liquid formulation of a long-acting oxyntomodulin derivative conjugate and method for preparing the

专利摘要:
LIQUID FORMULATION OF A PROTEIN CONJUGATE COMPRISING OXINTOMODULIN AND A FRAGMENT OF IMMUNOGLOBULIN. The present invention relates to an albumin-free liquid formulation comprising a long-acting oxyntomodulin conjugate, wherein an oxyntomodulin peptide comprising an oxyntomodulin derivative, variant, fragment or precursor is linked to an immunoglobulin Fc region, which can increase immunoglobulin duration of the long-lasting physiological activity of the oxyntomodulin conjugate and maintaining its in vivo stability for a longer period of time, compared to native oxyntomodulin, as well as a method for preparing the liquid formulation. The liquid formulation comprises a buffer, a sugar alcohol and a non-ionic surface active agent and does not contain a human serum albumin and factors that are potentially harmful to the human body, and therefore not susceptible to viral infections. Furthermore, the oxyntomodulin conjugate of the invention comprises oxyntomodulin linked to an immunoglobulin Fc region and therefore has a large molecular weight, prolonged physiological activity, and excellent storage stability compared to native oxyntomodulin.
公开号:BR112015010235B1
申请号:R112015010235-2
申请日:2013-11-06
公开日:2021-05-18
发明作者:Hyun Uk Kim；Hyung Kyu Lim；Myung Hyun Jang；Sang Yun Kim；Sung Min Bae；Se Chang Kwon
申请人:Hanmi Pharm. Co., Ltd；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[001] The present invention relates to an albumin-free liquid formulation comprising a long-acting oxyntomodulin conjugate in which an oxyntomodulin peptide comprising an oxyntomodulin derivative, variant, fragment or precursor is linked to an immunoglobulin Fc region, which can increase the duration of the physiological activity of the long-lasting oxyntomodulin conjugate and maintain its in vivo stability for an extended period of time compared to native oxyntomodulin. The present invention also relates to a method for preparing the liquid formulation. BACKGROUND OF THE INVENTION
[002] Obesity is defined as a condition of abnormal or excessive fat accumulation that can harm health and result from an energy imbalance in which energy consumption exceeds energy expenditure. Obesity was not a serious health problem in the past, but with economic growth, the obese population is increasing with increasing economic wealth, and the number of various obesity-related diseases is also increasing. According to the World Health Organization (WHO) report, more than 1.5 billion adults worldwide are overweight, more than 500 million of them are obese, and the obese population has increased by nearly twice between 1980 and 2008 (World Health Organization, Fact sheet on obesity and overweight, 2011). Not only in high-income countries, but also in low-income countries, the percentage of obese people is increasing. Overweight and obesity are responsible for increased blood pressure and cholesterol levels and cause or aggravate various diseases. In addition, the problem of obesity is more serious in children or adolescents, increases the incidence of diabetes, heart disease, hypertension or hyperlipidemia, and can also cause death or disability.
[003] As described above, obesity is a global disease and social problem, but in the past it was believed that obesity could be overcome through individual effort, and thus no particular emphasis was placed on the treatment of obesity. However, obesity is not easy to treat as it is a complex disease associated with appetite control mechanisms and energy metabolism. Consequently, the treatment of obesity requires not only the patient's own efforts, but also a method capable of treating the abnormal mechanisms associated with appetite control and energy metabolism. Thus, efforts have been made to develop drugs for the treatment of obesity.
[004] As a result of such efforts, drugs including Rimonabant (Sanofi-Aventis), sibutramine (Abbott), Contrave (Takeda), Orlistat (Roche) and the like have been developed, but these drugs have deficiencies as they show effects adverse fetal effects or have an insufficient effect on the treatment of obesity. Rimonabant (Sanofi-Aventis) was reported to have central nervous system disorders, Sibutramine (Abbott) and Contrave (Takeda) to have cardiovascular adverse effects, and Orlistat (Roche) to show a weight-reducing effect of only about 4 kg when administered for 1 year. Thus, there are currently few or no obesity therapeutic agents that can be safely prescribed to obese patients.
[005] Recently, glucagon derivatives have received a lot of attention. Glucagon is produced by the pancreas when blood glucose levels begin to drop due to medications, illnesses, hormones or enzyme deficiencies, or the like. Glucagon's function is to stimulate liver cells to break down stored glycogen into glucose, which is then released into the blood increasing the blood glucose level to a normal level. In addition to the effect of increasing blood glucose level, glucagon has been reported to suppress appetite and activate adipocyte lipase-sensitive hormone (HSL) to facilitate lipolysis, thus showing anti-obesity effects. Among glucagon derivatives, glucagon-like peptide-1 (GLP-1) is being developed as a therapeutic agent to reduce hyperglycemia in diabetic patients and works by stimulating insulin synthesis and secretion, inhibiting glucagon secretion, suppressing insulin gastric emptying, and increases glucose utilization by inhibiting food intake. It is known that exendin-4, which is isolated from lizard venom, has an amino acid homology of about 50% with GLP-1 and activates the GLP-1 receptor to reduce hyperglycemia in diabetic patients. However, therapeutic obesity drugs, including GLP-1, have been reported to cause adverse effects such as nausea and vomiting.
[006] Thus, as an alternative to GLP-1, oxyntomodulin capable of binding to both receptors of the two peptides (GLP-1 and glucagon) has been receiving attention. Oxyntomodulin is made from a pre-glucagon peptide, a precursor of glucagon, and is a potent anti-obesity agent because it inhibits food intake such as GLP-1, promotes satiety, and has lipolytic activity , similar to glucagon.
[007] Based on the dual function of the peptide oxyntomodulin, studies on the development of drugs for the treatment of obesity have been actively conducted. For example, Korean patent application No. KR 925,017 discloses an oral, parenteral, mucosal, rectal, subcutaneous or transdermal pharmaceutical composition for the treatment of human obesity, which comprises oxyntomodulin as an active ingredient. However, it has been reported that therapeutic agents against obesity comprising oxyntomodulin have a short half-life in vivo and show a lesser effect on the treatment of obesity, even when they are administered at a high dose, three times a day. Thus, efforts have been made to increase the short in vivo half-life or obesity treatment effect of oxyntomodulin by modifying oxyntomodulin.
[008] For example, the dual agonist of oxyntomodulin (Merck) is obtained by substituting L-serine for D-serine at amino acid 2 of oxyntomodulin to increase resistance to dipeptidyl peptidase-IV (DPP-IV) and joining a part from cholesterol to the C-terminus to increase the half-life in blood. ZP2929 (Zealand) is obtained by replacing L-serine with D-serine at amino acid 2 of oxyntomodulin to increase resistance to DPP-IV, replacing arginine with alanine at amino acid 17 to increase protease resistance, replacing methionine with lysine at amino acid 27 to increase oxidative stability, and replacing glutamines at amino acids 20 and 24 and asparagine at amino acid 28 with aspartic acid, alanine and serine, respectively, to increase deamidation stability. The dual agonist oxyntomodulin (Merck) has an in vivo increased half-life of 1.7 hours, which is longer than the half-life (8-12 minutes) of native oxyntomodulin, but still has a half-life. in vivo life is very short and is administered at a very high dose of several mg/kg. Thus, oxyntomodulin or its derivatives have two major disadvantages, namely, a short half-life and low medicinal effects. Due to these disadvantages, it must be administered daily in high doses. In order to overcome these disadvantages, a method of increasing the blood half-life of oxyntomodulin while maintaining its activity in vivo was studied, and, as a result, an oxyntomodulin derivative was developed. Furthermore, using this technology, a non-peptide polymer was prepared by conjugating a vehicle with the oxyntomodulin derivative, and it was found that the protein conjugate can exhibit a better anti-obesity effect as a result of the increased half-life in the blood from it while maintaining in vivo activity (Korean patent application No. KR 10-2012-0064110).
[009] Generally, proteins and peptides have a very short half-life, and suffer from denaturation such as precipitation through aggregation of monomers, and adsorption on vessel surfaces, after exposure to various factors such as unfavorable temperatures, interface air-water, high pressure, physical/mechanical stress, organic solvents and microbial contamination. This denaturation is irreversible, and therefore, denatured proteins and peptides lose intrinsic physicochemical properties and physiologically active effects. In addition, proteins and peptides are unstable and sensitive to extrinsic factors, such as temperature, humidity, oxygen, UV rays or the like, undergoing physical or chemical changes, including association, polymerization or oxidation, which results in substantial loss of activity ( Korean Patent Application No. KR 10-0.389,726).
[010] In addition, the adsorbed proteins and peptides are easily aggregated by the denaturation process, and the denatured proteins and peptides, when administered to the human body, act as the cause of the formation of antibodies in the human body, and for this reason, the proteins and peptides must be administered in a sufficiently stable form. Thus, several methods for preventing denaturation of proteins and peptides in solution have been studied (John Geigert, J. Parenteral Sci. Tech., 43, No5, 220-224, 1989; David Wong, Pharm. Tech. October, 34 -48, 1997; Wei Wang., Int. J. Pharm., 185, 129-188, 1999; Willem Norde, Adv. Colloid Interface Sci., 25, 267-340, 1986; Michelle et al., Int. . Pharm. 120, 179-188, 1995).
[011] Lyophilization is applied to some protein and peptide drugs to achieve the goal of stability. However, lyophilized products are inconvenient in that they have to be redissolved in water for injectable use. Furthermore, in the case of freeze-drying, huge investment in high-capacity freezer-dryers or the like is required, because the freeze-drying process is included in the production processes. In addition, a method for the production of proteins and peptides using a powder spray dryer is also being used, but in this case, the economic efficiency is diminished due to a low yield, and exposure to high temperatures can adversely affect the protein stability.
[012] In order to overcome such limitations, studies were performed in which stabilizers were added to proteins and peptides in solution to suppress the physicochemical changes of proteins and peptides, while maintaining efficiency in vivo even after long-term storage. Human serum albumin, a type of protein, has been widely used as a stabilizer for various protein drugs, and its performance has been proven (Edward Tarelli et al., Biologicals (1998) 26, 331-346).
[013] A process to purify human serum albumin includes the inactivation of biological contaminants such as mycoplasma, prions, bacteria and viruses and screening or analysis for one or more biological contaminants or pathogens. However, there is always a risk that patients will be exposed to biological contaminants that are not completely removed or inactivated. For example, the screening process includes examining whether human blood from donors contains a certain virus, but this process is not always reliable. In particular, a specific virus that exists in a small number of donors may go undetected.
[014] Different proteins can be gradually inactivated at different rates under different conditions during storage, due to their chemical differences. That is, the extension of storage time by a stabilizer is not identical for different proteins. For this reason, the appropriate proportion, concentration and type of stabilizing agent that is used to provide stability during storage varies depending on the physicochemical properties of the target protein. When stabilizers are used in combination, they can cause adverse effects different from the desired effects due to competition and interaction between them. Furthermore, due to the nature or concentration of proteins may change during storage, the stabilizers used may show effects other than those intended. Thus, a great deal of effort and precautions are required to stabilize proteins in solution.
[015] Particularly, a conjugate of oxyntomodulin and immunoglobulin Fc is a conjugate in which oxyntomodulin which is a physiologically active peptide is linked to an immunoglobulin Fc region. Thus, because the molecular weight and volume of the conjugate certainly differ from those of native oxyntomodulin, a special composition is needed for protein stabilization.
[016] Thus, because oxyntomodulin (which is a physiologically active peptide) and the Fc region of the immunoglobulin are peptides or proteins with different physicochemical properties, they must be simultaneously stabilized. However, as described above, different proteins or proteins can be gradually inactivated at different rates under different conditions during storage, due to their chemical differences, and when suitable stabilizers for the proteins or peptides are used in combination, they can cause adverse effects. different from the intended effects, due to the competition and interaction between them. Thus, in the case of a long-acting oxyntomodulin conjugate, there is no great difficulty in finding a composition for simultaneously stabilizing the oxyntomodulin, which is a physiologically active peptide, and the Fc region of the immunoglobulin.
[017] Under such circumstances, the present inventors have made extensive efforts to provide a stable liquid formulation that can be stored for a long period of time, without concern for viral contamination, and, as a result, have found that a stabilizer, which includes a buffer, a sugar alcohol and a nonionic surface active agent, and may further include an additive such as an isotonic agent or an amino acid, and a preservative for repeated use, may enhance the stability of a long-lasting oxyntomodulin derivative, and a stable, cost-effective liquid formulation can be prepared using the stabilizer, thus completing the present invention. DESCRIPTION OF THE INVENTION TECHNICAL PROBLEM
[018] It is an object of the present invention to provide a liquid formulation of a long-acting oxyntomodulin conjugate, which comprises a pharmacologically effective amount of a long-acting oxyntomodulin conjugate, wherein an oxyntomodulin that is a physiologically active peptide is linked to an immunoglobulin Fc region; and an albumin free stabilizer.
[019] Another object of the present invention is to provide a method for preparing the above liquid formulation.
[020] Yet another objective of the present invention is to provide a composition for the prevention or treatment of obesity or diabetes, comprising a liquid formulation of an oxyntomodulin oxyntomodulin conjugate comprising a physiologically active peptide linked to an immunoglobulin Fc region.
[021] Yet another object of the present invention is to provide a method for the prevention or treatment of obesity or diabetes, which comprises administering the above liquid formulation to a subject. SOLUTION OF THE PROBLEM
[022] To achieve the above objectives, in one aspect, the present invention provides a liquid formulation of a long-acting oxyntomodulin conjugate, comprising a pharmacologically effective amount of a long-acting oxyntomodulin conjugate, wherein an oxyntomodulin that is a physiologically active peptide is linked to an immunoglobulin Fc region; and an albumin-free stabilizer, wherein the stabilizer contains a buffer, a sugar alcohol and a nonionic surface active agent.
[023] As used herein, the term "liquid formulation" refers to a pharmaceutical formulation processed into a liquid form and is intended to include all liquid formulations for internal use and formulations for external use. In the prior art, the liquid formulation of the invention suitable for a pharmacologically effective amount of the oxyntomodulin conjugate which comprises oxyntomodulin linked to the Fc domain of the immunoglobulin has not been reported. Thus, the liquid formulation of the present invention may comprise a pharmacologically effective amount of the oxyntomodulin conjugate which comprises oxyntomodulin linked to the Fc domain of the immunoglobulin and an albumin-free stabilizer, wherein the stabilizer contains a buffer, a sugar alcohol and a surface-active agent non-ionic. Furthermore, the liquid formulation of the present invention may further comprise a preservative.
[024] In the present invention, the stabilizer can further comprise one or more components selected from the group consisting of isotonic agents, sugars, polyhydric alcohols, and amino acids. The sugar alcohol can be one or more selected from the group consisting of mannitol, sorbitol and glycerol, and the concentration of sugar alcohol in the liquid formulation can be 215% (w/v). Furthermore, the buffer can be one or more selected from the group consisting of citrate, acetate, histidine and phosphate buffers and can have a pH between 4.5 and 7.0. The isotonic agent can be sodium chloride, and the nonionic surfactant can be polysorbate or poloxamer and be present in a concentration of 0.001-0.1% (w/v). The amino acid can be methionine. Thus, the liquid formulation of the present invention may comprise a stabilizer containing a buffer having a pH between 4.8 and 6.0, one or more sugar alcohols selected from the group consisting of mannitol and sorbitol, and polysorbate 20.
[025] In addition, the liquid formulation of the present invention may further comprise one or more preservative agents selected from the group consisting of m-cresol, phenol and benzyl alcohol. The concentration of preservative in the liquid formulation can be 0.001-1% (w/v).
[026] Particularly, the liquid formulation of the present invention may comprise a pharmacologically effective amount of the long-acting oxyntomodulin conjugate, 5-50 mM histidine, 2-15% (w/v) mannitol, from 0.01-1 mg/ml methionine and 0.001-0.1% (w/v) polysorbate 20. In addition to these components, the liquid formulation may further comprise 0.001-1% (w/v) m-cresol.
[027] As used herein, the term "stabilizer" refers to a substance that maintains stable ingredients such as active ingredients for a certain period of time. For the purpose of the present invention, the term refers to a substance that allows the long-lasting oxyntomodulin conjugate to be stably stored. The storage stability of proteins such as the long-lasting oxyntomodulin conjugate is important not only to ensure an accurate dose, but also to inhibit the potential for producing an antigenic substance for the oxyntomodulin-derived conjugate.
[028] The stabilizer in the present invention preferably contains a buffer, a sugar alcohol and a nonionic surfactant, in order to provide stability to the long-lasting oxyntomodulin conjugate. In addition, the stabilizer may further comprise, Preferably, one or more components selected from the group consisting of isotonic agents, sugars, polyhydric alcohols and amino acids.
[029] The buffer serves to maintain the pH of the liquid formulation, so that the pH of the liquid formulation does not change quickly so as to make the long-lasting oxyntomodulin conjugate stable. Examples of the buffer may include pharmaceutically acceptable pH buffers, including an alkaline salt (sodium phosphate, potassium phosphate, or a hydrogen or dihydrogen phosphate salt thereof), sodium citrate, citric acid, sodium acetate, acetic acid , and histidine, or a mixture of these buffers can also be used. The buffer is preferably a citrate or histidine buffer, and more preferably a histidine buffer. The concentration of the buffer is preferably 5-100 mM, and more preferably 5-50 mM. The pH of the buffer is preferably 4.0-8.0, more preferably 4.5-7.0, and most preferably 5.0-6.0.
[030] In an example of the present invention, the stability of the long-lasting oxyntomodulin conjugate was measured according to the pH of the buffer of the liquid formulation. That is, after the long-lasting oxyntomodulin conjugate was stored at 25°C for 0-4 weeks during the buffer pH change, the remaining amount of the conjugate was analyzed, and as a result, it was found that the oxyntomodulin conjugate was more stable at pH 5.6, pH 5.8 and pH 6.0 (Example 3, Tables 2 to 5, Example 7, Tables 18 to 21, Example 8 and Tables 22 to 25). Thus, it has been found that the pH of the most stable buffer in the present invention ranges from 5.0 to 6.0. In an example of the present invention, the stability of the long-lasting oxyntomodulin conjugate was measured according to the type of buffer of the liquid formulation. Specifically, after the oxyntomodulin conjugate was stored with 0.02% polysorbate 20, 0.1 mg/ml methionine, and 5% mannitol at 25°C for 0-4 weeks, the remaining amount of the conjugate was analyzed. . The results of SE-HPLC analysis indicated that the remaining amount of conjugate does not differ greatly between buffers at the same pH. The results of analysis by IE-HPLC or by RP-HPLC indicate that histidine was more stable at the same pH (Example 8 and Tables 22 to 25).
[031] Sugar alcohol works to increase the stability of the long-lasting oxyntomodulin conjugate. In the present invention, the sugar alcohol can be one or more selected from the group consisting of mannitol, sorbitol and glycerol. Preferably, the sugar alcohol can be mannitol. The concentration of sugar alcohol in the liquid formulation is Preferably 1-20% (w/v), and more preferably 2-15% (w/v).
[032] In an example of the present invention, the influence of the type of sugar alcohol as a stabilizer on the stability of the long-lasting oxyntomodulin conjugate was analyzed. Specifically, the oxyntomodulin conjugate was stored in citrate buffer (pH 5.6) at 25°C for 0-4 weeks, and then analyzed by IE-HPLC, SE-HPLC and RP-HPLC. As a result, the conjugate was more stable in the presence of mannitol or sorbitol than in the presence of glycerol at the same concentration. The results of the RP-HPLC analysis indicated that the conjugate was somewhat more stable in the presence of mannitol compared to the presence of sorbitol (Example 4 and Tables 6 to 9). In other words, it was found that the addition of mannitol or sorbitol had excellent stability, but the conjugate was more stable in the presence of mannitol.
[033] In an example of the present invention, the influence of the concentration of sugar alcohol as a stabilizer on the stability of the long-lasting oxyntomodulin conjugate was analyzed. Specifically, the oxyntomodulin conjugate was stored at 25°C for 0-4 weeks, and then analyzed by IE-HPLC, SE-HPLC and RP-HPLC. As a result, in the presence of 2% mannitol or 15% mannitol, a protein precipitate was produced, and in the presence of 5% mannitol or 10% mannitol, the conjugate was stable (Example 5, Tables 10 and 13, Example 7 and Tables 18 to 21).
[034] The nonionic surfactant works to lower the surface tension of the protein solution to prevent the protein from being adsorbed onto a hydrophobic or aggregating surface. Preferred examples of a nonionic surface active agent that can be used in the present invention include nonionic surface active agents based on polysorbate and nonionic surface active agents based on poloxamer, which can be used alone or in combination of two or more. It is not convenient for the non-ionic surfactant to be used in high concentrations in the liquid formulation. The liquid formulation of the present invention contains the nonionic surfactant at a concentration of 0.2% (w/v) or less, and preferably 0.001-0.1% (w/v).
[035] The stabilizer of the present invention may contain an amino acid such as methionine. Methionine additionally works to stabilize the protein by inhibiting the production of impurities that can be caused by, for example, the protein's oxidative reaction.
[036] In an example of the present invention, the influence of the concentration of the nonionic surfactant as a stabilizer and the presence or absence of an amino acid on the stability of the long-lasting oxyntomodulin conjugate was tested. Specifically, the oxyntomodulin conjugate was stored in citrate buffer (pH 5.6) and 10% mannitol at 25°C for 0-4 weeks, and then analyzed by IE-HPLC, SE-HPLC and RP-HPLC . As a result, the oxyntomodulin conjugate was more stable in the presence of 0.02% polysorbate 20 and 0.1 mg/ml methionine (Example 6 and Tables 14 to 17).
[037] The isotonic agent serves to maintain the osmotic pressure at an adequate level when administering the long-lasting oxyntomodulin conjugate in the solution in vivo and may additionally work to further stabilize the long-lasting oxyntomodulin conjugate in the solution. Typical examples of the isotonic agent include water-soluble inorganic salts such as sodium chloride, sodium sulfate, sodium citrate and the like. The concentration of the isotonic agent is preferably 0-200 mM, and its content can be adequately controlled.
[038] The stabilizer of the present invention preferably does not contain albumin. Human serum albumin that can be used as a protein stabilizer is prepared from human blood, and therefore can be contaminated with a human pathogenic virus, and gelatin or bovine serum albumin can cause disease or can cause allergic reactions in some patients. The albumin-free stabilizer of the present invention does not contain a foreign protein, such as human or animal serum albumin, or purified gelatin, and therefore is not susceptible to viral infection.
[039] Preferred examples of sugars among the polyhydric sugars and alcohols that can additionally be used to increase the storage stability of the long-lasting oxyntomodulin conjugate include monosaccharides such as mannose, glucose, fucose and xylose, and polysaccharides, such as lactose, maltose, sucrose, raffinose and dextran, and preferred examples of the polyhydric alcohols include polypropylene, low molecular weight polyethylene glycol, low molecular weight glycerol, polypropylene glycol and the like. These polyhydric sugars and alcohols can be used alone or in combination of two or more.
[040] In addition to the above-described buffer, an isotonic agent, sugar alcohol, amino acid and nonionic surfactant, the liquid formulation of the present invention may further comprise other components or substances known in the art within a range that does not harm the effect of the present invention.
[041] The liquid formulation of a long-acting oxyntomodulin conjugate of the invention comprises a pharmacologically effective amount of the long-acting oxyntomodulin conjugate comprising a physiologically active oxyntomodulin peptide linked to an immunoglobulin Fc region, and an albumin-free stabilizer, in that the stabilizer may contain a buffer having a pH of 4.8 to 7.0, one or more sugar alcohol selected from the group consisting of mannitol and sorbitol, and polysorbate 20. More specifically, the stabilizer may contain a buffer having a pH ranging from 5.0 to 6.0, mannitol and polysorbate 20. In addition, the stabilizer may further comprise one or more components selected from the group consisting of isotonic agents, sugars, polyhydric alcohols and amino acids.
[042] The albumin-free liquid formulation of the invention containing a high concentration of the long-lasting oxyntomodulin conjugate, which provides stability to the long-lasting oxyntomodulin conjugate, is not susceptible to viral infection, is simple and has excellent storage stability, and thus can be provided in an economical manner compared to other stabilizers or lyophilized formulations.
[043] Furthermore, as the liquid formulation of the present invention comprises a long-lasting oxyntomodulin conjugate that has physiological activity for an extended period of time compared to native oxyntomodulin, it can maintain the protein activity in the human body during a long period of time compared to conventional formulations of oxyntomodulin, and thus it can be used as an efficient pharmaceutical formulation. Furthermore, the liquid formulation of the present invention provides excellent stability, even at a high concentration of the long-lasting oxyntomodulin conjugate.
[044] As used herein, the term "oxyntomodulin" refers to a peptide produced from pre-glucagon that is a precursor of glucagon. In the present invention, oxyntomodulin is intended to include native oxyntomodulin and its precursor, derivative, fragment and variant. Preferably, oxyntomodulin has an amino acid sequence of SEQ ID NO: 1 (HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA).
[045] As used herein, the term "oxyntomodulin derivative" is intended to include a peptide, a peptide derivative or a peptide mimetic that is obtained by the addition, deletion or substitution of amino acids in the amino acid sequence of oxyntomodulin and can activate glucagon and GLP-1 receptors at a higher level than activated by native oxyntomodulin. In the present invention, the oxyntomodulin derivative can have any of the amino acid sequences of SEQ ID NOs: 2 to 34. Preferably, the oxyntomodulin derivative can have an amino acid sequence of SEQ ID NOs: 23 or 25. More preferably, it may have an amino acid sequence of SEQ ID NO: 25.
[046] As used herein, the term "fragment of oxyntomodulin" refers to a fragment having one or more amino acids at the amino or carboxy terminus of native oxyntomodulin, wherein the added amino acids may also be non-naturally occurring amino acids (by example, amino acid type D). This oxyntomodulin fragment serves to regulate blood glucose levels in vivo.
[047] As used herein, the term "variant oxyntomodulin" is a peptide that has one or more amino acid residues different from the native amino acid sequence of oxyntomodulin and has an activating function of GLP-1 and glucagon receptors. The variant oxyntomodulin can be prepared by any one of substitution, addition, deletion, alteration, or a combination of a few amino acids in the native oxyntomodulin amino acid sequence.
[048] The methods for preparing the variant oxyntomodulin, derivatives and fragments can be used alone or in combination. For example, the present invention also includes a peptide, which has one or more amino acids other than native oxyntomodulin and deamination of the N-terminal residue of the amino acids and has an activating function of both the GLP-1 receptor and the glucagon receptor.
[049] The amino acids mentioned here are abbreviated according to the nomenclature rules of the IUPAC-IUB, as follows: Alanine A; Arginine R; Asparagine N; Aspartic Acid D; Cysteine C; Glutamic Acid E; Glutamine Q; Glycine G; Histidine H; Isoleucine I; Leucine L; Lysine K; Methionine M; Phenylalanine F Proline P; Serine S; Threonine T; Tryptophan W; Tyrosine Y; Valine V.
[050] In the present invention, the oxyntomodulin derivative encompasses any peptide, which is prepared by substitution, addition, deletion or post-translational modification (for example, methylation, acylation, ubiquitination, or intramolecular covalent bonding) of amino acids in the amino acid sequence of SEQ ID NO:1 and can activate both glucagon and GLP-1 receptors. For amino acid substitution or addition, not only the 20 amino acids commonly found in human proteins, but also atypical or unnaturally occurring amino acids can be used. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep Inc., and Genzyme Pharmaceuticals. Peptides, which include these amino acids, and atypical peptide sequences can be synthesized and purchased from commercial suppliers, for example, American Peptide Company or Bachem (USA) or Anygen (South Korea).
[051] In a specific embodiment of the present invention, the oxyntomodulin derivative of the present invention is a new peptide including the amino acid sequence of the following formula 1: Formula 1
[052] R1-X1-X2-GTFTSD-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22 -X23-X24-R2 (SEQ ID NO: 50)
[053] in which
[054] R1 is histidine, desaminohistidyl, dimethylhistidyl (N-dimethylhistidyl), beta-hydroxyimidazopropionyl, 4-imidazoacetyl, imidazopropionyl or tyrosine beta-carboxy;
[055] X1 is Aib (aminosiobutyric acid), d-alanine, glycine, Sar (N-methylglycine), serine or d-serine;
[056] X2 is glutamic acid or glutamine;
[057] X3 is leucine or tyrosine;
[058] X4 is serine or alanine;
[059] X5 is lysine or arginine;
[060] X6 is glutamine or tyrosine;
[061] X7 is leucine or methionine;
[062] X8 is aspartic acid or glutamic acid;
[063] X9 is glutamic acid, serine or alpha-methyl glutamic or is excluded;
[064] X10 is glutamine, glutamic acid, lysine, arginine or serine or is excluded;
[065] X11 is alanine, arginine or valine or is excluded;
[066] X12 is alanine, arginine, serine or valine or is excluded;
[067] X13 is lysine, glutamine, arginine or alpha-methyl-glutamic acid or is excluded;
[068] X14 is aspartic acid, glutamic acid or leucine or is excluded;
[069] X15 is phenylalanine or is excluded;
[070] X16 is isoleucine or valine or is excluded;
[071] X17 is alanine, cysteine, glutamic acid, lysine, glutamine or alpha-methyl-glutamic acid or is excluded;
[072] X18 is tryptophan or is deleted;
[073] X19 is alanine, isoleucine, leucine, serine or valine or is excluded;
[074] X20 is alanine, lysine, methionine, glutamine or arginine or is excluded;
[075] X21 is asparagine or is excluded;
[076] X22 is alanine, glycine or threonine or is excluded;
[077] X23 is cysteine or lysine or is excluded;
[078] X24 is a peptide with 2 to 10 amino acids consisting of a combination of alanine, glycine and serine or is excluded; and
[079] R2 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36), GPSSGAPPPSK (SEQ ID NO: 37), HSQGTFTSDYSKYLD (SEQ ID NO: 38), HSQGTFTSDYSRYLDK (SEQ ID NO: °:39), HGEGTFTSDLSKQMEEEAVK (SEQ ID NO:40) or is excluded (with the exception of the case where the amino acid sequence of formula 1 is identical to that of SEQ ID NO:1).
