HHS SBIR PA-09-179

Clostridium botulinum is an anaerobic bacterium that produces a neurotoxin thought to be the most lethal substance known (on a per molecule or per weight basis). Strains of C. botulinum have been identified that produce 7 different types of this neurotoxin, designated as types A through G. The toxin is synthesized as a single polypeptide of approximately 150 kD. Selective proteolysis is important in achieving the fully active toxin molecule consisting of two chains, designated as the heavy and light chains. The toxins are zinc metalloproteases that bind at specific receptor sites on cholinergic presynaptic terminals. The heavy chain is required for binding and entry into the neurons through interaction with the SNARE complex, and the light chain is required to prevent neurotransmitter release by cleaving synaptic terminal proteins (types A and E on SNAP-25; types B, D, F, and G on synaptobrevin; and type C on syntaxin). The botulinum toxin effectively produces a state of functional denervation through the failure of synaptic transmission at neuromuscular junctions (NMJ) by inhibiting release of acetylcholine neurotransmitter which is responsible for activation of muscle contraction. Eventually new nerve terminals are formed and this sprouting and formation of new NMJ is responsible for the recovery of muscle function and the consequent loss of effectiveness of the toxin over subsequent months.

Therapeutic applications of botulinum toxin have increased steadily over the years since the initial FDA approval in 1989 for its use in the treatment of strabismus and blepharospasm. Type A neurotoxin is presently in widespread clinical use although other serotypes are under investigation by various manufacturers, and one type B neurotoxin product is on the market. In addition to cosmetic indications, BoNT/A is employed effectively for the treatment of movement disorders and spasticity, particularly for focal dystonia, the most frequent occurring types of which include cervical dystonia or torticollis, blepharospasm, writers cramp, musicians dystonia, oromandibular dystonia, hemifacial spasm, occupational dystonia, and laryngeal dystonia.

The potency of botulinum toxins is currently being assessed with a mouse intraperitoneal injection assay which measures activity in Mouse Units (MU), with 1 MU of activity defined as the LD50 dose (death within 72 to 96 hours in 50% of the mice). However the LD50 assay does not allow for standardization of potency units for products made by different manufacturers because of the inherent variability in conducting the assay and species differences in sensitivity to the various toxins. This poses problems in product use and has potential safety consequences which are magnified as new products enter the market. Dramatic differences in the apparent potency confound clinical dose finding and could potentially result in over or under dosing of patients.

This Funding Opportunity Announcement (FOA) seeks applications that will develop cell-based or other alternative approaches to the current mouse LD50 assay that will reliably assess the potency of BoNT/A products. The proposed assay must be able to accurately quantify nanogram levels of active toxin in the finished product, which may contain a high concentration of bulking or stabilizing proteins. In addition, the approach must provide high sensitivity, with a limit of detection that resembles or exceeds that of the mouse LD50 assay. If both functions of the neurotoxin cannot be measured in a single assay, then the development of more than one assay is acceptable. The proposal should include development of a reference standard. Assays should have the following minimal characteristics:

• Accurate determination of BoNT/A potency in the therapeutic dose range used in humans
• Suitability for routine commercial release testing of BoNT/A therapeutics. For guidance on some of the requirements for commercial suitability, please see International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use Guidelines Q2(R1) Validation of Analytical Procedures: Text and Methodology. http://www.ich.org/cache/compo/276-254-1.html
• Suitability for use in determining stability of BoNT/A products over time
• Standard potency units that can be applied across different products