In Rats, the Chemotherapeutic Drug Vincristine-Induced Neuropathic Nociception is Suppressed When Cannabinoid CB1 and CB2 Receptors are Activated


Austin J Pharmacol Ther. 2023; 11(3): 1180.

“In Rats, the Chemotherapeutic Drug Vincristine-Induced Neuropathic Nociception is Suppressed When Cannabinoid CB1 and CB2 Receptors are Activated”

Hari Prasad Sonwani*

Apollo College of Pharmacy, Anjora, Durg CG, India

*Corresponding author: Hari Prasad Sonwani Apollo College of Pharmacy, Anjora, Durg CG, India. Email: [email protected]

Received: November 10, 2023 Accepted: December22, 2023 Published: December 29, 2023


Rats were used to test the potential of cannabis to reduce mechanical hypersensitivity, or mechanical allodynia, which was brought on by vincristine chemotherapy. After that, action sites were located. Method of experimentation: After ten daily injections of vincristine, mechanical hypersensitivity developed in comparison to those that received saline at the same periods. The effects on chemotherapy-induced neuropathy were assessed for the CB1/CB2 receptor agonist WIN55, 212-2, the receptor-inactive enantiomer WIN55, 212-3, the CB2-selective agonist (R,S)-AM1241, the opiate agonist morphine, and vehicle. To determine the locations of action, WIN55, 212-2 was injected either locally in the hind paw or intrathecally (i.t.). By employing competitive antagonists for either CB1 (SR141716) or CB2 receptors (SR144528), pharmacological selectivity was demonstrated. Vincristine-evoked mechanical allodynia was decreased when WIN55, 212-2, but not WIN55, 212-3, were administered systemically. A change in the dose-response curve to the left was noticed after WIN55, 212-2 in comparison to morphine therapy. Antibodies of CB1 (SR141716) and CB2 (SR144528) inhibited WIN55, 212-2’s anti-allodynic actions. Via a CB2 mechanism, (R,S)-AM1241 reduced c-induced mechanical hypersensitivity. Without causing catalepsy, both cannabinoid agonists reduced the mechanical hypersensitivity brought on by vincristine. Cannabis-induced neuropathy may be modulated by cannabinoids at spinal sites of action. When delivered intraperitoneally, WIN55, 212-2 but not WIN55,212-3 inhibited vincristine-evoked mechanical hypersensitivity at dosages that were inert after local hindpaw injection. Spinal co-administration of CB1 and CB2 antagonists inhibited WIN55,212-2’s anti-allodynic effects.By activating CB1 and CB2 receptors, cannabinoids inhibit the maintenance of vincristine-induced mechanical allodynia. The spinal cord is involved in the mediation of these anti-allodynic actions, at least partially.

