Engineering a long-acting, potent GLP-1 analog for microstructure-based transdermal delivery.

Antidiabetic treatments aiming to reduce body weight are currently gaining increased interest. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist administered twice daily via s.c. injection, improves glycemic control, often with associated weight reduction. To further improve the therapeutic efficacy of exendin-4, we have developed a novel peptide engineering strategy that incorporates a serum protein binding motif onto a covalent side-chain staple and applied to the peptide to enhance its helicity and, as a consequence, its potency and serum half-life. We demonstrated that one of the resulting peptides, E6, has significantly improved half-life and glucose tolerance in an oral glucose tolerance test in rodents. Chronic treatment of E6 significantly decreased body weight and fasting blood glucose, improved lipid metabolism, and also reduced hepatic steatosis in diet-induced obese mice. Moreover, the high potency of E6 allowed us to administer this peptide using a dissolvable microstructure-based transdermal delivery system. Pharmacokinetic and pharmacodynamic studies in guinea pigs showed that a single 5-min application of a microstructure system containing E6 significantly improved glucose tolerance for 96 h. This delivery strategy may offer an effective and patient-friendly alternative to currently marketed GLP-1 injectables and can likely be extended to other peptide hormones.

Transdermal delivery of macromolecules using solid-state biodegradable microstructures.

PURPOSE: The purpose of this work is to demonstrate the feasibility of using a proprietary technology called MicroCor™, based on solid-state, biodegradable microstructures (SSBMS), for transdermal delivery of macromolecules. METHODS: The proteins FITC-BSA (66 kDa) and recombinant protective antigen (rPA; 83 kDa) were incorporated into SSBMS arrays using a mold-based, liquid formulation casting and drying process. Arrays were applied to the skin with a custom applicator and then inspected to assess the extent of microstructure dissolution. In vitro FITC-BSA delivery to human cadaver skin was visualized using light and fluorescence microscopy and quantified by extracting and measuring the fluorescently labeled protein. rPA-containing SSBMS arrays were applied in vivo to Sprague-Dawley rats. The resulting serum IgG response was measured by ELISA and compared with responses elicited from intramuscular (IM) and intradermal (ID) routes of administration. RESULTS: FITC-BSA and rPA SSBMS arrays successfully penetrated the skin. Microstructure dissolution was observed over >95% of the array area and >75% of the microstructure length. FITC-BSA delivery correlated with protein content in the formulations. Antibody titers after transdermal delivery of rPA were comparable or higher than IM and ID titers. CONCLUSIONS: Transdermal delivery of macromolecules can be conveniently and effectively accomplished using the MicroCor technology.

Stabilization and Transdermal Delivery of an Investigational Peptide Using MicroCor® Solid-State Dissolving Microstructure Arrays.

The formulation of biotherapeutics presents unique challenges especially with regard to physical and chemical stability and often requires refrigerated storage conditions of final drug products. Peptide A is an example of a developmental compound which showed significant stability challenges when prepared as a liquid formulation for a subcutaneous injection. The aim of the present study was to evaluate whether Peptide A can be successfully formulated in MicroCor® microstructure arrays (MSAs) as an alternative delivery option. MSAs contain a high density of dissolving microstructures allowing for transdermal delivery. In the present work, a 5600-needle MSA (~200 µm long microstructures, 2 cm2 array) was prepared with a therapeutically-relevant dose of Peptide A. The array was shown to be stable under room-temperature storage conditions for 3 months. On in vivo application to Yucatan minipigs, Peptide-A-loaded MSAs demonstrated only mild and transient skin irritation and a very high efficiency of peptide transfer from dissolving microstructures into the skin resulting in absolute bioavailability of 74%. This transdermal bioavailability was very similar to the 73% bioavailability obtained from a subcutaneous injection. This technical feasibility study demonstrated that MicroCor® technology represents a viable option for delivery of Peptide A with significant improvements in peptide stability.

Plasmid DNA and siRNA transfection of intestinal epithelial monolayers by electroporation.

