Modification Of Endomorphin-1 To Enhance The Blood-brain Barrier Permeability: Design, Synthesis And Biological Activity Characterization

Opioid peptides have been designed for the treatment of pain, and the mediation of opioid analgesia has long been thought to occur exclusively within the central nervous system (CNS). In 1997, two tetrapeptides, endomorphin (EM)-1 (Tyr¹-Pro²-Trp³-Phe⁴-NH₂) and EM-2 (Tyr¹-Pro²-Phe³-Phe⁴-NH₂), have been isolated from bovine frontal cortex and were reported to be the endogenous ligands to u-opioid receptors. With high affinity and selectivity for the u-opioid receptors, these two neuropeptides were found to elicit equipotent analgesia to morphine but without some of its undesirable side effects. Unfortunately, both EM-1 and EM-2 undergo metabolic degradation by peptidases. Besides, the existence of the blood-brain barrier (BBB) also limits the transit of peptides into the CNS, thus further reducing their therapeutic benefits. One solution to these problems is to design and synthesize new and improved EM analogues by chemical modification. Herein, EM-1 was chosen as the parent peptide because there were evidences that EM-1 had a more favorable therapeutic profile than EM-2 and otherμ-opioids. The aim of the present study is to make EM-1 overcome the problems of enzymatic degradation and limited entry into the CNS by systematic chemical modification, thus being able to produce analgesia after systemic administration. A clear understanding of the potential contribution of these modifications to the physicochemical properties and biological activity of EM-1 will further our knowledge of the structural requirements that is necessary for its pharmacological effects, and will provide important evidences for the development of novel analgesics based on EM-1.Three groups of modification approaches, including N-terminal cationization by N^α-amidination on Tyr¹, unnatural amino acid (D-Ala, D-Pro and Sar) substitutions in position 2 and C-terminal chloro-halogenation, were introduced into the primary structure of EM-1. Ten EM-1 derived peptides were synthesized. Effects of each modification on enzymatic breakdown, lipophilicity and protein binding were determined and compared. For the determination of the biological activity profiles, we used a battery of test systems, including receptor binding assays, isolated tissue assays and in vivo assays. Both N-terminal cationization and C-terminal chloro-halogenation caused a decrease of affinity for the u-opioid receptor, but they increased protein binding ability of peptides. All of the synthetic peptides exhibited increased in vitro metabolic stability to the parent in both mouse brain homogenate and serum. The N^α-amidination contributed to the majority enhancement of brain stability, whereas chloro-halogenation, together with amino acid substitution in position 2, was more important for the serum stability enhancement. Determination of the octanol/buffer coefficient revealed that chloro-halogenation did compromise the decreased lipophilicity caused by N^α-amidination, and introduction of D-Ala as well as D-Pro-Gly, but not Sar, in place of L-Pro², also increased the overall lipophilicity to some extent. In the antinociceptive activity test, intracerebroventricular injection of GDAPC showed the strongest analgesia among all of the peptides tested, being 3 times more potent than EM-1. In addition, all of the synthetic peptides showed an increased duration of action compared with the parent peptide. We also found that in comparison with EM-1, the four D-Ala-containing tetrapeptides and the chloro-halogenated D-Pro-Gly-containing pentapeptide elicited significant and prolonged central-mediated analgesia upon both subcutaneous and intravenous administration, indicating that more peptides might reach the CNS, eliciting greater analgesic effect. When given intravenously to mice, these five EM-1 derived peptides exhibited different pharmacokinetic properties and had significantly increased elimination half-lives compared with the parent. They showed an apparent distribution to the brain after intravenous administration, with the blood-to-brain influx rates ranging from 0.241 to 0.894μl/g·min, thus providing preliminary evidence for the central mechanism in the antinociception test. We extended to explore the pharmacological characteristics of EM-1 and its five derived peptides in mouse colon. The five EM-1 derived peptides displayed a profile similar to the parent on the mouse colon in vivo. When given centrally, they all dose-dependently inhibited colonic bead expulsion and retarded the rate of large intestinal transit in mice in a naloxone-sensitive manner. They also induced dose-dependent contraction on the isolated longitudinal muscle strips of mouse distal colon, which were significantly antagonized by naloxone,β-funaltrexamine, tetrodotoxin and indomethacin. Differences among the synthetic tetrapeptides in both in vivo and in vitro studies suggested that N-terminal cationization had a greater influence on the regulation of the colonic motor actions relative to the C-terminal halogenation. We compared the inhibitory effects of morphine and these peptides on the electrical field stimulation (EFS)-induced cholinergic bronchoconstriction in normal rats. All of the drugs tested inhibited the EFS-induced cholinergic constriction in rat isolated bronchus in a concentration- and frequency-dependent and naloxone-sensitive manner. The inhibitory effect of EM-1 on the EFS-induced bronchoconstriction was stronger than that of the same dose of morphine as well as the five EM-1 derived peptides at the same frequency. Our results indicated that N-terminal cationization and C-terminal halogenation showed opposite influences on the inhibitory effects of the four D-Ala-containing peptides in rat isolated bronchus.