Peptide Biomarkers of drug exposure: from brain microdialysate to plasma

描述(由申请人提供)：本申请的目标是识别反映与成瘾过程的多个阶段相关的大脑区域神经可塑性的血浆肽签名，最初使用大鼠的简单条件位置偏爱(CPP)程序进行建模。提出了一种有重点的方法，在这种方法中，最初的搜索将从与成瘾过程有关的脑区的细胞外区域开始，使用与质谱学(高分辨率纳米孔LC-MS和数据依赖LC-MS2)相关联的微透析。然后，我们将使用LC-MS3和/或LC-MRM以有针对性的方式在血浆中搜索和量化这些已识别的候选多肽。该项目的发现阶段将确定候选多肽的阵列，这些多肽在被认为是成瘾行为中枢的大脑区域，随着长期接触和戒除两种不同药理类别的高度成瘾药物--即吗啡和可卡因--而发生细胞外变化。一个平行的策略将完全集中在容易释放的多肽上，通过构建在钾去极化与基础条件下脑透析液的差减筛选。在这两种方法中，将同时采集血浆样本。随后将有针对性地在血浆中搜索在这两个透析液筛查中确定的多肽。(A)在透析液和血浆中都能检测到的多肽，以及(B)作为药物历史或去极化产物的浓度变化将被选为验证阶段。在验证阶段，在发现阶段确定的候选者将在纵向实验中使用位置偏好范式进行研究，以将血浆含量的变化与条件反射实验的多个阶段相关联：药物条件作用、初始测试、孵化(退出)、重新测试、消失和恢复。我们希望识别血浆中反映成瘾过程不同阶段的单个多肽签名，如果它们是由CPP模拟的话。这些实验将重点放在可卡因和吗啡上，但将扩展到其他滥用药物和其他行为模式(例如自我给药)，在未来的实验中将超出本提案的范围。 公共卫生相关性：这些研究将提供一种简单成瘾行为模型的不同阶段所特有的血浆肽信号。一旦这一原则证明成立，该方法可以扩展到其他更复杂的成瘾模型，并最终扩展到人类，从而提供潜在成瘾过程的标记。这是开发治疗对多种滥用物质成瘾的有效药物的关键问题。

DESCRIPTION (provided by applicant): The goal of this application is to identify plasma peptide signatures that reflect neuroplasticity in regions of the brain associated with multiple stages of the addictive process, modeled initially using the simple conditioned place preference (CPP) procedure in rats. A focused approach is proposed in which the initial search will begin in the extracellular compartment of brain regions implicated in addictive processes, using microdialysis linked to mass spectrometry (high resolution nanobore LC-MS and data-dependent LC-MS2). We will then search for, and quantify, such identified candidate peptides in plasma in a targeted fashion, using LC-MS3 and/or LC-MRM. The discovery phase of the project will identify arrays of candidate peptides that, in brain regions considered central to addictive behavior, change extracellularly in response to chronic exposure to, and abstinence from, two highly addictive drugs of different pharmacological classes; namely morphine and cocaine. A parallel strategy will focus entirely on readily releasable peptides by constructing a subtractive screen of brain dialysates under potassium depolarization versus basal conditions. In both of these approaches blood plasma samples will be collected simultaneously. Peptides identified in either of these dialysate screens will subsequently be searched for in plasma in a targeted fashion. Peptides that are (a) detectable in both dialysate and plasma, and (b) change in concentration in tandem as a product of drug history or depolarization will be selected for the validation phase. In the validation phase, candidates identified in the discovery phase will be studied in longitudinal experiments using the place preference paradigm to correlate changes in plasma content with multiple phases of the conditioning experiment: drug conditioning, initial testing, incubation (withdrawal), re-testing, extinction and reinstatement. We expect to identify individual peptide signatures in plasma that reflect different stages of the addiction process to the extent that they are modeled by CPP. These experiments will focus on cocaine and morphine, but will be extended to other abused drugs and to other behavioral models (e.g. self-administration) in future experiments beyond the scope of this proposal. PUBLIC HEALTH RELEVANCE: These studies will provide plasma peptide signatures unique to different stages of one simple model of addictive behavior. Once this proof of principle is established the approach could be extended to other more complex addiction models, and ultimately to humans, thereby providing markers of underlying addictive processes. This is a key issue for the development of effective medications to treat addiction to multiple classes of abused substances.