Keywords: CB1/CB2 receptor; Hypersensitivity; Hypersensitivity; Chemotherapy


A common side-effect of several different kinds of chemotherapy drugs is painful peripheral neuropathy. Examples of these groups of drugs include vinca alkaloids (like vincristine), compounds derived from taxanes (like paclitaxel), and compounds derived from platinum (like cisplatin). According to Sandler et al. (1969), Polomano and Bennett (2001a), Bacon et al. (2003), Cata et al. (2006)b, and other studies, the incidence and severity of chemotherapy-induced neuropathy are influenced by the type of cancer, the dose schedule, the choice of chemotherapeutic drug, and the existence of concurrent medical issues.It has been suggested that vincristine causes cytoskeletal structural changes and microtubule disorientation to produce anti-tumor effects [56,58]. In the periphery, where the effects of disrupted axonal transport would first become apparent, neurofilament buildup in cell bodies and proximal axons may cause paraesthesiae and dysesthesiae (Topp) in 2000, et al. It has also been noted that chemotherapy-induced neuropathy occurs when primary afferents are not morphologically damaged; these subsequent investigations show that microtubule disruption is not a prerequisite for chemotherapy-induced neuropathy [48]. Dysregulation of cellular calcium homoeostasis due to aberrant mitochondrial action may be the cause of chemotherapy-induced neuropathy (Flatters and Bennett, 2006; Siau and Bennett, 2006). According to Jackson et al. (1988), vincristine-induced neuropathy restricts the duration and dosage of anti-cancer treatments that may save lives. Patients are frequently recommended aspirin, ibuprofen, and celebrex to treat chemotherapy-induced neuropathy; nevertheless, their effectiveness is limited [32]. The discovery of potent substitute analgesics is a critical medical necessity since there are currently no approved therapies for chemotherapy-induced neuropathy. In animal models of traumatic nerve injury, cannabinoids—drugs that have the same target as cannabis's psychoactive component, D9-tetrahydrocannabinol—suppress neuropathic nociception through mechanisms specific to cannabinoid CB1 and CB2 receptors [6,14,17,24,31,51,64]. The central nervous system has the highest density of CB1 receptors. [68] on the CNS. The majority of CB2 receptors are expressed [7,39], although they are not the only ones (Van Sickle et al., 2005; Outside the central nervous system [5]. After spinal nerve ligation, CB2 is significantly upregulated in the rat spinal cord and dorsal root ganglion [5,65,67], indicating that CB2-mediated antihyperalgesic effects in neuropathic pain states may be mediated by additional neuroanatomical substrates. Paclitaxel-induced neuropathic nociception is suppressed by the mixed CB1/CB2 receptor agonist WIN55,212-2 via a CB1 mechanism [45]. Nevertheless, little is known about the mechanisms behind the emergence of excruciating peripheral neuropathies brought on by various chemotherapy drugs (for a review, see Cata et al., 2006b). Different symptoms of neuropathic pain complied with the International Association for the Study of Pain's recommendations for treating animals [69]. Following the relevant institutional procedures, bedding containing metabolized vincristine was handled as biohazardous waste and disposed of.

Typical Experimental Techniques

In order to stop behavioral sensitization to cutaneous stimulation from developing, the effects of the drugs were assessed using just one stimulus modality. On day zero, baseline reactions to mechanical or thermal stimulation of the hindpaw were determined. After undergoing behavioral testing, the rats were given intraperitoneal (i.p.) injections of vincristine sulphate (0.1 ml/kg/day i.p.) or saline (1 ml/kg/day i.p.) every day for a period of 12 days. The five daily injections were part of the treatment paradigm, which was followed by a two-day break during which no injections were given, and then five more daily injections as explained earlier on. [63]. The experimenter was blinded to the drug condition in every study. Every day, weights were recorded.

