Chinese Journal of Pharmacology and Toxicology

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    2025, 39(2): 0.
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    • ORIGINAL ARTICLES
  • ORIGINAL ARTICLES
    Dopamine receptor 3 knockout inhibits acquisition and recall of fear memory induced by electric shocks
    DING Xiaoyan, WANG Zhiyuan, WU Ning, LI Jin, SONG Rui
    2025, 39(2): 81. https://doi.org/10.3867/j.issn.1000-3002.2025.02.001
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    OBJECTIVE  To investigate the effect of dopamine receptor 3 (D3R) on fear memory induced by intense electric shocks and the possible neurobiological mechanism. METHODS  ① To prevent pain threshold differences from influencing the effect of intense electric shocks, wild-type (WT) and D3R knockout mice (D3R-/-) were used in the Hargreaves test to evaluate their basal pain threshold, with the paw withdrawal latency (PWL) as the observation index. ② WT and D3R-/- mice were divided into control groups and model groups, respectively. On the training day (the first day, D 1), the model groups received inescapable electric shocks (1.5 mA, 10 s, 10 s interval, 15 cycle) while the control groups did not. Contextual fear tests were conducted on D2, D7, D10, D14, and D16 after training, with the percentage of freezing time (FT) as the observation index to evaluate fear memory acquisition induced by contextual cues. On D17, after the model groups showed no more fear responses to contextual cues, they were re-stimulated with low-intensity current (0.5 mA, 10 s, 10 s interval, 15 cycle) to evoke fear memory. The two control groups did not receive any shocks. Contextual fear tests were conducted on day 18, and the FT% of each group was observed to evaluate fear memory retrieval induced by contextual cues. ③ Another cohort of WT and D3R-/- mice was used to further investigate the underlying neural mechanism, with the same grouping and treatment as in ②. Real-time dynamic changes in calcium signals of dopamine (DA) neurons in the ventral tegmental area (VTA) of WT and D3R-/- mice were detected using fiber photometry during electric shocks. The fluorescence area under the curve (AUC) was used as the indicator to quantify the excitability of DA neurons. RESULTS  ① In the Hargreaves test, there was no significant difference in PWL between D3R-/- mice and WT mice, indicating the two genotype mice had no significant differences in the basal pain threshold. ② Compared with the WT control group, the percentage of FT of the WT model group significantly increased on D2, D7, D10, and D14 (P<0.05). Compared with the D3R-/- control group, the percentage of FT of the D3R-/- model group significantly increased only on D2 and D7 (P<0.01). Meanwhile, the percentage of the FT of D3R-/- model group was significantly lower than in the WT control group on D2, D7, D10, and D14 (P<0.05, P<0.01). During memory recall (D18), the percentage of FT of the WT model and D3R-/- model groups significantly increased compared to their respective control groups (P<0.01, P<0.05), while the percentage of FT of D3R-/- model mice was significantly lower than that of WT model mice (P<0.01). ③ In the fiber photometry test, during the shock period, the calcium signals of DA neurons in the VTA of WT model and D3R-/-model mice rapidly increased within the first 2 s, and then gradually decreased between 2 to 10 s. The AUC within the 2 to 10 s interval was significantly lower in D3R-/- model mice compared to WT model mice (P<0.05), indicating that the excitability of DA neurons in the VTA of D3R-/-model mice was significantly lower than that of WT-model mice. CONCLUSION  D3R knockout inhibits the acquisition and recall of long-term fear memory in mice, and its neurobiological mechanism may be related to the decreased excitability of DA neurons during electric shock.
