A quantitative bifunctional in vitro potency assay for botulinum neurotoxin serotype A.

INTRODUCTION: Botulinum neurotoxin type A (BoNTA) is one of seven serotypes produced by Clostridium botulinum (types A thru G) and is the serotype most widely used to treat both cosmetic and medical conditions. Potency for botulinum toxin preparations is expressed in mouse LD50 units. There is a need to develop a non-animal based replacement for this potency assay. METHODS: An in vitro potency assay measuring BoNTA activity has been developed that addresses both BoNTA heavy chain binding to its cell receptor SV2C and BoNTA light chain enzymatic activity in cleaving SNAP-25, an intracellular protein essential in neurotransmitter release. This bifunctional assay utilizes a 96 well microtiter format and well defined reagents. Assay characterization determined that the relative standard deviation for intermediate precision was less than 10%. RESULTS: The assay standard curve covers the range of BoNTA concentrations from 0.0624 to 32 ng/mL. Specificity was demonstrated with purified BoNTA heavy chain which inhibited the activity in a dose dependent manner. A correlation between this bifunctional assay and the mouse LD50 potency assay was demonstrated.

A novel botulinum neurotoxin topical gel: Treatment of allergic rhinitis in rats and comparative safety profile.

BACKGROUND: Rhinitis affects a significant proportion of adults and children with typically seasonal or chronic symptoms. Botulinum neurotoxin type A (BoNTA) is a well-known cholinergic antagonist widely used in a number of approved neurological and esthetic indications. This study was designed to assess the therapeutic effect of RT001, a novel topical gel formulation of BoNTA, in the treatment of allergic rhinitis using a rat model and to compare its safety profile with that of an aqueous formulation of BoNTA complex. METHODS: A rat model of allergic rhinitis was used involving induction of classic rhinitis signs (sneezing and nasal itch) in addition to nasal inflammatory pathology to assess the degree of therapeutic effect of RT001. Comparative safety of RT001 and BoNTA complex was assessed in guinea pigs based on lethality and body weight gain. RESULTS: Clinical signs of rhinitis were significantly (p < 0.01) relieved after a single intranasal administration of RT001 and resolved to normal baseline levels within 5 days after treatment. Mucosal inflammation characterized by edema, congestion, and vascular dilatation along with increased expression of vasoactive intestinal peptide was noted in control animals after allergy induction, whereas RT001 treatment resolved inflammation to essentially normal baseline levels. Safety studies in guinea pigs via intranasal dosing revealed ∼31-fold greater safety factor for RT001 when compared with BoNTA complex. CONCLUSION: These results suggest that topical intranasal application of RT001 is effective in relief of clinical signs and inflammatory pathology associated with allergic rhinitis in a rodent model and may provide a safe treatment for rhinitis.

Characterization of diffusion and duration of action of a new botulinum toxin type A formulation.

RT002, an injectable form of botulinum neurotoxin type A (BoNTA), comprised of a purified 150 kDa neurotoxin formulated in a novel formulation, is designed to limit the extent of diffusion and permit safe administration of longer acting doses. The aim of this study was to evaluate the degree of diffusion of RT002 in comparison to another commercially available BoNTA product, Botox® Cosmetic (OnabotulinumtoxinA, Allergan, Inc., Irvine, CA, USA), and establish the relative duration of effect for diffusion matched doses of the two BoNTA formulations using quantitative measurements in mice. Measurement of muscle paralysis by muscle force generation (MFG) in mice at the injected gastrocnemius muscles indicated that RT002 and Botox are equipotent. Measurements of MFG inhibition in an adjacent muscle, the tibialis anterior muscle, indicated significantly less diffusion for RT002 as compared to Botox. When RT002 and Botox were dosed using the established diffusion matched doses, RT002 treatment resulted in an extended duration of drug effect as compared to Botox by 58-100% as assessed by either partial or complete recovery endpoints. Use of a daily voluntary running activity model provided confirmation that the two BoNTA formulations are equipotent by daily running distance and further confirmed the diffusion matched dose ratio assessed by degree of drug effect on body weight gain. Using these diffusion matched doses, RT002 treatment resulted in an extended duration of drug effect as compared to Botox (100-126% increase in duration) as assessed by either partial or complete recovery endpoints. Use of this model provides further evidence that the RT002 formulation limits diffusion with equipotency and thereby may permit safe administration of higher and more efficacious doses. In summary, data from two murine models suggest that RT002 may represent a next generation of BoNTA drug formulation offering superior degree and duration of effect at the intended target while controlling the unwanted diffusion and accompanying adverse effects of BoNTA at neighboring muscles and distal systemic targets.

Production and characterization of clinical grade exosomes derived from dendritic cells.

We describe methods for the production, purification, and characterization of clinical grade (cGMP) exosomes derived from antigen presenting cells (APCs). Exosomes have been shown to have immunotherapeutic properties through their presentation of biologically relevant antigens [Nat. Med. 4 (1998) 594] and are being developed as an alternative to cellular therapies. Exosomes are 50-90-nm-diameter vesicles secreted from multivesicular bodies (MVBs) found in a variety of both hematopoietic and tumor cells. These particles contain antigen presenting molecules (MHC class I, MHC class II, and CD1), tetraspan molecules (CD9, CD63, CD81), adhesion molecules (CD11b and CD54), and costimulatory molecules (CD86); hence, providing them the necessary machinery required for generating a potent immune response [J. Biol. Chem. 273 (1998) 20121; J. Cell. Sci. 113 (2000) 3365; J. Immunol. Methods 247 (2001) 163; J. Immunol. 166 (2001) 7309]. Exosomes from monocyte-derived dendritic cells (MDDCs) were rapidly purified (e.g. 4-6 h of a 2-3 l culture) based on their unique size and density. Ultrafiltration of the clarified supernatant through a 500-kDa membrane and ultracentrifugation into a 30% sucrose/deuterium oxide (D2O) (98%) cushion (density 1.210 g/cm3) reduced the volume and protein concentration approximately 200- and 1000-fold, respectively. The percentage recovery of exosomes ranged from 40% to 50% based on the exosome MHC class II concentration of the starting clarified supernatant. This methodology was extended to a miniscale process with comparable results. Conversely, the classical differential centrifugation technique is a more lengthy and variable process resulting in exosomes being contaminated with media proteins and containing only 5-25% of the starting exosome MHC class II concentration; hence, making it difficult for their use in clinical development. Lastly, we developed the following quality control assays to standardize the exosome vaccine: quantity (concentration of MHC class II) and protein characterization (FACS). The combination of a rapid and reproducible purification method and quality control assays for exosomes has allowed for its evaluation as a cancer vaccine in clinical trials [Proc. Am. Soc. Oncol. 21 (2002) 11a].