The conotoxin Contulakin-G reverses hypersensitivity observed in rodent models of cancer-induced bone pain without inducing tolerance or motor disturbance
Data files
Feb 19, 2026 version files 30.57 KB
Abstract
As the incidence and survival rates of patients with cancer continues to grow, an increasing number of people are living with comorbidities, which often manifests as cancer-induced bone pain (CIBP). The majority of patients with CIBP report poor pain control from currently available analgesics. A conotoxin, Contulakin-G (CGX), has been demonstrated to be an antinociceptive agent in postsurgical and neuropathic pain states via a neurotensin receptor 2 (NTSR2)-mediated pathway. However, the efficacy and side effect profile of CGX have never been assessed in CIBP. Here, we evaluated CGX’s antinociceptive potential in a rodent model of CIBP. We hypothesized that CGX engages the NTSR2 pathway, providing pain relief with minimal tolerance and motor side effects. Our results demonstrated that CGX intrathecal injection in mice with CIBP attenuated both spontaneous pain behaviors and evoked mechanical hypersensitivity, regardless of their sex. Furthermore, the antinociceptive effect of CGX was dependent upon expression of NTSR2 and the R-type voltage-gated calcium channel (Cav2.3); gene editing of these targets abolished CGX antinociception without affecting morphine antinociception. Examination of the side effect profile of CGX demonstrated that, unlike morphine, chronic intrathecal infusion maintained antinociception with reduced tolerance in rats with CIBP. Moreover, at antinociceptive doses, CGX had no impact on motor behavior in rodents with CIBP. Finally, RNAScope and immunoblotting analysis revealed expression of NTSR2 in both dorsal and ventral horns, while Cav2.3 was minimally expressed in the ventral horn, possibly explaining the sensory selectivity of CGX. Together, these findings support advancing CGX as a potential therapeutic for cancer pain.
Dataset DOI: 10.5061/dryad.70rxwdcb7
Description of the data and file structure
There are 6 figures in this paper and the data in the excel sheet is organized by figure and panel in that figure for which there are numerical outputs.
Figure 1:
Panel A: Number of flinches were compared between female groups (saline, CGX, morphine) at each time point for up to 6 hours (pre, post, 1,2,4,6) units were Flinches (counts/2min).
Panel B: Area under the curve (AUC) from data obtained in panel A.
Panel C: Number of flinches were compared between male groups (saline, CGX, morphine) at each time point for up to 6 hours (pre, post, 1,2,4,6) units were Flinches (counts/2min).
Panel D: Area under the curve (AUC) from data obtained in panel C.
Panel E: vonfrey behavioral measurement. Mechanical weight thresholds (MWTs) (measured in grams of pressure) were compared between female groups at each time point. timepoints: in hours (pre,post,1,2,4,6).
Panel F: AUC from data obtained in panel E.
Panel G: von frey mechanical thresholds measured. units in grams
Panel H: AUC from data obtained in panel G.
Figure 2:
Panel B: Number of flinches before CIBP induction (baseline), after surgery, and over 6 hours after injections. Number of flinches were compared between 2 groups (Ctrl plasmid or nstr2 gRNA) of 2 variables (saline, CGX) at each time point for up to 6 hours (pre, post, 1,2,4,6) units were Flinches (counts/2min).
Panel C: AUC analysis of data presented in panel B.
Panel D: vonfrey behavioral measurement. Mechanical weight thresholds (MWTs) (measured in grams of pressure) were compared between groups (Ctrl plasmid or nstr2 gRNA) of 2 variables (saline, CGX)at each time point. timepoints: in hours (pre,post,1,2,4,6).
Panel E: AUC analysis of data presented in panel D.
Ctrl, control; MWT, mechanical withdrawal threshold; NTSR2, neurotensin receptor type 2.
Figure 3:
Panel B: Number of flinches before CIBP induction, after surgery, and over 6 hours after CGX injections. Number of flinches before CIBP induction (baseline), after surgery, and over 6 hours after injections. Number of flinches were compared between 2 groups (Ctrl plasmid or cacna1e gRNA) of 2 variables (saline, CGX) at each time point for up to 6 hours (pre, post, 1,2,4,6) units were Flinches (counts/2min).
