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Imiquimod, a toll-like receptor 7 (TLR7) agonist, is an active pharmaceutical ingredient (API) established for the topical treatment of several dermal cancerous and precancerous skin lesions. Within this work, the immunostimulatory effect of imiquimod is further exploited in a transcutaneous immunization (TCI) approach based on a solid nanoemulsion (SN) formulation. SN contains a combination of imiquimod with the model peptide antigen SIINFEKL as a novel approach to omit needle and syringe and optimize dermal antigen administration. Excipients including sucrose fatty acid esters and the pharmaceutically acceptable oils MCT (middle chain triglycerides), avocado oil, jojoba wax and squalene are high pressure homogenized together with the antigen SIINFEKL. Freeze drying was performed to eliminate water and to achieve spreadable properties of the formulation for dermal administration. The influence of the different oil components was assessed regarding in vitro drug permeation in a Franz diffusion cell model using a murine skin setup. In vivo performance in terms of cytotoxic T-cell response was assessed in a C57BL/6 mouse model. Whereas Aldara® cream contains imiquimod in a dissolved state, the SN formulations carry the active in a suspended state. This resulted in a reduction of imiquimod permeation across murine skin from the SN when compared to Aldara® cream. In spite of this permeation rate reduction, each SN induced an in vivo immune response by specific T-cell lysis. A stabilized solid nanosuspension containing squalene/tocopherol exhibited a significantly higher performance (p⩽0.05) in comparison with Aldara® cream. MCT based SN exerted an in vivo effect comparable to Aldara®. In conclusion, anhydrous highly dispersed vehicles containing imiquimod in a submicron particle size distribution can represent promising formulations for TCI. The choice of the oil component has a strong influence on SN performance, independent of in vitro drug permeation.

This work evaluates the transcutaneous in vitro and in vivo immunization efficacy of five commercially available 5% imiquimod containing formulations. The parameters included micro- scopic analysis, rheological properties, drug permeation across synthetic membranes of molecular weight cutoff 10kDa and ablated murine skin with both 0.1 M HCl and a phthalate buffer pH 3.6 Ph.Eur./methanol 3/7 (v/v) as receiver solutions in a Franz-diffusion cell model. For in vivo formu- lation characterization, the cytotoxic T-cell activity and interferon-g production in C57BL/6 mice was determined ex vivo 24h after transcutaneous administration. OVA257264 (SIINFEKL) from chicken al- bumin served as a target antigen. Microscopic images demonstrated differences with respect to the presence or absence of crystalline API. Rheological properties point to a roughly ten-fold difference between the products at low shear rates. With regard to drug permeation across synthetic membranes, only 'Nan Bo' demonstrated equality compared with Aldara™, resulting in f1 and f2 values of 5.25 and 59.58, respectively. The drug permeation rates were maximum from Aldara™ across ablated murine skin. Furthermore, differentiation between the formulations containing crystalline and dissolved states of active imiqui- mod was possible using this model. The in vivo results yielded significant immunomodulating activities (p

Introduction: Transcutaneous immunization (TCI) is a novel vaccination strategy to induce strong therapeutic cytotoxic T-lymphocyte (CTL) responses by directly targeting skin-resident professional antigen-presenting cells (APC). This vaccination approach is very promising to overcome current limitations of standard vaccination approaches that are mostly effective in prophylaxis, but not in the treatment of diseases. In this context, we have developed a TCI method based on a synthetic TLR7 agonist imiquimod that partial tumor protection in experimental rodent models. In our present work, we describe a novel optimized formulation of imiquimod in a solid suspension of crystalline imiquimod (IMI-Sol) that induces superior CTL responses and enhanced tumor protection. Methods: We determined imiquimod-penetration into the skin as well as systemic distribution by HPLC in C57BL/6 mice (at 6-12 weeks). For TCI, C57BL/6, Tlr7 (TLR7-/-) or Myd88 (MyD88-/-) gene deficient mice IMI-sol or imiquimod (in the commercially available formulation Aldara®) together with the CTL epitope SIINFEKL (derived from chicken ovalbumin) was applied on two consecutive days on the dorsum of the animals after hair removal by electric clippers. Induction of CTL responses was characterized after 7 days by the analysis of peptide-specific CTLs (ELISpot, tetrameric MHC I complexes) and in vivo cytolytic activity by flow cytometry. Tumor protection was assessed using the B16OVA tumor model. Results: Despite equal imiquimod penetration into the skin, we found superior vaccination capacity of IMI-Sol compared to Aldara® leading to enhanced tumor-protection. Using TLR7-/- or MyD88-/- mice, we confirmed that the induction of CTL responses with IMI-Sol was completely dependent on the TLR/MyD88 pathway. The enhanced immune response are due to more efficiently activated skin-derived DCs upon IMI-Sol treatment found in skin-draining lymph nodes. Conclusions: We developed of a superior imiquimod formulation for transcutaneous vaccination that boosts vaccination efficacy by the enhanced activation of skin-resident APCs leading to the induction of therapeutic CTL responses underscoring the importance of pharmaceutical aspects affecting vaccination potency beyond understanding of the underlying basic mechanisms. Combined efforts are necessary to establish TCI as a promising novel next generation vaccination platform that can be used for the treatment of persistent infections and cancer. Disclosures No relevant conflicts of interest to declare.