Pain Relieving and Neuroprotective Effects of Non-opioid Compound, DDD-028, in the Rat Model of Paclitaxel-Induced Neuropathy

Abstract

Chemotherapy-induced neuropathy (CIN) is a major dose-limiting side effect of anticancer therapy that can compel therapy discontinuation. Inadequate analgesic efficacy of current pharmacological approaches requires the identification of innovative therapeutics and, hence, the purpose of this study is to conduct a preclinical evaluation of the efficacy of DDD-028, a versatile pentacyclic pyridoindole derivative, against paclitaxel-induced neuropathic pain. In two separate experiments, DDD-028 was administered per os acutely (1-25 mg kg^-1) or repeatedly (10 mg kg^-1) in paclitaxel-treated rats. The response to mechanical noxious stimulus (paw pressure) as well as to non-noxious mechanical (von Frey) and thermal (cold plate) stimuli was investigated. Acute administration of DDD-028 induced a dose-dependent anti-neuropathic pain effect in all tests performed. Further, repeated daily treatment for 18 consecutive days (starting the first day of paclitaxel administration) significantly reduced the development of pain over time without the development of tolerance to the anti-hyperalgesic effect. Ex vivo analysis showed that DDD-028 was able to reduce oxidative damage of dorsal root ganglia as evidenced by the increase in the level of carbonylated proteins and the decrease in catalase activity. In the lumbar spinal cord, periaqueductal gray matter, thalamus, and somatosensory cortex 1, DDD-28 significantly prevented the activation of microglia and astrocytes. The pharmacodynamic study revealed that the pain-relieving effects of DDD-028 were fully blocked by both the non-selective nicotinic receptor (nAChR) antagonist mecamylamine and by the selective α7 nAChR antagonist methyllycaconitine. In conclusion, DDD-028 was active in reducing paclitaxel-induced neuropathic pain after single or repeated administrations without tolerance development and displaying a double symptomatic and neuroprotective profile. DDD-028 could represent a valuable candidate for the treatment of CIN.

Keywords: DDD-028; Glial cell; Neuroprotection; Nicotinic receptor; Oxidative stress; Paclitaxel.

Fig. 1

Structure of DDD-028

Fig. 2

Effect of single DDD-028 administrations on pain behavior induced by paclitaxel. Sensitivity to a noxious mechanical stimulus as measured by the paw pressure test (a). Pain threshold to a non-noxious mechanical stimulus as measured by the von Frey test (b). Pain threshold to a non-noxious thermal stimulus as measured by the cold plate test (c). Paclitaxel (2.0 mg kg⁻¹, i.p.) was administered on four days (1, 3, 5, and 8). Starting from day 10, DDD-028 was acutely per os administered (1–25 mg kg⁻¹) and measurements assessed before treatment and 15, 30, 45, 60, 90, and 120 min after injection. Results were expressed as mean ± SEM of 8 rats analyzed in 2 different experimental sets. **P < 0.01 vs vehicle + vehicle; ^^P < 0.01 vs paclitaxel + vehicle

Fig. 3

Study of DDD-028 pharmacodynamic profile. The nAChRs (a) and α7 nAChR (b) involvement in DDD-028 effects. Pain was induced by repeated treatment with paclitaxel. The hypersensitivity to a cold stimulus was measured by the Cold plate test. DDD-028 was administered per os at 25 mg/kg. The nAChR antagonist MECA (2 mg kg⁻¹) was administered intraperitoneally 15 min before DDD-028 administration. In a separate experiment, mecamylamine was administered for a second time 45 min after DDD-028. The α7 nAChR antagonist MLA (6 mg kg⁻¹) was administered intraperitoneally 15 min before DDD-028 injection. Results were expressed as mean ± SEM of 8 rats analyzed in 2 different experimental sets. **P < 0.01 vs vehicle + vehicle; ^^P < 0.01 vs paclitaxel + vehicle; °°P < 0.01 vs paclitaxel + DDD-028

Fig. 4

Effects of repeated administration of DDD-028 on pain behavior induced by paclitaxel. Sensitivity to a noxious mechanical stimulus as measured by the paw pressure test (a). Pain threshold to a non-noxious mechanical stimulus as measured by the von Frey test (b). Pain threshold to a non-noxious thermal stimulus as measured by the cold plate test. Behavioral tests were performed on days 10, 12, and 18 after the beginning of paclitaxel and DDD-028 administrations, 24 h after the last treatment. Paclitaxel (2.0 mg kg⁻¹, i.p.) was administered on four days (1, 3, 5, and 8) while DDD-028 (10 mg kg⁻¹, p.o.) was daily administered, starting from day 1 of paclitaxel injection. Results were expressed as mean ± SEM of 8 rats analyzed in 2 different experimental sets. **P < 0.01 vs vehicle + vehicle; ^^P < 0.01 vs paclitaxel + vehicle

Fig. 5

Carbonylated protein. Dorsal root ganglia. Densitometric analysis, data were normalized on the expression of beta-actin as housekeeping and expressed as mean ± SEM of 6 samples from 6 different animals analyzed twice. Representative western blot was also showed (3 samples of each treatment are shown). **P < 0.01 vs vehicle + vehicle; ^^P < 0.01 vs paclitaxel + vehicle

Fig. 6

Catalase activity. Dorsal root ganglia (a) and sciatic nerve (b) were analyzed. Enzymatic activity was expressed as percentage of control (vehicle + vehicle was considered as 100%). Data were expressed as mean ± SEM of 6 samples from 6 different animals analyzed in triplicate. *P < 0.05 vs vehicle + vehicle; ^P < 0.05 vs paclitaxel + vehicle

Fig. 7

Glial cells analysis. Spinal cord. Astrocytes (a) were studied by immunohistochemistry performed with a GFAP antibody. Representative image of the dorsal horn (lumbar level) at × 20 magnification; × 40 images were shown to highlight morphological alterations. Quantitative analysis was reported as number of GFAP-positive cells, and data were expressed as mean ± SEM of 3 different fields of 3 specimens for each of 6 samples from 6 different animals. *P < 0.05 vs vehicle + vehicle; ^P < 0.05 and ^^P < 0.01 vs paclitaxel + vehicle. Microglia (b) was studied by immunohistochemistry performed with a Iba1 antibody. Representative image of the dorsal horn (lumbar level) at × 20 magnification. Quantitative analysis was reported as number of Iba1-positive cells, and data were expressed as mean ± SEM of 3 different fields of 3 specimens for each of 6 samples from 6 different animals. *P < 0.05 vs vehicle + vehicle; ^P < 0.05 vs paclitaxel + vehicle. Brain. Astrocytes (c) and microglia (d) were studied by immunohistochemistry performed with GFAP and Iba1 antibodies, respectively. Analysis was performed on periaqueductal grey (PAG), thalamus, and somatosensory area 1 (S1). Quantitative analysis was reported as number of GFAP- and Iba1-positive cells, and data were expressed as mean ± SEM of 3 different fields of 3 specimens for each of 6 samples from 6 different animals. *P < 0.05 vs vehicle + vehicle; ^P < 0.05 vs paclitaxel + vehicle