Transcutaneous immunotherapy of pollinosis using solid-in-oil nanodispersions loaded with T cell epitope peptides.

Pollinosis, a typical seasonal allergy, is a serious public health problem. Limited numbers of patients receive curative immunotherapy instead of symptomatic therapy; however, there are still some concerns about the inconvenience and side effects of subcutaneous injections and sublingual administration caused by immunotherapy. Here, we propose a simple and safe transcutaneous immunotherapy using solid-in-oil (S/O) nanodispersions loaded with vaccine T cell epitope peptides derived from pollen allergen. S/O nanodispersions are oil-based dispersions of antigens coated with hydrophobic surfactants. They have a high potential to deliver biomolecules including peptides or proteins to immune cells in the skin, and to induce an immune response. The result of quantitative and qualitative analysis by in vitro permeation experiments demonstrated the effective permeation of T cell epitope peptides into the skin. Furthermore, in vivo experiments using a pollinosis mouse model indicated that the S/O nanodispersions loaded with T cell epitopes suppressed serum antibody IgE and cytokine production, and alleviated allergic symptoms to a similar therapeutic level to that observed for subcutaneous injection. These results indicate the potential of transcutaneous immunotherapy using S/O nanodispersions for the future treatment of pollinosis.

Transcutaneous Peptide Immunotherapy of Japanese Cedar Pollinosis Using Solid-in-Oil Nanodispersion Technology.

Peptide immunotherapy is an attractive approach to relieve allergic symptoms such as rhinitis and asthma. Treatment of Japanese cedar pollinosis (Cryptomeria japonica; Cj), from which over one quarter of Japanese population suffer, is becoming a great concern. Recently, oral feeding of a peptide (7crp) consisting of seven immunodominant human T cell epitopes derived from two enzymes present in Cj pollen was demonstrated to have a benefit in treating Cj pollinosis. In this work, we aimed to apply a novel transcutaneous administration system as a simple and easy peptide delivery for an immunotherapy using a T cell epitope peptide. A modified 7crp peptide (7crpR) which contained triarginine linkers between each epitopes was designed to increase water solubility and was encapsulated in a unique solid-in-oil (S/O) nanodispersion. The S/O nanodispersion consists of a nano-sized peptide-surfactant complex dispersed in an oil vehicle. The S/O nanopartilces having an average diameter of 230 nm facilitated the permeation of the peptide 7crpR into the skin and suppressed serum total IgE and antigen-specific IgE levels in a Cj pollinosis mouse model. Transcutaneous administration of the T cell epitope peptide using the S/O nanodispersion system has potential for future simple and easy immunotherapy of Cj pollinosis.

Transglutaminase-mediated synthesis of a DNA-(enzyme)n probe for highly sensitive DNA detection.

A new synthetic strategy for DNA-enzyme conjugates with a novel architecture was explored using a natural cross-linking catalyst, microbial transglutaminase (MTG). A glutamine-donor substrate peptide of MTG was introduced at the 5-position on the pyrimidine of deoxyuridine triphosphate to prepare a DNA strand with multiple glutamine-donor sites by polymerase chain reaction (PCR). A substrate peptide that contained an MTG-reactive lysine residue was fused to the N terminus of a thermostable alkaline phoshatase from Pyrococcus furiosus (PfuAP) by genetic engineering. By combining enzymatically the substrate moieties of MTG introduced to the DNA template and the recombinant enzyme, a DNA-(enzyme)(n) conjugate with 1:n stoichiometry was successfully obtained. The enzyme/DNA ratio of the conjugate increased as the benzyloxycarbonyl-L-glutaminylglycine (Z-QG) moiety increased in the DNA template. The potential utility of the new conjugate decorated with signaling enzymes was validated in a dot blot hybridization assay. The DNA-(enzyme)(n) probe could clearly detect 10(4) copies of the target nucleic acid with the complementary sequence under harsh hybridization conditions, thereby enabling a simple detection procedure without cumbersome bound/free processes associated with a conventional hapten-antibody reaction-based DNA-detection system.

Oral delivery of diclofenac sodium using a novel solid-in-oil suspension.

The present work reports on a new pharmaceutical formulation for oral delivery of diclofenac sodium (DFNa), a non-steroidal anti-inflammatory drug (NSAID). Although DFNa itself is water-soluble at neutral pH, it was readily suspended in soybean oil via complex formation with an edible lipophilic surfactant and a matrix protein. The resulting solid-in-oil (S/O) suspension containing stably encapsulated DFNa in an oil phase markedly reduced the risks for gastrointestinal ulcers upon oral administration even at the LD(50) level in rats (ca. 50 mg/kg DFNa). In addition, plasma concentration of DFNa upon administration of an S/O suspension was comparable with that of the aqueous counterpart at the same DFNa dose. These results indicate the potential use of S/O suspensions as novel oil-based pharmaceutical formulations for oral delivery of water-soluble drugs without causing severe mucitis.

An enteric-coated dry emulsion formulation for oral insulin delivery.

A novel oral dosage formulation of insulin consisting of a surfactant, a vegetable oil, and a pH-responsive polymer has been developed. First, a solid-in-oil (S/O) suspension containing a surfactant-insulin complex was prepared. Solid-in-oil-in-water (S/O/W) emulsions were obtained by homogenizing the S/O suspension and the aqueous solution of hydroxypropylmethylcellulose phthalate (HPMCP). A microparticulate solid emulsion formulation was successfully prepared from the S/O/W emulsions by extruding them to an acidic aqueous solution, followed by lyophilization. The insulin release from the resultant dry emulsion responded to the change in external environment simulated by gastrointestinal conditions, suggesting that the new enteric-coated dry emulsion formulation is potentially applicable for the oral delivery of peptide and protein drugs.