Pharmacokinetics of glycine–proline–glutamate, the N-terminal tripeptide of insulin-like growth factor-1, in rats D.C. Batchelor, a, * H. Lin, a J.-Y. Wen, a C. Keven, b P.L. Van Zijl, b B.H. Breier, b P.D. Gluckman, b and G.B. Thomas a a NeuronZ Ltd., P.O. Box 9923, Newmarket, Auckland 1031, New Zealand b Liggins Institute, Faculty of Medical and Health Science, The University of Auckland, Private Bag 92019, Auckland, New Zealand Received 29 April 2003 Abstract Glycine–proline–glutamate (GPE) is the N-terminal tripeptide of insulin-like growth factor-1 and has been shown to be neu- roprotective following ischemia-induced brain injury. The pharmacokinetics of GPE were studied in adult rats since GPE is a candidate for use in neuroprotection therapies. To measure plasma concentrations of GPE a novel radioimmunoassay was de- veloped whereby GPE was initially derivatized with Bolton and Hunter reagent before use in a standard homologous assay against the Bolton and Hunter iodinated form. The derivatized GPE radioimmunoassay showed a 83% recovery of unlabeled GPE and complete parallel displacement with rat plasma. The simplicity and speed of the assay described here indicate an exciting new use for a previously described technology. The pharmacokinetic studies were conducted in adult rats using a single bolus intravenous in- jection of GPE at 30 or 100 mg/kg and showed that GPE was rapidly cleared from the circulation. In addition, evidence of the route of the metabolic degradation of GPE is presented. The findings presented here are the first description of the pharmacokinetics of GPE and suggest that, because of its very short half-life in plasma, continuous intravenous infusion of GPE may be the preferred route of administration for use in future neuroprotection therapies. Ó 2003 Elsevier Inc. All rights reserved. Keywords: GPE; HPLC; Derivatization; Bolton and Hunter reagent; Radioimmunoassay GPE 1 is the N-terminal tripeptide cleaved from in- sulin-like growth factor-1 (IGF-1) by a putative acid protease in all tissues [1]. The small size of GPE (MW 301) makes it an ideal candidate for clinical neuronal rescue therapies. When administered by intracerebro- ventricular (i.c.v.), intraperitoneal (i.p.), and intrave- nous (i.v.) injections following acute ischemic brain injury, GPE has been shown to be neuroprotective in vivo, reducing both cortical damage and neuronal loss in the CA1-2 subregions of the hippocampus [2,3,5]. Furthermore, GPE had a stimulatory effect on the potassium-induced release of acetylcholine from rat cortical slices [4]. However, the pharmacokinetic prop- erties and metabolism of GPE have not been previously investigated. Unlike thyrotrophin-releasing hormone (TRH), which has an uncommon pyro-glutamate moiety that is ideal for generating antibodies, small molecules such as GPE often lack such suitable antigenic sites. This makes it very difficult to generate suitable antibodies for use in routine radioimmunoassay (RIA). There have been a number of attempts to measure by radioimmunoassay small peptides such as the metabolite of cholecystokinin, Gly-Trp-Met, using a variety of methods [6–8]. How- ever, many of these methods often require complex sample preparation or are difficult to measure accurately in samples because of cross-reactivity with similar small molecules [6,9–11]. Consequently to measure such small molecules accurately researchers have been forced to * Corresponding author. Fax: +64-9-3677186. E-mail address: david.batchelor@neuronz.com (D.C. Batchelor). 1 Abbreviations used: GPE, glycine–proline–glutamate; IGF-1, insulin-like growth factor-1; TRH, thyrotrophin-releasing hormone; RIA, radioimmunoassay; BSA, bovine serum albumin; PBS, phos- phate-buffered saline; DMSO, dimethyl sulfoxide. 0003-2697/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2003.08.032 Analytical Biochemistry 323 (2003) 156–163 ANALYTICAL BIOCHEMISTRY www.elsevier.com/locate/yabio