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. 2024 Jul 5;25(13):7410.
doi: 10.3390/ijms25137410.

A New Application for Cenicriviroc, a Dual CCR2/CCR5 Antagonist, in the Treatment of Painful Diabetic Neuropathy in a Mouse Model

Aleksandra Bober  1 , Anna Piotrowska  1 , Katarzyna Pawlik  1 , Katarzyna Ciapała  1 , Magdalena Maciuszek  1 , Wioletta Makuch  1 , Joanna Mika  1
Affiliations

A New Application for Cenicriviroc, a Dual CCR2/CCR5 Antagonist, in the Treatment of Painful Diabetic Neuropathy in a Mouse Model

Aleksandra Bober et al. Int J Mol Sci. .

Abstract

The ligands of chemokine receptors 2 and 5 (CCR2 and CCR5, respectively) are associated with the pathomechanism of neuropathic pain development, but their role in painful diabetic neuropathy remains unclear. Therefore, the aim of our study was to examine the function of these factors in the hypersensitivity accompanying diabetes. Additionally, we analyzed the analgesic effect of cenicriviroc (CVC), a dual CCR2/CCR5 antagonist, and its influence on the effectiveness of morphine. An increasing number of experimental studies have shown that targeting more than one molecular target is advantageous compared with the coadministration of individual pharmacophores in terms of their analgesic effect. The advantage of using bifunctional compounds is that they gain simultaneous access to two receptors at the same dose, positively affecting their pharmacokinetics and pharmacodynamics and consequently leading to improved analgesia. Experiments were performed on male and female Swiss albino mice with a streptozotocin (STZ, 200 mg/kg, i.p.) model of diabetic neuropathy. We found that the blood glucose level increased, and the mechanical and thermal hypersensitivity developed on the 7th day after STZ administration. In male mice, we observed increased mRNA levels of Ccl2, Ccl5, and Ccl7, while in female mice, we observed additional increases in Ccl8 and Ccl12 levels. We have demonstrated for the first time that a single administration of cenicriviroc relieves pain to a similar extent in male and female mice. Moreover, repeated coadministration of cenicriviroc with morphine delays the development of opioid tolerance, while the best and longest-lasting analgesic effect is achieved by repeated administration of cenicriviroc alone, which reduces pain hypersensitivity in STZ-exposed mice, and unlike morphine, no tolerance to the analgesic effects of CVC is observed until Day 15 of treatment. Based on these results, we suggest that targeting CCR2 and CCR5 with CVC is a potent therapeutic option for novel pain treatments in diabetic neuropathy patients.

Keywords: cenicriviroc; chemokines; diabetes; female; male; neuropathic pain.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflicts of interest.

