Preclinical to Clinical Translation of CNS Transporter Occupancy of TD-9855, a Novel Norepinephrine and Serotonin Reuptake Inhibitor

Background:

Monoamine reuptake inhibitors exhibit unique clinical profiles that reflect distinct engagement of the central nervous system (CNS) transporters.

Methods:

We used a translational strategy, including rodent pharmacokinetic/pharmacodynamic modeling and positron emission tomography (PET) imaging in humans, to establish the transporter profile of TD-9855, a novel norepinephrine and serotonin reuptake inhibitor.

Results:

TD-9855 was a potent inhibitor of norepinephrine (NE) and serotonin 5-HT uptake in vitro with an inhibitory selectivity of 4- to 10-fold for NE at human and rat transporters. TD-9855 engaged norepinephrine transporters (NET) and serotonin transporters (SERT) in rat spinal cord, with a plasma EC₅₀ of 11.7ng/mL and 50.8ng/mL, respectively, consistent with modest selectivity for NET in vivo.Accounting for species differences in protein binding, the projected human NET and SERT plasma EC₅₀ values were 5.5ng/mL and 23.9ng/mL, respectively. A single-dose, open-label PET study (4–20mg TD-9855, oral) was conducted in eight healthy males using the radiotracers [¹¹C]-3-amino-4- [2-[(di(methyl)amino)methyl]phenyl]sulfanylbenzonitrile for SERT and [¹¹C]-(S,S)-methylreboxetine for NET. The long pharmacokinetic half-life (30–40h) of TD-9855 allowed for sequential assessment of SERT and NET occupancy in the same subject. The plasma EC₅₀ for NET was estimated to be 1.21ng/mL, and at doses of greater than 4mg the projected steady-state NET occupancy is high (>75%). After a single oral dose of 20mg, SERT occupancy was 25 (±8)% at a plasma level of 6.35ng/mL.

Conclusions:

These data establish the CNS penetration and transporter profile of TD-9855 and inform the selection of potential doses for future clinical evaluation.     

携带靶向沉默LSD1基因表达shRNA慢病毒载体的构建与鉴定

目的:构建慢病毒干扰LSD1表达的载体并鉴定。方法:设计并合成3对针对LSD1基因的shRNA链,退火形成双链,与酶切的质粒连接,转化DH5a菌株,提取质粒,进行酶切鉴定和测序分析。脂质体法转染人胃癌MKN-28细胞,通过荧光倒置显微镜判定转染效率,并通过real time-PCR法检测细胞中LSD1基因的表达水平。结果:经酶切鉴定筛选出的重组质粒测序结果与目的序列相同,重组质粒构建成功;转染胃癌MKN-28细胞后,LSD1基因在mRNA水平的表达降低。结论:成功构建了靶向LSD1基因的shRNA慢病毒载体,并筛选出1种对LSD1基因有显著抑制作用的shRNA。     

Epigenetic silencing BCL6B induced colorectal cancer proliferation and metastasis by inhibiting P53 signaling

BCL6B, a homologue of BCL6, has been reported to be frequently methylated in human gastric cancer. The epigenetic change and the function of BCL6B remains to be elucidated in colorectal cancer. 7 colorectal cancer cell lines (RKO, HT-29, DLD1, LOVO, HCT116, SW480, SW620) and 102 cases of primary colorectal cancer samples were used in this study. Semi-quantitative RT-PCR, methylation specific PCR (MSP), Flow cytometry and western blot were employed. Loss of BCL6B expression was found in HT29, RKO LOVO, SW480, SW620 and DLD1 cells, and reduced expression was found in HCT116 cell line. Complete methylation was found in HT29, RKO, LOVO, SW480, SW620 and DLD1 cells, partial methylation was detected in HCT116 cells. Restoration of BCL6B expression was induced by 5-Aza treatment in these colorectal cancer cells. BCL6B was methylated in 79.4% (81/102) of primary human colorectal cancer and reduced expression was associated with promoter region hypermethylation (p < 0.05). Methylation of BCL6B is associated with late stage (p < 0.05) and lymph node metastasis (p < 0.05). Re-expression of BCL6B inhibited cell proliferation, invasion and migration in RKO and HT29 cells. BCL6B activated P53 signaling and induced apoptosis, Re-expression of BCL6B sensitized RKO and HT29 cells to 5-fluorouracil. In conclusion, BCL6B was frequently methylated in human colorectal cancer and its expression was regulated by promoter region methylation. Methylation of BCL6B is a prognostic and chemo-sensitive marker in colorectal cancer. BCL6B suppresses colorectal cancer growth by activating P53 signaling.     

104例成人急性白血病生存分析

目的 总结成人急性白血病患者的预后因素。方法 对我院3年来104例治疗和随访的成人急性白血病患者，采用对数秩检验(Logrank)和比例危险率(COX)回归模型分析其预后因素。结果 104例患者中有15例达5年以上长期生存，总的5年长期生存率为14．4％，急性髓细胞白血病(AML)为18．6％，急性淋巴细胞白血病(ALL)为1.8％，确诊后第1年病死率高；急性早幼粒细胞白血病(M3)的长期生存率较其他类型高。第1次化疗诱导缓解、骨髓移植、乳酸脱氢酶(LDH)正常的患者生存时间较长，核型结构或数目异常组生存概率均低于正常棱型组。结论 成人急性白血病5年长期生存率为14．4％，M3预后较好，第1次诱导是否缓解及是否骨髓移植是影响长期生存的主要预后因素，LDH可考虑作为急性白血病(AL)疗效及预后判断的一项参考指标，核型分析在AL中具有独立于其他临床指标的预后意义。     