Evaluation of Mechanical Withdrawal Limits

A digital Electrovonfrey Anesthesiometer (IITC model Alemo 2290-4; Woodland Hills, CA, USA) with a stiff tip was used to measure mechanical withdrawal thresholds. Rats were arranged on a raised mesh platform beneath plastic cages that had been turned inside out. Prior to testing, the rats were given ten to fifteen minutes to acclimate to the chamber. The floor of the mesh platform was used to apply stimulation to the midplantar area of the hind paw. As paw withdrawal ended mechanical stimulation, there was no higher threshold limit established at which a trial may end. Each paw's mechanical withdrawal threshold was measured twice before and 24 hours after each vincristine or saline infusion. On day 11, the final injection of either saline or vincristine was given. Baseline mechanical withdrawal thresholds were measured on test day (day 12), which was around 24 hours after the last vincristine or saline injection. Additionally, the effects of pharmacological interventions were tested. When vincristine was administered to rats at pressures (g) that did not produce withdrawal symptoms prior to chemotherapeutic treatment, nocifensive responses were seen. Measuring mechanical paw withdrawal thresholds with the Electrovonfrey Anesthesiometer, vincristine-induced reductions were thus classified as mechanical allodynia. The animals treated with vincristine were given systemic injections of either vehicle (n ¼ 8) or WIN55, 212-2 (0.75, 1.5, or 2.5 mg kg—1 i.p.; n ¼ 8 per group) after their baseline mechanical withdrawal thresholds were assessed on day 12. Various groupings were given The available options are the CB2-selective agonist AM1241, the receptor-inactive enantiomer WIN55, 212-3 (2.5 mg kg—1 i.p.; n ¼ 8), or the opiate agonist morphine (2.5 or 8 mg kg—1 i.p.; n ¼ 8 and 4, respectively). The low-dose morphine was chosen since it was shown to both elicit antinociception [25] and decrease neuropathic pain behavior in a spinal nerve ligation model [27,31]. The dosage of AM1241 used was comparable to the amount that, after spinal nerve ligation, restored mechanical paw withdrawal thresholds [50]. Groups were given WIN55,212-2 (2.5 mg kg—1 i.p.) in conjunction with either SR141716 (2.5 mg kg—1 i.p.; n ¼ 8) or SR144528 (2.5 mg kg—1 i.p.) to ascertain the pharmacological specificity. (i.p.; n ¼ 8) and AM1241 (2.5 mg kg—1 i.p.) were jointly administered An antagonist given alone (n ¼ 8 per group), SR141716 (2.5 mg kg—1 i.p.; n ¼ 8) or SR144528 (2.5 mg kg—1 i.p.; n ¼ 8). In every study, mechanical withdrawal thresholds were assessed on day 12, about 24 hours after the final vincristine injection. The drug or vehicle withdrawal thresholds were measured prior to injection (baseline), as well as thirty and sixty minutes thereafter. 31 days after the last vincristine injection, rats treated with vincristine and given a vehicle were also assessed for the existence of mechanical allodynia as a potential indicator of a remission of the painful peripheral neuropathy caused by the drug.

Evaluation of Thermal Latencies during Paw Withdrawal

Paw withdrawal latencies to radiant heat were measured for each paw in duplicate using a commercially available device and the Hargreaves test [16]. The IITC model 33 plantar stimulation machine, located in Woodland Hills, California, USA. Rats were arranged on an elevated glass platform, under inverted plastic cages. Before testing, rats were given ten to fifteen minutes to become accustomed to the equipment. The floor of the glass platform allowed radiant heat to reach the midplantar area of the hind paw. To avoid damaging the tissue, stimulation was stopped when the paw was removed or after twenty seconds. The report presents the average of two sets of duplicate results, averaged across paws, for thermal paw withdrawal latencies. Thermal withdrawal latencies were measured prior to (day 0) and after (days 3, 6, 9, and 12) after either As previously mentioned, either saline (n ¼ 6) or vincristine (n ¼ 12). The same animals were then examined for the mechanical allodynia (on day 12) as determined by the previously mentioned techniques.

Catheter Insertion Intrathecal

Through an incision in the atlanto-occipital membrane, intrathecal catheters (PE10 tubing, Clay Adams, Parsippany, NJ, USA) were surgically implanted under pentobarbital/ketamine anesthesia into the spinal subarachnoid space [22,66]. The distal end of the catheters was heat-sealed after they were inserted to a depth of 8.5 cm and fastened to the skull. Any indicators of motor impairment caused by catheter implantation, such as difficulty walking on a wire cage cover or impaired righting reflex, were instantly fatal to the animals. Ten percent or so of the animals that had catheters implanted had motor impairment, and as a result, they were never tested again or given vincristine or saline treatment. Animals had permission to must heal for a minimum of five days after surgery before baseline paw withdrawal thresholds are established and vincristine or saline is started.

