Glucagon-like peptide-2 (GLP-2) is a 33 amino acid peptide hormone which is produced by the intestinal endocrine L cell upon nutrient ingestion. GPL-2 stimulates a mucosal growth in the small and large intestines (D G Burrin et al., Am J Physiol Gastrointest Liver Physiol 279(6): G1249-1256, 2000) and suppresses apoptosis of intestinal cells and crypt cells (Bernardo Yusta et al., Gastrpenterology 137(3): 986-996, 2009). Furthermore, GLP-2 enhances absorption of nutrients in the small intestine (PALLE B J et al., Gastrpenterology 120: 806-815, 2001) and reduces intestinal permeability (Cameron H L et al., Am J Physiol Gastrointest Liver Physiol 284(6): G905-12, 2003). In addition, GLP-2 suppresses gastric emptying and gastric acid secretion (Meier J J et al., Gastrpenterology 130(1): 44-54, 2006), while increasing an intestinal blood flow rate (Bremholm L et al., Scand J Gastroenterol. 44(3): 314-9, 2009) and relaxing intestinal smooth muscle (Amato A et al., Am J Physiol Gastrointest Liver Physiol. 296(3): G678-84, 2009).
Since GLP-2 has capabilities to absorb and protect energy and activate the function of intestinal cells, it has demonstrated a high therapeutic potential in various in vivo models of intestinal diseases and injuries. As a hormone for regulating nutrient absorption, GLP-2 has a therapeutic of great promise for the treatment of short bowel syndrome (SBS). SBS is caused by a congenital reason or an acquired reason such as the surgical removal of the intestine, and leads to nutritional deficiencies due to the decrease in the absorption area of the small intestine. It has been reported that GLP-2 improves nutrient uptake and absorption in the digestive tract in rat models having SBS (Ljungmann K et al., Am J Physiol Gastrointest Liver Physiol. 281(3): G779-85, 2001).
Further, Crohn's disease is a chronic inflammatory intestinal disease that can be caused in any region of the digestive tract ranging from the mouth to the anus. The cause of Crohn's disease is not yet known, but it is believed to be caused by an excessive inflammatory response of the body towards bacterial cells normally present within the digestive tract along with environmental and genetic reasons. It has been known that GLP-2 can prevent or relieve the damage in mucosal epithelial cells when mucositis, colitis or inflammatory intestinal disease is developed by chemotherapy or genetic reasons (Qiang Xiao et al., Am J Physiol Regul Integr Comp Physiol. 278(4): R1057-R1063, 2000).
Chemotherapy-induced diarrhea (CID) is one of the factors that limit the dose of an anticancer agent and is the most common side effect of anticancer chemotherapy. About 10% of the patients had advanced cancer. About 80% of the patients with advanced cancer received single/combination therapy of 5-FU (5-fluorouracil, Adrucil)/irinotecan (Camptosar) experience CID and about 30% of them show serious diarrhea symptoms of grade 3 to 5. In addition, if mucositis or neutropenia occurs simultaneously in the patients with CID, there is a possible risk of death. In the CID-induced rat models, GLP-2 shows the effect of alleviating the reduction of intestine weight, villus height and crypt depth that are induced by 5-FU, thereby demonstrating its therapeutic potential for CID treatment (A. Tavakkolizadeh et al., J Surg Res. 91(1): 77-82, 2000).
Despite this high therapeutic potential, GLP-2 still has limitations in being developed into a commercial drug. Peptides such as GLP-2 can be easily transformed due to low stability, are apt to be degraded by protease in the body and lose activity, and are easily removed through the kidney due to their relatively small size. Therefore, in order to maintain optimal blood concentrations and titers of peptide drugs, there is a need to administer the peptide drug more frequently. However, most peptide drugs are administered in various types of injections, and frequent injections are required to maintain the blood concentration of the peptide drug, which causes severe pain in patients. In this regard, there have been many attempts to solve these problems, one of which has developed a method of increasing membrane permeability of a peptide drug, leading to the delivery of the peptide drug to the body through an oral or a nasal route. But this method had a limitation of a low delivery efficiency of the peptide drug as compared with the injection thereof, and thus it still remains difficult to retain sufficient biological activity of the peptide drug for therapeutic use.
In particular, GLP-2 has extremely short in vivo half-life (7 minutes or shorter) due to its inactivation by dipeptidyl peptidase-IV (DPP IV) which cleaves between the amino acids at position 2 (Ala) and at position 3 (Asp) of GLP-2 (Bolette H. et al., The Journal of Clinical Endocrinology & Metabolism. 85(8): 2884-2888, 2000). It has been tried to increase the in vivo half-life of GLP-2 by amino acid substitution.
Currently, NPS Pharmaceuticals Inc. (U.S.A.) is developing as a therapeutic agent for Crohn's disease, SBS and gastrointestinal disease a GLP-2 analog “Teduglutide” in which the amino acid at position 2 (Ala) of native GLP-2 is substituted with asparagines (Asp). Teduglutide is resistant to the cleavage by DPPIV through the substitution of the amino acid at position 2, which in turn increases the stability and efficacy. However, since the increase in resistance to DPPIV cleavage is insufficient to extend the in vivo half-life of Teduglutide. Thus, Teduglutide also needs to be administered through injection once in a day, which is still a huge burden to the patient (WO 2005/067368).