心肌梗死冠状动脉介入治疗107例健康知识水平调查分析

目的:调查心肌梗死患者行冠状动脉介入治疗(PC I)后健康知识的掌握情况及其影响因素。方法:采用自行设计的调查表,以问卷调查的方式,调查患者对健康知识的了解情况。结果:患者缺乏相关的健康知识,其知识水平与住院的次数及文化程度有关。结论:对初次住院和文化程度较低的患者应加强健康教育指导,对相关的医学知识应给予重点指导。     

中药威麦宁改善晚期肺癌患者生活质量及免疫功能的作用

目的:观察中药威麦宁对晚期肺癌患者生活质量和免疫功能的影响。方法:收集2002-01/2004-04哈尔滨医科大学附属肿瘤医院内科明确病理诊断并预计生存期>3个月的中晚期(Ⅲ,Ⅳ)肺癌患者为观察对象。随机数字表法分为化疗 威麦宁胶囊治疗组(n=32)和单纯化疗组(n=33)。化疗 威麦宁胶囊组在化疗的同时,给予威麦宁胶囊饭后口服,3次/d,6～8粒/次,2个月为一个疗程;单纯化疗组单纯化疗,每4周为1个周期,重复3个周期。治疗前后采用生活质量调查表进行生活质量评估。该量表有12个项目。每个项目采用5级(1～5分)评分,得分越高表示生活质量越好。满分为60分,最差为12分。临床受益情况评估以KPS评分和体质量变化为指标。KPS评分为反应患者体力变化情况的百分制评分方案。治疗后KPS评分增加≥10分为改善;减少≥10分为降低;变化<10分为稳定。体质量变化:治疗后增加≥1.0kg为改善;减少≥1.0kg为降低;增加或减少<1.0kg为稳定。于化疗3个周期后进行评估。毒性反应按世界卫生组织抗癌药物毒性分级0-IV度标准进行评价,同时进行T细胞亚群的检测和咳嗽、气促等临床表现的观察。结果:按实际处理分析,进入结果分析患者63例。①生活质量:化疗 威麦宁胶囊治疗组生活质量改善高于单纯化疗组(P<0.01)。②临床受益情况:化疗 威麦宁胶囊治疗组KPS评分稳定率明显高于单纯化疗组(65%,38%,P<0.05);化疗 威麦宁胶囊治疗组体质量变化稳定率明显高于单纯化疗组,差异有显著意义(68%,34%,P<0.01)。③免疫功能:化疗 威麦宁胶囊治疗组T细胞亚群细胞数较化疗前提高[(2.23±0.7),(1.90±0.6),P<0.05];单纯化疗组化疗后T细胞亚群细胞数较化疗前明显下降[(1.10±0.9),(1.80±0.7),P<0.05]。④副反应与血液学毒性:单纯化疗组白细胞减少以及恶心、呕吐反应比化疗 威麦宁胶囊治疗组严重(P<0.05以及P<0.01),两组均未发生严重毒性反应。⑤两组患者临床表现咳嗽,气促等症状缓解。结论:威麦宁胶囊对晚期肺癌患者的毒副反应小,患者的生活质量改善明显,免疫功能提高。     

免疫磁性分选系统大量分选CD34+细胞的实验研究

CD3 4 细胞的分离纯化在自体外周血干细胞、异基因骨髓移植 /外周血干细胞移植及干细胞研究中具有重要的应用价值。为了摸索大量分选CD3 4 细胞的方法 ,本研究应用Isolex3 0 0i磁性分选系统富集CD3 4 细胞 ,采用流式细胞术监测分选前后细胞表面标志 ,经CD3 4 细胞体外增殖实验及克隆形成实验验证分选获得的CD3 4 细胞生物学活性。结果显示 ,所完成的 5例次自体外周血富集CD3 4 细胞时 ,先收获单个核细胞约 ( 3 .5 -6.0 )× 10 10 ,CD3 4 细胞占单个核细胞百分率为 ( 0 .5 5 - 1.2 ) % ;收获的CD3 4 阳性细胞总数为 ( 2 - 3 )× 10 8,纯度为 ( 75 - 85 ) % ,回收率为 ( 4 0 - 65 ) %。体外实验表明 ,在SCF IL 3 FL TPO EPO存在下 ,经过 3 - 4天培养 ,可扩增 2 - 3倍 ;经CFU GM、BFU E集落形成实验显示具有形成集落的能力 ,证实分选后细胞具有造血祖细胞生物活性。结论 :应用Isolex3 0 0iCD3 4 细胞分选仪可以高效大量富集CD3 4 细胞 ,适于临床应用。     

对分课堂在《中医诊断学》教学中的应用及探索

探讨对分课堂在《中医诊断学》教学中的应用。将对分课堂授课模式引入《中医诊断学》教学过程中，通过学生的课堂表现及课后对学生的问卷调查，评价对分课堂教学模式对《中医诊断学》教学效果的影响。学生普遍认可对分课堂教学模式，学生课堂活跃程度提高、主观能动性增加，自主学习能力增加，学习效率提高。通过对分课堂教学，有助于提高学生对《中医诊断学》的学习热情，提高学习成绩，提升教学质量。具有一定的推广意义。