Location of the Incident

A preliminary study was conducted to ascertain whether intraperitoneal injection (i.t.) of the b-cyclodextrin vehicle (n ¼ 6) affected mechanical withdrawal thresholds in comparison to groups that had catheter implantation surgery but did not receive an i.t. injection (n ¼ 4). An additional vincristine-treated groups were given either WIN55,212-3 (10 mg i.t., n ¼ 6) or WIN55,212-2 (10 mg or 30 mg i.t.; n ¼ 6 each group). To ascertain In order to determine the pharmacological specificity of cannabis activities, two different groups were given WIN55,212-2 (30 mg intravenously) in combination with either SR141716 (30 mg intravenously; n ¼ 8) or SR144528 (30 mg intravenously; n ¼ 8)), WIN55,212-2 (30 mg intravenously) coadministered utilizing both SR141716 (30 mg intramuscular) and SR144528 (30 mg intramuscular) given simultaneously (n ¼ 6) or SR144528 (30 mg intramuscular; n ¼ 6) or SR141716 (30 mg intramuscular; n ¼ 5) given separately. Mechanical paw withdrawal thresholds were assessed every day in all of the investigations as previously said to confirm that the administration of vincristine caused mechanical allodynia in comparison to groups that were given saline (n ¼ 9) at the same periods. After testing, a post-mortem injection of Fast green dye and subsequent dissection were used to confirm the catheter's location.

No animals showed signs of tissue injury from the catheter implantation. Mechanical withdrawal thresholds were assessed in every study (on the day12) around twenty-four hours after the previous vincristine injection. The baseline paw withdrawal thresholds were tested in duplicate, as well as five, thirty, and sixty minutes after the injection. of a substance or car. In order to assess potential cannabinoid activity peripheral areas, WIN55, 212-2 or vehicle was applied topically to the paw. On the day of the test, each animal received a unilateral intraplantar ( injection into the plantar surface of the hindpaw (day 12). Rats treated with vincristine were given either WIN55, 212-2 (30 or 150 mg; n ¼ 9 per group) or vehicle locally in the hindpaw (n ¼ 7). The subjects received injections into their right and left paws in equal amounts. All animals had their thresholds measured before (baseline) and 30 minutes after injection, in both the injected and non-injected paws.

Catalepsy Examination

On test day 12, catalepsy testing was carried out on rats that had previously been assessed for their reactivity to temperature stimuli using the bar test [38,46]. After the measurement of thermal paw withdrawal latencies, the rats were placed back into their home cages for a minimum of half an hour before the baseline catalepsy assessment was initiated.

The animals were positioned as previously mentioned (Martin et al., 1996) with their forepaws hanging over a stainless steel bar that was suspended 9 cm above a level platform. In animals treated with vincristine and given either a vehicle (n ¼ 6) or WIN55, 212-2 (2.5 mg kg—1 i.p.; n ¼ 6), catalepsy was reassessed. A different set of mice treated with vincristine (who did not have thermal test) were given AM1241, which is 2.5 mg kg-1 i.p.; n ¼ 6. For example, WIN55, 212-2 (2.5 or 10 mg kg—1 i.p.; n ¼ 6 per group) was given to two groups of otherwise naive rats. The time I stood there at the bar was measured for each group in triplicate at 30, 45, and 60 minutes after the medication injection.

Examinations of Statistics

For repeated measures, Analysis of Variance (ANOVA) or planned comparison unpaired t-tests were used for data analysis when applicable. The Greenhouse-Geissner adjustment was implemented for every element that was repeated. Additionally, post-drug thresholds and pre-vincristine thresholds were compared using paired t-tests. (baseline) cutoff points. Using the following formula, the percent (%) reversal of mechanical allodynia was determined at the moment of maximal cannabis anti-allodynic efficacy: Using Fisher's protected least significant difference (PLSD) test, post hoc comparisons were executed. It was decided that Po0.05 was statistically significant.

