DOI: 10.5061/dryad.70rxwdcb7
title: The conotoxin Contulakin-G reverses hypersensitivity observed in rodent models of cancer-induced bone pain without inducing tolerance or motor disturbance

description: 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.
description: # The conotoxin Contulakin-G reverses hypersensitivity observed in rodent
 models of cancer-induced bone pain without inducing tolerance or motor
 disturbance Dataset DOI:
 [10.5061/dryad.70rxwdcb7](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](https://datadryad.org/downloads/pre_submit/4606816 "Download from Dryad") **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.](javascript:void\(0\);) ##### ## Code/software software used to collect and analyze data. Microsoft excel Graph pad Prism ImageJ  Any-MAZE