and the combination group (P<0.05).The level of Lp-PLA2 in combination group was significantly reduced (P<0.05). The results of liver HE staining and oil red O staining showed that both of them could improve the hepatic lesion and lipid deposition after drug treatment, and the improvement of the combination group was better than that of the two groups alone. CONCLUSION: Simvastatin combined with Cortex Mori alkaloids can effectively decrease lipid level and inflammatory level of golden hamstrel with hyperlipidemia.Cortex Mori alkaloids can be used as the auxiliary drug of simvastatin, which is expected to provide a new choice for clinical treatment of hyperlipidemia.

Resveratrol protects human retinal pigment epithelial cells from toxicity of high glucose by up-regulation of miR-26a

ZHOU Haiyan, WANG Kailing

2020, 25(3): 285-292. doi:10.12092/j.issn.1009-2501.2020.03.007

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AIM: To study the protective effects and mechanism of resveratrol (RES) on the injury of human retinal pigment epithelial cells (ARPE-19) induced by high glucose (HG). METHODS: ARPE-19 cells were cultured in HG to simulate injury. Cell viability, apoptosis, ROS generation and miR-26a level were examined by CCK-8 assay, flow cytometry assay, DCFH-DA staining and RT-qPCR, respectively. Expression of proteins associated with viability, apoptosis and oxidative stress was measured by Western blot analysis. In addition, the involvements of the ERK and Wnt/β-catenin pathways were analyzed by Western blot analysis.RESULTS:HG reduced cell viability while promoted apoptosis and oxidative stress in ARPE-19 cells. RES ameliorated HG-induced cell injury. The expression of miR-26a was up-regulated by RES in HG-treated cells, and miR-26a inhibition obviously reversed the effects of RES on HG-treated cells. Finally, we found the ERK and Wnt/β-catenin pathways were inhibited by RES through up-regulation of miR-26a. CONCLUSION: RES protected ARPE-19 cells against HG-induced injury through up-regulating miR-26a, along with inhibition of the ERK and Wnt/β-catenin pathways. RES might be a potential therapeutic drug for diabetic retinopathy.

Stimulation of Yes-associated protein activity attenuates lung injury repair induced by LPS in mice

HUANG Tianpeng, HAO Jiale, YU Yan, WANG Yaoyao, FAN Xiaofang, GONG Yongsheng, MAO Sunzhong

2020, 25(3): 293-298. doi:10.12092/j.issn.1009-2501.2020.03.008

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AIM: To observe the effect and possible mechanism of stimulation of Yes-associated protein (YAP) activity on acute lung injury and repair induced by LPS in mice. METHODS: Ninety adult male ICR mice were divided into two groups: acute lung injury model group induced by LPS (7.5 mg/kg, i.p.) and LPS+XMU treatment (1 mg·kg-1·d-1) group. At 0 h, 24 h, 48 h and 72 h after LPS injection, the contents of TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF) were detected by ELISA method. The activity of myeloperoxidase (MPO) and the content of protein in BALF were evaluated by chemical technique. The protein level of nuclear YAP, cytosol phosphorylated YAP (P-YAP), connective tissue growth factor (CTGF) and proliferating cell nuclear antigen (PCNA) in lung were analyzed by Western blot. RESULTS:The protein level of nuclear YAP at 24 h, 48 h and 72 h in lung of LPS+XMU group were up-regulated than those of LPS group by 39.2%, 148.1% and 42.9%, and the protein level of CTGF in lung were up-regulated than those of LPS group by 186.6%, 10.1% and 146.1% (P<0.05), respectively, while the protein level of cytosol P-YAP was down-regulated. The content of IL-6 and TNF-α at 24 h in BALF of LPS+XMU group were lower than those of LPS group by 28.4% and 23.7%, and the activity of MPO and the content of TNF-α at 48 h in BALF were lower by 13.5% and 39.5%, and the content of IL-6 and TNF-α at 72 h in BALF were lower by 42.8% and 16.7% (P<0.05), respectively. The protein level of PCNA at 48 h and 72 h in lung of LPS+XMU group were up-regulated than those of LPS group by 44.2% and 14.9% (P<0.05), respectively, while there was no significant difference at 24 h. The content of protein at 24 h, 48 h and 72 h in BALF of LPS+XMU group were lower than those of LPS group by 32.4%, 46.0% and 26.3% (P<0.05), respectively. The pathological changes showed a significantly attenuated tissue injury and accelerated recovery from lung injury in LPS+XMU group compared with mice injected with LPS alone at each times point. CONCLUSION: Stimulation of YAP activity attenuates lung injury and promotes lung recovery by alleviating lung inflammation and injury at injury phase, but by promoting inflammation resolution and stimulating cell proliferation at repair phase.