[080] In order to increase the wild-type oxyntomodulin activity for the glucagon receptor and the GLP-1 receptor, the oxyntomodulin derivative of the present invention can be substituted with 4-imidazoacetyl obtained by deleting the alpha carbon of histidine at the position 1 of the amino acid sequence of SEQ ID NO: 1, desamino-histidyl obtained by exclusion of the terminal N-amino group, dimethyl-histidyl (N-dimethyl-histidyl) obtained by modification of the N-terminal amino group with two methyl groups , beta-hydroxy imidazopropionyl obtained by replacing the N-terminal amino group with a hydroxyl group, or beta-carboxy imidazopropionyl obtained by replacing the N-terminal amino group with a carboxyl group. In addition, the GLP-1 receptor binding region can be replaced with amino acids that enhance hydrophobic and ionic bonds or a combination thereof. In addition, a portion of the oxyntomodulin sequence can be replaced with the amino acid sequence of GLP-1 or exendin-4 to increase GLP-1 receptor activity.
[081] In addition, a portion of the oxyntomodulin sequence can be replaced with a sequence that enhances the alpha helix. Preferably, the amino acids at positions 10, 14, 16, 20, 24 and 28 of the amino acid sequence of formula 1 can be substituted by amino acids or amino acid derivatives consisting of Tyr (4-Me), Phe, Phe (4-Me) , Phe (4-Cl), Phe (4-CN), Phe (4-NO2), Phe (4-NH2), Phg, Pal, Nal, Ala (2-thienyl) and Ala (benzothienyl) which are known as stabilizing the alpha helix, and the type and number of alpha helix stabilizing amino acids or amino acid derivatives to be inserted is not limited. Preferably, the amino acids at positions 10 and 14, 12 and 16, 16 and 20, 20 and 24, and 24 and 28 of the amino acid sequence can also be replaced by glutamic acid or lysine, so as to form rings, and the number of rings to be inserted is not limited. More preferably, the oxyntomodulin derivative may have an amino acid sequence selected from the following formulas 2-6.
[082] In a specific embodiment, the oxyntomodulin derivative of the present invention is a new peptide including the amino acid sequence of the following formula 2, obtained by replacing the amino acid sequence of oxyntomodulin with that of exendin or GLP-1: Formula 2 R1-A-R3 (SEQ ID NO: 51)
[083] In another specific embodiment, the oxyntomodulin derivative of the present invention is a novel peptide including the amino acid sequence of the following formula 3, which is prepared by linking a portion of the amino acid sequence of oxyntomodulin and a portion of the sequence of exendin or GLP-1 amino acids via a suitable linker amino acid: Formula 3 R1-BC-R4 (SEQ ID NO: 52)
[084] In yet another specific embodiment, the oxyntomodulin derivative of the present invention is a novel peptide including the amino acid sequence of the following formula 4, wherein a portion of the oxyntomodulin amino acid sequence is replaced with a hydrophobic amino acid that improves binding to the GLP-1 receiver. For example, it is a peptide in which Leu at position 26 is replaced by the amino acid Ile or Val, which increases hydrophobicity. Formula 4 R1-SQGTFTSDYSKYLD-D1-D2-D3-D4-D5-LFVQW-D6-D7-N-D8-R3 (SEQ ID NO: 53)
[085] In yet another specific embodiment, the oxyntomodulin derivative of the present invention is a novel peptide including the amino acid sequence of the following formula 5, wherein a portion of the native oxyntomodulin amino acid sequence is deleted, added or replaced by other amino acids in order to increase the ability of native oxyntomodulin to activate the GLP-1 receptor and glucagon receptor: Formula 5 R1-E1-QGTFTSDYSKYLD-E2-E3-RA-E4-E5-E6-FV-WLMNT-E7-R5 ( SEQ ID NO: 54)
[086] In formulas 2-5, R1 is as described in formula 1;
[087] A is selected from the group consisting of SQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO: 41), SQGTFTSDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO: 42), SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQ ID NO: 43), SEQ ID NO. 44), GQGTFTSDYSRQMEEEAVRLFIEWLKNG (SEQ ID NO: 45), GEGTFTSDLSRQMEEEAVRLFIEWAA (SEQ ID NO: 46), and SQGTFTSDYSRQMEEEAVRLFIEWLMNG (SEQ ID NO: 47);
[088] B is selected from the group consisting of SQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO: 41), SQGTFTSDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO: 42), SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQ ID NO: 43), SEQ ID NO. 44), GQGTFTSDYSRQMEEEAVRLFIEWLKNG (SEQ ID NO: 45), GEGTFTSDLSRQMEEEAVRLFIEWAA (SEQ ID NO: 46), SQGTFTSDYSRQMEEEAVRLFIEWLMNG (SEQ ID NO: 47), GEGTFTSDLSRQMEEEAVRLFIEWKA (SEQ ID NO: 46), SQGTFTSDYSRQMEEEAVRLFIEWLMNG (SEQ ID NO: 47), GEGTFTSDLSRQMEEEAVRLFIEW (SEQ ID NO: 48), : 49);
[089] C is a peptide with 2 to 10 amino acids consisting of a combination of alanine, glycine and serine;
[090] D1 is serine, glutamic acid or arginine;
[091] D2 is arginine, glutamic acid or serine;
[092] D3 is arginine, alanine or valine;
[093] D4 is arginine, valine or serine;
[094] D5 is glutamine, arginine or lysine;
[095] D6 is isoleucine, valine or serine;
[096] D7 is methionine, arginine or glutamine;
[097] D8 is threonine, glycine or alanine;
[098] E1 is serine, Aib, Sar, d-d-alanine or serine;
[099] E2 is serine or glutamic acid;
[100] E3 is arginine or lysine;
[101] E4 is glutamine or lysine;
[102] E5 is aspartic acid or glutamic acid;
[103] E6 is glutamine, cysteine or lysine;
[104] E7 is cysteine or lysine or is excluded;
[105] R3 is KRNRNNIA (SEQ ID NO:35), GPSSGAPPPS (SEQ ID NO:36) or GPSSGAPPPSK (SEQ ID NO:37);
[106] R4 is HSQGTFTSDYSKYLD (SEQ ID NO:38), HSQGTFTSDYSRYLDK (SEQ ID NO:39) or HGEGTFTSDLSKQMEEEAVK (SEQ ID NO:40); and,
[107] R5 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36) or GPSSGAPPPSK (SEQ ID NO: 37) or is excluded (except in the case where the amino acid sequences of formulas 2-5 are identical to that of SEQ ID NO: 1).
[108] Preferably, the oxyntomodulin derivative of the present invention may be a novel peptide with the following formula 6: Formula 6
[109] R1-X1-X2-GTFTSD-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22 -X23-X24-R2 (SEQ ID NO: 55)
[110] where R1 is histidine, desaminohistidyl, 4-imidazoacetyl or tyrosine;
[111] X1 is Aib (aminosiobutyric acid), glycine or serine;
[112] X2 is glutamic acid or glutamine;
[113] X3 is leucine or tyrosine;
[114] X4 is serine or alanine;
[115] X5 is lysine or arginine;
[116] X6 is glutamine or tyrosine;
[117] X7 is leucine or methionine;
[118] X8 is aspartic acid or glutamic acid;
[119] X9 is glutamic or alpha-methyl glutamic acid or is excluded;
[120] X10 is glutamine, glutamic acid, lysine or arginine or is excluded;
[121] X11 is alanine or arginine or is excluded;
[122] X12 is alanine or valine or is excluded;
[123] X13 is lysine, glutamine, arginine or alpha-methyl-glutamic acid or is excluded;
[124] X14 is aspartic acid, glutamic acid, or leucine or is excluded;
[125] X15 is phenylalanine or is excluded;
[126] X16 is isoleucine or valine or is excluded;
[127] X17 is alanine, cysteine, glutamic acid, glutamine or alpha-methyl glutamic acid or is excluded;
[128] X18 is tryptophan or is excluded;
[129] X19 is alanine, isoleucine, leucine, or valine or is excluded;
[130] X20 is alanine, lysine, methionine or arginine or is excluded;
[131] X21 is asparagine or is excluded;
[132] X22 is threonine or is excluded;
[133] X23 is cysteine, lysine or is excluded;
[134] X24 is a peptide with 2 to 10 amino acids that consists of glycine or is excluded; and
[135] R2 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36), GPSSGAPPPSK (SEQ ID NO: 37), HSQGTFTSDYSKYLD (SEQ ID NO: 38), HSQGTFTSDYSRYLDK (SEQ ID NO: °: 39) or HGEGTFTSDLSKQMEEEAVK (SEQ ID NO: 40) or is excluded (with the exception of the case where the amino acid sequence of formula 6 is identical to that of SEQ ID NO: 1).
[136] More preferably, the oxyntomodulin derivative of the present invention may be selected from the group consisting of the peptides of SEQ ID Nos: 2 to 34. Even more preferably, the oxyntomodulin derivative of the present invention may be a derivative of oxyntomodulin described in Table 1 of Example 1.
[137] Oxintomodulin has the activities of two peptides, GLP-1 and glucagon. GLP-1 has the effect of lowering blood glucose levels by secreting insulin, but glucagon has the effect of raising blood glucose levels. In addition, GLP-1 inhibits food intake and suppresses gastric emptying, and glucagon has the effect of reducing body weight, facilitating lipolysis and increasing energy metabolism. Thus, GLP-1 and glucagon have different biological effects. Thus, in case of two peptides present as a conjugate, if the effect of any one of the two peptides is greater than that of the other, an adverse effect can occur. For example, if the effect of glucagon is greater than that of GLP-1, blood glucose levels may rise, and if the effect of GLP-1 is greater than that of glucagon, side effects such as nausea and vomiting may occur. Also, the effect of the two peptides can vary depending on the ratio of the activities of the two peptides. Thus, oxyntomodulin derivatives and their conjugates are not limited only to derivatives having enhanced activities.
[138] As used herein, the term "oxyntomodulin conjugate" refers to a conjugate comprising oxyntomodulin and another element. The other element can be any substance that has beneficial functions, including increasing the blood half-life of oxyntomodulin or delaying the release of oxyntomodulin from the kidneys. The conjugate of the present invention can covalently bind to oxyntomodulin or form microspheres to increase the serum stability of oxyntomodulin or to delay the release of oxyntomodulin from the kidney or to alter the binding activity of oxyntomodulin to its receptor. The carrier that can form a conjugate comprising oxyntomodulin can be selected from the group consisting of albumin, transferrin, antibodies, antibody fragments, elastin, heparin, polysaccharide such as chitin, fibronectin, and the like, which can be linked to oxyntomodulin to increase the serum stability of oxyntomodulin. Preferably, the carrier is an immunoglobulin Fc region.
[139] The immunoglobulin Fc that can be used in the present invention may be a human immunoglobulin Fc, an immunoglobulin Fc having the sequence of an analogue thereof, or an immunoglobulin Fc derived from animals, including cows, goats, pigs, rats, rabbits, hamsters, rats and guinea pigs. Furthermore, the immunoglobulin Fc region can be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. Furthermore, each domain of the Fc region of the immunoglobulin of the present invention may be a hybrid of domains derived from different immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. Alternatively, the immunoglobulin Fc region is a dimer or multimer consisting of single-chain immunoglobulins composed of domains of the same origin. Preferably, the Fc region of the immunoglobulin is a derivative of IgG or IgM, which is richer in human blood. More preferably, it is an IgG-derived immunoglobulin Fc known to increase the half-life of ligand binding proteins. Immunoglobulin Fc can be prepared either by treating IgG with a native or specific protease from transformed cells using the recombination technique. Preferably, it is a recombinant human immunoglobulin Fc prepared in E. coli.
[140] Meanwhile, IgG can also be subclassified into IgG1, IgG2, IgG3 and IgG4, and in the present invention, a combination or hybrid of these subclasses is also possible. Preferably, the IgG is an IgG2 and IgG4 subclass, and more preferably, it is the Fc region of IgG4 that substantially lacks effector functions, such as complement dependent cytotoxicity (CDC). In other words, the most preferred immunoglobulin Fc region that is used as a pharmaceutical carrier in the present invention is a non-glycosylated Fc region derived from human IgG4. The Fc region of human origin is more preferable than a Fc region of non-human origin, which can act as an antigen in the human body and trigger unwanted immune responses, such as the production of a new antibody against the antigen.
[141] In the present invention, the oxyntomodulin conjugate can be prepared by using a non-peptide polymer or by genetic recombination technique. Preferably, the conjugate can be prepared by linking oxyntomodulin to the immunoglobulin Fc region by a non-peptide polymer.
[142] The non-peptide polymer can be linked to each of the oxyntomodulin and Fc region of the immunoglobulin. Each end of the non-peptide polymer can be linked to the Fc region of the immunoglobulin and the amino or thiol group of the oxyntomodulin derivative, respectively.
[143] As used herein, the term "oxyntomodulin conjugate" refers to one having a greater long-lasting effect compared to native oxyntomodulin. Examples of the long-lasting conjugate include, but are not limited to, a conjugate in which an oxyntomodulin derivative resulting from the modification, substitution, addition or deletion of amino acids in the native oxyntomodulin amino acid sequence is linked to a biodegradable polymer such as polyethylene glycol (PEG), a conjugate, in which a protein that has excellent long-lasting properties, such as albumin or immunoglobulin, is linked to oxyntomodulin, a conjugate in which a fatty acid having the ability to bind albumin in vivo is linked to oxyntomodulin, or a conjugate in which oxyntomodulin is encapsulated in biodegradable nanoparticles.
[144] As used herein, the term "non-peptide polymer" refers to a biocompatible polymer, including two or more repeating units linked together by any covalent bond in place of a peptide bond. In the present invention, the non-peptide polymer can be used interchangeably with the non-peptide linker.
[145] A peptide binding agent that is used in a fusion protein obtained by a conventional inframe fusion method has disadvantages in that it is easily cleaved by proteinase in vivo, and thus the desired half-increasing effect. serum life of the active drug by a carrier cannot be obtained. However, in the present invention, the protease-resistant polymer can be used to maintain its serum peptide half-life, similar to the carrier. Therefore, any non-peptide polymer can be used without limitation of the present invention, as long as it is a polymer with the aforementioned function, i.e., a polymer that has resistance to protease in vivo. The non-peptide polymer has a molecular weight in the range of 1 to 100 kDa, and preferably 1 to 20 kDa. The non-peptide polymer of the present invention, which is linked to the Fc region of the immunoglobulin, can be one type of polymer or a combination of different polymers.
[146] The non-peptide polymer that is used in the present invention may have a reactive group capable of binding to the Fc region of the immunoglobulin and to protein drug. The reactive group at both ends of the non-peptide polymer is preferably selected from the group consisting of a reactive aldehyde group, a propionaldehyde group, a butyraldehyde group, a maleimide group and a succinimide derivative.
[147] The succinimide derivative can be succinimidyl propionate, hydroxy succinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate. Particularly, when the non-peptide polymer has a reactive aldehyde group at both ends of it, non-specific reactions can be minimized, and a physiologically active polypeptide and an immunoglobulin can be effectively attached to both ends of the non-peptide polymer, respectively. . An end product generated by reductive alkylation with an aldehyde linker is much more stable than one linked by an amide linkage. The reactive aldehyde group selectively binds to an N-terminus at low pH and can form a covalent bond with a lysine residue at high pH, such as pH 9.0.
[148] The reactive groups at both ends of the non-peptide polymer can be the same or different. For example, the non-peptide polymer can have a maleimide group at one end and an aldehyde group, propionaldehyde group, or butyraldehyde group at the other end. When a polyethylene glycol with a reactive hydroxy group at both ends thereof is used as the non-peptide polymer, the hydroxyl group can be activated by various reactive groups by known chemical reactions, or a polyethylene glycol having a commercially available modified reactive group can be used in order to prepare the long acting conjugate of the present invention.
[149] The conjugate of the present invention can be one, wherein each end of the non-peptide polymer is linked to the Fc region of the immunoglobulin and the amino or thiol group of the oxyntomodulin derivative, respectively.
[150] However, in the present invention, both ends of the non-peptide polymer include reactive groups to which an immunoglobulin Fc region and a protein drug can bind. Examples of the reactive groups include, but are not limited to, an aldehyde group, a propionaldehyde group or a butyraldehyde group, a maleimide group, a succinimide derivative (succinimidyl propionate, hydroxyl succinimidyl, carboxymethyl succinimidyl or succinimidyl carbonate) and the like.
[151] The reactive groups at both ends of the linker, the non-peptide polymer, can be the same or different. For example, the non-peptide polymer can have a maleimide group at one end and an aldehyde group, a propionaldehyde group, or a butyraldehyde group at the other end. For example, when the non-peptide polymer has a reactive aldehyde group at one end and a reactive maleimide group at the other end, non-specific reactions can be minimized, and a physiologically active polypeptide and an immunoglobulin can be effectively attached to both ends of the non-peptide polymer. The non-peptide polymer that can be used in the present invention can be selected from the group consisting of polyethylene glycol, polypropylene glycol, an ethylene glycol/propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, acetate of polyvinyl ether, biodegradable polymers such as PLA (poly (lactic acid)) and PLGA (lactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof. Preferably, the non-peptide polymer is polyethylene glycol. Furthermore, derivatives thereof known in the art and derivatives which can be easily prepared by a method known in the art are included within the scope of the present invention.
[152] In one example of the present invention, a conjugate was synthesized by linking oxyntomodulin or a derivative thereof to the Fc region of the immunoglobulin via a covalent bond via non-peptide polymer to PEG including a propionaldehyde group alone or both a maleimide group and a group aldehyde.
[153] The conjugate of the present invention has excellent GLP-1 receptor and glucagon receptor activity compared to native oxyntomodulin. Furthermore, it has attached to it an Fc region that increases the half-life in vivo in blood thereof to maintain its activity in vivo for an extended period of time.
[154] As used herein, the term "preservative" refers to a substance that is used to prevent abnormal reactions or decomposition from being caused by microbial contamination. The liquid formulation of the present invention may further comprise a preservative. A preservative is generally used in multi-dose formulations that are likely to be contaminated with, but not limited to, microorganisms and can also be used in lyophilized formulations or single-dose formulations to prevent microbial contamination. The liquid formulation of the present invention may comprise one or more preserving agents selected from m-cresol, phenol and benzyl alcohol. The concentration of preservative in the liquid formulation can be 0.001-1% (w/v). Particularly, the preservative that is included in the liquid formulation of the present invention is preferably m-cresol. The liquid formulation of the present invention can be a multiple dose formulation.
[155] In another aspect, the present invention provides a method for preparing a liquid formulation of a long-acting oxyntomodulin conjugate.
[156] Specifically, in one embodiment of the present invention, the method for preparing the liquid formulation may comprise the steps of: a) preparing a long-acting oxyntomodulin conjugate; and b) mixing the prepared long-acting oxyntomodulin conjugate with a stabilizer containing a buffer, a sugar alcohol and a nonionic surfactant.
[157] In another embodiment of the present invention, the method for preparing the liquid formulation may comprise the steps of: a) preparing a long-acting oxyntomodulin conjugate; and b) mixing the prepared long-acting oxyntomodulin conjugate with a stabilizer, which contains a buffer, a sugar alcohol and a nonionic surfactant, and preservative.
[158] Preferably, the stabilizer in step b) may further comprise one or more selected from the group consisting of isotonic agents, sugars, polyhydric alcohols and amino acids.
[159] In yet another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of obesity or diabetes, which comprises the above liquid formulation.
[160] As used herein, the term "prevention" refers to all actions that inhibit or delay the development of a target disease. As used herein, the term "prevent" means administration of the conjugate of the present invention to inhibit or delay the development of diabetic conditions, such as abnormal blood glucose levels or abnormal isolation secretion, obesity, or conditions such as an increase in body weight or body fat.
[161] As used herein, the term "treat" refers to all actions that alleviate, ameliorate, or alleviate the symptoms of the developed disease. As used herein, the term "treatment" means administering the conjugate of the present invention to alleviate, ameliorate or alleviate the above diabetic conditions or obesity conditions to normalize blood glucose levels and insulin secretion and reduce body weight. or body fat.
[162] As used herein, the term "obesity" refers to an excessive amount of body fat. A body mass index (= weight (kg) divided by height (m)) of 25 or more is defined as obesity. Obesity usually results from an energy imbalance in which energy consumption exceeds energy expenditure. Obesity is a metabolic disease that affects the entire body and is highly susceptible to diabetes and hyperlipidemia. Furthermore, obesity is related to sexual dysfunction, arthritis, and an increased risk of developing cardiovascular disease, and is also related to the development of cancer in some cases.
[163] As used herein, the term "diabetes" is a type of metabolic disease in which insulin secretion is insufficient or normal functions are not performed. Diabetes is characterized by increased blood glucose levels and causes various health problems. In the case of diabetes, glucose is excreted in the urine.
[164] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent. As used herein, the term "pharmaceutically acceptable" means an amount that is sufficient to exhibit therapeutic effects and does not cause side effects. The dose of the active ingredient of the pharmaceutical composition of the present invention can be easily determined by those skilled in the art depending on the type of disease, patient age, weight, health condition, sex and drug sensitivity, the administration route, the administration mode , frequency of administration, duration of treatment, drugs used in combination or coinciding with the composition of the present invention, and other factors known in the field of medicine.
[165] In yet another aspect, the present invention provides a method for preventing or treating obesity or diabetes, which comprises administering the liquid formulation to a subject.
[166] In this, the liquid formulation, obesity and diabetes are as described above.
[167] As used herein, the term "subject" refers to an individual suspected of having obesity or diabetes. Specifically, the term means mammals, including humans, rats and domestic animals, that have or are at risk of developing the above disease. Furthermore, the subject can be any subject that can be treated by the liquid formulation derivative of the present invention.
[168] The therapeutic method of the present invention may comprise administering a pharmaceutically effective amount of the pharmaceutical composition comprising the liquid formulation. The total daily dose of the composition can be determined by appropriate medical judgment by a physician, and the composition can be administered once or several times. However, in view of the purpose of the present invention, the therapeutically effective dose of the specific composition for any particular patient may vary depending on a number of factors well known in the field of medicine, including the type and degree of response to be achieved, specific compositions of according to whether other agents are used with the same or not, the age, body weight, health status, sex and diet of the patient, the time and route of administration, the secretion rate of the composition, the duration of treatment, other drugs used in combination or coincidental with the composition of the present invention, and other factors known in the field of medicine. ADVANTAGEOUS EFFECTS OF THE INVENTION
[169] The liquid formulation of the invention comprising the long-acting oxyntomodulin conjugate comprises a buffer, a sugar alcohol and a nonionic surfactant and does not contain a human serum albumin and factors that are potentially harmful to the human body, and therefore not susceptible to viral infection. Furthermore, the oxyntomodulin conjugate of the present invention comprises oxyntomodulin linked to an immunoglobulin Fc region and therefore has a large molecular weight, prolonged physiological activity, and excellent storage stability compared to native oxyntomodulin. BRIEF DESCRIPTION OF THE FIGURES
[170] FIG. 1a is a graph showing the results obtained by purification of a mono-PEGylated oxyntomodulin derivative (SEQ ID NO: 25) through a SOURCE S purification column.
[171] FIG. 1b is a graph showing the results obtained by purification of a conjugate of a mono-PEGylated oxyntomodulin derivative (SEQ ID NO: 25) and an immunoglobulin Fc by means of a SOURCE 15Q purification column.
[172] FIG. 1C is a graph showing the results obtained purification of a conjugate of a mono-PEGylated oxyntomodulin derivative (SEQ ID NO: 25) and an immunoglobulin Fc by means of a SOURCE ISO purification column.
[173] FIG. 2a is a graphical diagram showing the results obtained by evaluating the stability of a long-lasting oxyntomodulin conjugate according to pH by IE-HPLC in Example 3, after 0-2 weeks storage at 25°C. Each graph in FIG. 2a shows the percentage retention of the long-acting oxyntomodulin conjugate relative to the initial value.
[174] FIG. 2b is a graphical diagram showing the results obtained by evaluating the stability of a long-lasting oxyntomodulin conjugate according to pH by SE-HPLC in Example 3, after 0-2 weeks storage at 25°C. Each graph in FIG. 2b shows the percent retention rate of the long-acting oxyntomodulin conjugate relative to the initial value.
[175] FIG. 2c is a graphical diagram showing the results obtained by evaluating the stability of the long-lasting oxyntomodulin conjugate according to pH by RP-HPLC in Example 3, after 0-2 weeks storage at 25°C. Each graph in FIG. 2c shows the percent retention of the long-acting oxyntomodulin conjugate from baseline.
[176] FIG. 3a is a graphic diagram showing the results obtained by evaluating the stability of a long-acting oxyntomodulin conjugate according to the type of sugar alcohol and the presence or absence of an isotonic agent by IE-HPLC in Example 4, after 0 -4 weeks of storage at 25 °C. Each graph in FIG. 3A shows the percent retention of the long-acting oxyntomodulin conjugate from baseline.
[177] FIG. 3b is a graphic diagram showing the results obtained by evaluating the stability of a long-acting oxyntomodulin conjugate according to the type of sugar alcohol and the presence or absence of an isotonic agent by SE-HPLC in Example 4, after 0 -4 weeks of storage at 25 °C. Each graph in FIG. 3b shows the percent retention of the long-acting oxyntomodulin conjugate relative to the initial value.
[178] FIG. 3c is a graphic diagram showing the results obtained by evaluating the stability of a long-acting oxyntomodulin conjugate according to the type of sugar alcohol and the presence or absence of an isotonic agent by RP-HPLC in Example 4, after 0 -4 weeks of storage at 25 °C. Each graph in FIG. 3C shows the percent retention of long-acting oxyntomodulin conjugate from baseline.
[179] FIG. 4a is a graphic diagram showing the results obtained by evaluating the stability of long-acting oxyntomodulin conjugate according to sugar alcohol concentration by IE-HPLC in Example 5, after 0-4 weeks of storage at 25°C. Each graph in FIG. 4a shows the percent retention of the long-acting oxyntomodulin conjugate relative to the initial value.
[180] FIG. 4b is a graphical diagram showing the results obtained by evaluating the stability of a long-acting oxyntomodulin conjugate according to the sugar alcohol concentration by SE-HPLC in Example 5, after 0-4 weeks of storage at 25°C . Each graph in FIG. 4b shows the percent retention of the long-acting oxyntomodulin conjugate from baseline.
[181] FIG. 4c is a graphical diagram showing the results obtained by evaluating the stability of a long-acting oxyntomodulin conjugate according to sugar alcohol concentration by means of RP-HPLC in Example 5, after 0-4 weeks of storage at 25°C. °C. Each graph in FIG. 4c shows the percent retention of the long-acting oxyntomodulin conjugate from baseline.
[182] FIG. 5a is a graphic diagram showing the results obtained by evaluating the stability of a long-acting oxyntomodulin conjugate according to the concentration of a surfactant and the presence or absence of an amino acid by IE-HPLC in Example 6, after 0- 4 weeks of storage at 25 °C. Each graph in FIG. 5a shows the percent retention of the long-acting oxyntomodulin conjugate relative to the initial value.
[183] FIG. 5b is a graphic diagram showing the results obtained by evaluating the stability of a long-acting oxyntomodulin conjugate according to the concentration of a surfactant and the presence or absence of an amino acid by SE-HPLC in Example 6, after 0- 4 weeks of storage at 25 °C. Each graph in FIG. 5b shows the percent retention of the long-acting oxyntomodulin conjugate relative to the initial value.
[184] FIG. 5c is a graphical diagram showing the results obtained by evaluating the stability of a long-acting oxyntomodulin conjugate according to the concentration of a surfactant and the presence or absence of an amino acid by RP-HPLC in Example 6, after 0- 4 weeks of storage at 25 °C. Each graph in FIG. 5c shows the percent retention of the long-acting oxyntomodulin conjugate from baseline.
[185] FIG. 6a is a graphical diagram showing the results obtained by evaluating the stability of long-lasting oxyntomodulin conjugate according to pH and sugar alcohol concentration by IE-HPLC in Example 7 after 0-4 weeks storage at 25°C . Each graph in FIG. 6a shows the percent retention of the long-acting oxyntomodulin conjugate relative to the initial value.
[186] FIG. 6b is a graphical diagram showing the results obtained by evaluating the stability of long-lasting oxyntomodulin conjugate according to pH and sugar alcohol concentration by SE-HPLC in Example 7 after 0-4 weeks storage at 25°C . Each graph in FIG. 6b shows the percent retention of the long-acting oxyntomodulin conjugate from baseline.
[187] FIG. 6c is a graphical diagram showing the results obtained by evaluating the stability of long-lasting oxyntomodulin conjugate according to pH and sugar alcohol concentration by means of RP-HPLC in Example 7 after 0-4 weeks storage at 25°C °C. Each graph in FIG. 6c shows the percent retention of the long-acting oxyntomodulin conjugate from baseline.
[188] FIG. 7a is a graphic diagram showing the results obtained by evaluating the stability of long-lasting oxyntomodulin conjugate according to pH and alcohol buffer type by IE-HPLC in Example 8 after 0-4 weeks of storage at 25° Ç. Each graph in FIG. 7a shows the percent retention of the long-acting oxyntomodulin conjugate relative to the initial value.
[189] FIG. 7b is a graphic diagram showing the results obtained by evaluating the stability of long-lasting oxyntomodulin conjugate according to pH and alcohol buffer type by SE-HPLC in Example 8, after 0-4 weeks of storage at 25° Ç. Each graph in FIG. 7b shows the percent retention of the long-acting oxyntomodulin conjugate from baseline.
[190] FIG. 7c is a graphic diagram showing the results obtained by evaluating the stability of long-lasting oxyntomodulin conjugate according to pH and alcohol buffer type by RP-HPLC in Example 8 after 0-4 weeks of storage at 25° Ç. Each graph in FIG. 7c shows the percent retention of the long-acting oxyntomodulin conjugate from baseline.
[191] FIG. 8a is a graphical diagram showing the results obtained by evaluating the stability of a long-acting oxyntomodulin conjugate according to the presence or absence of a preservative and the concentration of long-acting oxyntomodulin by IE-HPLC in Example 9 after 0- 4 weeks of storage at 25 °C. Each graph in FIG. 8a shows the percent retention of the long-acting oxyntomodulin conjugate relative to the initial value.
[192] FIG. 8b is a graphical diagram showing the results obtained by evaluating the stability of a long-acting oxyntomodulin conjugate according to the presence or absence of a preservative and the concentration of long-acting oxyntomodulin by SE-HPLC in Example 9 after 0- 4 weeks of storage at 25 °C. Each graph in FIG. 8b shows the percent retention of the long-acting oxyntomodulin conjugate from baseline.
[193] FIG. 8c is a graphical diagram showing the results obtained by evaluating the stability of a long-acting oxyntomodulin conjugate according to the presence or absence of a preservative and the concentration of long-acting oxyntomodulin by RP-HPLC in Example 9 after 0- 4 weeks of storage at 25 °C. Each graph in FIG. 8c shows the percent retention of the long-acting oxyntomodulin conjugate from baseline. MODE FOR INVENTION
[194] Hereinafter, the present invention will be described in greater detail with reference to examples. It should be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Example 1: Synthesis of Oxyntomodulin and Oxyntomodulin Derivatives
[195] In order to measure the stabilities of oxyntomodulin and oxyntomodulin derivatives of the liquid formulation of the present invention, oxyntomodulin derivatives having the amino acid sequences shown in Table 1 below were synthesized. Table 1 [Table 1] Oxintomodulin and oxyntomodulin derivatives




