Centrally acting opioids, such as morphine, are the most frequently used analgesic agents for the treatment of severe pain. However, their usefulness is limited by the production of a range of adverse effects such as constipation, respiratory depression, tolerance and physical dependence. In addition, opioids generally exhibit poor efficacy against neuropathic pain. Neuropathic pain is a complex, chronic pain state that may develop secondary to damage to, or dysfunction of, the peripheral or central nervous system.
Endomorphin-1 and -2 are endogenous opioid peptides, which have been shown to produce potent antinociception in rodent models of acute and neuropathic pain. These peptides cause less undesired side effects compared with opioid alkaloids. Like all small peptides, endomorphins suffer from short duration of action even after central administration, poor metabolic stability and a relative inability to be transported across the gastro-intestinal mucosa and the blood-brain-barrier. Therefore, before being considered for clinical applications, it is essential to improve their membrane permeability, resistance to enzymatic degradation and central nervous system entry. In order to enhance the drug like properties of endomorphin-1 (Endo-1), the N-terminus of this peptide was modified with an 8- or 10-carbon chain lipoamino acid, LAA, (C8-Endo-1 or C10-Endo-1, respectively) and with 2,6-dimethyltyrosine (Dmt) replacement of Tyr1 (C8-Dmt-Endo-1 or C10-Dmt-Endo-1, respectively), or lactose succinamic acid (Lac-Endo-1). All currently and previously performed in vitro studies revealed moderate to potent µ-opioid (MOP) binding affinities and agonist activity of all peptide derivatives in the nanomolar range. C10-Endo-1 and Lac-Endo-1 did not elicit any significant increase in binding affinity at δ-opioid (DOP) receptors, in contrast to C10-Dmt-Endo-1, which showed enhanced DOP receptor binding affinity. That was consistent with a previous report for C8-Endo-1 and C8-Dmt-Endo-1.
All peptides were tested in a rat-chronic constriction injury (CCI) model of neuropathic pain following intravenous (i.v.) administration and produced dose-dependent analgesic activity. The obtained mean (± SEM) ED50 values from dose-response curves were between 0.99 ± 0.89 µmol/kg for C10-Dmt-Endo-1 and 6.58 ± 1.22 µmol/kg for C8-Endo-1. In spite of notably higher MOP receptor binding affinity of C8-Dmt-Endo-1 and C10-Dmt-Endo-1 compared to C8-Endo-1 and C10-Endo-1 in in vitro experiments, Dmt modification did not improve the in vivo pain-relieving activity of LAA-modified endomorphin-1 derivatives in CCI rats significantly, p > 0.05. Furthermore, the oral analgesic potential of Lac-Endo-1 was also evaluated in CCI rats. This glycopeptide produced significant dose-dependent pain-relief (ED50 = 19.6 ± 1.2 µmol/kg) comparable to that of morphine (ED50 = 20.7 ± 3.6 µmol/kg) after oral administration. Anti-neuropathic effects of C10-Endo-1 and Lac-Endo-1 peptides were significantly attenuated by naloxone hydrochloride confirming the opioid receptor-mediated analgesia. In contrast to morphine, no significant constipation was produced by i.v. C8-Endo-1, C10-Endo-1 and oral Lac-Endo-1 at 16 μmol/kg, in naïve rats. Furthermore, there was less tolerance developed to the analgesic effect of C10-Endo-1 derivative than morphine at equipotent doses. In agreement with previous findings, hypercapnic-stimulated respiratory ventilation was not affected significantly by C8-Endo-1 at analgesic doses, in contrast to morphine which produced significant depression of the ventilatory response at the antinociceptive dose (p> 0.05).
Conjugation of C-terminal fragments of substance P (SP) to morphine and opioid peptides has previously produced chimeric peptides with strong analgesic activity without development of tolerance. Four hybrid peptides were synthesized by conjugating the N-terminal domain of Endo-1 and C-terminal fragments of SP (4 or 5 amino acids, SP8-11 or SP7-11, respectively) with an overlapping Phe residue. That resulted in peptides designated E1SP8 and E1SP7, respectively. C10 was attached to the N-terminal domain of E1SP8 and E1SP7 to achieve C10-E1SP8 and C10-E1SP7. The hybrid peptides were synthesized via Fmoc solid phase peptide synthesis and tested for their in vitro biological activity. Both C10-modified hybrid peptides showed improved stability in Caco-2 cell homogenates and membrane permeability across Caco-2 cell monolayers. In radioligand competitive binding assays, hybrid peptides, E1SP8, E1SP7 and C10-E1SP7, produced high MOP receptor binding affinities (Kiµ 0.73 ± 0.20, 1.6 ± 0.20 and 3.87 ± 0.51 nM, respectively), similar to that of parent peptide, Endo-1 (Kiµ 0.76 ± 0.19 nM). None of the hybrid peptides exhibited improved affinity for DOP receptors, with Kiδ >1000. All four hybrid peptide analogues showed some agonist activity in forskolin-stimulated MOP receptor expressing cells. However, E1SP7 and C10-E1SP7 inhibited the accumulation of cAMP significantly (p<0.05) with IC50 values of 12.0 ± 2.1 and 45 ± 13 nM, respectively.
In conclusion, our findings suggest that the LAA- and lactose-modified derivatives of endomorphin-1 have the potential to be developed as novel opioid analgesic agents to treat neuropathic pain with a wider therapeutic window with respect to constipation and respiratory depression. In vitro experiments on the hybrid Endo-1/SP analogues revealed that C10-E1SP7 derivative is a promising candidate to be further examined for in vivo pain modulating activity.