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Comparison of in vivo and in vitro mouse bioassays for botulinum toxin antagonists
R E Sheridan1, S S Deshpande, T Smith
1Pharmacology Division, USAMRICD, Aberdeen Proving Ground, MD 21010-5425, USA. robert.sheridan@amedd.army.mil
Abstract:
Measurements of the efficacy of novel botulinum toxin antagonists can be based on classical bioassays of toxin concentration. However, the relative sensitivities of in vivo and in vitro assays to the effects of antagonists are not necessarily correlated with the sensitivities of the assays to toxin. Comparisons of the sensitivity of an in vitro mouse muscle contraction assay with an in vivo mouse survival assay indicated that the in vivo assay was more sensitive to botulinum toxin serotype A by more than one order of magnitude at equivalent molar concentrations. However, in studies of toxin neutralization with equine antisera, the in vitro muscle assay was more than three times more sensitive to the presence of antisera than the equivalent mouse survival assay. For the development of new drugs to treat botulism, antagonist sensitivity is a primary consideration in determining relative efficacy during structure-activity studies. Thus, in studies of toxin antagonists, the in vitro assay appears to be superior for initial testing.
Insights
For botulinum toxin research, in vitro muscle assays are more sensitive to antagonists than in vivo survival assays. This makes in vitro methods superior for initial drug development and structure-activity studies.
Area of Science:
- Biochemistry
- Pharmacology
- Toxicology
Background:
- Botulinum toxin antagonist efficacy is often measured using bioassays.
- In vivo and in vitro assay sensitivities to antagonists may not correlate with toxin sensitivities.
- Botulism treatment drug development requires sensitive antagonist efficacy assays.
Purpose of the Study:
- To compare the sensitivity of in vitro and in vivo assays for botulinum toxin antagonists.
- To determine the optimal assay for initial structure-activity studies of novel botulinum toxin antagonists.
Main Methods:
- Comparison of an in vitro mouse muscle contraction assay with an in vivo mouse survival assay.
- Evaluation of assay sensitivity to botulinum toxin serotype A.
- Assessment of assay sensitivity to toxin neutralization by equine antisera.
Main Results:
- The in vivo assay was over ten times more sensitive to botulinum toxin serotype A than the in vitro assay.
- The in vitro assay was over three times more sensitive to equine antisera than the in vivo assay.
- In vitro assays demonstrated superior sensitivity to toxin antagonists.
Conclusions:
- In vitro muscle contraction assays are more sensitive to botulinum toxin antagonists than in vivo survival assays.
- In vitro assays are preferable for initial structure-activity studies in botulinum toxin antagonist development.
- Assay choice is critical for accurately determining antagonist efficacy in drug development.