Proteomic Biosignatures of Chronic Drug Exposure

描述（由申请人提供）：基于长期血浆蛋白变化的生物标记，与慢性药物暴露特异性相关，可用于识别易重复用药的患者，从而促进早期干预。我们将通过长期暴露于成瘾性药物和差异蛋白质组学表达来测试发现这种特征的可行性。大鼠将接受可卡因或非成瘾性多巴胺转运抑制剂、吗啡或生理盐水的治疗，根据14天的剂量递增计划（慢性暴露），并在最后一次药物治疗后75天收集血浆。蛋白质组学分析将包括逐步耗尽丰富的血浆蛋白，然后通过液相色谱联用在线质谱分析耗尽的蛋白质组，包括非常低丰度的蛋白质和中等丰度的结合蛋白。如果蛋白质的表达改变可以维持数周，例如30天，并最终恢复到基线水平，那么这些蛋白质就可以作为候选的生物标志物，在成瘾药物不能再容易测量的时候，用于检测最近的暴露情况。首选候选药物将在不需要蛋白质抗体的定量多重检测中通过多重反应监测进行验证。蛋白质组学变化将被检查与慢性药物治疗的行为和神经化学后遗症的时间相关性，这将被并行测量。统计分析和数据挖掘将用于识别具有所需属性的生物签名。一项成功的可行性研究将刺激并为人类受试者样本的类似研究提供信息。

DESCRIPTION (provided by applicant): A biosignature based on long-term plasma protein changes specifically associated with chronic drug exposure can be used to identify patients predisposed to repeated drug use and thereby facilitate early intervention. We will test the feasibility of finding such a signature using chronic exposure of rats to addictive drugs and differential proteomic expression. Rats will receive treatment with cocaine or a non-addictive dopamine transporter inhibitor, or morphine, or saline according to a 14 day escalating dose schedule (chronic exposure) and plasma collected up to 75 days following last drug treatment. Proteomic analysis will involve stepwise depletion of abundant plasma proteins followed by liquid chromatography coupled online with mass spectrometry to analyze both the depleted proteome consisting of very low abundance proteins and the bound proteins of intermediate abundance. Proteins whose altered expression is maintained for several weeks, e.g. 30 days, and eventually return to baseline would be candidate biomarkers that could be used to detect recent exposure at a time when the addictive drug can no longer be readily measured. Top candidate will be validated by multiple reaction monitoring in a quantitative multiplexed assay that does not require antibodies to the proteins. Proteomic changes will be examined for temporal correlation with behavioral and neurochemical sequelae of chronic drug treatment that will be measured in parallel. Statistical analysis and data mining will be used to identify a biosignature with the desired properties. A successful feasibility study would stimulate and inform similar studies on human subject samples. PUBLIC HEALTH RELEVANCE: There is an unmet need for analytical biomarkers, a biosignature, of chronic drug exposure that would help to identify patients predisposed to repeated drug. An effective test could become part of routine medical care that would identify patients in need of treatment for substance use disorders and mitigate the personal and societal burdens of drug addiction.