1. Antigen-Adjuvant Dose Response in a Rabbit Model using 3M's Microstructured Transdermal System J. Wolter, D. Brandwein, H. Choi, R. Ferber, J. Gysbers, P. Johnson, J. Li, J. Raeder-Devens 3M Drug Delivery Systems, 3M Center, St. Paul, MN 55144 3M Drug Delivery Systems Division has developed a platform for the delivery of vaccines that is based on the use of antigen coated microstructures for localized deposition of antigen in the epidermis. The objective of the study was to map the antigen-adjuvant specific IgG dose response surface for aluminum adjuvanted tetanus toxoid formulations delivered intracutaneously to rabbits using 3M’s Microstructured Transdermal System (MTS). The region of parameter space that was mapped included low levels of tetanus toxoid relative to the amount necessary for effective intramuscular (IM) vaccination. MTS is a platform for the delivery of vaccines to the epidermal region of the skin, an area that naturally has a high concentration of antigen presenting cells (including Langerhans cells). MTS is capable of transcutaneous delivery of macromolecules by creating transient micro-channels through the stratum corneum. The MTS system is comprised of an array of microstructures that is injection molded from polycarbonate with defined microstructure height and spacing that is laminated to a backing to form a patch that is applied to the skin with an applicator. MTS patches adhere to the skin with a ring of adhesive. Rabbits are an industry standard immune model for toxinogenic bacterial infection and provide a challenging model to demonstrate the high performance and robustness of the MTS delivery system. Introduction Results and Discussion Conclusions References The antigen-adjuvant dose response surfaces obtained in this study indicate that IgG responses in rabbits are relatively linear with respect to the amount of tetanus toxoid and nonlinear with respect to the amount of aluminum. The response surfaces indicate there are formulations that provide extremely high IgG response relative to the amount of tetanus toxoid deposited in the subcutaneous region of the skin. All coatings and formulations other than formulation 47S elicited specific IgG levels greater that the protective titer of 0.2 IU. Formulation 46R, which deposited approximately 1/10 of the IM tetanus toxoid dose, generated specific IgG responses above the protective titer. ANOVA testing of Day 28 anti-TT-IgG levels indicates that formulation 47S generated statistically lower anti-TT-IgG titers than all other formulations at the 95% confidence level. None of the other groups were statistically differentiable. There were two non-responding animals, both belonging to group 47S. If one assumes the lack of response is due to “special cause” and these zero values are removed from the data, the pooled standard deviation drops significantly. In this case, formulations 43Q and 94C join 47S as statistically different from the other MTS and control formulations at the 95% confidence level. Animal treatments of sedation, clipping and shaving were well tolerated, leaving a low background of irritation. Dose 1 irritation scores were uniformly very low. Dose 2 scores were low but edema was more persistent for a few formulations. Edema did not track with efficacy. Formulation options that gave higher efficacy with low irritation were identified. Dose 2 site-specific skin responses were consistent with a delayed type hypersensitivity reaction suggesting that Dose 1 delivered a potent primary immunization. Pathology report of biopsies from several Dose 2 sites on day 28 found that “the skin responses were consistent with a delayed type hypersensitivity reaction”. Hair density was higher in general, but hair length was about the same for Dose 2 compared to Dose 1. MTS systems were easy to handle and robustly applied to the animals. 3 Drug Delivery Systems Two types of aluminum adjuvanted tetanus toxoid formulations were coated on MTS arrays and tested for IgG response in rabbits over a six-week period. The tetanus toxoid was fully recoverable and remained stable throughout the study period. All formulations elicited anti-tetanus toxoid IgG levels, as shown Figure 1. Antigen-adjuvant dose response surfaces are shown in Figures 3 and 4. All samples had anti-tetanus toxoid IgG levels sufficient to provide prophylaxis, and high enough on the assay curve where the results of direct binding ELISA are known to correlate with in vivo toxin challenge (2). The commercial tetanus toxoid vaccine used as a control demonstrated that the animal model is capable of generating and detecting anti-tetanus toxoid IgG 140 patches were used over the course of the study. All patches were successfully applied; adhesion was excellent and was operator independent. A protocol was developed to remove as much hair from the MTS application site as possible without causing irritation. Hair removal was well tolerated as evidenced by low irritation scores after the first dose. Sedation was sufficient to keep the animals in an awake but quiescent state for the 20-minute dosing period. Erythema and edema was scored on the Draize scale at 20 minutes and then daily for 4 days after patch removal. The first application was well tolerated; irritation was typically non-existent. After the second application, erythema was generally slight to non-existent while edema was moderate to non-existent, depending on the formulation. Irritation was typically resolved by the end of the observation period, but edema was more persistent in three of the groups. End of study histology results of several second-dose skin sites was consistent with a delayed-type hypersensitivity reaction. Acknowledgments David Wirtanen, Dennis Ferguson, Frank Frederickson, Marla Emery, Ronald Krienke, Kevin Puckett, Rebecca Severson, Mindy Hoey, Thomas Keene, Nathan Johnson 1. European Directorate for Quality Medicines, European Pharmacopedia Commission, BRP Batch 1, Catalog Number C2425600 2. O. Simonsen, M.W. Bentzon, and I. Heron, J. Biol. Standardization (1986) 14, 231- 239