Chemicals and Drugs

Tocris Cookson provided the vincristine sulphate (Ellisville, MO, USA). R(þ)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl] WIN55,212-23-de pyrrolo[1,2,3]One (1) 1,4-benzoxazin-ylWIN55, 212-3 (S(–)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]), -(1-naphthalenyl)methanone mesylate3-de pyrrolo[1,2,3]One (1) 4,4-benzoxazinylSigma Aldrich (St. Louis, MO, USA) provided the morphine sulfate, b-cyclodex-trin, and -(1-naphthalenyl)metha-none mesylate salt. (S, R)AM1241, ((R,S)-(2-iodo-5-nitro-phenyl)-[l-(l-methyl-piperidin-2-ylmethyl)-lH-indol-3-yl]-methanone) was produced. sized at one of the authors' laboratories (AM). Asymmetric pyrazole-3-carboxamide N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)- 4-methyl-1H-pyrazole and N-[(1S)- endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl] SR144528-5-(4-chloro-3- methylphenyl) NIDA supplied the -1-(4-methylbenzyl)-pyrazole-3-carboxamide. A 0.9% saline solution was used to dissolve vincristine sulfate. Every other medication was dissolved in awith one exception, a systemic delivery vehicle consisting of 10% ethanol, 10% emulation, and 80% saline delivered at a volume of 1 ml/kg bodyweight. Because of solubility restrictions, antagonists were supplied in conjunction with AM1241 in certain trials, with a total injection volume of 1.5 ml/kg. For intraperitoneal and intrapleural administration, drugs were dissolved in 45% b-cyclodextrin according to the earlier description [22]. For intraperitoneal and intrapleural administration, the drug or vehicle was given in amounts of 10 and 50 ml, respectively.


Overall outcomes: Before either saline or vincristine was administered, there was no difference in body weight between the groups. In rats treated with saline, normal weight growth was noted throughout the injection period (F1,40 ¼ 41.515, Po0.0002; Figure 1a). In contrast, individuals receiving vincristine demonstrated a lack of gaining weightat all times after injection (F11,440, 23.32, Po0.0002, Po0.001 for every comparison; Figure 1a). Figure 1a displays variations in body weight for the groups depicted in Figure 1b during vincristine or saline treatment. In vincristine-treated mice receiving vehicle (i.p.), mechanical hypersensitivity had fully gone by 31 days after the last injection, and normal weight growth was noted (data not shown).The way that subjects responded to mechanical and thermal stimuli did not change in research using systemic or intrathecal injections. For each group on any given day, the right and left paws; as a result, withdrawal thresholds are shown as the average of the repeated measurements, averaged across paws. Results for the injected and non-injected paws are presented separately in trials using unilateral Injections. Vincristine reduced paw withdrawal thresholds to mechanical stimulation (i.e., in each paw) in every study (Po0.0002 for all experiments; Figures 1b, 2, 5a, and 7). In a subset of groups, there were slight baseline variations in paw withdrawal thresholds prior to vincristine delivery (Po0.01 for each study; Figures 3a, c, and 6a). On test day, however, there was no difference in mechanical withdrawal thresholds prior to pharmacological interventions in any research between vincristine-treated groups. Three animals were not employed because they did not experience vincristine-induced hypersensitivity. In the pharmacological tests that followed. Evaluation of mechanical allodynia after WIN55, 212-2 is administered systemically WIN55, 212-2 caused a dose-dependent rise in mechanical withdrawal thresholds in rats given vincristine. The day 12 (preinjection) paw withdrawal thresholds established before to pharmacological treatments (F6, 56 6.628, Po0.0002) and in relation to the vehicle (F3, 28 ¼ 5.141, Po0.006, Figure 3a). The effects of the intermediate (1.5 mg kg—1 i.p.) and low (0.75 mg kg—1 i.p.) doses of WIN55, 212-2 were outlasted by the high dose (2.5 mg kg—1 i.p.) of the drug (Po0.02 for all comparisons). The high dose also generated the maximum suppression of mechanical hypersensitivity. Mechanical withdrawal thresholds were efficiently normalized in relation to previncristine levels by the high dose of WIN55, 212-2 (one-tailed t-test, P ¼ 0.059). Thirty minutes after the medication injection, WIN55, 212-2 caused a dose-dependent reversal of mechanical allodynia (F3,28 14.829, Po0.0002; Figure 3b). More than 50% of mechanical allodynia was reversed by the intermediate and low doses of WIN55, 212-2 (0.75 and 1.5 mg kg—1 i.p.) (Po.01 for all comparisons). The large amount 30 minutes after injection, the highest suppression of mechanical hypersensitivity was achieved with a dose of WIN55, 212-2 (2.5 mg kg—1 i.p.) (Po0.002 for all comparisons; Figure 3b).