This study was conducted to evaluate the ability of electroporation to efficiently transfect differentiated intestinal epithelial monolayers with plasmid DNA and to determine whether electroporation can transfect these monolayers with short-interfering RNA (siRNA) to cause gene silencing. Confluent T84 monolayers were transfected with reporter plasmids expressing luciferase or green-fluorescent protein or with siRNA directed against the nuclear envelope proteins lamin A/C using electroporation. Optimized electroporation conditions resulted in luciferase and GFP expression. Both intracellular uptake of fluorescently labeled plasmid and expression of the reporter genes increased with increasing electroporation strength and DNA concentration. When monolayers were transfected by lipofection with the reporter plasmids, expression and DNA uptake were less than for electroporation. Electroporation was also found to transfect monolayers with siRNA, which resulted in up to 90% inhibition of targeted protein production. Silencing occurred within 24h of transfection and increased with increasing siRNA concentration. These results suggest that electroporation can provide a valuable research tool for transfection of intestinal epithelial monolayers and other differentiated cell systems, and may ultimately be useful for clinical gene therapy applications.

Increased permeability of intestinal epithelial monolayers mediated by electroporation.

This study assessed whether electroporation enhances transport across intact intestinal epithelial monolayers that mimic the intestinal epithelium. Confluent Caco-2 monolayers were exposed to electroporation pulses and then monitored over time for transepithelial transport of calcein, a small fluorescent tracer, or fluorescein-labeled bovine serum albumin, a large protein. Cumulative transport of both molecules across the monolayers increased significantly (up to 34-fold) after electroporation and depended on electroporation voltage and pulse length and on molecular size. Increased transport was accompanied by a decrease in the transepithelial electrical resistance of the monolayers. Further analysis of these results suggests that the increase in transport observed after electroporation is due, at least in part, to the killing of a small fraction of cells, which increased transport across "leaky" dead cells that remained adherent and increased transport through small, temporary holes left by dead cells that detached, but appeared to reseal within minutes by monolayer restitution. These findings could form the basis for the development of electroporation as a clinical tool to increase intestinal permeability and, thereby, increase the absorption of poorly absorbed drugs.

Fabrication of cell culture-derived influenza vaccine dissolvable microstructures and evaluation of immunogenicity in guinea pigs.

Microstructure patches provide an opportunity for simple, effective, and safe vaccine administration, while achieving the desired immune response. We have evaluated the MicroCor transdermal system for cell culture-derived trivalent influenza vaccine administration. Influenza monovalent purified bulk vaccines (monobulks) (H1N1, H3N2, B) were concentrated by tangential flow filtration, lyophilized, and formulated with biocompatible excipients to form the microstructure array dissolvable tips. Standard single radial immunodiffusion (SRID) determined that the influenza antigens retained potency through the formulation and microstructure array fabrication processes. Array stability was evaluated for storage in both refrigerated and room temperature conditions. Microstructure mechanical strength was confirmed by application to excised pig skin, resulting in successful skin penetration and tip dissolution within 5 min of microstructure insertion. Guinea pigs immunized with influenza vaccine-loaded microstructures had hemagglutinin inhibition (HI) and IgG titers comparable to those obtained by intramuscular injection. After two immunizations, serum HI titers for all immunized groups were greater than 40 (>4-fold higher than the untreated group). These data demonstrate the feasibility for the development of skin delivery technologies that are compatible with cell culture-derived influenza vaccines.

Electroporation-mediated delivery of molecules to model intestinal epithelia.

This study was conducted to determine if electroporation can deliver membrane-impermeant molecules intracellularly to intact, physiologically competent monolayers that mimic the intestinal epithelium. In addition, the long-term effects of electroporation on these monolayers were studied to determine the kinetics with which monolayers recover barrier function. Caco-2 and T84 cells were electroporated as monolayers using calcein and fluorescein-labeled bovine serum albumin as marker molecules for measuring delivery into cells. Confocal microscopy and flow cytometry were used, respectively, to visualize and quantify uptake of these molecules. Transepithelial resistance was used as a measure of physiologic barrier function. We found that intracellular uptake of calcein and bovine serum albumin occurred uniformly throughout both types of model epithelia and increased as a function of voltage, pulse length, and pulse number. There was no significant difference in uptake resulting from single and multiple pulses of the same total exposure time. We also observed that monolayers exposed to electroporation that induced uptake of up to 10(6) molecules/cell were able to recover normal barrier function within one day. These findings suggest that electroporation may be useful for intracellular delivery into monolayers to study epithelial biology and, possibly, for drug delivery to intestinal epithelium.