[196] In Table a above, the amino acid residues indicated by bold letters in each of SEQ ID Nos: 9, 20, 22, 25, 26, 27, 32, 33, and 34, taken together, form rings , and the amino acid residues indicated by X means alpha-methyl glutamic acid which is an unnatural amino acid. In addition, CA indicates 4-imidazoacetyl, DA indicates desaminohistidyl, and (d)S indicates d-serine. Example 2: Preparation of conjugate comprising oxyntomodulin derivative (SEQ ID NO: 25) and immunoglobulin Fc (oxyntomodulin derivative (SEQ ID NO: 25) linked to immunoglobulin Fc region)
[197] First, in order to PEGylate MAL-10K-ALD-PEG at a cysteine residue at amino acid position 30 of an oxyntomodulin derivative (SEQ ID NO: 25), the oxyntomodulin derivative (SEQ ID NO: °: 25) and MAL-10K-ALD-PEG were kept reacting with each other at a molar ratio of 1:3 and a protein concentration of 3 mg/ml at room temperature for 3 hours. Here, the reaction was carried out in 50 mM Tris buffer (pH 8.0) in the presence of 1 M guanidine. After completion of the reaction, the reaction solution was applied to a SOURCE S column to purify an oxyntomodulin derivative mono-PEGylated on cysteine (SOURCE S column, flow rate: 2.0 ml/min, gradient: A 0 ^100% 50 min B (A: 20 mM Na-citrate, pH 3.0 + 45% ethanol, B: A + 1M KCl)) (Figure 1a). FIG. 1a is a graph showing the results obtained by purification of the mono-PEGylated oxyntomodulin derivative (SEQ ID NO: 25) through the SOURCE S purification column.
[198] Then, the purified mono-PEGylated oxyntomodulin derivative (SEQ ID NO: 25) and an immunoglobulin Fc were reacted together at a molar ratio of 1:5 and a protein concentration of 20 mg/ ml at 4°C for 16 hours. The reaction was carried out in 100 mM potassium phosphate buffer (pH 6.0) in the presence of 20 mM SCB as a reducing agent. After completion of the reaction, the reaction was applied to a SOURCE 15Q purification column (column: 2.0 ml/min, gradient: A 0 ^ 4% 1 min B ^ 20% 80 min B (A: 20mM Tris-HCl, pH 7.5, B: A + 1M NaCl)) (Fig 1b) and a SOURCE ISO purification column (column: SOURCE ISO, flow rate: 2.0 ml/min, gradient: B 0 ^ 100% 100 min A (A: 20mM Tris-HCl, pH 7.5, B: A + 1.1M AS)) (figure 1c) to purify a conjugate comprising the oxyntomodulin derivative (SEQ ID NO: 25) and the immunoglobulin Fc. FIG. 1b is a graph showing the results obtained from purification of the conjugate comprising the oxyntomodulin derivative (SEQ ID NO: 25) and the immunoglobulin Fc by means of the SOURCE 15Q purification column, and FIG. 1C is a graph showing the results obtained from the purification of the conjugate, comprising the oxyntomodulin derivative (SEQ ID NO: 25) and the immunoglobulin Fc, by means of the SOURCE ISO purification column.
[199] Oxyntomodulin conjugate prepared as described above was developed to increase the half-life of oxyntomodulin in blood. It comprises the Fc region of immunoglobulin, the non-peptide polymer and oxyntomodulin, covalently linked together in a site-specific manner, and has a significantly increased half-life in blood. Example 3: Evaluation of the stability of long-lasting oxyntomodulin conjugate according to pH
[200] In order to assess the stability of the long-acting oxyntomodulin conjugate (prepared in Example 2) in liquid formulations, the long-acting oxyntomodulin conjugate was stored in the compositions shown in Table 2, at 25 °C for 0-2 weeks, and then analyzed by high performance ion exchange liquid chromatography (IE-HPLC), high size exclusion liquid chromatography (SE-HPLC) and high performance reverse phase liquid chromatography (RP-HPLC). For the storage of the oxyntomodulin conjugate, citrate buffer as a buffer, mannitol as a sugar alcohol, and polysorbate 20 as a nonionic surfactant were used. In Tables 3, 4 and 5 below, IE-HPLC (%), SE-HPLC (%) and RP-HPLC (%) indicate the area%/initial area%, which indicates the retention percentage of the oxyntomodulin conjugate long-lasting compared to the initial value. Table 3 shows the IE-HPLC area (%) of the long-lived oxyntomodulin conjugate after storage, Table 4 shows the SE-HPLC area (%) of the long-lived oxyntomodulin conjugate after storage, and the Table 5 shows the RP-HPLC Area (%) of the long-lasting oxyntomodulin conjugate after storage. Table 2 [Table 2]