Figures

Figure 1
Figure 1
Changes in weight, blood glucose levels, and pain hypersensitivity on the 7th day after vehicle or STZ treatment in male and female mice. The effects of a single STZ injection on weight (A), blood glucose level (B), and pain hypersensitivity (C,D). The behavioral tests were performed on Day 7 after STZ injection. Mechanical and thermal hypersensitivity were measured by von Frey (C) and cold plate (D) tests, respectively. The data are presented as the mean ± SEM. The total number of animals included 70 mice: 32 males (n = 14–17 per group) and 38 females (n = 18–20 per group). The results were statistically evaluated using a t-test; ^^ p < 0.01 and ^^^ p < 0.001 indicate changes compared with non-diabetic mice injected with water. Abbreviations: ns, not significant; STZ, streptozotocin; CTRL, control.
Figure 2
Figure 2
Changes in the mRNA levels of CCR2 and CCR5 ligands on the 7th day after vehicle or STZ treatment in male and female mice. The effects of a single STZ injection on the mRNA levels of Ccl2 (A), Ccl3 (B), Ccl4 (C), Ccl5 (D), Ccl7 (E), Ccl8 (F), Ccl11 (G), and Ccl12 (H) were measured by RT–qPCR. The data are presented as the fold change relative to that of control mice (non-diabetic mice injected with water for injection) ± SEM, except for (A), where data are presented as a relative expression ± SEM. The total number of animals was 43 mice, with 23 males (n = 10–12 per group) and 20 females (n = 8–11 per group), except for (D), in which females (n = 5–8 per group) were included. The results were statistically evaluated using a t-test; ^ p < 0.05, ^^ p < 0.01, and ^^^ p < 0.001 indicate changes compared with the control. Abbreviations: ns, not significant; STZ, streptozotocin; CTRL, control.
Figure 3
Figure 3
Changes in the protein levels of CCR2 and CCR5 ligands (CCL2, CCL5, CCL7, and CCL8) on the 7th day after vehicle or STZ treatment in male and female mice. The effects of a single STZ injection on the protein levels of CCL2 (A), CCL5 (B), CCL7 (C), and CCL8 (D) were measured by ELISA. The data are presented as the fold change relative to that of control mice (non-diabetic mice were injected with water for injection) ± SEM. The total number of animals was 40 mice: 20 males (n = 7–10 per group) and 20 females (n = 8–10 per group). The results were statistically evaluated using a t-test; ^ p < 0.05 indicates changes compared with the control. Abbreviations: ns, not significant; STZ, streptozotocin; CTRL, control.
Figure 4
Figure 4
Changes in the CCR2 and CCR5 mRNA and protein levels on the 7th day after vehicle or STZ treatment in male and female mice. The effects of a single STZ injection on Ccr2 and Ccr5 mRNA (A,B) and CCR2 and CCR5 protein (C,D) levels were measured by RT-qPCR and Western blot, respectively. The data are presented as the fold change relative to that of control mice (non-diabetic mice were injected with water for injection) ± SEM. The total number of animals was 43 mice: 23 males (n = 9–12 per group) and 20 females (n = 7–11 per group). The results were statistically evaluated using a t-test; ^ p < 0.05 indicates changes compared with the control. Abbreviations: ns, not significant; STZ, streptozotocin; CTRL, control.
Figure 5
Figure 5
Effect of i.p. injection of CVC on pain hypersensitivity measured on the 7th day after vehicle or STZ treatment in male and female mice. Pretests were performed before CVC administration, and behavioral tests were performed 2, 4, 6, and 24 h after CVC injection (A). Thermal and mechanical hypersensitivity were measured by von Frey (B,C) and cold plate (E,F) tests, respectively. The AUC was calculated for each test (D,G). The data are presented as the mean ± SEM. The total number of animals: 64 mice: 23 males (n = 7–8 per group at each time point) and 41 females (n = 11–15 per group at each time point except 24 h, where n = 4–6 per group). The results were statistically evaluated using one-way ANOVA with the Bonferroni correction; # p < 0.05, ## p < 0.01, and ### p < 0.001 indicate changes compared with those in the V1 group, & p < 0.05 indicates changes between mice treated with 2 and 5 mg/kg CVC, and @ p < 0.05 indicates changes between male and female mice. Abbreviations: AUC, area under the curve; CVC, cenicriviroc; intraperitoneal, i.p.; STZ, streptozotocin; V1, vehicle (1% DMSO).
Figure 6
Figure 6
Effect of repeated i.p. CVC injections on pain hypersensitivity and morphine tolerance on Days 6–22 after STZ treatment in female mice. Pretests were performed on Day 6 after STZ injection. (A). Behavioral tests were performed on Days 8, 9, 11, 13, 15, 17, 20, and 22. In diabetic mice V2 or M was administered twice daily 0.5 h after V1 or CVC injection, and behavioral tests were performed 0.5 h after the last injection (A). Thermal and mechanical hypersensitivity were measured by von Frey (B) and cold plate (E) tests, respectively. The early (Days 8–15) and late (Days 15–22) phase AUC were calculated (C,D,F,G). The data are presented as the mean ± SEM. The total number of animals: 57 mice, n = 9–12 per group. The results were statistically evaluated using one-way ANOVA with the Bonferroni correction; * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate changes compared with the naïve group; # p < 0.05, ## p < 0.01, and ### p < 0.001 indicate changes compared with the V1 + V2 group; $ p < 0.05, $$ p < 0.01, and $$$ p < 0.001 indicate changes compared with the V1 + M-group; and %% p < 0.01 and %%% p < 0.001 indicate changes compared with the CVC + M group. Abbreviations: AUC, area under the curve; CVC, cenicriviroc; intraperitoneal, i.p.; STZ, streptozotocin; V1, vehicle (1% DMSO); V2, vehicle (water for injection); M, morphine; N, naïve.
Figure 7
Figure 7
Effect of repeated i.p. CVC injections on weight, blood glucose levels, and motor functions on Days 21–22 after STZ treatment in female mice. Weight (A) and blood glucose level (B) were measured on the last (22) day of behavioral testing. Motor functions were measured on Day 21, 0.5 h after morphine injection by the rotarod test (C). The data are presented as the mean ± SEM. The results were statistically evaluated using one-way ANOVA with the Bonferroni correction; * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate changes compared with the naïve group; # p < 0.05 and ## p < 0.001 indicate changes compared with the V1 + V2 group; $ < 0.05 indicates changes compared with the V1 + M-group; and % p < 0.05 and %% p < 0.01 indicate changes compared with the CVC + M group. Abbreviations: CVC, cenicriviroc; intraperitoneal, i.p.; STZ, streptozotocin; V1, vehicle (1% DMSO); V2, vehicle (water for injection); M, morphine; N, naïve.
Scheme 1
Scheme 1
Study design and procedures used in the experiment.
See this image and copyright information in PMC

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