Botulinum neurotoxin type A (BoNT/A) is intramuscularly injected for the treatment of, e.g., spasticity, cervical dystonia or facial lines. Several BoNT/A products with or without complexing proteins, with non-interchangeable dose units and various duration of effect claims, are approved but hard to compare. The goal of this study was to compare the complexing protein-free approved BoNT/A products IncobotulinumtoxinA (INCO), DaxibotulinumtoxinA (DAXI) and RelabotulinumtoxinA (RELA) in vitro and in vivo. BoNT/A protein content per 100 U was lowest in INCO and highest in DAXI (INCO 0.44, RELA 0.46, DAXI 0.58 ng/100 U). Relative bioactivity of INCO, DAXI and RELA was comparable (116, 104 and 117 U/100 labeled units). INCO and DAXI caused a maximum mouse digit abduction score (DAS) 2–3 days after IM injection of 20 or 40 U/kg. The DAS after 20 U/kg INCO was higher and showed a 10 days longer paralysis than DAXI at equivalent dosing. The in vivo spread of DAXI in the mouse gastrocnemius muscle was indistinguishable from that after INCO, and the spread of RELA ex vivo in porcine muscle was larger than INCO but equal to 0.9% NaCl. These results show the differences between 150 kDa botulinum type A toxin products beyond the published claims.

The neurotoxin botulinum toxin type A (BoNT/A), produced by the bacteria Clostridium botulinum, is commonly injected intramuscularly (IM) for the management of chronic muscle hyperactivity, such as post-stroke spasticity. New indications such as peripheral neuropathic pain require alternative routes of BoNT/A administration, such as subcutaneous (SC) or intradermal (ID). While IM BoNT/A injections may elicit anti-drug-antibodies (ADAs), their occurrence following SC or ID administration is unknown. Therefore, we investigated whether repeated SC or ID injections of 150 kDa BoNT/A can elicit ADAs in a dose-dependent manner, and whether these differ depending upon the route of administration. Mice were injected 5 times ID or SC with 150 kDa BoNT/A or, for higher doses, inactive mutant BoNT/A (DRBoNT/A) or inactivated toxoid (IA-BoNT/A). Total ADAs were analyzed by an immunoassay and the subgroup of neutralizing ADAs by an in vivo digit abduction score (DAS) assay following a challenge of 0.6 U BoNT/A IM. DRBoNT/A and IA-BoNT/A injections elicit ADAs (22.7 U/mL vs. 460.5 U/mL for ID; 4.7 U/mL vs. 339.4 U/mL for SC), while therapeutic doses of 150 kDa BoNT/A do not. Whereas mice with repeated 150 kDa BoNT/A injections at therapeutic dose show an unrestricted DAS of 3.7 (ID) or 3.4 (SC), mice injected repeatedly with 1.8 µg/kg DRBoNT/A or 500 µg/kg IA-BoNT/A show only a minimal DAS of ≤0.7, indicating a high titer of neutralizing ADAs. No differences were observed between administration routes. Accordingly, repeated ID or SC injections of pure 150 kDa BoNT/A at therapeutic doses fail to induce ADA formation in mice. On the other hand, DRBoNT/A ID injections induce higher ADA concentrations than SC, but generate similar amounts of neutralizing ADAs. IA-BoNT/A injections induce ADAs and neutralizing ADAs similarly after ID and SC injections. ADA development at intermediate BoNT/A doses can be higher after ID injection, but does not lead to differences in neutralizing ADAs. Our data demonstrate that the antibody response to botulinum toxin depends predominantly on the protein load, and less on the administration route.