[201] As can be seen from the IE-HPLC results (%) in Table 3 above, the oxyntomodulin conjugate was more stable at lower pH. In the SE-HPLC results in Table 4, the oxyntomodulin conjugate was more stable at a pH of 5.2, and in the RP-HPLC results in Table 5, the oxyntomodulin conjugate was more stable at a pH of 5.6 . Although the pH stability differs between the analysis methods, the difference in retention between pHs was greater in the RP-HPLC analysis method. This suggests that the oxyntomodulin conjugate was more stable at pH 5.6. Example 4: Evaluation of the stability of the long-acting oxyntomodulin conjugate according to the type of sugar alcohol and the presence or absence of an isotonic agent
[202] The present inventors tested the influences of the type of sugar alcohol as a stabilizer and the presence or absence of sodium chloride as an isotonic agent on the stability of the long-lasting oxyntomodulin conjugate. Specifically, using the citrate buffer (pH 5.6) selected in Example 3, the long-acting oxyntomodulin conjugate was stored in the compositions shown in Table 6 below, at 25°C for 0-4 weeks, and then analyzed for IE-HPLC, SE-HPLC and RP-HPLC. In Tables 7, 8 and 9 below, IE-HPLC (%), SE-HPLC (%) and RP-HPLC (%) indicate the area%/initial area%, which indicates the retention percentage of the oxyntomodulin conjugate from long duration relative to the initial value. Table 7 shows the IE-HPLC area (%) of the long-acting oxyntomodulin conjugate after storage, Table 8 shows the SE-HPLC area (%) of the long-acting oxyntomodulin conjugate after storage, and in Table 9 shows the RP-HPLC Area (%) of the long-lasting oxyntomodulin conjugate after storage. Table 6 [Table 6]



