Structure-Activity Study And Characterization Of Opioid Activities Of Endomorphin-1 And Endomorphin-2 Analogs With C-Terminal Modifications

The endogenous peptides ligands forμ-opioid receptor, endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂, EM-1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂, EM-2) were discovered in 1997 by Zadina et al. Both endomorphins (EMs) exhibited the highest affinity forμ-opioid receptor and extraordinarily high selectivity relative toδ-opioid receptor andκ-opioid receptor systems of all known opioid substances. These tetrapeptides are involved in the regulation of several behavioral, emotional, cardiovascular, gastrointestinal and respiratory functions. Furthermore, these tetrapeptides have a strong antinociceptive effect on acute pain, similar to that of morphine, but without some of the undesirable side effects. They are also more effective than the majority of the opioid peptides against neuropathic pain even at low doses, opening the possibility of using the two peptides as drugs. However, development of opioid peptides including EMs as therapeutic agents has historically been limited due to pharmacokinetic issues including stability and blood-brain barrier (BBB) permeability.EM-2 is a putative endogenousμ-opioid receptor ligand. To get insight into the important role of C-terminal amide group of EM-2, we investigated herein a series of EM-2 analogs (see Chapter 2) by substitution of the C-terminal amide group with -NHNH₂, -NHCH₃, -N(CH₃)₂, -OCH₃, -OCH₂CH₃, -OC(CH₃)₃ and -CH₂-OH. Their binding affinity and bioactivity were determined and compared. Despite similar (analogs 1, 4 and 7) or decreased (analogs 2,3, 5 and 6)μaffinity in binding assays, all analogs showed low guinea pig ileum (GPI) and mouse vas deferens (MVD) potencies compared to their parent peptide. Interestingly, as for analogs 2 and 3 (a single and double N-methylation of C-terminal amide), the potency order with the K_i(μ) values was 2＞3; for the C-terminal esterified analogs 4, 5 and 6, the potency order with the K_i(μ) values was 4＞5＞6. Thus, we concluded that the steric hindrance of C-terminus might play an important role in opioid receptor affinity. We further investigated the conformational properties of these analogs by 1D and 2D ¹H NMR spectroscopy and molecular modeling. Evaluating the ratios of cis- and trans-isomers, aromatic interactions, dihedral angles and stereoscopic views of the most convergent conformers, we found that modifications at the C-terminal amide group of EM-2 affected these analogs' conformations markedly, therefore changed the opioid receptor affinity and in vitro bioactivity.Moreover, to further our knowledge of the influence of C-terminal esterified modification on the pharmacological activities, we investigated the in vitro and in vivo opioid activities of C-terminal esterified EM-2 analogs 1-3 (see Chapter 3). Our results showed that the ED₅₀ values on contractions of the longitudinal muscle of distal colon induced by analogs 1, 2 and 3 were about 1.5-fold more potent, 2- and 8-fold less potent than EM-2, respectively. In addition, intravenous (i.v.) injections of analogs 1 and 2 dose-dependently decreased the system arterial pressure (SAP) and heart rate (HR) in anesthetized rats, but the degree of the hypotension and bradycardia was significantly smaller relative to the parent. Moreover, analog 3 was almost ineffective. Nevertheless, all these analogs produced potent antinociception in the tail-flick test after intracerebroventricular (i.c.v.) injection, and this antinociception was inhibited by naloxone, indicating an opioid mechanism. In summary, these results gave the evidence that C-terminal amide to esterifition conversion may play an important role in the regulation of opioid affinities and pharmacological activities.EMs can't be delivered into the central nervous system (CNS) in sufficient quantity to elicit antinociception when given systemically because they still suffers from several limitations including lack of oral activity, poor metabolic stability, short duration of action, and relative inability to cross the blood-brain barrier (BBB) to CNS. In the present study, we investigated herein a series of EM-1 analogs (see Chapter 4) with C-terminal linked by oligoarginine in order to improve the brain delivery and antinociception after systemic administration. Indeed, all these analogs decreased the opioid receptor affinity and in vitro pharmacological activity. Moreover, analogs 4, 7-9 produced a less potent antinociceptive activity after intracerebroventricular (i.c.v.) administration, with the ED₅₀ values about 11- to 13-fold lower potencies than that of EM-1. Nevertheless, our results revealed that EM-1 failed to induce any significant antinociception at a dose of 50μmol/kg after subcutaneous (s.c.) administration, whereas equimolar dose of these four analogs produced low but potent antinociceptive effects. Naloxone (10 nmol/kg, i.c.v.) significantly blocked the antinociceptive effects, indicating an opioid and central mechanism. These results demonstrated that C-terminal of EM-1 linked to oligoarginine improved the brain delivery, eliciting potent antinociception following peripheral administration.