BRAIN TARGETING OF THYROTROPIN RELEASING HORMONE ANALOGS

促甲状腺激素释放激素（TRH）类似物为中枢神经系统的各种疾病（主要与运动神经元疾病、阿尔茨海默病、电休克治疗等引起的胆碱能功能减退有关）提供了潜在的治疗方法。该项目的主要目标是开发和评估通过化学酶方法靶向中枢活性类似物进入中枢神经系统（CNS）的化学递送系统。由于共价连接的亲脂官能团（1,4-二氢trigonellyl和亲脂部分），“包装”肽通过被动运输穿过血脑屏障，一旦进入中枢神经系统，转化为保留在目标位点的离子trigonellyl衍生物。然后，通过顺序代谢得到具有生物活性的肽。新的脑靶向系统将基于系统修饰先导化合物（[Leu2]TRH）来设计和合成，以改善大脑中类似物的隔离和/或增强生物活性肽的递送后稳定性。这些修改将允许减少系统给药剂量，也允许增加实验或治疗药物在中枢神经系统中的停留时间。我们的假设是，由于酯酶裂解保护酯功能后，酶的生物活化率更高，因此利用α -羟甘氨酸通过肽酰氨基乙酸裂解酶（PGL, EC 4.3.2.5）的作用实现羧基端酰胺化可以提高cns隔离的效果。其中羧基末端脯氨酸酰胺被l -胡椒果酸取代或氨基末端焦谷氨酰残基被非天然片段取代的类似物也将纳入适当的靶向系统中。设计和开发将由理论计算支持。新设计的类似物将被测试与大脑TRH受体的结合，以比较它们与先导化合物的内在活性。体外稳定性和代谢实验将解决优化和实际开发。脑组织的稳定性研究将解决肽激活和/或裂解的速率、位点和程度，以探测cns隔离的关键步骤。体内分布和代谢研究将评估该策略在大脑中运输和隔离TRH类似物的功效。我们将研究脑递送的药代动力学，预测前体的“锁定”，以及合成靶向系统外给药后生物活性肽的释放。脑传递类似物效果的比较药效学评估将通过体内脑微透析研究来解决，其中将分析治疗引起的乙酰胆碱水平的变化。最终，药理学实验将在动物身上进行，以调查这种方法治疗与胆碱能功能丧失相关的疾病的潜力。戊巴比妥诱导睡眠的拮抗作用将被用作评估脑靶向化合物急性效应的一般范例。行为观察、剂量依赖性和作用时间也将通过适当的方法或研究设计来解决。

Thyrotropin-releasing hormone (TRH) analogues offer potential treatment of various maladies of the central nervous system (associated mainly with cholinergic hypofunction due to motorneuron diseases, Alzheimer's disease, electroconvulsive shock therapy, etc.). The main objective of the project is to develop and evaluate chemical delivery systems for targeting centrally active analogues into the central nervous system (CNS) by a chemical-enzymatic approach. Due to covalently attached lipophilic functional groups (a 1,4-dihydrotrigonellyl and a lipophilic moiety) a "packaged" peptide crosses the BBB by passive transport and, once in the CNS, converts to an ionic trigonellyl derivative that is retained at the target site. Then, the biologically active peptide is obtained by sequential metabolism. New brain-targeting systems will be designed and synthesized based on systematically modifying a lead compound ([Leu2]TRH) to improve sequestration of the analog in the brain and/or enhance post-delivery stability of the biologically active peptide. These modifications will allow for a decrease of the systemically administered dose and also for an increase in the residence time of the experimental or therapeutic agent in the CNS. Our hypothesis is that the efficacy of CNS-sequestration can be improved by using alpha-hydroxyglycine to achieve carboxy-terminal amidation via peptidylamidoglycolate lyase (PGL, EC 4.3.2.5) action because of the higher rate of enzymatic bioactivation after esterase cleavage of the protecting ester function. Analogues in which the carboxy-terminal prolinamide is replaced by L-pipecolic acid or the amino-terminal pyroglutaminyl residue is subtituted by an unnatural moiety will also be incorporated into appropriate targeting systems. The design and development will be supported by theoretical calculations. The newly designed analogs will be tested for binding to brain TRH receptors to compare their intrinsic activity with that of the lead compound. In vitro stability and metabolism experiments will address optimization and practical development. Stability studies in brain tissue will address rates, sites and extent of peptide activation and/or cleavage to probe crucial steps in the CNS-sequestration. In vivo distribution and metabolism studies will assess the efficacy of the strategy to transport and sequester the TRH analogs in the brain. We will examine pharmacokinetics of brain-delivery, "lock-in" of the predicted precursors, and the release of the biologically active peptide after parenteral administration of the synthesized targeting systems. Comparative pharmacodynamic evaluation of the effect of brain-delivered analogues will be addressed via in vivo cerebral microdialysis studies in which changes in acetylcholine levels due to treatment will be assayed. Ultimately, pharmacological experiments will be carried out in animals to survey the potential of the approach to treat maladies associated with the loss of cholinergic functions. The antagonism of pentobarbital-induced sleeping will be used as general paradigm to assess the acute effects of brain-targeted compounds. Behavioral observations, dose-dependence and duration of action will also be addressed by appropriate method or study designs.