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血浆肽特征，这些特征反映了与成瘾过程的多个阶段相关的大脑区域的神经可塑性，最初使用大鼠的简单条件位置偏好（CPP）程序建模。本文提出了一种重点研究方法，使用与质谱（高分辨率纳米孔LC-MS和数据依赖LC-MS2）相关的微透析技术，从与成瘾过程有关的大脑区域的细胞外隔室开始初步研究。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并量化这些已确定的候选肽。该项目的发现阶段将确定候选肽阵列，这些肽被认为是成瘾行为的核心大脑区域，在长期接触和戒断两种不同药理学类别的高成瘾性药物时，细胞外会发生变化；即吗啡和可卡因。一个平行的策略将通过构建钾去极化与基础条件下脑透析液的减法筛选，完全关注易于释放的肽。在这两种方法中，血浆样本将同时收集。在这些透析液筛选中确定的肽将随后在血浆中以有针对性的方式进行搜索。验证阶段将选择(a)在透析液和血浆中均可检测到的肽，以及(b)作为药物史或去极化产物的浓度串联变化的肽。在验证阶段，在发现阶段确定的候选药物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作用实验的多个阶段（药物条件作用、初始测试、孵育（退出）、重新测试、消失和恢复）联系起来。我们期望在血浆中识别出反映成瘾过程不同阶段的个体肽特征，以达到CPP建模的程度。这些实验将集中在可卡因和吗啡上，但在未来的实验中，将扩展到其他滥用药物和其他行为模式（如自我给药），超出本提案的范围。

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.