Structure-guided discovery of potent and oral soluble epoxide hydrolase inhibitors for the treatment of neuropathic pain

Soluble epoxide hydrolase (sEH) is related to arachidonic acid cascade and is over-expressed in a variety of diseases, making sEH an attractive target for the treatment of pain as well as inflammatory-related diseases. A new series of memantyl urea derivatives as potent sEH inhibitors was obtained using our previous reported compound 4 as lead compound. A preferential modification of piperidinyl to 3-carbamoyl piperidinyl was identified for this series via structure-based rational drug design. Compound A20 exhibited moderate percentage plasma protein binding (88.6%) and better metabolic stability in vitro. After oral administration, the bioavailability of A20 was 28.6%. Acute toxicity test showed that A20 was well tolerated and there was no adverse event encountered at dose of 6.0 g/kg. Inhibitor A20 also displayed robust analgesic effect in vivo and dose-dependently attenuated neuropathic pain in rat model induced by spared nerve injury, which was better than gabapentin and sEH inhibitor (±)-EC-5026. In one word, the oral administration of A20 significantly alleviated pain and improved the health status of the rats, demonstrating that A20 was a promising candidate to be further evaluated for the treatment of neuropathic pain.

Correlation of IDO-1, LAG-3 and TIM-3 with clinicopathological features and prognosis of differentiated thyroid cancer / 中华核医学与分子影像杂志

Objective:To explore the expression of indoleamine 2, 3-dioxygenase 1(IDO-1), lymphocyte-activation gene 3 (LAG-3) and T cell immunoglobulin domain and mucin domain-containing molecule 3 (TIM-3) in differentiated thyroid cancer (DTC), and the value of them on prognosis.Methods:From May 2014 to November 2015, 119 DTC patients (33 males, 86 females, media age: 42 years) who underwent surgical treatment in Shanghai Sixth People′s Hospital were retrospectively analyzed. The expressions of IDO-1, LAG-3 and TIM-3 in the specimens were analyzed by immunohistochemistry and the expression differences between cancer tissues and normal tissues were analyzed by χ2 test. The correlation of IDO-1, LAG-3 and TIM-3 with clinical characteristics were analyzed using logistic regression analysis. The patients were followed up for 5 years, and the relationships of the progression-free survival (PFS) rate with the expressions of the three immune checkpoints were analyzed by Kaplan-Meier method, log-rank test and Cox proportional hazard models. Results:The overall 5-year PFS rate for 119 DTC patients (median follow-up time: 55(2-66) months) was 76.47%(91/119). The positive expression rates of LAG-3 and TIM-3 in cancer tissues were 21.85%(26/119) and 78.15%(93/119) respectively, which were significantly higher than those in normal thyroid tissues (7.34%(8/109) and 62.39%(68/109); χ2 values: 9.43, 6.81, both P<0.05). While the positive expression rate of IDO-1 was 70.59%(84/119) in cancer tissues, which did not show a significant difference from that in normal thyroid tissues (64.22%(70/109); χ2=1.05, P>0.05). Factors associated with the positive expression of LAG-3 included tumors with a single lesion (odds ratio ( OR)=0.248, 95% CI: 0.086-0.716, P=0.010). Log-rank test ( χ2=4.96, P=0.026) and multivariate Cox regression analysis (hazard ratio ( HR)=2.239, 95% CI: 1.013-4.592, P=0.046) suggested that LAG-3 positive expression was an independent risk factor of PFS. The same analysis of TIM-3 found no clinicopathological factors related to TIM-3 positive expression ( OR: 0.309-3.084, all P>0.05) and no association between TIM-3 positive expression and PFS ( χ2=0.008, P=0.929). Conclusion:The expressions of LAG-3 and TIM-3 are significantly increased in DTC tissues, and the higher expression of LAG-3 is associated with the worse prognosis, suggesting that LAG-3 may be a potential target for immunotherapy in DTC patients.

Efficacy evaluation of percutaneous osteoplasty combined with 131I therapy and survival analysis in patients with bone metastasis from differentiated thyroid carcinoma / 中华核医学与分子影像杂志

Objective:To explore the efficacy of percutaneous osteoplasty (POP) combined with 131I therapy in patients with bone metastases from differentiated thyroid carcinoma (DTC) and assess the survival. Methods:From Januray 2008 to January 2020, 29 DTC patients with bone metastases (16 males, 13 females, age range: 24-64 years) who received POP combined with 131I therapy in Shanghai Sixth People′s Hospital were retrospectively analyzed. Clinical data and characteristics of patients were analyzed. The efficacy and prognosis were evaluated based on the changes of serum thyroglobulin (Tg) and relieving condition of bone pain after the combined treatment. χ2 test was used to determine the association between clinical characteristics and efficacy, and Kaplan-Meier analysis was used to estimate the overall survival (OS) rate. Results:The biochemical response rate of serum Tg was 68.97% (20/29) after the combined treatment. For the influence on changes of serum Tg, whether or not combined with non-osseous distant metastasis, and cumulative dose of 131I treatment(≥22.2 vs <22.2 GBq) were statistically significant ( χ2 values: 5.448 and 4.371, both P<0.05). The rate of bone pain relief was 65.52%(19/29). Age (≥55 vs <55 years) and the cumulative dose of 131I treatment had statistically influences on bone relief ( χ2 values: 7.486 and 5.154, both P<0.05). The 5-years OS rate of patients was 87.68%, while the 10-years OS rate was 65.76%. Conclusion:POP combined with 131I therapy is effective on relieving the pain, reducing the serum Tg to some extent, and improving the long-term survival of DTC patients with bone metastasis.

