Materials

Cisplatin injection vials (100 mg/ 100 mL) were from The Wesley Hospital Pharmacy (Brisbane, Australia). The NLRP3 inflammasome inhibitor, MCC950 (N-(1,2,3,5,6,7-Hexahydro- s-indacen-4-ylcarbamoyl)-4-(2-hydroxy-2-propanyl)-2-furansulfonamide) as the sodium salt (purity 99.75%), was from MedChemExpress (Monmouth Junction, New Jersey, USA) and supplied by their Australian agent, Focus Bioscience (Sydney, Australia). Pregabalin was from Toronto Research Chemicals INC (North York, Ontario, Canada). Sterile water for injection (WFI) ampoules and sterile normal saline ampoules, were from Pfizer Inc. (Sydney, NSW, Australia). Topical antibiotic powder was from Apex Laboratories Pty Ltd (Somersby, NSW, Australia).

Animals

Ethics approval was from The University of Queensland Animal Ethics Committee (Approval number: 2023/AE000699) and this work was undertaken in compliance with the National Health and Medical Research Council’s Australian Code for the Care and Use of Animals for Scientific Purposes (8th edition, 2013). Male Sprague–Dawley rats (n = 17; 160 to 180 g) were from Ozgene-ARC (Perth, Australia). Rats were housed in groups of two to three in individually ventilated cages in a purpose-built, temperature-controlled holding facility (21 ± 2 °C, mean ± SD) with a fixed 12 h/12 h light–dark cycle at the Centre for Integrated Preclinical Drug Development. Rats had free access to rodent chow and water, and they were acclimatized for at least three days before initiation of experimentation. Rodent hutches, chewsticks, and Kimwipes were used for environmental enrichment.

Induction of cisplatin-induced CIPN in rats

CIPN was induced by intraperitoneal (IP) injection of cisplatin (3 mg/kg) once weekly for four weeks on days 0, 7, 14 and 21 with a cumulative dose of 12 mg/kg. This cisplatin dosing regimen evoked temporal development of mechanical hypersensitivity in the bilateral hindpaws which was fully developed by day 21 after the first dose of cisplatin and it persisted for at least 42 days. At 5 min before each cisplatin injection, rats received a subcutaneous injection of sterile saline (2 mL) to hyperhydrate the animals and avoid nephrotoxicity.

General health monitoring

For each rat, body weight was measured and urinalysis was performed at once-weekly intervals commencing prior to the first cisplatin dose. Haematocrit was assessed once fortnightly on the day before cisplatin injection. Baseline von Frey paw withdrawal thresholds (PWTs) were measured on the day prior to each cisplatin injection (Days -1, 6, 13, 20). For haematocrit measurements, blood samples were taken from the tail vein and placed into labelled haematocrit tubes (75 mm; Drummond Scientific Co., Broomall, Pennsylvania, USA). Using a haematocrit rotor set at 10,000 g (Damon/IEC division, IEC MB centrifuge, Micro Haematocrit; Hawksley & Sons Ltd, West Sussex, UK), blood samples were centrifuged for 5 min at room temperature. In male Sprague–Dawley rats, the normal range of values for haematocrit is 34–57% (Han et al. 2014). For urinalysis, urine dipsticks (Multi reagent strips 10SG, Livingstone) were used to monitor renal function, as cisplatin frequently causes nephrotoxicity.

Assessment of von Frey Paw withdrawal thresholds in the hindpaws

On the day prior to each cisplatin injection, rats were housed individually in cages with wire mesh floors and given at least 30 min to acclimatise to the testing environment. Calibrated Von Frey filaments that delivered graded forces of 2, 4, 6, 8, 10, 12, 16, 18 and 20 g were applied to each of the hindpaws and PWTs were assessed using the up-down method. Specifically, the 6 g filament was applied first to the plantar surface of the hindpaw for 3 s until the filament buckled slightly. If no response was evoked after three seconds of applying the filament, the next filament in an ascending sequence (2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 g) was used until hindpaw withdrawal was elicited. If no response was elicited by the 20 g filament, rats were assigned a measurement of 20 g arbitrarily. Conversely, if application of the 6 g filament evoked a hindpaw withdrawal response, the next filament in the descending sequence was used until the filament that delivered the lowest force that evoked a hindpaw withdrawal response within three seconds of filament application, was identified. Hindpaw PWTs were measured in the left hindpaw first, then in the right hindpaw five min later. There were three repetitions of this sequence, with a 5-min interval between each sequence. The mean of the three measurements for each hindpaw served as the baseline PWT on each testing occasion. For post-dosing, one measurement per time point was performed.

Treatment groups

Commencing on day 28 after the first dose of cisplatin, rats exhibiting fully developed mechanical hypersensitivity in the bilateral hindpaws were randomized to receive a single oral dose of MCC950 in approximate half-log units (10, 30, 50, 100 or 300 mg/kg) to define the dose–response relationship, oral pregabalin (30 mg/kg) as the positive control, or oral vehicle (negative control). Dosing solutions were prepared using sterile WFI as the vehicle. Each rat received up to five single doses of the test or reference compound or vehicle according to a ‘washout’ protocol, such that there were at least two days of washout between successive doses. Just prior to dosing on each testing occasion, PWTs in the bilateral hindpaws were measured using von Frey filaments and then at 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 3, and 4 h post-dose. Von Frey PWTs in the bilateral hindpaws of CIPN-rats were assessed by a tester blinded to treatment group to avoid unconscious bias.

Adverse effects

A standardized set of approximately 40 clinical signs in categories including (i) skin and fur (piloerection, lack of grooming, fur loss, irritation or sores), (ii) eyes and mucus membranes (cloudy eyes, ocular or nasal discharge, diarrhoea etc.), (iii) respiratory and circulatory function (respiratory rate, gasping etc.), (iv) gait and posture (hunched posture, walking on tiptoe etc.), (v) behaviour (reduced/increased activity, circling, repetitive sniffing, Straub tail etc.), (vi) clonic tremors (tremors, myoclonic jerks, wet dog shakes etc.), (vii) tonic tremors (head and body rigidly arched etc.). The observations graded as mild, moderate and severe, if any, were used to assess adverse effects.

Statistical analysis

For the baseline PWT versus time curve data showing the temporal development of mechanical hypersensitivity in the bilateral hindpaws of CIPN-rats, a one-way ANOVA with Dunnett’s multiple comparisons test was performed using GraphPad Prism (v11.0.0). Mean (± SEM) PWT vs time curves for each oral dose of MCC950, pregabalin, and vehicle were also generated. For each individual animal, the increase in PWT above baseline values was calculated by subtracting the pre-dosing PWT value from each post-dosing PWT value to give ΔPWT versus time curves. Trapezoidal integration as implemented in GraphPad Prism (v11.0.0) was utilised to estimate the area under the ΔPWT versus time curve values (ΔPWT AUCs) for individual animals. Dose–response curves were generated by plotting mean (± SEM) ΔPWT AUC values versus log dose. Nonlinear regression in GraphPad Prism (v11.0.0) was used to determine the ED50 (95% confidence intervals) for oral MCC950 in CIPN-rats. A two-way ANOVA with Dunnett’s multiple comparisons test was used to compare mean (± SEM) ΔPWT AUC values for each dose level relative to that for the vehicle-treated group. For one-way ANOVA, F values are expressed as Fdf of treatment, residual. For 2-way ANOVA, F values are expressed as Fdf of treatment, time, interaction/residual. An unpaired t-test was used to compare the ΔPWT AUC values for single oral doses of MCC950 at 100 mg/kg and pregabalin at 30 mg/kg. The statistical significance criterion was p < 0.05.