Anti-nociceptive effect of a conjugate of substance P and light chain of botulinum neurotoxin type A

Abstract

Neuropathic pain is a debilitating condition resulting from damage to sensory transmission pathways in the peripheral and central nervous system. A potential new way of treating chronic neuropathic pain is to target specific pain-processing neurons based on their expression of particular receptor molecules. We hypothesized that a toxin-neuropeptide conjugate would alter pain by first being taken up by specific receptors for the neuropeptide expressed on the neuronal cells. Then, once inside the cell the toxin would inhibit the neurons' activity without killing the neurons, thereby providing pain relief without lesioning the nervous system. In an effort to inactivate the nociceptive neurons in the trigeminal nucleus caudalis in mice, we targeted the NK1 receptor (NK1R) using substance P (SP). The catalytically active light chain of botulinum neurotoxin type A (LC/A) was conjugated with SP. Our results indicate that the conjugate BoNT/A-LC:SP is internalized in cultured NK1R-expressing neurons and also cleaves the target of botulinum toxin, a component-docking motif necessary for release of neurotransmitters called SNAP-25. The conjugate was next tested in a murine model of Taxol-induced neuropathic pain. An intracisternal injection of BoNT/A-LC:SP decreased thermal hyperalgesia as measured by the operant orofacial nociception assay. These findings indicate that conjugates of the light chain of botulinum toxin are extremely promising agents for use in suppressing neuronal activity for extended time periods, and that BoNT/A-LC:SP may be a useful agent for treating chronic pain.

Keywords: Botulinum neurotoxin; Neurokinin receptor; Neuropathic pain.

Figure 1

Synthesis of BoNT/A-LC:SP. A Schematic representation of the procedure used to synthesize BoNT/A-LC:SP. B. Western blots of the final BoNT/A-LC:SP product. The concentrated conjugate was run on western blot and probed with both anti-BoNT/A-LC and anti SP.

Figure 2

In situ evaluation of BoNT/A-LC:SP. A. Cultured neuronal cells from the diencephalon of rat embryos were treated with either BoNT/A-LC:SP (50 ng/ml) or BoNT/A-LC (50 ng/ml) only. The cells were incubated at 25 °C overnight. The immunocytochemistry of the cells was performed using antibodies against LC/A and NK1R. The arrow head indicates the area where LC/A localized after internalization. B. Cleavage of SNAP-25 by BoNT/A-LC:SP. Cultured neuronal cells were incubated with either BoNT/A-LC (100 ng/ml) or BoNT/A-LC:SP (100 ng/ml) at 25 °C for 48 hours. A control was run treating the cells with PBS only. The unbound toxin or toxin conjugate was washed away with PBS. The cells were sonicated and the SNAP-25 cleavage was analyzed by western blot using anti-SNAP-25 antibodies.

Figure 3

Hyperalgesic effect of Taxol in mice at 46 °C. A. Mice (N=16) received a cumulative 2 mg/kg dose of Taxol and were tested in a thermal operant system as described in the methods section. A * indicates p<0.05 and a ** indicates p<0.01 for week 3 and week 4 respectively in a Repeated Measures One-Way ANOVA followed by Dunnett’s test. B. Mice (N=8) received vehicle for Taxol and tested in the same device. There was no significant change in behavior at 46 °C for the vehicle group as evidenced by P>0.05 in a One-Way Repeated Measures ANOVA followed by Dunnett’s test. C and D. Thermal operant response at 37 °C for the above Taxol receiving group (N=16) and vehicle group (N=8) with a P>0.05 for both groups indicating no significant change in behavior at non-nociceptive temperature.

Figure 4

Antinociceptive effect of SP-LC/A. A. Mice received BoNT/A-LC:SP (N=9) or PBS (N=3) by i.c.m were analyzed for their behavioral response at noxious temperature (46 °C) in the thermal operant system after 2 weeks of training at 37 °C and 46 °C alternatively. A* indicates p<0.05 in an unpaired t-test with Welch’s correction. B. Both the BoNT/A-LC:SP (N=8) and PBS group (N=8) were treated with Taxol and their behavior to a noxious temperature (46 °C) was measured with the thermal operant system for two weeks. After 2 weeks each mouse of the LC-SP group received 1μg of BoNT/A-LC:SP in 2μl i.c.m and each mouse in the PBS group received 2 μl of PBS i.c.m. After 48 hours both groups were tested for pain response in thermal operant system for up to two consecutive weeks. A* indicate P<0.05 and a ** indicates P<0.001 in a Repeated Measures One-Way ANOVA followed by Dunnett’s test. A ## indicates significant comparison in an unpaired t-test (P< 0.0001) between the BoNT/A-LC:SP and PBS treated group 2 days after the treatment.