Delivery of Soluble Tumor Necrosis Factor Receptor from In-Situ Forming PLGA Implants: In-Vivo Rom E. Eliaz, 1,2 David Wallach, 3 and Joseph Kost 1,4 Received August 23, 2000; accepted September 15, 2000 KEY WORDS: biodegradable polymers; injectable implant delivery system; in-situ; protein/peptide delivery system; in-vivo/in-vitro cor- relation; TNF soluble receptors. INTRODUCTION Many harmful effects of tumor necrosis factor (TNF) result from chronic formation of this cytokine at certain dis- tinct loci in the body. The soluble forms of the TNF receptors p55 and p75, which have the ability to block TNF action by competing for it with the cell surface TNF receptors (1–4), can protect against harmful pathological effects of TNF in chronic inflammatory diseases. Chronic inflammatory diseases are as- sociated with increased endogenous formation of the soluble TNF receptors, in part resulting from stimulation of their synthesis by TNF (5–12). These receptors are formed by pro- teolytic processing of the cell surface TNF receptors and cor- respond to the cysteine-rich ligand-binding regions in their extracellular domains. Since sp55-R are cleared rather rapidly from the blood, repeated injections may not maintain them at the required site, at high enough concentrations. Due to the short half-life (6 hours) of this protein in-vivo, a controlled delivery system based on implants of ethylene vinyl-acetate (EVAc) or injec- tions of microspheres of poly (lactic-co-glycolic) acid (PLGA) copolymers containing human soluble p55 TNF receptors (13), could enhance the therapeutic potential of the natural soluble TNF receptors in chronic diseases. The objective of this study was to develop a drug delivery system with the benefits of an implant but the ease of admin- istration of an injection to the site of need. This delivery system involves simple preparation procedures and avoids an invasive technique such as surgery in its implantation or re- moval. We have developed an in-situ forming implant system that can be administered as a liquid using standard syringes and needles. A biodegradable injectable delivery system was described recently by Dunn et al. (14). Upon contact with body fluids, the liquid system coagulates to form a solid im- plant for drug delivery. We looked for materials that have been approved for human application. We present a system comprised of a bioactive agent and a water insoluble biode- gradable polymer (poly lactic-co-glycolic acid, PLGA) dis- solved in a pharmaceutically acceptable water-miscible sol- vent (glycofurol). PLGA is a commercially available product approved by the FDA for human application. Glycofurol has been used as a solvent in parenteral products for intravenous, intranasal or intramuscular injection (15–21). We show that the injectable in-situ-forming controlled release system used in-vitro (22), is able to increase the soluble TNF receptor serum concentrations such as occurs in chronic inflammatory diseases in a controlled manner in-vivo. Furthermore, we demonstrate that this system can be used as a long-term protection devise against the harmful pathologi- cal effects of TNF. MATERIALS AND METHODS Preparation of PLGA Formulations The copolymer PLGA 75:25 (Boheringer, Resomer RG 755, i.v. 0.59 dl/g) was dissolved in glycofurol (-[(tetrahydro- 2-furanyl) methyl]-w-hydroxy-poly(oxy-1,2-ethanediyl); Sigma Chemical Co., USA) at room temperature. The pro- teins were incorporated into the PLGA solution in a dis- persed form. Powders of sp55-R (the human recombinant protein, Mw36 kD, produced in Chinese hamster ovary cells (CHO), InterPharm Laboratories Ltd., Israel), BSA (Sigma Chemical Co., USA) or a mixture of sp55-R and BSA (sp55-R/BSA solution containing sp55-R, BSA and 0.5 mM sodium phosphate buffer, pH 6.0, was freeze dried using a Labconco Lyph lock-6 freeze-drier) were sieved, yielding par- ticles of 75–100 m. The protein particles were dispersed into a 3 ml PLGA solution in a 15 ml tube using vortex (Press-to- mix, 34524, Snijders) at maximum speed for 3 seconds and probe sonication (Model VC-250, Sonics and Material Inc.) at output 4 (50 W) for 30 seconds, while the solution was kept on ice. Determination of the Release Characteristics In Vitro In vitro release profiles were obtained by injecting 350 l of the suspensions through a needle directly into 10 ml of phosphate buffered saline (PBS, pH 7.4) contained in a vial (20 ml capacity) to form a solid device. The study was carried out at 37°C in a shaker bath. Samples of the release media were withdrawn periodically and BSA release was quantified by absorbance at 280 nm using a spectrophotometer (Spec- tronic Model 1201, Milton Roy Ltd.). sp55-R levels were de- termined by a 2-site capture Enzyme Linked Immuno Sor- bent Assay (23). After every sampling the PBS solution was decanted from the solid device and replaced with fresh PBS. The amount of protein in PLGA implants reflect the actual percentage of protein entrapped in the system allowed to age in PBS as determined by two methods: directly by recovering the protein from PLGA implants and indirectly by measuring the residual unentrapped protein in the outer water phase. 1 Department of Chemical Engineering, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel. 2 Present address: Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, School of Pharmacy, University of Cali- fornia, San Francisco, CA 94143-0446. 3 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100, Israel. 4 To whom correspondence should be addressed. (email: kost@ bgumail.bgu.ac.il) ABBREVIATIONS: TNF, Tumor necrosis factor; sp55-R, soluble p55 TNF receptors; EVAc, ethylene vinyl-acetate; PLGA, poly (lac- tic-co-glycolic) acid. Pharmaceutical Research, Vol. 17, No. 12, 2000 Short Communication 1546 0724-8741/00/1200-1546$18.00/0 © 2000 Plenum Publishing Corporation