HK1192261B - Novel oxyntomodulin derivatives and pharmaceutical composition for treating obesity comprising the same - Google Patents

Description

Novel oxyntomodulin peptide derivative and pharmaceutical composition containing the same for treating obesity

Technical Field

The present invention relates to a novel peptide showing superior activity on glucagon-like peptide-1 receptor and glucagon receptor compared to natural oxyntomodulin, and a composition for preventing or treating obesity comprising the peptide as an active ingredient.

Background Art

Recently, economic growth and changes in lifestyle have led to changes in eating habits. The main reason for the increase in contemporary overweight and obesity rates is the consumption of high-calorie foods such as fast food and lack of exercise. The World Health Organization (WHO) estimates that more than 1 billion people are overweight in the world, and at least 300 million of them are clinically obese. Specifically, 250,000 people die in Europe and more than 2.5 million people die in the world every year due to overweight (World Health Organization, Global Strategy on Diet, Physical Activity and Health, 2004).

Overweight and obesity increase blood pressure and cholesterol levels, causing or worsening a variety of diseases such as cardiovascular disease, diabetes, and arthritis. They are also the main cause of increased incidence of arteriosclerosis, hypertension, hyperlipidemia, or cardiovascular disease in children, adolescents, and adults.

Obesity is a serious condition that causes many diseases in the world. It is believed that it is overcome by personal efforts, and it is also believed that obese patients lack self-control. However, obesity is difficult to treat because obesity is a complex disease involving appetite regulation and energy metabolism. With regard to obesity treatment, the abnormal manifestations related to appetite regulation and energy metabolism should be treated in combination with the efforts of obese patients. Multiple attempts have been made to develop drugs that can treat abnormal manifestations. Based on these efforts, drugs such as rimonabant (Rimonabant, Sanofi-Aventis), sibutramine (Sibutramin, Abbott), Contrave (Takeda) and orlistat (Orlistat, Roche) have been developed, but they have serious side effects or the defect of very weak anti-obesity effect. For example, it is reported that rimonabant (Sanofi-Aventis) shows the side effect of central nervous system disease, sibutramine (Abbott) and Contrave (Takeda) show cardiovascular side effects, and orlistat (Roche) shows that taking it for 1 year only reduces weight by 4kg. Unfortunately, there is no obesity treatment agent that can be safely dispensed for obese patients.

Many studies have been conducted to develop obesity therapeutic agents that do not have the problems of conventional anti-obesity drugs. Recently, glucagon derivatives have received a lot of attention. When the glucose level in the blood decreases due to other drugs or diseases, hormones or enzyme deficiencies, the pancreas produces glucagon. Glucagon stimulates the decomposition of glycogen in the liver and promotes glucose release so that blood sugar levels are raised to a normal range. In addition to the effect of increasing blood sugar levels, glucagon also suppresses appetite and activates hormone-sensitive lipase (HSL) of fat cells to promote lipolysis, thereby showing an anti-obesity effect. One of the glucagon derivatives, glucagon-like peptide-1 (GLP-1), is being developed as a hyperglycemic therapeutic agent for diabetic patients, and it plays a role to stimulate insulin synthesis and secretion, inhibit glucagon secretion, slow down gastric emptying, increase glucose utilization, and suppress food intake. Exendin-4, isolated from a lizard toxin, shares approximately 50% amino acid homology with GLP-1 and has also been reported to activate the GLP-1 receptor, thereby alleviating hyperglycemia in diabetic patients. However, anti-obesity drugs containing GLP-1 have been reported to exhibit side effects such as vomiting and nausea.

Therefore, as an alternative to GLP-1, considerable attention has focused on oxyntomodulin—a peptide derived from the glucagon precursor, proglucagon, that binds to receptors for both GLP-1 and glucagon. Oxyntomodulin is an effective anti-obesity treatment because, like GLP-1, it inhibits food intake and promotes satiety, and, like glucagon, has lipolytic activity.

Based on the dual function of oxyntomodulin, it has been actively studied as a medicine for treating obesity. For example, Korean Patent No. 925017 discloses a pharmaceutical composition comprising oxyntomodulin as an active ingredient for treating overweight people, which is administered by oral, parenteral, mucosal, rectal, subcutaneous or transdermal routes. However, it has been reported that the half-life in vivo of this anti-obesity drug comprising oxyntomodulin is short and the therapeutic efficacy is weak - even if administered three times a day at high doses. Therefore, multiple efforts have been made to improve the half-life in vivo of oxyntomodulin or the therapeutic effect on obesity by modifying it.

For example, the dual agonist oxyntomodulin (Merck) was prepared by replacing the L-serine at position 2 of oxyntomodulin with D-serine to increase resistance to dipeptidyl peptidase-IV (DPP-IV); and simultaneously attaching a cholesterol moiety at the C-terminus to increase blood half-life. ZP2929 (Zealand) was prepared by replacing the L-serine at position 2 with D-serine to enhance resistance to DPP-IV; replacing the arginine at position 17 with alanine to enhance resistance to proteases; replacing the methionine at position 27 with lysine to enhance oxidative stability; and replacing the glutamine at positions 20 and 24 with aspartic acid and alanine and replacing the asparagine at position 28 with serine to enhance deamidation stability. However, even if the half-life of the dual agonist oxyntomodulin (Merck) is increased to show a half-life 8 to 12 minutes longer than that of native oxyntomodulin, it still has a very short in vivo half-life of 1.7 hours, and its administration dose is still as high as several mg/kg. Unfortunately, due to its short half-life and low efficacy, oxyntomodulin or its derivatives have the disadvantage of being administered in high daily doses.