[203] As can be seen from Tables 6 to 9 above, the long-acting oxyntomodulin conjugate was more stable in mannitol or sorbitol than in glycerol at the same concentration. The RP-HPLC results indicate that the long-acting oxyntomodulin conjugate was somewhat stable in mannitol than sorbitol. Furthermore, the stability of the long-acting oxyntomodulin conjugate did not differ significantly between the presence and absence of sodium chloride as an isotonic agent. Example 5: Evaluation of the stability of long-lasting oxyntomodulin conjugate according to sugar alcohol concentration
[204] The present inventors tested the influence of sugar alcohol concentration as a stabilizer on the stability of the long-lasting oxyntomodulin conjugate. Specifically, using the citrate buffer (pH 5.6 and mannitol selected in the examples above, the long-acting oxyntomodulin conjugate was stored in the compositions shown in Table 10 below, at 25°C for 0-4 weeks, and then analyzed). by IE-HPLC, SE-HPLC and RP-HPLC. In Tables 11, 12 and 13 below, IE-HPLC (%), from SE-HPLC (%) and RP-HPLC (%) indicate the area % / initial area %, which indicates the percentage retention of the long-term oxyntomodulin conjugate in relation to the initial value. Table 11 shows the IE-HPLC area (%) of the long-term oxyntomodulin conjugate after storage, Table 12 shows the SE-HPLC area (%) of the long-acting oxyntomodulin conjugate after storage, and Table 13 shows the RP-HPLC area (%) of the long-acting oxyntomodulin conjugate after storage.