Development and application progress of physiologically based pharmacokinetic modeling and its combined use with other modeling methods

CHEN Wenjun, RUAN Zourong, XIANG Xiaoqiang

2020, 25(3): 299-305. doi:10.12092/j.issn.1009-2501.2020.03.009

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Physiologically based pharmacokinetics (PBPK) is one of the main research fields of pharmacometrics, and it plays an important role at all the stages of drug development and clinical practice. In early drug discovery and development, human pharmacokinetics (PK) could be predicted by PBPK modeling using in silico, in vitro and preclinical in vivo data. During clinical studies, PBPK model could be used to investigate the effects of various physiological and pathological factors on PK, such as age, gender, liver/kidney impairment, and to guide dose adjustment of special population (pregnant women, children, etc.). Furthermore, PBPK modeling is now becoming more appealing with the ability to predict drug-drug interaction (DDI) in the case of co-administration of multiple drugs. In recent years, the application of PBPK modeling in industry has increased widely. Also, regulatory agencies have recognized the potential of PBPK and its impact on labeling recommendations. As the popularity of model-informed drug development, the combination of PBPK modeling with other commonly used modeling methods, such as population pharmacokinetics (PopPK), pharmacokinetic/pharmacodynamic (PK/PD) modeling and model-based meta-analysis (MBMA), has shown attractive advantages. In this paper, the origin and development, as well as the application status of PBPK are introduced briefly, and the application of PBPK modeling merged with PopPK, PK/PD and MBMA is reviewed.

Bioequivalence of apixaban tablets in healthy Chinese subjects

NING Xiaoyi, KUANG Yun, HUANG Jie, LIU Yanan, YANG Xiaoyan, YANG Shuang, GUO Can, YANG Guoping

2020, 25(3): 306-311. doi:10.12092/j.issn.1009-2501.2020.03.010

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AIM: To evaluate the bioequivalence of the two preparations of apixaban tablets administered once orally under fasting and fed conditions. METHODS: The study was designed as randomized, open, self-crossover, and twenty four healthy volunteers were recruited respectively in fasting and fed conditions. Subjects were assigned to receive a single oral of the test or reference formulation per period at a dose of 2.5 mg. The plasma apixaban concentration was analyzed by LC-MS/MS. The major pharmacokinetic parameters were calculated by WinNonlin 6.4 and the bioequivalence was evaluated.RESULTS:The main pharmacokinetic parameters of a single oral apixaban under fasting condition for T and R were as follows: Cmax (80±14) and (87±21) ng/mL, AUC0-t (713±136) and (733±142) h·ng·mL-1, AUC0-∞ (722±143) and (741±142) h·ng·mL-1, tmax 2.5 h and 2.5 h, t1/2 (9±8) and (8±6) h. The relative bioavailability was 97.16% for AUC0-t, 97.20% for AUC0-∞. The main pharmacokinetic parameters of a single oral apixaban under fed condition for T and R were as follows: Cmax (70±13), (72±13) ng/mL; AUC0-t (642±130), (690±135) h·ng·mL-1; AUC0-∞ (652±129), (704±138) h·ng·mL-1. tmax 2.5 h and 2.5 h, t1/2 (8±4) and (12±9) h. The relative bioavailability was 93.03% for AUC0-t, 92.62% for AUC0-∞. CONCLUSION: The test preparation of apixaban tablets is bioequivalent to the reference preparation under both fasting and fed conditions.