Panel C: AUC analysis of data presented in panel B.
Panel D: MWTs were assessed over 6 hours in each condition. Mechanical weight thresholds (MWTs) (measured in grams of pressure) were compared between groups groups (Ctrl plasmid or cacna1e gRNA) of 2 variables (saline, CGX) at each time point for up to 6 hours (pre, post, 1,2,4,6)
Panel E: AUC analysis of data presented in panel D. .
Ctrl, control; MWT, mechanical withdrawal threshold, cacna1e; CaV2.3 ion channel.
Figure 4: CGX’s antinociceptive effect is devoid of motor block and tolerance.
Panel B: Distance travelled and maximum speed were measured during open field sessions. Results represent errors per step and number of errors while mice are crossing the beam composed of 4 mesh-covered segments decreasing in size, 30 minutes after injection. measurements included distance traveled, max speed and errors per segment. groups are Saline, CGX 0.15nmol, CGX 0.5nmol and bupivacaine 12.5ug.
Panel C: CIBP rats open field test. Results represent distance travelled (meters) and maximum speed, meters per second (m/s) during the open field sessions. groups are (CIBP -, CGX -; CIBP +, CGX -; CIBP +, CGX +).
Panel D: tolerance assessment assessing antinociceptive effect. 2 groups (CGX and Morphine) results are represented as antinociceptive effect percent of first day of infusion (day 1). x axis values is the day assessed. days ( 1, 3, 6). AUC represents the overall effect of morphine or CGX during the 6 days of infusion.
Panel E: von frey machanical data represents the curves for MWT (grams) over 8 days during morphine or CGX infusion, used to calculate the percentage of antinociception between days 1 and 6. (x axis Pre-, post- CIBP, day 1,2,3,4,5,6,7,8).
AUC, area under the curve; CGX, Contulakin-G; CIBP, cancer-induced bone pain; i.th., intrathecal; MWT, mechanical withdrawal threshold.
figure 5:
Panel C: results from quantification of western blot data. CaV2.3 expression assessed (% relative to dorsal and ventral horn beta III tubulin expression). values represented in %. and results from quantification of western blot data. NTSR2 expression assessed (% relative to dorsal and ventral horn beta III tubulin expression). values represented in %
Figure 6:
Panel C: the percentage of cells expressing Ntsr2 and/or Cacna1e, in GABAergic neurons or other subtypes or neurons. data set is the percentage of each labeled group. groups (VGAT+ Cacna1e+ Ntsr2+, VGAT+ Cacna1e+, Ntsr2-, VGAT+ Cacna1e- Ntsr2+, VGAT+ Cacna1e- Ntsr2-, VGAT- Cacna1e+ Ntsr2+, VGAT- Cacna1e+ Ntsr2-, VGAT- Cacna1e- Ntsr2+, VGAT- Cacna1e- Ntsr2-)
Panel D: data represent the quantification of puncta in each cell. (N = 3, n = 13, neurons expressing Ntsr2, to 192, GABAergic neurons expressing only Cacna1e). Values represent mean ± SEM. units are the number of puncta measured in image. groups ( vgat- ntsr2+cacna1e+ ntPunct vgat- ntsr2+cacna1e+ caPunct vgat- ntsr2+cacna1e- ntPunct vgat- ntsr2-cacna1e+ caPunct vgat+ ntsr2+cacna1e+ ntPunct vgat+ ntsr2+cacna1e+ caPunct vgat+ ntsr2+cacna1e- ntPunct vgat+ ntsr2-cacna1e+ caPunct)
Files and variables
File: Raw_Data_for_CGX_reverses_hypersensitivity_observed_in_rodent_model_of_CIBP.xlsx
Description: All data is organized by figure (1-6) and by panel in the figures which are part of this publication: The conotoxin Contulakin-G reverses hypersensitivity observed in rodent models of cancer-induced bone pain without inducing tolerance or motor disturbance.
Code/software
software used to collect and analyze data.
Microsoft excel
Graph pad Prism
ImageJ
Any-MAZE