[205] As can be seen from Tables 10 to 13, the long-acting oxyntomodulin conjugate was stable in the presence of 5% mannitol or 10% mannitol. However, a protein precipitate was formed in the presence of 2% mannitol or 15% mannitol. IE-HPLC or SE-HPLC results indicate that the stability of the long-acting oxyntomodulin conjugate was similar between 10% mannitol and 5% mannitol. The RP-HPLC results indicate that the stability of the long-acting oxyntomodulin conjugate was more stable in 10% mannitol than 5% mannitol. Example 6: Evaluation of the stability of the long-lasting oxyntomodulin conjugate according to the concentration of surfactant and the presence or absence of amino acid
[206] The present inventors tested the influence of the concentration of a surface-active agent as a stabilizer and the presence or absence of an amino acid on the stability of the long-lasting oxyntomodulin conjugate. Using the citrate buffer (pH 5.6) and citrate buffer and 10% mannitol selected in the examples above, the long-lasting oxyntomodulin conjugate was stored in the compositions shown in Table 14 below, at 25°C for 0-4 weeks , and then analyzed by IE-HPLC, SE-HPLC and RP-HPLC. In Tables 15, 16 and 17 below, IE-HPLC (%), SE-HPLC (%) and RP-HPLC (%) indicate the area%/initial area%, which indicates the percentage retention of long-term oxyntomodulin conjugate duration relative to the initial value. Table 15 shows the IE-HPLC area (%) of the long-acting oxyntomodulin conjugate after storage, Table 16 shows the SE-HPLC area (%) of the long-acting oxyntomodulin conjugate after storage, and the Table 17 shows the RP-HPLC Area (%) of the long-lasting oxyntomodulin conjugate after storage. Table 14 [Table 14]


