Design And Opioid Activity Research Of Novel EM-1 Analogs With CPPs Linked At C-terminus

Morphine is the most frequently used as analgesic agent for the treatment of severe pain. However the use of morphine is limited by some severe side effects such as constipation, respiratory depression and addiction. In 1997, people found and isolated two endogenous opioid peptides named endomorphins(EMs) from bovine brain which have shown potent binding affinity and selection to μ-opioid receptor. These two peptides, named endomorphin-1(EM-1) and-2(EM-2), have shown to produced potent antinociception effect with less side effects which makes them potential clinical analgesic agents in the future. However, EMs are peptides constructed by four amino acids, so they also have some limits due to poor metabolic stability and relative inability to penetrate blood-brain-barrier(BBB). This research aimed to enhance the metabolic stability and ability to penetrate BBB of EM-1 by chemical modification. First, we added 2 and 5 arginines to C-terminal of EM-1 because short sequence of arginines were cationic cell-penetrate peptides(CPPs) which could enhance the ability to penetrate BBB. We also substituted Pro2 amino acids with D-Ala and Sar at the second place of EM-1 to improve the metabolic stability because Pro2 is the specific recognition site for Dipeptidyl aminopeptidase IV(DPP IV) which is the most common enzyme in the degradation of EM-1. At last we examined the antinociception effect of our analogs and also investigated the influence on gastrointestinal tract in mice,respiratory tract and cardiovascular issues in rats.Our results indicated: All the analogs showed great binding affinity to μ-opioid receptor and better metabolic stability. GPI and MVD tests indicated that analogs showed better agonist activity and reaction efficiency to isolated preparation than EM-1.Agonist activity and reaction efficiency of analogs 1, 3 was better than analogs 2, 4;Analogs 1-4 also showed better metabolic stability than EM-1.Metabolic stability of analogs 2, 4 was better than analogs 1, 3. Analogs 1-4 showed significant antinociception effect in mice both by intracerebroventricular(i.c.v.) administration and subcutaneous(s.c.) administration. Analogs 1, 3 showed better antinociception effect than analogs 2, 4. Antinociception effects of analogs 1-4 could be blocked byopioid receptor antagonists which indicated that analogs 1-4 produced potent antinociception effect via central nervous system.Colonic bead expulsion test showed that analogs 1-4 and EM-1 all produced inhibition effect on colonic contraction in mice. But analogs 1-4 reduced the inhibition effect of the colonic contraction. Our isolated colon test showed that analogs 1-4induced less contraction effect on isolated colons of mice. Both tests indicated that analogs 1-4 produced less side effect on gastrointestinal tract than EM-1; Analogs 1, 3produced less inhibition effect on isolated bronchus contraction caused by electrical stimulation which indicated that analogs 1, 3 produced less side effect than EM-1 on respire system; Analogs 1,3 also produced less side effect than EM-1 on cardiovascular issues in rats.All the results showed that analogs 1,3 produced less side effects than EM-1.This study demonstrated that the combination of chemical modification was important for improving the stability of EM-1 and the transport capacity to the central nervous system, and it had important clinical analgesic value.And side effects of these analogs were significantly decreased compared with EM-1, which is conducive to develop safe and non-toxic analgesic drugs, with critical clinical application value.