In inflammation, the immune system produces a specific and site directed analgesic mechanism against nociception. This is mediated through the activation of opioid receptors on peripheral terminals of sensory neurons and through their endogenous ligands, opioid peptides. Inflammation enhances the migration of opioid-containing immunocytes from the circulation to the site of inflammation resulting in an accumulation of opioid peptides at the site of injury. Meanwhile, enhanced synthesis of opioid receptors in dorsal root ganglia and their axonal transport to sensory nerve terminals increases the amount of peripheral opioid receptors available for opioid activity. The cumulative effect of these mechanisms is an increase in the interaction of endogenous opioids with peripheral opioid receptors, producing analgesia.
Beta endorphin (β-END (1-31)) is considered to be the most potent endogenous opioid peptide. The analgesic capability of β-END (1-31) is greater than that of many currently available analgesics. However, as a consequence of in vivo metabolism, it has limited efficacy in alleviating severe pain and, in the case of inflamed tissue, the acidic environment produced during the inflammation process is likely to enhance the metabolism of β-END (1-31). It is plausible that metabolic fragments of β-END (1-31) may possess opioid action, either as an analgesic or as an opioid receptor antagonist. Therefore, the summation of endogenous analgesia that occurs in inflamed tissue may not be due to β-END (1-31) alone but may also arise due to fragments, either alone or in combination. The identification of opioid-active β-END (1-31) fragments in inflamed tissue may provide insights into new therapeutic strategies or targets in the treatment of inflammatory pain. This thesis focuses on the analgesic effects of rat beta endorphin (β-ENDrat (1-31)) and in particular, its fragments in inflamed tissue, with the aim of identifying novel opioid-active fragments.
There is a dearth of information or insight into the degradation of β-END (1-31) within inflamed tissue. Therefore, studying the degradation of β-ENDrat (1-31) to identify the fragments produced in inflamed tissue was the first aim of this study. As the pH of the inflamed tissue is lower (pH 5.5) than that of normal tissue, metabolism was studied by incubating β-ENDrat (1-31) in rat inflamed tissue extracts at pH 5.5 as well as 7.4. As speculated (above), degradation was found to be more extensive at acidic pH, producing 26 fragments at pH 5.5 compared to 22 at pH 7.4.
Based on the amounts produced and their stability, size, and commercial availability, three N-terminal fragments; β-ENDrat (1-20), β-ENDrat (1-17), β-ENDrat (1-9) and two C-terminal fragments; β-ENDrat (19-31), β-ENDrat (29-31) produced via metabolism at pH 5.5 were selected for further efficacy studies using the cell-based assay for cyclic adenosine monophosphate (cAMP). Evaluation of the efficacy of these fragments in terms of cAMP inhibition was performed by the cAMP AlphaScreen assay method utilising Human Embryonic Kidney 293 cells (HEK cells) expressing μ opioid receptors (MOR). A concentration dependent increase in cAMP inhibition was observed for the N-terminal fragments; β-ENDrat (1-20), β-ENDrat (1-17) and β-ENDrat (1-9). The cAMP inhibition potencies of β-ENDrat (1-20) and β-ENDrat (1-17) were similar to that of β-ENDrat (1-31) but that of β-ENDrat (1-9) was lower. Naloxone, the opioid receptor antagonist, was shown to reverse β-ENDrat (1-31), β-ENDrat (1-20) and β-ENDrat (1-17) inhibition of cAMP. This finding confirmed the opioid specific nature of cAMP inhibition potencies of β-ENDrat (1-31), β-ENDrat (1-20) and β-ENDrat (1-17). C-terminal fragments; β-ENDrat (19-31) and β-ENDrat (29-31) did not show opioid agonist or antagonist activity.
The anti-nociceptive effects of β-ENDrat (1-20), β-ENDrat (1-17) and β-ENDrat (1-9) were assessed in an in vivo animal model of inflammatory pain. The paw withdrawal responses on inflamed rat paws after injecting β-ENDrat (1-31) and these N-terminal fragments were assessed over a 24 hr period. Compared to the saline control, a significant anti-nociceptive effect for β-ENDrat (1-17) was observed during the first 60 min following intra-plantar bolus dosing. Moreover, the potency of β-ENDrat (1-17) was similar to that observed with an approximately three-fold larger dose of fentanyl at 30 min post administration. However, the anti-nociceptive effects of β-ENDrat (1-20) and β-ENDrat (1-9) fragments were found to be not significant at the dose tested.
The studies in this thesis provide evidence that the acidic environment generated as a result of the inflammatory process enhances the metabolism of β-ENDrat (1-31). Cell based studies suggest that the N-terminal fragments β-ENDrat (1-17) and β-ENDrat (1-20) are capable of inhibiting cAMP. Preliminary in-vivo studies further demonstrated the anti-nociceptive efficacy of β-ENDrat (1-17) which may exhibit potential as a lead peptide in future research into therapeutic targets for the treatment of inflammatory pain.