Research paper Physicochemical and pharmacological characterization of novel vasoactive intestinal peptide derivatives with improved stability Satomi Onoue a, * , Shingen Misaka a , Yuki Ohmori a , Hideyuki Sato a , Takahiro Mizumoto c , Mariko Hirose c , Sumiko Iwasa b , Takehiko Yajima b , Shizuo Yamada a a Department of Pharmacokinetics and Pharmacodynamics, University of Shizuoka, Shizuoka, Japan b Department of Analytical Chemistry, Toho University, Chiba, Japan c Department of Product Development, ILS Inc., Ibaraki, Japan article info Article history: Received 27 June 2008 Accepted in revised form 25 May 2009 Available online 28 May 2009 Keywords: VIP Stability Receptor binding Neurite outgrowth UPLC–MS abstract Previously, [R 15,20,21 , L 17 ]-VIP-GRR (IK312532), a long-acting VIP derivative, was proposed as potential drug candidate for the treatment of asthma/COPD. The present work is aimed to elucidate solution- state stability of IK312532 and to develop further stabilized derivative with equipotent or higher bio- logical functions. A stability study on IK312532 was carried out in solution state, and degradation mechanism was deduced by UPLC–MS and amino acid analyses. Three novel VIP derivatives were designed and chemically synthesized on the basis of stability data, being subjected to physicochemical and pharmacological characterization. Solution-state stability studies revealed the gradual degradation of IK312532, following pseudo-first-order kinetics. Chemical modification of IK312532, mainly position at 24, resulted in marked improvement of stability, although the chemical modification had no influ- ence on the secondary structure, receptor binding, and activation of adenylate cyclase in rat lung cells. Novel derivatives also exhibited more potent neurite outgrowth in rat pheochromocytoma PC12 cells when compared to VIP and IK312532, possibly due to improved stability. Deamination of Asn at posi- tion 24 might be responsible for degradation of VIP derivative, and stability and chemical modification studies led us to the successful development of novel VIP derivatives with higher stability and biolog- ical functions. Ó 2009 Elsevier B.V. All rights reserved. 1. Introduction Vasoactive intestinal peptide (VIP) [1], an octacosapeptide, is a member of glucagon/secretin superfamily [2]. VIP and its receptor are widely distributed in the body, such as in the heart, lung, diges- tive and genitourinary tract, eye, skin, ovaries, and thyroid gland [3]. VIP is one of the major peptide transmitters in the central and peripheral nervous systems, being involved in a wide range of biological functions in organisms, including metabolic processes, exocrine and endocrine secretions, cell differentiation, relaxation of smooth muscle [4], secretion of regulatory hormones [5], and regulation of immune response [6]. On the basis of its numerous biological actions, VIP has been considered to be a potential candi- date for pharmaceutical agents for several diseases, including dia- betes [7], asthma [4], impotence [8], and rheumatism [9]. While the pharmacological potency of a peptide itself may be of general importance, chemical and physical stability is also a critical factor in the drug design of peptide for overall therapeutic success. In particular, the rapid degradation of VIP after systemic adminis- tration is part of the reason for the limitation of its clinical applica- tions. Therefore, for the clinical application of VIP as a therapeutic agent, a metabolically stable analogue of VIP needs to be devel- oped, and many structure–activity relationship (SAR) studies focusing on VIP stabilization have been carried out [10,11]. Our group previously conducted chemical modification studies to investigate the SARs of VIP by using synthetic VIP analogues. The replacement of Lys residue at the positions 15, 20, and 21 by Arg, and Met at position 17 by Leu ([R 15,20,21 , L 17 ]-VIP) in VIP resulted in a significant improvement in metabolic stability [11]. In addi- tion, C-terminally extended VIP derivative, IK312532, [R 15,20,21 , L 17 ]-VIP-GRR, exhibited much higher biological activity than natu- ral VIP in vitro and in vivo [12]. Interestingly, IK312532 showed a higher stability against enzymatic digestion and even higher bio- logical activity, including relaxant effect and protective effect against the cytotoxicity of CSE and deactivation of CSE-evoked cas- pase-3, compared to VIP [13,14]. By combining the clinical advan- tages of bronchodilation and protection of the alveolar wall, 0939-6411/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejpb.2009.05.013 * Corresponding author. Department of Pharmacokinetics and Pharmacodynam- ics and Global Center of Excellence (COE) Program, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. Tel.: +81 54 264 5633; fax: +81 54 264 5635. E-mail address: onoue@u-shizuoka-ken.ac.jp (S. Onoue). European Journal of Pharmaceutics and Biopharmaceutics 73 (2009) 95–101 Contents lists available at ScienceDirect European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb