CR4056, a new analgesic I2 ligand, is highly effective against bortezomib-induced painful neuropathy in rats

Figures & data

Figure 1 Flow charts of in vivo experiments: Experiments 1, 2, and 3.

Abbreviations: BTZ, bortezomib; CTRL, control; i.v., intravenously; p.o., orally; s.c., subcutaneously.

Figure 2 Body weight changes along the study period: Experiment (Exp) 1, phase (A) 1 and (B) 2; in Exp 1, bortezomib (BTZ)-treated animals do not show any significant difference in weight gain with respect to the control (CTRL) at the end of the 8-week treatment. Exp 2, phase (C) 1 and (D) 2; Exp 3, phase (E) 1 and (F) 2; in Exps 2 and 3, BTZ significantly affected body weight growth (C and E) (P < 0.01); CR4056 when coadministered with BTZ neither worsened nor improved the toxicity induced by BTZ (E). No significant changes were observed in each group between the values measured during phase 2 (B, D, and F).

Note: Data presented as mean plus or minus standard deviation.
Abbreviations: BTZ-fu, bortezomib-fake-manipulated but untreated; Bupre, buprenorphine; CR4056 0.6, CR4056 at 0.6 mg/kg/day; CR4056 2, CR4056 at 2 mg/kg/day; CR4056 20, CR4056 at 20 mg/kg/day; CR4056 6, CR4056 at 6 mg/kg/day; CR4056 60, CR4056 at 60 mg/kg/day; Gaba, gabapentin; ws, weeks.

Figure 3 Results of nerve conduction velocity (NCV) study performed on the tail of control (CTRL) and bortezomib (BTZ)-treated rats: Experiment (Exp) 1, phase (A) 1 and (B) 2; Exp 2, phase (C) 1 and (D) 2; Exp 3, phase (E) 1 and (F) 2. BTZ induced a significant reduction in NCV in all BTZ-treated rats after 8 weeks of treatment and at the end of Exps 1, 2, and 3.^a The analgesic compounds tested did not significantly counteract the effect of BTZ on NCV.

Notes: ^aMean values plus or minus standard deviation; *Significantly different from CTRL (P < 0.001).
Abbreviations: BTZ-fu, bortezomib-fake-manipulated but untreated; Bupre, buprenorphine; CR4056 0.6, CR4056 at 0.6 mg/kg/day; CR4056 2, CR4056 at 2 mg/kg/day; CR4056 20, CR4056 at 20 mg/kg/day; CR4056 6, CR4056 at 6 mg/kg/day; CR4056 60, CR4056 at 60 mg/kg/day; Gaba, gabapentin; ws, weeks.

Figure 4 Plantar test phase 1 (A) and 2 (B) in Experiment 1: bortezomib (BTZ) and CR4056 (CR) did not induce any significant modification in thermal threshold with respect to the control (CTRL) rats at any experimental time point evaluated in this study.

Note: Data presented as mean values plus or minus standard deviation.
Abbreviations: Bupre, buprenorphine; CR 20, CR4056 at 20 mg/kg/day; CR 6, CR4056 at 6 mg/kg/day; CR 60, CR4056 at 60 mg/kg/day; sec, second.

Table 1 Dynamic test data (mechanical nociceptive threshold in grams) at baseline (ie, before starting bortezomib [BTZ] treatment) and at the end of phase 1 (8–6 weeks)a

Experiment	Phase	Group
		CTRL	BTZ	CR4056	CR4056 + BTZ
1	Basal	29.33 ± 3.51 (n = 20)	27.33 ± 3.32 (n = 50)	NA	NA
	End of phase 1 (8 weeks)	30.69 ± 1.99	24.20 ± 2.96§	NA	NA
2	Basal	28.50 ± 1.88 (n = 12)	28.51 ± 2.26 (n = 50)	NA	NA
	End of phase 1 (8 weeks)	33.51 ± 2.45	27.62 ± 4.59§	NA	NA
3	Basal	28.52 ± 2.11 (n = 9)	28.95 ± 3.61 (n = 25)	28.54 ± 2.40 (n = 9)	28.93 ± 3.31 (n = 8)
	End of phase 1 (6 weeks)	34.52 ± 4.14	30.16 ± 6.96§	36.77 ± 4.63	34.28 ± 3.05#

Notes:

a Data presented as mean plus or minus standard deviation;

§ P < 0.05, compared with CTRL group;

# P < 0.05, compared with BTZ group.

Abbreviations: CTRL, control; NA, not applicable.