Simultaneous Determination of Shikonin, Acetylshikonin and β, β-dimethylacrylshikonin in Arnebia euchroma by RP-HPLC / 中国药房

OBJECTIVE: To establish a method for the simultaneous determination of shikonin, acetylshikonin and β, β-dimethylacrylshikonin in Arnebia euchroma. METHODS: RP-HPLC method was adopted. The determination was performed on Kromasil 100-5 C18 column with mobile phase consisted of acetonitrile-0. 1％ formic acid solution (80: 20, V/V) at the flow rate of 1. 0 mL/min. The detection wavelength was set at 516 nm, column temperature was 25 ℃, and sample size was 10 μL. RESULTS: The linear ranges of shikonin, acetylshikonin and β, β-dimethylacrylshikonin were 0. 404-10. 100 μg/mL(r=0. 999 8), 5. 350-107. 000 μg/mL(r=0. 999 6), 2. 035-40. 700 μg/mL(r=0. 999 8), respectively. The limit of quantitation was 0. 40, 2. 91, 1. 34 μg/mL, and the limit of detection was 0. 12, 0. 87, 0. 40 μg/mL. RSDs of precision, stability and reproducibility tests were all lower than 2. 0％ (n=6). The recovery rate were 99. 12％-104. 18％ (RSD=1. 85％, n=6), 96. 51％-100. 21％ (RSD=1. 43％, n=6), 98. 11％-102. 51％ (RSD=1. 42％, n=6), respectively. CONCLUSIONS: The method is simple, precise, stable and reproducible. It can be used for simultaneous determination of shikonin, acetylshikonin and β, β-dimethylacrylshikonin in A. euchroma.

Targeted inhibition of microRNA-200c on expression of AP-2α to enhance the proliferation of colon cancer cells in vitro / 肿瘤研究与临床

Objective To investigate the impact of miR-200c overexpression on colon cancer cell proliferation ability and the related mechanism.Methods MicroRNAs which may combined with the transcription factor AP-2α were screened and forecasted by the bioinformatics database,while its eukaryotic expression plasmids and specific inhibitor were synthesized.Plasmids PEZX-miR-200c,PEZX-NC,pmirGLO-AP-2α3'UTR,pmir-GLO and the specific inhibitors miR-67-inhibtor,miR-200c-inhibitor were transfected in vitro into colon cancer HCT-116 and SW480 cells and the HEK293T cell by Lipofectamine2000.The expression of AP-2α mRNA and protein in colon cancer cells was analyzed by qRT-PCR,Westem blot and immunocytochemical staining.CCK-8 assay and flow cytometry were adopted to observe the effect of miR-200c on colon cancer cells proliferation and apoptosis.Dual-Luciferase assay experiments were performed to observe the relative luciferase activity induced by miR-200c.Results The proliferation activity was significantly decreased in anti-miR-200c/SW480 group,while in PEZX-miR-200c/HCT-116 group,it was higher than that in PEZX-NC/HCT-116 group.The apoptosis ability was significantly increased in anti-miR-200c/SW480 group [(78±0.7) ％ vs (66±1.1) ％,P ＜ 0.05].The expression of AP-2o both in mRNA and protein levels was decreased in PEZX-miR-200c/HCT-116 group,while the protein level was increased in Anti-miR-200c/SW480 group.The relative luciferase activity inhibited by miR-200c was decreased in HEK-293T cells transfected with PEZX-miR-200c and pmir-GLO-AP-2α3' UTR (0.51±0.09 vs 0.98±0.04,P ＜ 0.01).Conclusion MicroRNA-200c could promote cell proliferation ability by targeting transcriptional factor AP-2α in human colorectal cancer cells.

Silencing AP-2α induces chemoresistance to oxaliplatin of HCT-116 cell in colorectal carcinoma HCT-116 cells / 肿瘤研究与临床

Objective To investigate the effect of AP-2α on the chemoresistance to oxaliplatin of colorectal carcinoma cell and its related mechanism.Methods Plasmid of GV102-AP-2α-RNAi (experimental group) and control plasmid GV102-NC (negative control group) were transfected into HCT-116 using Lipofectamine 2000 respectively.The AP-2α expression levels of mRNA and protein of experimental group,control group and HCT-116 blank group were detected by qRT-PCR and Western blot.Cell proliferation assay was performed using the CCK-8 and the apoptosis assays were preformed with Annexin V-PE Apoptosis Kit.Results The AP-2α expression levels of mRNA and protein both decreased after transfection of AP-2α-RNAi plasmid,moreover,the effect produced by subsequence 1 was the most significant.After treatment by oxaliplatin,AP-2α protein levels increased with time while mRNA did not change significantly.Western blot results suggested that the level of AP-2α protein in experimental group which was maintained in oxaliplatin was lower than the negative control group.CCK-8 results suggested that cell proliferation ability was significantly higher for the experimental group maintained in oxaliplatin [(88±3) ％] than the negative control group maintained in oxaliplatin [(57±3) ％] and the blank group maintained in oxaliplatin [(73t4) ％].Flow cytometry showed that the apoptosis rate of the experimental group maintained in oxaliplatin [(15.07±1.20) ％] was lower than the control group maintained in oxaliplatin [(24.93±0.90) ％] and the blank group maintained in oxaliplatin [(23.71±1.32) ％].Conclusion AP-2α may be related to the sensitivity of colon cancer cells to oxaliplatin.