[207] As can be seen from the results in Tables 4 to 17, the long-acting oxyntomodulin conjugate was more stable in the composition containing 0.02% polysorbate 20 and 0.1 mg/ml methionine. Example 7: Evaluation of the stability of long-lasting oxyntomodulin conjugate according to pH and sugar alcohol concentration
[208] The present inventors tested the influences of pH and sugar alcohol concentration as a stabilizer on the stability of long-lasting oxyntomodulin conjugate. Specifically, using 0.02% of the polysorbate 20 and 0.1 mg/ml of methionine selected in the examples above, the long-acting oxyntomodulin conjugate was stored in the compositions shown in Table 18 below, at 25°C for 0-4 weeks , and then analyzed by IE-HPLC, SE-HPLC and RP-HPLC. In Tables 19, 20 and 21 below, IE-HPLC (%), SE-HPLC (%) and RP-HPLC (%) indicate the area%/initial area%, which indicates the retention percentage of the long oxyntomodulin conjugate duration relative to the initial value. Table 19 shows the IE-HPLC area (%) of the long-acting oxyntomodulin conjugate after storage, Table 20 shows the SE-HPLC area (%) of the long-acting oxyntomodulin conjugate after storage, and the Table 21 shows the RP-HPLC Area (%) of the long-lasting oxyntomodulin conjugate after storage. Table 18 [Table 18]




[209] As can be seen from the results in the Tables above, the IE-HPLC results indicate that the stability of the long-lasting oxyntomodulin conjugate was higher at pH on the order of pH 5.2, pH 5.6 and pH 6.0. The RP-HPLC results indicate that the stability of the long-lasting oxyntomodulin conjugate was higher on the order of pH 6.0, pH 5.6 and pH 5.2. The SE-HPLC results indicate that the stability of the long-lasting oxyntomodulin conjugate did not differ significantly between pH 5.2, pH 5.6 and pH 6.0. In other words, the IE-HPLC, RP-HPLC and SE-HPLC results indicate that the long-acting oxyntomodulin conjugate was stable at pH 5.6.
[210] However, IE-HPLC and SE-HPLC results indicate that the long-acting oxyntomodulin conjugate did not differ significantly between mannitol concentrations at pH 5.6. However, in RP-HPLC results, the long-acting oxyntomodulin conjugate was more stable in 5% mannitol than 10% or 15% mannitol at pH 5.6. Example 8: Stability evaluation of long-lasting oxyntomodulin conjugate according to pH and buffer type
[211] The present inventors tested the influences of pH and buffer type as a stabilizer on the stability of long-lasting oxyntomodulin conjugate. Specifically, using the 0.02% polysorbate 20, 0.1 mg/ml methionine and 5% mannitol selected in the Examples above, the long-lasting oxyntomodulin conjugate was stored in the compositions shown in Table 22 below, at 25°C. °C for 0-4 weeks, and then analyzed by IE-HPLC, SE-HPLC and RP-HPLC.
[212] In Tables 23, 24 and 25 below, IE-HPLC (%), SE-HPLC (%) and RP-HPLC (%) indicate the area%/initial area%, which indicates the percentage of conjugate retention of long-acting oxyntomodulin relative to baseline. Table 23 shows the IE-HPLC area (%) of the long-acting oxyntomodulin conjugate after storage, Table 24 shows the SE-HPLC area (%) of the long-acting oxyntomodulin conjugate after storage, and the Table 25 shows the RP-HPLC Area (%) of the long-lasting oxyntomodulin conjugate after storage. Table 22 [Table 22]



