Development of a New Analgesic Based on Metabolism of Endomorphin, an Endogenous Opioid Peptide

内吗啡肽-2（Tyr-Pro-Phe-PheNH_2）是μ-阿片受体的内源性配体。内吗啡肽-2作为神经递质或神经调质的生理功能可能与其他神经肽一样，通过脑突触中的快速酶促过程而终止。本研究旨在研究内吗啡肽-2在小鼠脑突触膜上的代谢。主要代谢产物为游离酪氨酸、游离苯丙氨酸、Tyr-Pro和PheNH_2。内吗啡肽-2的降解和主要代谢产物的积累都被二肽基肽酶IV的特异性抑制剂如diprotin A和B抑制。而苯丙氨酸和苯丙氨酸氨解氨酶抑制剂bestatin的存在则使Phe-PheNH_2和Pro-Phe-PheNH_2的积累增加，而使游离苯丙氨酸和PheNH_2的积累减少。此外，纯化的二肽基肽酶IV在切割位点Pro^2-Phe^3键处水解内吗啡肽-2。因此，脑突触膜对内吗啡肽-2的降解似乎主要是通过二肽基肽酶IV切割Pro^2-Phe^3键，然后通过氨肽酶从释放的片段Phe-Phe NH_2中释放游离苯丙氨酸和Phe NH_2来进行的。我们还在小鼠缩爪实验中检测了双蛋白A对侧脑室注射内吗啡肽-2诱导的抗伤害性感受的作用。同时注射内吗啡肽-2可增强内吗啡肽-2诱导的抗伤害作用。这些结果表明，二肽基肽酶IV可能是一个重要的肽酶负责终止内吗啡肽-2诱导的抗伤害性在脊髓上水平的小鼠。这些发现也表明，选择性二肽基肽酶IV抑制剂或二肽基肽酶IV抗性内吗啡肽-2类似物具有作为镇痛剂的临床应用的潜力。

Endomorphin-2 (Tyr-Pro-Phe-PheNH_2) was discovered as an endogenous ligand for the mu-opioid receptor. The physiological function of endomorphin-2 as a neurotransmitter or neuromodulator may cease through the rapid enzymatic process in the synapse of brain, as for other neuropeptides. The present study was conducted to examine the metabolism of endomorphin-2 by synaptic membranes prepared from mouse brain. Major metabolites were free tyrosine, free phenylalanine, Tyr-Pro and PheNH_2. Both the degradation of endomorphin-2 and the accumulation of major metabolites were inhibited by specific inhibitors of dipeptidyl peptidase IV, such as diprotin A and B. On the other hand, the accumulation of Phe-PheNH_2 and Pro-Phe-PheNH_2 was increased in the presence of bestatin, an aminopeptidase inhibitor, whereas that of free phenylalanine and PheNH_2 was decreased. Furthermore, purified dipeptidyl peptidase IV hydrolyzed endomorphin-2 at the cleavage site, Pro^2-Phe^3 bond. Thus, degradation of endomorphin-2 by brain synaptic membranes seems to take place mainly through the cleavage of Pro^2-Phe^3 bond by dipeptidyl peptidase IV, followed by release of free phenylalanine and Phe NH_2 from the liberated fragment, Phe-Phe NH_2 by aminopeptidase. We have also examined that the effect of diprotin A on the antinociception induced by intracerebroventricularly administered endomorphin-2 in the mouse paw withdrawal test. Diprotin A simultaneously injected with endomorphin-2 enhanced endomorphin-2-induced antinociception. These results indicate that dipeptidyl peptidase IV may be an important peptidase responsible for terminating endomorphin-2-induced antinociception at the supraspinal level in mice. These findings also suggest that selective dipeptidyl peptidase IV inhibitors or dipeptidyl peptidaseIV-resistant endomorphin-2 analogues have the potential for the clinical use as analgesics.