Distributions of MTHFR C677T gene polymorphism among cerebral stroke patients in southern Anhui province

YUAN Xiaolong, SUN Hua, WANG Zhongfang, CHEN Yajuan, ZHOU Lulu, YIN Qin, XU Jinying

2020, 25(3): 312-316. doi:10.12092/j.issn.1009-2501.2020.03.011

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AIM: To investigate and analyze the distribution characteristic of 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism among cerebral stroke patients and to provide prevention and treatment for stroke patients in southern Anhui Province. METHODS: A total of 114 patients with cerebral stroke in the Second Affiliated Hospital of Wannan Medical College from January 2018 to October 2019 were included. The MTHFR C677T genotype was performed by fluorescence in situ hybridization analysis. The MTHFR C677T gene polymorphism distribution data of the cerebral stroke population in southern Anhui Province was compared with the reported gene distribution data of Han Chinese stroke population in other parts of China. RESULTS:The frequencies distribution of TT, CT and CC genotypes of MTHFR C677T were 28.90%, 50.00%, 21.10%. The frequencies of C and T alleles were 53.95% and 46.05%. There was no gender difference in the distribution of this gene. There were significant difference in CC genotype between Chongqing area, Heilongjiang area and Guangzhou area (P<0.05). There were significant difference in TT genotype between Chongqing area, northern Henan area and Heilongjiang area (P<0.05). CONCLUSION: The distribution of MTHFR C677T gene polymorphism in the Han population of southern Anhui Province is different from other areas. It can provide a scientific basis for the prevention and treatment of cerebral stroke in high-risk population in southern Anhui Province by genetic testing technology.

Effects of low-dose baclofen adjuvant therapy on patients with trigeminal neuralgia and its effects on inflammatory factors and oxidative stress

PI Zhibing, YANG Xuezhi, PENG Yu, ZHANG Junkai, ZHANG Lei, LIN Hai

2020, 25(3): 317-322. doi:10.12092/j.issn.1009-2501.2020.03.012

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AIM: To study the effect of low-dose baclofen on inflammatory factors and oxidative stress in patients with trigeminal neuralgia. METHODS: A total of 112 patients with trigeminal neuralgia treated in our hospital from Jan. 2017 to Jan. 2019 were selected as the subjects; they were divided into test group (n=56) and control group (n=56) according to random number table method. The control group was treated with oxcarbazepine, while the test group was treated with low-dose baclofen on the basis of the control group; the clinical total effective rate, visual analogue scale (VAS), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) levels and the incidence of adverse reactions were compared between the two groups. RESULTS:The clinical total effective rate of the test group was significantly higher than that of the control group (P<0.05); the VAS score of the two groups was significantly lower than that before treatment (P<0.05), and the VAS score of the test group was significantly lower than that of the control group (P<0.05); the levels of TNF-α, IL-6 and in the two groups were significantly lower than those before treatment (P<0.05), and the levels of TNF-α, IL-6 and in the test group were significantly lower than those in the control group (P<0.05); the levels of MDA in the two groups were significantly lower than those before treatment (P<0.05), SOD and GSH-Px were significantly higher than those before treatment (P<0.05), and the levels of MDA in the test group were significantly lower than those in the control group (P<0.05), while the levels of SOD and GSH-Px were significantly higher than those in the control group (P<0.05); there was no significant difference in the incidence of adverse reactions between the two groups (P>0.05). CONCLUSION:Low-dose baclofen adjuvant therapy has good clinical efficacy for patients with trigeminal neuralgia, which can effectively relieve pain, reduce inflammation and oxidative stress.

COVID-19：Emergency response measures for GCP in Hunan province under the first level response of major public health emergencies（2.0 Version）

QIN Qun, LIU Xiaobao, YANG Guoping, LI Zhuo, LI Kunyan, HUANG Zhijun, LIU Zhaoqian

2020, 25(3): 323-328. doi:10.12092/j.issn.1009-2501.2020.03.013

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According to the epidemic situation in Hunan province, COVID-19: Emergency response measure for GCP in Hunan Province under the first-level response of major public health emergencies (version 2.0) as the consensus is reached by experts with years of rich experience in the field of drug clinical trials. We hope this paper can provide reference for GCP practitioners.

Ethical issues in the prevention and control of Coronavirus Disease 2019

WANG Xiaomin, YANG Guoping, WANG Bing, ZHONG Yuqiong, WU Ying, LIU Xing

2020, 25(3): 329-333. doi:10.12092/j.issn.1009-2501.2020.03.014

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