[213] As can be seen from the results in Tables 23 to 25, the SE-HPLC or RP-HPLC results indicate that the stability of the long-acting oxyntomodulin conjugate did not differ significantly between pH 5.6 and pH 5. 8. IE-HPLC results indicated that the long-acting oxyntomodulin conjugate was more stable at pH 5.6 than at pH 5.8. The SE-HPLC results show that the stability of the long-lasting oxyntomodulin conjugate did not differ significantly between buffers at the same pH. Furthermore, IE-HPLC or RP-HPLC results indicate that the long-acting oxyntomodulin conjugate was more stable in histidine at the same pH. Example 9: Evaluation of the influence of the presence or absence of preservative and the concentration of the long-acting oxyntomodulin conjugate on the stability of the long-acting oxyntomodulin conjugate
[214] The present inventors tested the influences of the presence or absence of a preservative such as a stabilizer and the concentration of the long-acting oxyntomodulin conjugate on the stability of the long-acting oxyntomodulin conjugate. Specifically, using histidine buffer (pH 5.6), 0.02% polysorbate 20, 0.1 mg/ml methionine and 5% mannitol, selected in the examples above, the long-lasting oxyntomodulin conjugate was stored in the presented compositions in Table 26 below, at 25°C for 0-4 weeks, and then analyzed by IE-HPLC, SE-HPLC and RP-HPLC. In Tables 27, 28 and 29 below, IE-HPLC (%), SE-HPLC (%) and RP-HPLC (%) indicate the area%/initial area%, which indicates the percentage of oxyntomodulin conjugate retention of long duration relative to the initial value. Table 27 shows the IE-HPLC area (%) of the long-acting oxyntomodulin conjugate after storage, Table 28 shows the SE-HPLC area (%) of the long-acting oxyntomodulin conjugate after storage, and the Table 29 shows the RP-HPLC area (%) of the long-lasting oxyntomodulin conjugate after storage. Table 26 [Table 26]



[215] As can be seen in Tables 26 to 2S SE-HPLC or RP-HPLC indicate that the long-acting stab oxyntomodulin did not change preservative and did not differ according to its c, IE-HPLC results, ility of the conjugate of u even in the presence of the concentration.
[216] While preferred embodiments of the present invention have been described for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the appended claims.

权利要求:
Claims (34)
[0001]
1. Liquid formulation of a long-acting oxyntomodulin derivative conjugate, characterized in that it comprises a pharmacologically active amount of a long-acting oxyntomodulin derivative conjugate, wherein the oxyntomodulin derivative conjugate comprises: an oxyntomodulin derivative , which is a physiologically active peptide, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 24, 25, 26, and 28; an immunoglobulin Fc region; and a non-peptide polymer, wherein the non-peptide polymer covalently links the oxyntomodulin derivative and the Fc region of the immunoglobulin; and an albumin-free stabilizer, where the stabilizer contains a buffer, a sugar alcohol and a non-ionic surfactant, where the buffer has a pH between 4.5 to 7.
[0002]
2. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to claim 1, characterized in that the stabilizer further contains one or more compounds selected from the group consisting of isotonic agents, sugars, polyhydric alcohols and amino acids.
[0003]
3. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to claim 1 or 2, characterized in that the oxyntomodulin derivative comprises the amino acid sequence of SEQ ID NO: 24.
[0004]
4. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to claim 1 or 2, characterized in that the oxyntomodulin derivative comprises the amino acid sequence of SEQ ID NO: 25.
[0005]
5. Liquid formulation of a long-acting oxyntomodulin derivative conjugate, according to claim 1 or 2, characterized in that the oxytomodulin derivative comprises the sequence of an amino acid of SEQ ID NO: 26.
[0006]
6. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to claim 1 or 2, characterized in that the oxyntomodulin derivative comprises the amino acid sequence of SEQ ID NO: 28.
[0007]
7. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to any one of claims 1 to 6, characterized in that the Fc region of the immunoglobulin is an Fc region derived from IgG, IgA, IgD, IgE or IgM.
[0008]
8. Liquid formulation of a long-acting oxyntomodulin derivative conjugate, according to claim 7, characterized in that each domain of the Fc region of the immunoglobulin is a hybrid of domains from different immunoglobulins selected from the group consisting of IgG , IgA, IgD, IgE and IgM.
[0009]
9. Liquid formulation of a long-acting oxyntomodulin derivative conjugate, according to claim 7, characterized in that the Fc region of the immunoglobulin is a dimer or multimer consisting of single-chain immunoglobulins composed of domains of the same origin .
[0010]
10. Liquid formulation of a long-acting oxyntomodulin derivative conjugate, according to claim 7, characterized in that the Fc region of immunoglobulin is an Fc region of IgG4.
[0011]
11. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to claim 10, characterized in that the immunoglobulin Fc region is a non-glycosylated human IgG4 Fc region.
[0012]
12. Liquid formulation of a long-lasting oxyntomodulin derivative conjugate according to any one of claims 1 to 11, characterized in that the non-peptide polymer is selected from the group consisting of polypropylene-glycol, ethylene-glycol copolymer /propylene glycol, polyoxyethylated polyol, poly(vinyl alcohol), polysaccharides, dextran, poly(ether-ethyl-vinyl), biodegradable polymers including poly-lactic acid (PLA) and glycolic-poly-lactic acid (PLGA), polymers of lipids, chitins, hyaluronic acid, and their combinations.
[0013]
13. Liquid formulation of a long-lasting oxyntomodulin derivative conjugate according to any one of claims 1 to 12, characterized in that the non-peptide polymer is polyethylene glycol.
[0014]
14. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to any one of claims 1 to 13, characterized in that the sugar alcohol is one or more alcohols selected from the group consisting of mannitol, sorbitol and glycerol.
[0015]
15. Liquid formulation of a long-acting oxyntomodulin derivative conjugate, according to claim 14, characterized in that the concentration of sugar alcohol in the liquid formulation is 2 to 15% (w/v).
[0016]
16. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to any one of claims 1 to 15, characterized in that the buffer is one or more buffers selected from the group consisting of citrate, acetate, histidine and phosphate buffer.
[0017]
17. Liquid formulation of a long-acting oxyntomodulin derivative conjugate, according to any one of claims 2 to 16, characterized in that the isotonic agent is sodium chloride.
[0018]
18. Liquid formulation of a long-lasting oxyntomodulin derivative conjugate, according to any one of claims 1 to 17, characterized in that the non-ionic surfactant is poloxamer or polysorbate.
[0019]
19. Liquid formulation of a long-acting oxyntomodulin derivative conjugate, according to claim 18, characterized in that the concentration of the nonionic surfactant in the liquid formulation is from 0.001 to 0.1% (w/v) .
[0020]
20. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to any one of claims 2 to 19, characterized in that the amino acid is methionine.
[0021]
21. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to any one of claims 1 to 20, characterized in that the stabilizer contains a buffer having a pH between 4.8 and 6.0, one or plus sugar alcohols selected from the group consisting of mannitol and sorbitol, and polysorbate 20.
[0022]
22. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to any one of claims 1 to 21, characterized in that it further comprises one or more preservative agents selected from the group consisting of m-cresol, phenol and benzilic alcohol.
[0023]
23. Liquid formulation of a long-acting oxyntomodulin derivative conjugate, according to claim 22, characterized in that the concentration of preservative in the liquid formulation is from 0.001 to 1% (w/v).
[0024]
24. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to claim 22, characterized in that the preservative is m-cresol.
[0025]
25. Liquid formulation of a long-acting oxyntomodulin derivative conjugate, according to any one of claims 1 to 24, characterized in that the formulation is for administration of multiple doses.
[0026]
26. Liquid formulation of a long-acting oxyntomodulin derivative conjugate, characterized in that it comprises: a pharmacologically effective amount of a long-acting oxyntomodulin derivative conjugate, wherein the oxyntomodulin derivative conjugate comprises: a derivative of oxyntomodulin which is a physiologically active peptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 24, 25, 26 and 28; an immunoglobulin Fc region; and a non-peptide polymer, wherein the non-peptide polymer covalently links the oxyntomodulin derivative and the Fc region of the immunoglobulin; and from 5 to 50 mM histidine; from 2 to 15% (w/v) of mannitol; from 0.01 to 1 mg/ml methionine; and from 0.001 to 0.1% (w/v) of polysorbate 20, where the buffer has a pH between 4.5 and 7.
[0027]
27. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to claim 26, characterized in that the oxyntomodulin derivative comprises the amino acid sequence of SEQ ID NO: 24.
[0028]
28. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to claim 26, characterized in that the oxyntomodulin derivative comprises the amino acid sequence of SEQ ID NO: 25.
[0029]
29. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to claim 26, characterized in that the oxyntomodulin derivative comprises the amino acid sequence of SEQ ID NO: 26.
[0030]
30. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to claim 26, characterized in that the oxyntomodulin derivative comprises the amino acid sequence of SEQ ID NO: 28.
[0031]
31. Liquid formulation of a long-acting oxyntomodulin derivative conjugate according to any one of claims 26 to 30, characterized in that it further comprises from 0.001 to 1% (w/v) of m-cresol.
[0032]
32. Method for preparing the liquid formulation of a long-acting oxyntomodulin derivative conjugate as defined in any one of claims 1 to 21 and 26 to 30, characterized in that it comprises the steps of: a) preparing a long-acting oxyntomodulin derivative conjugate; b) mixing the long-acting oxyntomodulin derivative conjugate, prepared in step a), with a stabilizer containing a buffer, a sugar alcohol and a nonionic surfactant, in which the buffer has a pH between 4.5 and 7.
[0033]
33. Method for the preparation of the liquid formulation as defined in any one of claims 22 to 25 and 31, characterized in that it comprises the steps of: a) preparing a long-lasting oxyntomodulin derivative conjugate; b) mixing the long-acting oxyntomodulin derivative conjugate, prepared in step a), with a stabilizer, which contains a buffer, a sugar alcohol and a nonionic surfactant, and a preservative, where the buffer has a pH between 4.5 and 7.
[0034]
34. Method according to claim 32 or 33, characterized in that the stabilizer also contains one or more compounds selected from the group consisting of isotonic agents, sugars, polyhydric alcohols and amino acids.

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EA201992760A2|2020-03-31|

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TW201740924A|2017-12-01|

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US20170360939A1|2017-12-21|

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AU2017279583A1|2018-01-18|

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CL2015001206A1|2015-07-03|

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AU2017279583B2|2019-11-21|

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-06| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-13| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-20| B06I| Publication of requirement cancelled [chapter 6.9 patent gazette]|Free format text: ANULADA A PUBLICACAO CODIGO 6.6.1 NA RPI NO 2462 DE 13/03/2018 POR TER SIDO INDEVIDA. |

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2019-02-12| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-10-15| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-12-08| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-03-23| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-05-18| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 06/11/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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KR10-2012-0124725|2012-11-06|

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KR20120124725|2012-11-06|

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PCT/KR2013/009986|WO2014073842A1|2012-11-06|2013-11-06|Liquid formulation of protein conjugate comprising the oxyntomodulin and an immunoglobulin fragment|

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