Figure 5 Dynamic test in phase 2 of (A) Experiment (Exp) 1^a; (B) Exp 2^b; and (C) Exp 3^c. The persistent and significant antiallodynic effect of oral CR4056 6 mg/kg was evident in all studies, in comparison with buprenorphine (Bupre), gabapentin (Gaba), and the other doses of CR4056. Before CR4056 administration, in Exps 1, 2, and 3, bortezomib (BTZ)-treated rats showed a significant difference compared with control (CTRL) (P < 0.05).

Notes: ^aExp 1: P < 0.001 for BTZ + CR4056 20 and 60 compared with BTZ-fu at day 1; P < 0.05 for BTZ + Bupre and BTZ + CR4056 6 compared with BTZ-fu at day 1; P < 0.001 for BTZ + CR4056 6, 20 and 60 compared with BTZ-fu at day 3; P < 0.05 BTZ + CR4056 6 compared with BTZ-fu at days 7 and 14. ^bExp 2: P < 0.01 for BTZ + CR4056 2 and 6 compared with BTZ-fu at day 1; P < 0.05 for BTZ + CR4056 0.6 compared with BTZ-fu at day 1; P < 0.01 for BTZ + CR4056 2 and 6 compared with BTZ-fu at day 4; P < 0.05 for BTZ + CR4056 0.6 and BTZ + Gaba compared with BTZ-fu at day 4; P < 0.05 for BTZ + CR4056 6 compared with BTZ-fu at day 14; P < 0.01 for BTZ + CR4056 6 compared with BTZ-fu at day 21. ^cExp 3: P < 0.05 for all groups co-treated with CR4056 and BTZ (BTZ-CR4056, BTZ-CR4056 2ws, BTZ-CR4056 4ws), as compared with BTZ, starting from days 0, 7, and 21, respectively.
Abbreviations: admin, administration; BTZ-fu, bortezomib-fake-manipulated but untreated; CR4056 0.6, CR4056 at 0.6 mg/kg/day; CR4056 2, CR4056 at 2 mg/kg/day; CR4056 20, CR4056 at 20 mg/kg/day; CR4056 6, CR4056 at 6 mg/kg/day; CR4056 60, CR4056 at 60 mg/kg/day; Gaba, gabapentin; IV, intravenously; OS, orally; tiw, three times a week; ws, weeks.

Figure 6 Cytotoxicity effect of bortezomib (BTZ) and CR4056 in (A) A549, (B) H460, (C) RPMI 8226, and (D) H929 cell lines.

Notes: Results are expressed as mean of three independent experiments (percentage of cell survival, mean plus or minus standard deviation, with respect to the control); control value was set as 100%; **Significantly different from control (P < 0.01); ***Significantly different from control (P < 0.001); ^#Significantly different from BTZ (P < 0.01).
Abbreviation: DMSO, dimethyl sulfoxide.

Figure S1. Chemical structure of CR4056.

Figure S2. (A) Porsolt forced swimming test: CR4056 (3, 10, and 30 mg/kg) was administered orally (per os) to male CD1 mice 1 hour before the test and both the immobility time and the latency to immobility were evaluated. (B) Tail suspension test: CR4056 was administered per os to male CD1 mice 30 minutes before the test; two independent experiments were performed to cover a broad range of doses (from 0.1 to 30 mg/kg).

Abbreviations: ns, not significant; sec, second.

Notes: *P < 0.05; ** P < 0.01.

Figure S3. (A) Rota-rod assay: rats were allowed a 30-minute acclimatization period in the testing room and then placed on a 9 cm diameter rod, which increased the speed from 0 to 20 rpm over a 60-second period. The time required for the rat to fall from the rod was recorded, with a maximum score of 60 seconds.^a (B) Open field test: CR4056 effects in the open field assay data (evaluated 1 and 6 hours after oral administration) are expressed as the distance traveled and as the rearing activity following an acute (left) and a subchronic treatment period (right).^b

Notes: ^aMean plus or minus standard error of the mean (n = 5 rats/group); ^bmean plus or minus standard error of the mean (n = 6 rats/group).

Abbreviations: h, hour; os, orally; o.a.d., once a day.