Background & Aims

Our previous work and that by others has implicated a role for angiotensin II (Ang II) signalling via the angiotensin II type 2 (AT2) receptor in the pathobiology of neuropathic pain (see reviews, (1,2). Intriguingly, Shepherd and colleagues3 showed that intraplantar (i.pl.) injection of Ang II into the mouse hindpaw evoked a prolonged period of mechanical allodynia in the hindpaw that was attenuated by i.pl. co-injection of the AT2 receptor antagonist, PD123319, but not the angiotensin II type 1 (AT1) receptor antagonist, losartan. Additionally, the Ang II-evoked mechanical hypersensitivity in the hindpaw was abolished in AT2 receptor knockout mice confirming the AT2 receptor as the target mediating this effect (3). Hence, our aim was to assess the efficacy of EMA401, a highly selective, orally active, small molecule AT2 receptor antagonist, relative to PD123319 and the AT1 receptor antagonist, losartan, for the relief of i.pl. Ang II-evoked mechanical allodynia in the mouse hindpaw.

Methods

Animal ethics approval (2023/AE000145) was from The University of Queensland. Experiments complied with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes5 and the IASP guidelines. Groups of male C57BL/6 mice (n=6-7/group) received single i.pl. injections (10 µL) of angiotensin II (Ang II; 100 pmol) in combination with vehicle (0.1% bovine serum albumin in water for injection containing 0.1% DMSO), PD123319 (300 pmol), EMA401 (300 pmol) or losartan (300 pmol). Dosing was unilateral and given according to a ‘washout’ protocol such that there was 7 days of washout between successive doses. Additionally, successive doses were given by i.pl. injection into the left or the right hindpaw on alternative dosing occasions. Hindpaw PWTs were assessed using calibrated von Frey filaments pre-dose and at 0.5, 1, 3, 5, 7, 24, 48 and 72 h post-dose by a tester blinded to treatment group. PWT versus time curves were generated using GraphPad Prism (v9.5.1).

Results

Following unilateral i.pl. injection of Ang II (100 pmol) into one hindpaw of individual mice, mechanical hypersensitivity was evoked such that the mean (±SEM) PWT decreased from 1.02 (±0.05) g pre-dose to a peak response of 0.26 (± 0.15) g at 1 h post-dosing which persisted for 48 h. Mean (± SEM) hindpaw PWTs returned to pre-dosing baseline levels by 72 h post-dosing. For mice that received i.pl. co-injection of Ang II with either PD123319 or EMA401 at 300 pmol, mechanical allodynia in the hindpaws was markedly attenuated so that mean (±SEM) PWTs matched that evoked by i.pl. vehicle injections (0.97 ± 0.15 g). By contrast, i.pl. co-injection of losartan (300 pmol) with Ang II did not attenuate the Ang II-evoked mechanical hypersensitivity response in the hindpaw. Specifically, the mean (± SEM) peak PWT response for mice co-administered Ang II and losartan was 0.23 (± 0.08) g at 1 h post-dosing which persisted for 48 h in a manner similar to Ang II co-administered with vehicle.

Conclusions

Single i.pl. doses of Ang II (100 pmol) administered unilaterally into the mouse hindpaw evoked a prolonged period of mechanical hypersensitivity (48 h) in the hindpaw. I.pl. co-injection of each of the AT2 receptor antagonists, PD123319 (300 pmol) and EMA401 (300 pmol) fully attenuated i.pl. Ang II-evoked mechanical hypersensitivity in the mouse hindpaws to match the corresponding i.pl. vehicle-evoked responses. By contrast, i.pl. co-injection of the AT1 receptor antagonist, losartan (300 pmol), did not attenuate Ang II-evoked mechanical hypersensitivity in the mouse hindpaw relative to the corresponding response evoked by i.pl. Ang II co-administered with vehicle. Our present data further implicate a role for Ang II signalling via the AT2 receptor in the pathobiology of chronic pain and provide additional impetus for the discovery of novel, highly efficacious and well-tolerated AT2 receptor antagonists for the relief of chronic pain.

References

1.Smith MT. Nonopioid analgesics discovery and the Valley of Death: EMA401 from concept to clinical trial. Pain 2022;163(Suppl 1):S15-S28.
2.Shepherd AJ, Rice ASC, Smith MT. Angiotensin II type 2 receptor signalling as a pain target: Bench, bedside and back-translation. Curr Opin Pharmacol 2023; 102415,1-8.
3.Shepherd AJ, Copits BA, Mickle AD, Karlsson P, Kadunganattil S, Haroutounian S, Tadinada SM, de Kloet AD, Valtcheva MV, McIlvried LA, Sheahan TD, Jain S, Ray PR, Usachev YM, Dussor G, Krause EG, Price TH, Gereau RW 4th, Mohapatra DP. J Neurosci 2018;38:7032-7057.

Presenting Author

Maree T Smith

Poster Authors

Maree Smith BS Pharm, PhD

PhD

UQ

Lead Author

Mohammad Zafar Imam; PhD

The University of Queensland

Lead Author

Andy Kuo

The University of Queensland

Lead Author

Topics

  • Treatment/Management: Pharmacology: Non-opioid