The neuroprotective effect of a novel 1,4‐Dihydroquinazolin‐3(2H)‐yl benzamide derivative against chronic constriction injury‐induced neuropathic pain in rats
Authors: Rezq Samar , Sakr Asmaa , Kothayer Hend , Hassan Nancy H. , Alsemeh Amira 
Published: May 2022

Background

Neuropathic pain (NP) is a severe, chronic inflammatory condition affecting both the central and peripheral nervous systems and is resistant to standard treatment. Nonsteroidal anti‐inflammatory drugs (NSAIDs) are commonly used to treat NP, but they are not the best option due to their limited penetration of the blood‐brain barrier and the gastrointestinal side effects associated with long‐term use. We recently reported a new series of benzamide derivatives that inhibit cyclooxygenase 2 (COX2) with comparable selectivity to celecoxib and tolerable side effects. Among these derivatives, 4‐chloro‐N‐(2‐(4‐chlorophenyl)‐1‐methyl‐4‐oxo‐1,4‐dihydroquinazolin‐3(2H)‐yl)benzamide (4b) demonstrated potent anti‐inflammatory and analgesic properties, as well as 32‐fold greater CNS accessibility than celecoxib. We aim to investigate the anti‐inflammatory and anti‐nociceptive effects of 4b against NP utilizing chronic constriction injury (CCI) model of neuropathy, elucidating the molecular mechanisms involved in its effect on both the peripheral (sciatic nerve) and central (brainstem) levels.

Methods

The CCI was performed unilaterally in male Wistar rats (275–300 g, n=5/group) given 4b (5 and 10 mg/kg, p.o), the standard drug for NP pregabalin (30 mg/kg, p.o), or vehicle for 14 days. Pain‐related behaviors, including mechanical and thermal hyperalgesia, were performed prior to and on days 7 and 14 post‐surgery. Sciatic nerve and brainstem oxidative stress [NADPH oxidase2 (NOX2) and catalase] and inflammatory [inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2), COX2, and 5‐lipoxygenase] markers were detected by ELISA. Myelin sheath integrity was assessed in paraformaldehyde‐fixed sciatic nerve sections (4μm) stained with osmic acid, and the ratios of myelinated to total nerve area were calculated using ImageJ software.

Immunohistochemistry was used to detect the expression of the microglial marker CD68 and the astrocyte marker GFAP as indicators of glial cell activation in the brainstem.

Results

CCI rats had increased mechanical and thermal hypersensitivity, which peaked 14 days post‐surgery. Compared to sham‐operated controls, CCI‐rats had significantly higher levels of sciatic nerve and brainstem inflammatory (3‐8‐fold, p<0.05) and oxidative stress (7‐10‐fold higher NOX2) markers, but lower catalase levels (sciatic nerve: 26.60 ± 4.15 vs. 203.08 ± 16.4 mU/mg protein, and brainstem: 25.90 ± 2.56 vs. 216.40 ± 7.50 mU/mg protein). 4b mitigated pain behaviors, indicating a strong central analgesic effect. Furthermore, all biochemical measurements were dose‐dependently attenuated in the 4b‐treated groups. CCI‐induced increases in microglial cell infiltration (CD68 expression) and astrocyte activation (GFAP expression) were attenuated in 4b‐treated CCI‐rats. Osmic acid stain of the sciatic nerve revealed that 4b administration restored the normal nerve architecture and integrity that had been lost due to CCI. Excitingly, 4b (10 mg/kg) was superior to pregabalin in terms of behavioral, biochemical, and structural effects.

Conclusion and significance

Our data show that the novel compound tested has potent peripheral and central analgesic and anti‐inflammatory properties, highlighting the potential of its use as a promising therapeutic approach for treating NP.