Deamidation of Model b-Turn Cyclic Peptides in the Solid State STEPHANIE L. KROGMEIER, D. SRINIVASA REDDY, DAVID VANDER VELDE, GERALD H. LUSHINGTON, TERUNA J. SIAHAAN, C. RUSSELL MIDDAUGH, RONALD T. BORCHARDT, ELIZABETH M. TOPP Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Avenue Lawrence, Kansas Received 31 January 2005; revised 8 July 2005; accepted 15 July 2005 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20468 ABSTRACT: To investigate the importance of secondary structure on peptide deamida- tion in the solid state, two cyclic b-turn peptides and their linear analogs were used as models of Asn residues in structured and unstructured domains, and incorporated into poly(vinyl pyrrolidone) (PVP)-based lyophilized solids. The secondary structure of the model peptides was determined in solution and the solid state using a combination of nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD), and Fourier transform infrared (FTIR) spectroscopy. The model b-turn cyclic peptides were found to be type II b-turns while the linear analogs were determined to be predominantly unstructured. Quantitatively, the cyclic peptides consisted of approximately 80% b-turn while the linear analogs contained only 30%–35% b-turn. To characterize the solid environment, T g , and moisture content of the solid-state formulations were determined. Accelerated stability studies were conducted in the solid state at 378C using formulations lyophilized from solutions at pH 8.8 (0.1 M borate buffer). The effect of matrix mobility on solid-state deamidation was investigated by altering the moisture content through variation of relative humidity or the addition of a plasticizer. Cyclic peptides degraded 1.2–8 times slower than the linear analogs under all of the conditions studied. The observed rate constants, however, for all of the peptides decreased dramatically (four orders of magnitude) in the glassy solids. This suggests the greater importance of matrix mobility in solid-state degradation. Molecular dynamics (MD) simulations were also performed to explore the low energy, preferred state of the peptides, and determine the structure around the b-turn. ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:2616 – 2631, 2005 INTRODUCTION Deamidation of asparagine (Asn) residues is a common route of degradation for peptides and proteins. In solution, the mechanism of deamida- tion has been well established. 1 At neutral to basic pH, the reaction involves the formation of a cyclic imide intermediate with subsequent hydro- lysis to aspartate (Asp) and isoaspartate (isoAsp) containing products. Local conformation strongly influences deamidation since the backbone amide nitrogen must be close enough in space to attack the side chain carbonyl. 2 The effect of secondary structure on deamidation in solution has been extensively explored. 3–6 In solution, the rate of Asn deamidation decreases with increased a- helical content 4 and when the Asn residue is present in a b-turn. 7 The effects of secondary structure on deamidation in the solid state, however, have not been established. Maintaining native secondary structure in the solid state has been proposed as necessary for the chemical stability of peptides and proteins, although little evidence has been presented that directly sup- ports this hypothesis. 8 2616 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 12, DECEMBER 2005 Correspondence to: Elizabeth M. Topp (Telephone: 785-864- 3644; Fax: 785-864-5875; E-mail: topp@ku.edu) Journal of Pharmaceutical Sciences, Vol. 94, 2616–2631 (2005) ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association