The safety of botulinum neurotoxin type A's intraarticular application in experimental animals

Abstract

In vivo studies of botulinum neurotoxin type A (BoNT-A) enabled characterization of its activity in the nociceptive sensory system separate from its preferred action in motor and autonomic nerve terminals. However, in the recent rodent studies of arthritic pain which employed high intra-articular (i.a.) doses (expressed as a total number of units (U) per animal or U/kg), possible systemic effects have not been conclusively excluded. Herein we assessed the effect of two pharmaceutical preparations, abobotulinumtoxinA (aboBoNT-A, 10, 20, and 40 U/kg corresponding to 0.05, 0.11, and 0.22 ng/kg neurotoxin) and onabotulinumtoxinA (onaBoNT-A, 10 and 20 U/kg corresponding to 0.09 and 0.18 ng/kg, respectively) injected into the rat knee, on safety-relevant readouts: digit abduction, motor performance and weight gain during 14 days post-treatment. The i. a. toxin produced dose-dependent impairment of the toe spreading reflex and rotarod performance, which was moderate and transient after 10 U/kg onaBoNT-A and ≤20 U/kg aboBoNT-A doses, and severe and long-lasting (examined up to 14 days) after ≥20 U/kg of onaBoNT-A and 40 U/kg aboBoNT-A. In addition, lower toxin doses prevented the normal weight gain compared to controls, while higher doses induced marked weight loss (≥20 U/kg of onaBoNT-A and 40 U/kg aboBoNT-A). Commonly employed BoNT-A formulations, depending on the doses, cause local relaxation of the surrounding muscles and systemic adverse effects in rats. Thus, to evade possible toxin unwanted local or systemic spread, careful dosing and motor testing should be mandatory in preclinical behavioral studies, irrespective of the sites and doses of toxin application.

Keywords: Motor effects; Rats; Systemic spread; abobotulinumtoxinA; onabotulinumtoxinA.

Graphical abstract

Fig. 1

Time-course of the effect of i. a. injections of different doses of two pharmaceutical BoNT-A formulations on motor impairment (balance maintenance and fatigue), measured by rotarod performance. Saline (20 μl) or different doses of abobotulinumtoxinA (aboBoNT-A) or onabotulinumtoxinA (onaBoNT-A) were injected i. a. into the knee joint. The toxin doses are expressed as mouse LD₅₀-based units per kg (U/kg) or nanograms of 150 kDa neurotoxin per kg (ng/kg). The balance maintenance times (latency) at the rotating rotarod device (10 rpm, 180 s cutoff) were measured at baseline (day 0) and 1, 3, 5, 7, and 14 days post-i. a. Treatments. The results (average of 3 measurements per session) are presented as mean ± SEM (n = 3/treatment group), c = p < 0.001 vs. control (normal saline), two-way RM ANOVA followed by Bonferroni's multiple comparisons tests.

Fig. 2

The time-course of the effect of i. a. injections of different doses of two pharmaceutical BoNT-A formulations on toe spreading reflex impairment assessed by digit abduction score (DAS) assay. Saline (0.9% NaCl, 20 μl) or different doses of abobotulinumtoxinA (aboBoNT-A) or onabotulinumtoxinA (onaBoNT-A) were injected i. a. into the knee joint. The doses are expressed as mouse LD₅₀-based units per kg (U/kg) or nanograms of 150 kDa neurotoxin per kg (ng/kg). The DAS score was measured at baseline and 1, 3, 5, 7, and 14 days post-i. a. treatments. The results (average of 3 measurements per session) are presented as mean ± SEM (n = 3/treatment group), a = p < 0.05, b = p < 0.01, c = p < 0.001 vs. saline control), two-way RM ANOVA followed by Bonferroni's multiple comparisons tests.

Fig. 3

The time course of the effect of i. a. injections of different doses of two pharmaceutical BoNT-A formulations on the rat weight. Saline (0.9% NaCl, 20 μl) or different doses of abobotulinumtoxinA (aboBoNT-A) or onabotulinumtoxinA (onaBoNT-A) were injected i. a. into the knee joint. The doses are expressed as mouse LD₅₀-based units per kg (U/kg) or nanograms of 150 kDa neurotoxin per kg (ng/kg). The percent weight change shown at 1, 3, 5, 7, and 14 days post-i. a. treatments were expressed as percent from baseline. The results are presented as mean ± SEM (n = 3/treatment group), a = p < 0.05, b = p < 0.01, c = p < 0.001 vs. control (normal saline), two-way RM ANOVA followed by Bonferroni's multiple comparisons tests.

Fig. 4

The effect of i. a. injections of different doses of two pharmaceutical BoNT-A formulations on the diameter of rat gastrocnemius. Saline (0.9% NaCl, 20 μl) or different doses of abobotulinumtoxinA (aboBoNT-A) or onabotulinumtoxinA (onaBoNT-A) were injected i. a. into the knee joint. The doses are expressed as mouse LD₅₀-based units per kg (U/kg) or nanograms of 150 kDa neurotoxin per kg (ng/kg). The mediolateral gastrocnemius diameter (in mm) was measured 14 days post-i. a. treatments. The results are presented as mean ± SEM (n = 3/treatment group), a = p < 0.05 vs. control (normal saline), one-way ANOVA followed by Bonferroni's multiple comparisons tests.