GLP2-2G-XTEN: A Pharmaceutical Protein with Improved Serum Half-Life and Efficacy in a Rat Crohn’s Disease Model

et al. (2012) GLP2-2G-XTEN: A Pharmaceutical Protein with Improved Serum Half-Life and Efficacy in a Rat Crohn's Disease Model. PLoS ONE 7(11): e50630. doi:10.1371/journal.pone.0050630 GLP2-2G-XTEN: A Pharmaceutical Protein with Improved Serum Half-Life and Efficacy in a Rat Crohn's Disease Model Susan E. Alters 0 Bryant McLaughlin 0 Benjamin Spink 0 Tigran Lachinyan 0 Chia-wei Wang 0 Vladimir Podust 0 Volker Schellenberger 0 Willem P. C. Stemmer 0 Partha Mukhopadhyay, National Institutes of Health, United States of America 0 Amunix Inc., Mountain View , California , United States of America Objectives: Glucagon-like peptide 2 (GLP2) is an intestinal growth factor that has been shown to stimulate intestinal growth and reduce disease severity in preclinical models of short bowel syndrome and inflammatory bowel disease. Teduglutide, a recombinant human GLP2 variant (GLP2-2G), has increased half-life and stability as compared to the native GLP2 peptide, but still requires twice daily dosing in preclinical models and daily dosing in the clinic. The goal of this study was to produce and characterize the preclinical pharmacokinetic and therapeutic properties of GLP2-2G-XTEN, a novel, long-acting form of GLP2-2G. Methodology and Results: A GLP2-2G-XTEN fusion protein with extended exposure profile was produced by genetic fusion of GLP2-2G peptide to XTEN, a long, unstructured, non-repetitive, hydrophilic sequence of amino acids. The serum half-life of GLP2-2G-XTEN in mice, rats and monkeys was 34, 38 and 120 hours, respectively. Intestinotrophic effects were demonstrated in normal rats, where GLP2-2G-XTEN administration resulted in a significant increase in both small intestine weight and length. Efficacy of the GLP2-2G-XTEN protein was compared to that of GLP2-2G peptide in a rat Crohn's disease model, indomethacin-induced inflammation. Prophylactic administration of GLP2-2G-XTEN significantly increased the length, reduced the number of trans-ulcerations and adhesions, and reduced the TNFa content of the small intestine. GLP22G-XTEN demonstrated greater in vivo potency as compared to GLP2-2G peptide, and improvement in histopathology supported the GLP2-2G-XTEN treatment effects. Conclusions and Significance: GLP2-2G-XTEN is intestinotrophic and demonstrates efficacy in a rat Crohn's disease model requiring a lower molar dose and less frequent dosing relative to GLP2-2G peptide. Allometric scaling based on pharmacokinetics from mouse, rat and monkey projects a human half-life of 240 hours. These improvements in preclinical pharmacokinetics and dosing indicate that GLP2-2G-XTEN may offer a superior therapeutic benefit for treatment of gastrointestinal diseases including Crohn's disease. - Competing Interests: All authors are employed by or have an ownership interest in Amunix, Inc. The project was supported by funding from Amunix, Inc. There are no patents, products in development or marketed products to declare. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. Glucagon-like peptide 2 (GLP2) is a 33 amino acid peptide derived from the post-translational processing of glucagon. First identified by Drucker [1], GLP2 is secreted from the enteroendocrine L cells of the small intestine and colon in response to nutritional stimulation. A specific gastrointestinal growth factor, GLP2 has been shown to increase the ability of the intestine to digest and absorb nutrients. In addition, it was found to possess potent intestinotrophic properties as well as significant reparative activity for the mucosal epithelium of the small and large intestine [2]. These features indicate that the GLP2 receptor is an attractive target for development of treatment interventions to promote intestinal adaptation and repair. Teduglutide (NPS Pharmaceuticals) is a recombinant human GLP2 variant with a stabilizing alanine to glycine substitution at the second amino acid residue (GLP2-2G). Preclinical studies have shown that GLP2, or GLP2-2G injection, causes an increase in intestinal length and weight, villus height, crypt depth and crypt cell proliferation in both normal rodents and during intestinal adaptation in surgical models of short bowel syndrome [17]. Clinical studies have confirmed the significant enterotrophic effects [811]. Teduglutide is pending approval by the US FDA and has received a recommendation for approval by the European Medicines Agency as treatment for short bowel syndrome. GLP2 therapy may also be beneficial for treatment of other gastrointestinal diseases involving malabsorption, inflammation, or mucosal damage of the small intestine, including Crohns disease. Mucosal GLP2 concentrations are reduced in areas of colonic inflammation in mice with inflammatory bowel disease [12], and treatment with GLP2 or GLP2-2G has been shown to reduce inflammation and severity of disease in a variety of relevant models. These include indomethacin-induced enteritis [13], HLAB27 [14], DSS- induced ulcerative colitis [15,16], and TNBSinduced colitis [17]. In these models GLP2 or GLP2-2G reduced mucosal damage, crypt cell apoptosis, intestinal lesions, inflammation, and mucosal cytokine production including TNFa, IL-1b, and IFNc. Given the enterotrophic and anti-inflammatory effects of GLP2 in preclinical models, and the preferential location of GLP2 receptors within the areas of intestine most affected by Crohns disease [18], a clinical trial of Teduglutide in Crohns disease was undertaken. While further study is necessary, results from this study revealed that Teduglutide may be an effective therapy for inducing mucosal healing in patients with moderate to severe Crohns disease. In this pilot study, subjects received once or twice daily injections (dependent on body weight); the frequent injections were said to contribute to injection site reactions and high subject withdrawal rate, and it was suggested that future trials use a formulation that allows for less frequent injections [19]. Therefore, additional GLP2 therapeutics with better pharmacokinetic properties are needed. Native GLP2 has a short half-life in human circulation of about seven minutes due to rapid proteolytic degradation by dipeptidyl peptidase IV (DPPIV) [20] as well as removal from circulation by kidney filtration. Both the intact and DPPIV cleaved forms are subject to renal clearance [21]. Teduglutide, the GLP2-2G variant, has increased resistance to DPPIV, and shows a modest increase in half-life to 35 hours in healthy humans [22]. In this study, we show that the GLP2-2G peptide genetically fused to XTEN yields a therapeutically active fusion protein with a greatly improved half-life and lower dose requirement in a rat Crohns disease model. XTEN is a long, unstructured, nonrepetitive, hydrophilic sequence of amino acids [23,24]. Fusion of XTEN to the C-terminus of GLP2-2G increases the hydrodynamic radius of the peptide (...truncated)