  • ORIGINAL ARTICLES
    Ginsenosides targeting P-glycoprotein enhance the inhibitory effect of paclitaxel on colon cancer
    ZHU Xiaohui, ZHAO Yuanyuan, LI Nanxi, GUO Jinnan, TIAN Yunfei, ZHAI Huiting, WANG Shanshan, YANG Dexuan, DOU Guifang, FENG Suxiang, MENG Zhiyun
    2025, 39(2): 89. https://doi.org/10.3867/j.issn.1000-3002.2025.02.002
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    OBJECTIVE  To investigate the effects of ginsenosides as P-glycoprotein (P-gp) substrates in combination with paclitaxel on the proliferation and migration of colon cancer Caco-2 cells.  METHODS  Bio-layer interferometry (BLI) technology was used to detect the constants of ginsenosides and P-gp. Network molecular docking was adopted to predict the binding affinity energy of ginsenosides and P-gp. Caco-2 cells were divided into paclitaxel 0, 6.25, 12.5, 25, 50, 100 and 200 mg·L-1 groups, ginsenoside Rg3 0, 6.25, 12.5, 25, 50, 100 and 200 mg·L-1 groups, and paclitaxel 5 mg·L-1+ginsenoside Rg3 0, 25, 50, 100 and 200 mg·L-1 groups. After 48 h of incubation, the growth inhibition rate of Caco-2 cells was detected by MTT assay, and the interaction between the two drugs was quantitatively evaluated using the "one-belt, one-line" modle. Caco-2 cells were divided into the cell control group, paclitaxel 5 mg·L-1 group, ginsenoside Rg3 50 and 100 mg·L-1 groups, and paclitaxel 5 mg·L-1+ginsenoside Rg3 50 and 100 mg·L-1 groups. After 24 h of incubation, the proliferation and migration ability of the cells were detected by colony assay and Transwell migration assay. Caco-2 cells were then divided into the cell control group, quinidine 12.5 mg·L-1 group, and ginsenoside Rg3 6.25 and 12.5 mg·L-1 groups. After  4 h of incubation, the expression levels of P-gp and total protein were detected by ELISA. RESULTS  The affinity constants of ginsenoside Rb1, Rg3, Rg5 with P-gp were all less than 10-3 mol·L-1, while that of ginsenoside CK with P-gp was 10-2 mol·L-1. There was no typical binding dissociation curve between ginsenoside Re and P-gp. The absolute binding affinities of ginsenosides Rg3 and Rg5 to P-gp were determined to be 8.5 kcal·mol-1 and 7.6 kcal·mol-1, respectively. Ginsenosides mixed with PTX 5 mg·L-1 inhibited the growth of colon cancer cells through synergy and addition, and the dose range of the synergistic effect was [0+5, 43.15+5] mg·L-1; [164.51+5, 200+5] mg·L-1, the additive effect dose ranged from [43.15+5, 164.51+5] mg·L-1. The combination of the two drugs could significantly reduce the proliferation and migration ability of Caco-2 cells (P<0.01). The ELISA results showed a decrease in total protein and P-gp content in both the ginsenoside and quinidine groups (P<0.05). CONCLUSION  Ginsenoside bind to and inhibit the activity of P-gp, synergizing with paclitaxel to reduce the proliferative and migratory abilities of Caco-2 cells. The combination of ginsenosides and paclitaxel enhances the sensitivity of Caco-2 cells to paclitaxel induced inhibition. The combined use of these two substances is expected to achieve better anticancer effects compared to paclitaxel alone.
  • ORIGINAL ARTICLES
    Development and tissue distribution assessment of a qPCR-based detection method for VGM-R02b in cynomolgus monkeys
    GAO Caihong, REN Xinyi, LUO Wenjing, ZHANG Yufei, CHENG Yuanguo
    2025, 39(2): 100. https://doi.org/10.3867/j.issn.1000-3002.2025.02.003
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    OBJECTIVE  To develop a highly sensitive and specific quantitative real-time polymerase chain reaction (qPCR) method for detecting the VGM-R02b (a gene therapy drug for glutaric acidemia typeⅠ) gene in cynomolgus monkeys and analyze the biological distribution of VGM-R02b. METHODS  A standard curve was constructed using the VGM-R02b standard plasmid [an adeno-associated virus serotype 9 (AAV9) capsid ] on a qPCR platform. The detection method was optimized and validated for key parameters, including the quantitative range, accuracy, precision, dilution linearity, selectivity, specificity, stability, and parallelism. The established method was used to determine the target gene of VGM-R02b in the blood, brain, stomach, heart, liver, spleen, lung, kidney, thymus, and duodenum of cynomolgus monkeys on day 29 (D29) and D92 after a single, unilateral intraventricular injection of VGM-R02b. The