Inhibition of SPRK1 in vivo in mice reduces nociceptive behaviour in a model of chemotherapeutic peripheral neuropathy.

Lucy F Donaldson¹, Matthew Swift¹, James Hudson¹, Jonathan C Morris², Hamish Toop², Beth Coyle¹, Nigel Mongan^1
1University of Nottingham, ²University of New South Wales

Introduction/Background & aims Chemotherapy-induced peripheral neuropathy (CIPN) is found during and following treatment with most currently used chemotherapy classes including taxanes, vinca alkaloids and platinum based approaches. Painful CIPN is a dose limiting factor in chemotherapy regimens and can persist or recur following cessation of treatment. Recently, we identified a VEGF splice variant, VEGF-A₁₆₅b as having both neuroprotective and anti-nociceptive actions on neurons exposed in vitro to hyperglycaemic conditions similar to those in diabetic neuropathy [1]. VEGF-A splicing is dependent on the phosphorylation of splicing factor SRSF1 by the splicing kinase SRPK1 [2].  SRPK1 inhibition causes a change in splice site selection resulting in expression of the neuroprotective, anti-nociceptive VEGF-A₁₆₅b isoform, and SRPK1 inhibition reduces sensory neuronal sensitisation resulting from chemotherapy treatment in vitro. Thus, we hypothesised that SRPK1 inhibition could be used as a prophylactic treatment to prevent increased nociception associated with CIPN in mice in vivo.

Method/Summary of work All procedures using experimental animals were performed with licenced authority under the Animals (Scientific Procedures) Act 1986/ASPA Amendment Regulations 2012. Animals were housed in groups of 4, with ad libitum food and water. Twenty four adult male mice (C57Bl6, ~25g weight) were included in the study randomly allocated to 4 groups that received: 1. SPHINX31 (S31: N-(2-(4-(pyridin-2-ylmethyl)piperazin-1-yl)-5-(trifluoromethyl)phenyl)-5-(pyridin-4-yl)furan-2-carboxamide); 2. 1%DMSO; 3. Vincristine (Vinc) + S31; 4. Vinc + 1%DMSO (n=6/gp). Sample size estimates were based on a prior pilot study. S31 (0.8mg/kg) or DMSO vehicle were administered i.p. twice weekly throughout the study. Vinc was administered i.p in weeks 1 and 2, in 2 cycles of 5 daily injections interrupted by 2 days. Each Vinc dose was 50µg/kg with a maximum cumulative dose of 50µg. Welfare checks were performed daily during vincristine administration. In weeks 1 &2, S31 or DMSO were given in the same injection/volume as Vinc. All animals received the same total volume in all injections throughout the study up to a maximum of 500µl per injection. von Frey withdrawal threshold behavioural measures were determined at baseline and after Vinc treatment starting at week 3, and weekly thereafter for a further 4 weeks. All behavioural tests, data extraction and analyses were done blinded to treatment.

Results/Discussion Vinc treatment only resulted in a change in nociceptive hypersensitivity compared to baseline values from weeks 5-7 i.e. 2 weeks after the cessation of Vinc treatment: baseline von Frey (vF) threshold Vinc 0.9±0.04g (mean ±SEM) vs. week 3: 0.81±0.1g ns; week 5: 0.5±0.08g, p=0.004, wk 6: 0.6±0.1g, p=0.06; wk 7: 0.5±0.06g, p=0.01. Kruskal-Wallis + Dunn’s test on raw data).

Prophylactic co-treatment with S31 reduced the Vinc-induced hypersensitivity up to week 7 (week 5: 0.8±0.1 p=0.04 compared to Vinc; week 6: 0.7±0.09g, ns ; week 7: 0.8±0.1g, ns). The effect of Vinc+S31 compared to Vinc alone did not reach statistical significance in weeks 6 and 7 despite the vF thresholds of Vinc+S31 treated animals not being significantly different from both control groups (at 5 weeks S31=1±0.03g, DMSO = 0.99±0.04g). S31 treatment alone had no effect on nociceptive behaviour. The study post-hoc calculated power was 79%.

Conclusion(s) In this model of CIPN, nociceptive changes became evident with some delay after the cessation of chemotherapy treatment, suggesting that ‘coasting’ may be a feature in this model. Systemic prophylactic treatment with SPHINX31 had no effect on nociception in control animals, but SRPK1 inhibition may prevent increased nociception in vincristine-induced neuropathy in mice.

Reference(s)

1. R. P. Hulse, Beazley-Long N. Ved N, Bestall SM, H. Riaz, P. Singhal, Ballmer Hofer K, S. J. Harper, D. O. Bates and L. F Donaldson. Vascular Endothelial Growth Factor (VEGF-A)₁₆₅b prevents diabetic neuropathic pain and hyperglycaemia-induced sensory neuronal degeneration. Clinical Science 129(8) (2015) 741-56.

2. S. Oltean, Gammons M, Hulse R, Hamdollah-Zadeh M, Mavrou A, Donaldson L, Salmon AH, Harper SJ, Ladomery MR, Bates DO. SRPK1 inhibition in vivo: modulation of VEGF splicing and potential treatment for multiple diseases. Biochemical Society Trans. 40(4) (2012) 831.