Establishment, optimisation and pPharmacological characterisation of rat model of postherpetic neuralgia pain with varicella zoster virus

Abstract

Post-herpetic neuralgia (PHN) is an extremely painful condition that may develop as a long-term consequence of the shingles that occurs due to reactivation of latent varicella zoster virus (VZV) residing in the dorsal root ganglia after a bout of chicken pox, usually in childhood. PHN is defined as pain that persists for more than 3-months after crusting of the last shingles lesion. PHN is often poorly alleviated by clinically available analgesic and adjuvant drugs. Hence, there is a large unmet medical need for new analgesics to improve relief of this chronic pain condition. Although rodent models of VZV-induced neuropathic pain have been described, there is considerable between-laboratory variability. Hence, there is a need for an optimised rat model of VZV-induced neuropathic pain for use in in vivo efficacy profiling of novel molecules as potential new analgesic agents for the improved relief of PHN. Initially, I assessed the in vitro infectivity of the Schenke, Ellen and AV-92:3L strains of VZV propagated in cultured human foreskin fibroblasts (HFFs) and/or MRC-5 cells in order to identify VZV strains for progression to in vivo establishment of a rat model of PHN. As the Schenke and Ellen strains of VZV exhibited similar infectivity in HFFs and MRC-5 cells, the latter was selected for VZV propagation. VZV-infection in MRC-5 cells was confirmed by the detection of viral proteins IE-62 and VZVgE. Collectively, my in vitro data showed that MRC-5 cells infected with either the Schenke or Ellen strain of VZV were suitable for progression to in vivo establishment of a rat model of PHN. Briefly, groups of adult male Wistar rats received unilateral intraplantar (i.pl.) injections (50 mL) of (i) phosphate buffered saline (pH 7.4, 1 mM, n=3-8; control-group), (ii) uninfected MRC-5 cells (7x105 cells, n=3-8; sham-group) or (iii) VZV-infected MRC-5 cells containing 2x105, 1x105, 2x104, 1x104, 2x103 cells of the Schenke strain of VZV. Following hindpaw inoculation, temporal development of hindpaw hypersensitivity was assessed by measuring paw withdrawal thresholds (PWTs), paw pressure thresholds (PPTs) and paw thermal (noxious heat) thresholds (PTTs). For rats administered unilateral i.pl. injections of 2x105 to 2x103 Schenke-infected MRC-5 cells, bilateral mechanical allodynia developed by ~day 7 and was maintained until day 21, with spontaneous resolution by ~day 56. For rats administered Schenke-infected MRC-5 cells, persistent mechanical hyperalgesia or persistent thermal hyperalgesia did not develop in either hindpaw. For rats administered 2x104 Ellen-infected MRC-5 cells, bilateral mechanical allodynia was fully developed by 2-3 weeks and maintained until at least day 56. Hence, as both MRC-5 cells and the Ellen strain of VZV are available commercially, I standardized my rat model of VZV-induced neuropathic pain using Ellen-infected MRC-5 cells. To pharmacologically characterize my afore-mentioned rat model, I administered clinically available analgesic and adjuvant drugs to VZV-rats with fully developed mechanical allodynia in the bilateral hindpaws. Briefly, VZV-rats received single bolus doses of gabapentin (10-60 mg/kg; s.c.), amitriptyline (5-30 mg/kg; i.p.), morphine, (0.1-3 mg/kg; s.c.), meloxicam (5-20 mg/kg; i.p.) or vehicle, according to a lwashoutr protocol with 2-3 days between successive doses. PWTs in the hindpaws were measured immediately pre-dose and at regular intervals for up to 3 h post-dosing to generate mean ( SEM) PWT versus time curves. Dose-response curves were constructed by plotting mean ( SEM) area under the change in PWT versus time curve (PWT AUC) values versus log dose. The ED50 for single s.c. bolus doses of gabapentin and morphine for the relief of mechanical allodynia in the ipsilateral hindpaws were 44.9 mg/kg and 1.6 mg/kg respectively. Amitriptyline and meloxicam lacked efficacy in the doses tested. Finally, I assessed the pain-relieving efficacy of several novel small molecule angiotensin II type 2 receptor (AT2R) antagonists as previous work by our laboratory had shown that these compounds were efficacious in rat models of peripheral neuropathic pain induced by mechanically and chemically-induced peripheral nerve injury. Hence, VZV-rats exhibiting fully developed mechanical allodynia in the ipsilateral hindpaws (PWTsl6g) received single bolus doses of intravenous (i.v.) PD123319 (0.03-3 mg/kg), subcutaneous (s.c.) EMA300 (0.3-5 mg/kg), oral (p.o.) EMA401 (0.03-1.0 mg/kg) or vehicle, according to a lwashoutr protocol. PWTs measurement in the hindpaws and dose-response curves were performed as described above. The mean ED50rs for i.v. PD123319, s.c. EMA300 and p.o. EMA401 were 1 mg/kg, 2.9 mg/kg and 0.8 mg/kg respectively. In summary, I have established and pharmacologically characterized an optimized rat model of VZV-induced neuropathic pain. I used this model to show that novel small molecule AT2R antagonists produce dose-dependent pain relief mirroring the recent clinical trial success of EMA401 for producing significant analgesia above placebo in patients with PHN.