biodistribution of VGM-R02b in cynomolgus monkeys was analyzed. RESULTSA qPCR method for quantifying the VGM-R02b target gene in cynomolgus monkeys was established and validated. The standard curve demonstrated a quantitative range of 5.00×109 to 5.00×10^1 copies·μg-1 DNA, with excellent precision, accuracy, dilution linearity, selectivity, and specificity. The target gene in tissues remained stable after being stored at room temperature for 4 h, -15 to -25 ℃ for 9 d, -60 to -80 ℃ for 90 d and after five freeze-thaw cycles. Similarly, the target gene in whole blood remained stable after being stored at room temperature for 4 h, -15 to -25 ℃ for 93 d, -60 to -80 ℃ for 93 d and after five freeze-thaw cycles. Extracted nucleic acid samples also showed stability after being stored at room temperature for 4.25 h, 2-8 ℃ for 24.16 h, -60 to -80 ℃ for 109 d and after five freeze-thaw cycles. The method was also applied to evaluate the biological distribution of the target gene in cynomolgus monkeys. In the control group, the target gene was undetectable on D29 and D92 post-administration, but in the drug administration group, the target gene was distributed across the tissues, with higher concentrations observed in the brain, liver, spleen, and spinal cord, and there were no significant differences in the target gene content across the tissues between D29 and D92. CONCLUSION  The established qPCR method is robust, reliable and suitable for determination of VGM-R02b target gene in cynomolgus monkeys.
  • ORIGINAL ARTICLES
    Prediction of PD-1 monoclonal antibody human pharmacokinetic characteristics based on PK in cynomolgus monkeys
    XU Yanjun, HAN Zijun, WANG Liang, YANG Fang, LOU Beilei, YAN Shaoyu, ZHU Jiman, BAI Lihui, GAO Yong
    2025, 39(2): 109. https://doi.org/10.3867/j.issn.1000-3002.2025.02.004
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    OBJECTIVE  To establish a population pharmacokinetic (PopPK) model to predict the PK characteristics of GLS-010 in humans. METHODS  Fifty-eight cynomolgus monkeys were used, 18 of which were randomly divided into three groups and received a single intravenous infusion of GLS-010 at doses of 2, 6, and 18 mg·kg-1, respectively. The rest were randomly assigned to four groups and received multiple intravenous infusions of GLS-010 at doses of 0, 5, 25, and 100 mg·kg-1, respectively, once a week (quaque week, qw) for five consecutive weeks. Blood samples were collected before and after administration. The concentrations of GLS-010 in the monkey serum were measured using a validated enzyme-linked immunosorbent assay, while those of anti-drug antibodies (ADA) in the cynomolgus monkey serum were determined by ultra-sensitive electrochemiluminescence immunoassay. The PK data on GLS-010 in cynomolgus monkeys was obtained, and the drug-time curves were plotted. A PopPK model was constructed using non-compartmental analysis and evaluated by goodness-of-fit plots and visual predictive checks. The constructed PopPK model was used to predict the PK characteristics in humans, which were finally compared with actual PhaseⅠclinical study results for validation. RESULTS  The predictive results of the PopPK model were highly consistent with the actual PhaseⅠclinical study results. The model was able to predict the human PK characteristics under various dosing regimens, including 1 mg·kg-1 quaque 2 weeks (q2w), 4 mg·kg-1 (q2w), 240 mg (q2w), 240 mg (q3w), and 10 mg·kg-1 (q2w). The predicted maximum plasma concentrations (Cmax) were 24.8, 99.1, 85.0, 85.0, and 247.8 mg·L-1, respectively, and the AUC0-336 h was 4 902.0, 20 060.0, 17 147.7, 22 145.7 (AUC0-504 h), and 50 817.6 mg·h·L-1, respectively. The safety risks for the corresponding dosing regimens were 47.3, 11.6, 13.5, 10.5, and 4.6, respectively. The predicted receptor occupancy at steady state (ROss) at Cmax, average plasma concentration (Cavg), and minimum plasma concentration (Cmin) were 38.8%, 72.7%, 69.4%, 64.1% and 87.2%, 29.1%, 63.8%, 60.0%, 49.8% and 82.1%, 21.9%, 55.5%, 51.3%, 36.3% and 76.7%, respectively. CONCLUSION  The PopPK model can effectively predict the human PK characteristics under different dosing regimens with high consistency with actual PhaseⅠclinical study results, which can serve as an important reference for selection of safe and effective doses for first-in-human research.
