The in vivo rat muscle force model is a reliable and clinically relevant test of consistency among botulinum toxin preparations.

To ensure safety and predictable clinical efficacy, the biological activity of type A botulinum toxin (BoNT-A) preparations must remain consistent. Several methods have been employed to assess consistency but lack clinical applicability and/or are associated with animal welfare concerns. Here, we describe a novel in vivo rat muscle force model for evaluating the biological activity of formulated BoNT-A product (Dysport) prepared from bulk toxin batches manufactured at different facilities. Toxin activity was assessed by measuring muscle force generation over time in the triceps surae muscles in the rat hind leg. Animals received 0.1 ml gelatine phosphate buffer (negative vehicle control) or 0.1 or 1.0 LD50 units of BoNT-A in phosphate buffer. Batch equivalence and consistency were confirmed by the lack of significant differences in muscle force generation and duration of effect between each test batch and the reference preparation tested in the same series of experiments. The reduction in muscle force generation was dose-related and reproducible for all active treatment groups. At appropriate dose levels, the rat muscle force model is a reliable tool for measuring biological activity in bulk toxin batches used to formulate clinical product and demonstrates the consistency of batches manufactured over many years.

Heat shock protein 70 overexpression does not attenuate atrophy in botulinum neurotoxin type A-treated skeletal muscle.

Botulinum neurotoxin type A (BoNT/A) is used clinically to induce therapeutic chemical denervation of spastically contracted skeletal muscles. However, BoNT/A administration can also cause atrophy. We sought to determine whether a major proteolytic pathway contributing to atrophy in multiple models of muscle wasting, the ubiquitin proteasome system (UPS), is involved in BoNT/A-induced atrophy. Three and ten days following BoNT/A injection of rat hindlimb, soleus muscle fiber cross-sectional area was reduced 25 and 65%, respectively. The transcriptional activity of NF-κB and Foxo was significantly elevated at 3 days (2- to 4-fold) and 10 days (5- to 6-fold). Muscle RING-finger protein-1 (MuRF1) activity was elevated (2-fold) after 3 days but not 10 days, while atrogin-1 activity was not elevated at any time point. BoNT/A-induced polyubiquitination occurred after 3 days (3-fold increase) but was totally absent after 10 days. Proteasome activity was elevated (1.5- to 2-fold) after 3 and 10 days. We employed the use of heat shock protein 70 (Hsp70) to inhibit NF-κB and Foxo transcriptional activity. Electrotransfer of Hsp70 into rat soleus, before BoNT/A administration, was insufficient to attenuate atrophy. It was also insufficient to decrease BoNT/A-induced Foxo activity at 3 days, although NF-κB activity was abolished. By 10 days both NF-κB and Foxo activation were abolished by Hsp70. Hsp70-overexpression was unable to alter the levels of BoNT/A-induced effects on MuRF1/atrogin-1, polyubiquitination, or proteasome activity. In conclusion, Hsp70 overexpression is insufficient to attenuate BoNT/A-induced atrophy. It remains unclear what proteolytic mechanism/s are contributing to BoNT/A-induced atrophy, although a Foxo-MuRF1-ubiquitin-proteasome contribution may exist, at least in early BoNT/A-induced atrophy. Further clarification of UPS involvement in BoNT/A-induced atrophy is warranted.