Zealand Pharma (Denmark) is currently developing GLP-2 analogs by substitution of one or more amino acids at positions 3, 8, 16, 24, 28, 31, 32 and 33 of native GLP-2. These substitutions not only enhance the stability and efficacy of the peptide, but also allow for selective treatment of symptoms by making the growth promoting activity higher in the small intestine relative to in the colon depending on the position of substitution or vice versa. In addition, Zealand Pharma is developing Elsiglutide (ZP1846) as a GLP-2 analog targeting gastrointestinal (GI)-mucositis and CID, and Elsiglutide is undergoing in a Phase I clinical trial. However, the above analog also does not have sufficient in vivo half-life, and thus it needs to be administered through injection once a day (WO 2006/117565).
Polyethylene glycol (PEG) non-specifically binds to a specific site or various sites of a target peptide and increases the molecular weight thereof, thereby preventing renal clearance and hydrolysis of the target peptide without causing any side effect. For example, U.S. Pat. No. 4,179,337 describes a method of linking calcitonin with PEG to enhance in vivo half-life and membrane permeability of calcitonin. WO 2006/076471 describes a method for increasing in vivo half-life of a B-type natriuretic peptide (BNP), which has been used as a therapeutic agent for Congestive heart failure, by linking with PEG. In addition, U.S. Pat. No. 6,924,264 discloses a method for increasing in vivo half-life of exendin-4 by linking PEG with a lysine residue thereof. Although these methods can extend in vivo half-lives of the peptide drugs by increasing the molecular weight of PEG to be linked therewith, there are several problems in that as the molecular weight of PEG is increased, the titer of the peptide drug is reduced and the reactivity of PEG with the peptide drug is also decreased, leading to the reduction of yield.
WO 02/46227 describes a method for preparing a fusion protein of GLP-1, exendin-4 or an analog thereof with human serum albumin or an immunoglobulin Fc region. U.S. Pat. No. 6,756,480 also describes a method for preparing a fusion protein of a parathyroid hormone (PTH) or an analog thereof with an immunoglobulin Fc region. These methods can overcome the problems of pegylation such as low yield and non-specificity, but the effect of increasing in vivo half-lives of the drug peptide is not noticeable as expected, and sometimes the titers thereof are also still low. In order to maximize the effect of increasing in vivo half-life of a drug peptide, various kinds of peptide linkers can be used, but there is a risk of inducing an immune response. Further, if a peptide drug having a disulfide bond such as BNP is used, there is a high probability of misfolding. Finally, if a peptide drug including non-naturally occurring amino acids is employed, it is impossible to produce its fusion protein by genetic recombination.
In the previous study, the present inventors have developed a method for preparing a GLP-2 conjugate with extended in vivo half-life, in which the GLP-2 conjugate is prepared by covalently linking GLP-2 or its derivative to an immunoglobulin Fc fragment through a non-peptidyl polymer. In this method, the GLP-2 derivatives, such as beta-hydroxy-imidazo-propionyl GLP-2, where the N-terminal amine group of GLP-2 is substituted with a hydroxyl group, des-amino-histidyl GLP-2, where the N-terminal amine group thereof is deleted, and an imidazo-acetyl-GLP-2, where the alpha carbon of the first histidine and the N-terminal amine group linked thereto are deleted, showed increased resistance to DPPIV cleavage while maintaining their bioactivities and thus in vivo half-lives of the GLP-2 conjugates were remarkably increased.
However, in the case where the immunoglobulin Fc fragment was site-specifically linked to the lysine residue of these GLP-2 derivatives, thus obtained GLP-2 conjugates showed a significantly reduced binding affinity to GLP-2 receptor and thus had a problem in that in vivo efficacy and duration thereof would be reduced.
In this light of investigating a method that can increase in vivo half-life of GLP-2 in blood and maximize the duration of in vivo efficacy, the present inventors have developed a GLP-2 conjugate by conjugating a GLP-2 derivative into which a thiol group is introduced at the C-terminal thereof to a non-peptidyl polymer and an immunoglobulin Fc fragment by covalent linking, and found that the GLP-2 conjugate exhibits an increased binding affinity to GLP-2 receptor and a remarkably enhanced duration of in vivo efficacy. In particular, it has been found that when the non-peptidyl polymer and immunoglobulin Fc fragment are site-specifically linked to the cysteine residue introduced into the C-terminal end of an imidazo-acetyl-GLP-2 where the α-carbon of the first histidine and the N-terminal amine group linked thereto are deleted, leading to an increase in resistance to DPPIV, in vitro efficacy of the imidazo-acetyl-GLP-2 conjugate is significantly increased.
USPTO Backgrounds
USPTO Backgrounds