  • ORIGINAL ARTICLES
    Mechanism of γ-bungarotoxin induced respiratory disorder in mice
    HUO Mingyang, CHEN Wei, ZHAO Na, SUN Chengbiao, DONG Mingxin, WANG Yan, XU Na, LIU Wensen
    2025, 39(2): 118. https://doi.org/10.3867/j.issn.1000-3002.2025.02.005
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    OBJECTIVE  To investigate the roles and mechanisms of γ-bungarotoxin (γ-BGT) in inducing respiratory distress in mice. METHODS  Six male Kunming mice were selected and anesthetized before tracheal intubation and respiratory recording. After stabilizing respiration, the mice were intraperitoneally injected with γ-BGT at a dose of 6 mg·kg-1. Once a decrease in respiratory frequency was observed, the mice were intravenously injected with nikethamide at a dose of 12.5 mg·kg-1. Respiratory frequency was monitored using the BL420 signal acquisition and processing system. Male Kunming mice were randomly divided into the normal control group (saline, ip), γ-BGT group (6 mg·kg-1, ip), and γ-BGT+nikethamide group (γ-BGT 6 mg·kg-1, ip, followed by nikethamide 12.5 mg·kg-1, ip, when shallow breathing and enhanced abdominal respiration were observed). The levels of Glu and GABA in the medulla oblongata were measured using ELISA. The protein expression levels of GAD65 and GAD67 in the medulla oblongata were determined by Western blotting. Primary mouse medullary neurons were cultured in vitro and divided into the following groups: cell control group, γ-BGT group, carbachol group, gallamine group, γ-BGT+H-89 group, and γ-BGT+Y-27632 group. The γ-BGT group, carbachol group, and gallamine group were incubated with γ-BGT (40 mg·L-1), carbachol (100 mmol·L-1), and gallamine (100 mmol·L-1), respectively, for 4 h. The γ-BGT+H-89 and γ-BGT+Y-27632 groups were pretreated with γ-BGT (40 mg·L-1) for 4 h, followed by incubation with the protein kinase A (PKA) inhibitor H-89 (50 mmol·L-1) and the Ca2+ channel inhibitor Y-27632 (50 mmol·L-1) for another 2 h, respectively. ELISA was used to measure the levels of Glu, GABA, cAMP, and calpain in the primary mouse medullary neurons. Western blotting was employed to assess the protein expression levels of GAD65 and GAD67, and PKA phosphorylation levels. Fluo-4 fluorescent probe was used to detect the intracellular Ca2+ level. RESULTS  The respiratory rate of mice significantly decreased after iv administration of γ-BGT (γ-BGT group) (P<0.05). After treatment with nikethamide (nikethamide group), the respiratory rate significantly recovered (P<0.05). Compared with the normal control group, the γ-BGT group exhibited a significant decrease in Glu content (P<0.05), a significant increase in GABA content (P<0.05), and a significant decrease in the Glu/GABA ratio. Additionally, the protein expression levels of GAD65 and GAD67 were significantly elevated (P<0.05). Compared with the γ-BGT group, the γ-BGT+nikethamide group showed a significant increase in Glu content (P<0.05), a significant decrease in GABA content (P<0.05), a significant increase in the Glu/GABA ratio, and a significant reduction in GAD65 and GAD67 protein expression levels (P<0.05). Compared to the cell control group, the γ-BGT group demonstrated a significant decrease in Glu content (P<0.05), a significant increase in GABA content (P<0.05), and a significant reduction in the Glu/GABA ratio. Furthermore, the protein expression levels of GAD65 and GAD67 were significantly elevated (P<0.05). Additionally, cAMP content, PKA phosphorylation levels, Ca2+ levels, and calpain activity were all significantly increased (all P<0.05). Glu, GABA, Glu/GABA ratio, and GAD expression levels in the γ-BGT group changed in the same way as in the gallamine group; In the γ-BGT+Y-27632 group, calpain activity and expression levels of GAD65 and GAD67 were all significantly decreased (all P<0.05). In the γ-BGT+H-89 group, Ca2+ levels and calpain activity were significantly reduced (all P<0.05). CONCLUSION  γ-BGT-induced poisoning can lead to respiratory distress in mice, possibly through the antagonism of M2 muscarinic acetylcholine receptors in medullary neurons, activation of the cAMP/PKA signaling pathway, elevation of intracellular Ca2+ levels, and increased expression and activity of GAD, resulting in an imbalance of Glu and GABA in the medulla.
