Background & Aims
The biologically active fragment of neurotensin, NT(8-13), induces non-opioid analgesia by activating NTS1 and NTS2 receptors, making it a promising candidate for pain management(1). Contulakin-G, a 16-amino acid conopeptide from Conus geographus, shares a C-terminal sequence with neurotensin and exhibits potent analgesia upon intrathecal (i.t.) administration in preclinical pain models(2). Although it binds to NTS1 and NTS2 receptors(3), systemic injection of contulakin-G does not induce analgesia. To enhance blood-brain barrier (BBB) penetration, enzymatic stability, analgesic efficacy, and selectivity for NTS2 (to mitigate the hypotension and hypothermia associated with NTS1 activation), we designed and synthesized three structural analogs of contulakin-G. These analogs incorporate lipophilic moieties in place of the disaccharide on Thr10.
Methods
Before testing contulakin-G and its analogs in preclinical pain models, we first assessed the binding affinity of these new derivatives to NTS1 and NTS2 using a competitive radioligand binding assay. We then determined their potency and efficacy in activating the G??q, G??13, G??o and B-arrestin 2 signaling pathways using BRET-based biosensors. The metabolic stability of these contulakin-G analogs was also evaluated in rat plasma for 24 hours and their hypotensive effects were determined following intravenous administration. The analgesic properties of contulakin-G and its analogs were then assessed via i.t. administration in male Sprague-Dawley rats in acute (tail-flick), tonic (formalin test), and postoperative (Brennan paw incision model) pain models. To confirm that the analgesic effects of contulakin-G were not due to opioid receptor activation, we co-administered contulakin-G with naloxone in rats and measured pain behaviors in the formalin test.
Results
The binding data revealed that our analogs have slightly better affinity for NTS2 than for NTS1 compared to contulakin-G. Similarly, the BRET-based assay demonstrated that our analogs exhibit a lower EC50 for activating the G??q, G??13, G??o and B-arrestin 2 pathways compared with contulakin-G. Moreover, contulakin-G and its analogs exhibited significantly longer half-lives (>24h) in rat plasma than NT(8-13) (2 min). Some of the analogs were more effective than contulakin-G in reducing thermal pain in the tail-flick test, with equivalent efficacy in the tonic and postoperative pain models. Importantly, rats treated with contulakin-G and a derivative featuring Glu-cycloheptylamide in position 10 showed no spontaneous pain behaviors two hours post-incision. All analogs also induced a significant drop in systemic blood pressure after intravenous administration. Finally, coadministration of naloxone did not reverse the analgesic effects of contulakin-G, implicating a nonopioid component.
Conclusions
In conclusion, our study introduces enhanced contulakin-G analogs with better NTS2 affinity, improved activation of G??q, G??13, G??o and B-arrestin 2 pathways and extended half-lives in rat plasma compared to NT(8-13). Administered intrathecally, these analogs show promising analgesic potential in various pain models. Future research should explore their BBB permeability and their analgesic effects following systemic delivery. Given the hypotensive effects observed following intravenous administration, we aim to develop more selective NTS2 analogs with lower affinity for NTS1. These modified analogs not only exhibit similar analgesic efficacy but also a more cost-effective synthesis approach than contulakin-G. In summary, our study paves the way for the development of more effective centrally-acting analgesics with a potentially safer clinical profile.
References
1. Chartier, M., Desgagné, M., Marc, S., Côté, J., Longpré, J.-M., Marsault, É., & Sarret, P. (2021). Design, Structural Optimization, and Characterization of the First Selective Macrocyclic Neurtensin Receptor Type 2 Non-opioid Analgesic. Medicinal Chemistry, 2110-2124.
2. Martin, L., Ibrahim, M., Gomez, K., Yu, J., Cai, S., Chew, L., . . . Patwardhan, A. (2022). Conotoxin contulakin-G engages a neurotensin receptor 2 /R-type calcium channel (Cav2.3) pathway to mediate spinal antinociception. PAIN.
3. Craig, A. G., Norberg, T., Griffin, D., Hoeger, C., Akhtar, M., Schmidt, K., Olivera. (1999). Contulakin-G, an O-Glycosylated Invertebrate Neurotensin. biological chemistry, 13752-13759.
4. Sang, C. N., Barnabe, K. J., & Kern, S. E. (2016). Phase IA Clinical Trial Evaluating the Tolerability, Pharmacokinetics, and Analgesic Efficacy of an Intrathecally Administered Neurotensin A Analogue in Central Neuropathic Pain Following Spinal Cord Injury. Clinical pharmacology in drug development, 5(4), 250–258.
Presenting Author
Annik Lanoie
Poster Authors
Annik Lanoie
BSc
Université de Sherbrooke
Lead Author
Sitan Diarra MSc
Institut National de la Recherche Scientifique, Centre Armand-Frappier , Université du Québec
Lead Author
Julianna Dallagnol PhD
Institut National de la Recherche Scientifique, Centre Armand-Frappier , Université du Québec
Lead Author
Léa Théroux BSc
Institut de pharmacologie de Sherbrooke, Université de Sherbrooke
Lead Author
Emile Breault
Université de Sherbrooke
Lead Author
Marc-André Dansereau PhD
Institut de pharmacologie de Sherbrooke, Université de Sherbrooke
Lead Author
Isabelle Brochu MSc
Institut de pharmacologie de Sherbrooke, Faculté de médecine et des sciences de la santé, Université de Sherbrooke
Lead Author
Jean-Michel Longpré PhD
Institut de pharmacologie de Sherbrooke, Université de Sherbrooke
Lead Author
David Chatenet PhD
Institut National de la Recherche Scientifique, Centre Armand-Frappier , Université du Québec
Lead Author
Philippe Sarret PhD
Institut de pharmacologie de Sherbrooke, Université de Sherbrooke
Lead Author
Topics
- Treatment/Management: Pharmacology: Novel Targets