RESEARCH PAPER Two-Layered Dissolving Microneedles for Percutaneous Delivery of Peptide/Protein Drugs in Rats Keizo Fukushima & Ayaka Ise & Hiromi Morita & Ryo Hasegawa & Yukako Ito & Nobuyuki Sugioka & Kanji Takada Received: 19 October 2009 / Accepted: 17 February 2010 / Published online: 19 March 2010 # Springer Science+Business Media, LLC 2010 ABSTRACT Purpose Feasibility study of two-layered dissolving micro- needles for percutaneous delivery of peptide/proteins using recombinant human growth hormone (rhGH) and desmo- pressin (DDAVP). Methods Two-layered dissolving microneedles were admin- istered percutaneously to the rat skin. Plasma rhGH and DDAVP concentrations were measured by EIA and LC/MS/ MS. In vivo dissolution and diffusion rates of drugs in the skin were studied using tracer dyes, lissamine green B (LG) for rhGH and evans blue (EB) for DDAVP. Diffusion of drugs vertically into the skin was studied using FITC-dextran (MW= 20 kDa)-loaded dissolving microneedles. Stability experiments were performed at -80°C and 4°C. Results The absorption half-lives, t 1/2a , of rhGH and DDAVP from dissolving microneedles were 23.7±4.3–28.9±5.2 and 14.4±2.9–14.1±1.1 min; the extents of bioavailability were 72.8±4.2–89.9±10.0% and 90.0±15.4–93.1±10.3%, respectively. LG and EB disappeared from the administered site within 2 h and 3 h after administration. Five green fluorescein spots were detected at 15 s and enlarged transversally at 30 s. FITC-dextran was delivered into the microcapillaries at 5 min and 10 min. The rhGH and DDAVP were stable in dissolving microneedles for one month at -80°C and 4°C. Conclusions Results suggest that the two-layered dissolving microneedles are useful as an immediate-release transdermal DDS for peptide/protein drugs. KEY WORDS bioavailability . desmopressin (DDAVP) . dissolving microneedles . recombinant human growth hormone (rhGH) . transdermal delivery INTRODUCTION Although the possibility of synthesized peptide and recom- binant protein drugs becoming available to treat intractable ailments is increasing, the complex structures of these peptide/proteins require special formulation as a pharma- ceutical preparation. The current list of approved thera- peutic peptide/protein drugs is growing rapidly because automated drug discovery and biotechnology production methods are creating new biochemical drugs for an expanding number of intractable ailments (1). Demand for delivery of such macromolecular biopharmaceuticals as peptide/protein drugs is increasing (2). Because oral administration of peptide/protein drugs is the most preferred mode, oral delivery of peptide/protein drugs has been attempted using protease inhibitors (3,4), absorp- tion enhancers (5–7), bioadhesive polymer-modified lip- osomes (8), chitosan capsules (9) and water-in-oil-in-water double emulsions (10–13), bioadhesive DDS (14), and cell- penetrating peptides (15,16). However, oral peptide/pro- tein DDS has not been developed yet because of low oral bioavailability (BA). Consequently, no such oral prepara- tion has been launched onto the pharmaceutical market. To date, most are administered intravenously or subcuta- neously by injection. On the other hand, a percutaneous administration route is an attractive alternative method for the delivery of these drugs because of its many advantages: (1) less or no degradation by hydrolytic enzymes compared to that in the GI tract, (2) no first-pass effects of the liver associated K. Fukushima (*) : A. Ise : H. Morita : R. Hasegawa : Y. Ito : N. Sugioka : K. Takada Department of Pharmacokinetics, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan e-mail: keizo@mb.kyoto-phu.ac.jp K. Takada e-mail: takada@mb.kyoto-phu.ac.jp Pharm Res (2011) 28:7–21 DOI 10.1007/s11095-010-0097-7