    • REVIEWS
  • REVIEWS
    Research progress in nanomaterials induced mitochondrial biogenesis dysfunction and mechanism
    XU Chang, YANG Xiaoya, GUO Jiabin, LI Yujie
    2025, 39(2): 129. https://doi.org/10.3867/j.issn.1000-3002.2025.02.006
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    Nanomaterials have been used in a variety of industries recently, involving foods, chemicals and biomedicine. There are multiple routes through which humans are exposed to nanomaterials, and their toxic effects deserve more attention. In vivo studies have confirmed that nanomaterials exposure can lead to toxicity in such target organs as the heart, liver, kidney, skin and nerve. The toxicity mechanism is related to changes in organelles such as the endoplasmic reticulum, lysosomes and mitochondria. Increasing studies suggest that mitochondria are critical targets for the toxicity of nanomaterials. Mitochondrial biogenesis serves as an important mechanism for maintaining mitochondrial homeostasis, which plays a vital role in the process of nanomaterials-induced cellular toxicity. This article summarizes the current research on the effects of nanomaterials on mitochondrial biogenesis, and elaborates the mechanism through which nanomaterials disrupt mitochondrial biogenesis by triggering oxidative stress, upsetting the homeostasis of calcium ion and disturbing toxicity pathways. This article is expected to provide a reference for toxicity testing and risk assessment of nanomaterials.
  • REVIEWS
    Advances in quantitative determination methods and pharmacokinetics of melatonin and its metabolites
    WANG Mengran, ZHUANG Xiaomei
    2025, 39(2): 138. https://doi.org/10.3867/j.issn.1000-3002.2025.02.007
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    Sleep disorders are becoming a social problem that poses a danger to human health and an obstacle to economic development. As an endogenous hormone, melatonin plays an important role in regulating circadian rhythm. Research has shown that exogenous melatonin holds promising prospects in the treatment of insomnia disorders and thus merits in-depth exploration and development. Given the close relationship between pharmacokinetic properties and drug efficacy as well as toxicity, this paper reviews the quantitative analysis methods for melatonin and its major metabolites, especially the  pretreatment methods for biological samples and the related LC-MS/MS analysis methods. Studies related to the absorption, distribution, metabolism, excretion and drug-drug interactions of melatonin both in vivo and in vitro, as well as the level and species differences of endogenous melatonin are also outlined. Meanwhile, the druggability of melatonin and corresponding solutions are explored in the hope of providing data for the development of exogenous melatonin into novel sleep-regulating drugs.
  • REVIEWS
    Current research and development of anti-pulmonary fibrosis drugs
    WANG Yuanying, YE Qiao
    2025, 39(2): 146. https://doi.org/10.3867/j.issn.1000-3002.2025.02.008
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    Along with more research into the mechanisms of pulmonary fibrosis, anti-pulmonary fibrosis drugs under development are becoming diversified. Over the past decade, multiple phase Ⅱ/Ⅲ clinical trials have been terminated due to insufficient efficacy, with seven single-target monoclonal antibody drugs failing to meet expectations. In contrast, multi-target drugs such as nintedanib and pirfenidone have been successfully marketed, demonstrating favorable clinical outcomes. However, drugs 
    directly targeting transforming growth factor-β have raised safety concerns. Ongoing phase Ⅲ candidates, such as lysophosphatidic acid receptor 1 antagonists and phosphodiesterase 4B inhibitors, do not directly intervene in the transforming growth factor-β signaling pathway. Given the potential limitations of single-target drugs, future drug development is expected to prioritize multi-target and multi-cell strategies while exploring synergistic multi-drug therapies. This article reviews the current clinical trials for anti-pulmonary fibrosis drugs worldwide and the challenges they face in order to provide references for the research and development of new anti-pulmonary fibrosis drugs.