Effect of Polyanions on the Structure and Stability of Repifermin TM (Keratinocyte Growth Factor-2) TIFFANY DERRICK, 3 ADEOLA O. GRILLO, 2 SAMADHI N. VITHARANA, 1 LATOYA JONES, 4 JASON REXROAD, 5 AMBARISH SHAH, 6 MELISSA PERKINS, 2 THOMAS M. SPITZNAGEL, 2 C. RUSSELL MIDDAUGH 1 1 Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047 2 Human Genome Sciences, Inc., Rockville, Maryland 20850 3 GlaxoSmithKline, King of Prussia, Pennsylvania 19406 4 Department of Pharmaceutical Sciences, UCHSC, Denver, Colorado 80262 5 Allergan, Irvine, California 92612 6 MedImmune, Gaithersburg, Maryland 20878 Received 7 June 2006; revised 11 August 2006; accepted 28 August 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20797 ABSTRACT: The interaction of several of the fibroblast growth factors (FGFs) with polyanions is thought to be of physiological significance and has been exploited to create more stable pharmaceutical formulations of FGF-1 and -2. The extent of such phenomena throughout the 23-member FGF family is, however, unknown. In these studies, we examine the effect of several polyanions on the structure and stability of keratinocyte growth factor 2 (KGF-2, FGF-10), a candidate for use as a wound-healing agent. Employing a variety of methods sensitive to the protein’s structure including circular dichroism (CD), intrinsic fluorescence, derivative near-UV absorption spectroscopy, bis- ANS (4,4 0 -dianilino-1,1 0 -binaphthyl-5,5-disulfonic acid) fluorescence, differential scan- ning calorimetry (DSC), and dynamic light scattering (DLS), we find that a variety of polyanions (e.g., heparin, sucrose octasulfate (SOS), and inositol hexaphosphate (IHP)) stabilize KGF-2 by increasing the thermal-unfolding temperature by approximately 9–158C. Negatively charged liposomes produce a similar effect, arguing for relatively nonspecific interactions of polyanions with KGF-2. Unlike some other FGFs, no evidence for the presence of a molten globule state is found during thermal perturbation of this growth factor. The generality of this polyanion/protein interaction is discussed as well as its potential role in various cellular events such as protein folding and transport. ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:761– 776, 2007 Keywords: FGF-10; KGF-2; polyanions; structure; stability; heparin; phytic acid; sucrose octasulfate; heparan sulfate INTRODUCTION Many proteins can exist in alternate conforma- tional forms with each distinguishable state exhibiting unique functional properties. One class of proteins in which some members display this property is the fibroblast growth factors (FGFs). For example, FGF-1 exists under physiological conditions (pH 5–8, 0–0.2 M salt, 30–508C) in a molten globule-like state with near-native sec- ondary structure content, disrupted tertiary con- tacts, and the ability to bind apolar dyes. 1 This form of the protein binds to membranes and may be involved in the unusual transport properties of the protein, 2 which includes exit from cells with- out the use of a conventional signal peptide. Many FGFs bind various polyanions which may be involved in their functional presentation to JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 4, APRIL 2007 761 Correspondence to: C. Russell Middaugh (Telephone: 785- 864-5813; Fax: 785-864-5814.; E-mail: middaugh@ku.edu) Journal of Pharmaceutical Sciences, Vol. 96, 761–776 (2007) ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association