Summary of the Invention

Technical issues

Therefore, the inventors have developed Oxintomodulin derivatives prepared by modifying the amino acid sequence of natural Oxintomodulin to enhance its therapeutic effect on obesity and reduce its dosage. Consequently, they discovered that Oxintomodulin derivatives exhibit superior activity at the glucagon receptor and GLP-1 receptor compared to natural Oxintomodulin, thereby completing the present invention.

Problem Solution

An object of the present invention is to provide novel peptides that exhibit excellent therapeutic effects on obesity.

Another object of the present invention is to provide a composition for preventing or treating obesity comprising the peptide.

Another object of the present invention is to provide a method for preventing or treating obesity by administering the peptide or composition to a subject.

Another object of the present invention is to provide use of the peptide in preparing a medicament for preventing or treating obesity.

Beneficial effects of the invention

Unlike natural oxyntomodulating peptides, the novel peptides of the present invention reduce food intake, inhibit gastric emptying, and promote lipolysis without side effects and also exhibit excellent receptor activation. Therefore, they can be widely used to safely and effectively treat obesity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing changes in food intake according to the administered dose of Oxyntomodulin or an Oxyntomodulin derivative.

Best Mode for Carrying Out the Invention

In one aspect to achieve the above object, the present invention provides a novel peptide comprising an amino acid sequence of the following formula 1.

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 (Formula 1)

wherein R1 is histidine, desamino-histidyl, dimethyl-histidyl (N-dimethyl-histidyl), β-hydroxyimidazolylpropionyl, 4-imidazolylacetyl, β-carboxyimidazolylpropionyl or tyrosine;

X1 is Aib (aminoisobutyric acid), d -alanine, glycine, Sar (N -methylglycine), serine, or d -serine;

X2 is glutamate or glutamine;

X3 is leucine or tyrosine;

X4 is serine or alanine;

X5 is lysine or arginine;

X6 is glutamine or tyrosine;

X7 is leucine or methionine;

X8 is aspartic acid or glutamic acid;

X9 is glutamic acid, serine, α-methyl-glutamate, or deleted;

X10 is glutamine, glutamic acid, lysine, arginine, serine, or deleted;

X11 is alanine, arginine, valine, or deleted;

X12 is alanine, arginine, serine, valine, or deleted;

X13 is lysine, glutamine, arginine, α-methyl-glutamate, or deleted;

X14 is aspartic acid, glutamic acid, leucine, or deleted;

X15 is phenylalanine or deleted;

X16 is isoleucine, valine, or deleted;

X17 is alanine, cysteine, glutamic acid, lysine, glutamine, α-methyl-glutamate, or deleted;

X18 is tryptophan or is deleted;

X19 is alanine, isoleucine, leucine, serine, valine, or deleted;

X20 is alanine, lysine, methionine, glutamine, arginine, or deleted;

X21 is asparagine or deleted;

X22 is alanine, glycine, threonine, or deleted;

X23 is cysteine, lysine, or deleted;

X24 is a peptide having 2 to 10 amino acids consisting of a combination of alanine, glycine, and serine, or is deleted; and

R2 is KRNRNNIA (SEQ ID NO.32), GPSSGAPPPS (SEQ ID NO.33), GPSSGAPPPSK (SEQ ID NO.34), HSQGTFTSDYSKYLD (SEQ ID NO.35), HSQGTFTSDYSRYLDK (SEQ ID NO.36), HGEGTFTSDLSKQMEEEAVK (SEQ ID NO.37) or is deleted (if the amino acid sequence of Formula 1 is the same as SEQ ID NO.1, it is excluded).

As used herein, the term "peptide" means a compound of two or more α-amino acids linked by a peptide bond. In the context of the present invention, it refers to a peptide that activates the GLP-1 receptor and the glucagon receptor to exhibit an anti-obesity effect. The peptides according to the present invention include peptides, peptide derivatives, or peptide analogs that are prepared by adding, deleting, or replacing amino acids of oxyntomodulin, thereby activating the GLP-1 receptor and the glucagon receptor at higher levels than native oxyntomodulin.

The amino acids mentioned in this article are abbreviated according to the IUPAC-IUB naming rules, as follows:

Alanine A Arginine R

Asparagine N Aspartic acid D

Cysteine C Glutamate 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

In the present invention, peptides include any peptides that are prepared by substitution, addition, deletion, or post-translational modification (e.g., methylation, acylation, ubiquitination, intramolecular covalent bonding) of the amino acid sequence of oxyntomodulin (HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA, SEQ ID NO. 1) to simultaneously activate the glucagon and GLP-1 receptors. When substituting or adding amino acids, any of the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids, can be used. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep Inc., and Genzyme Pharmaceuticals. Peptides comprising these amino acids and atypical peptide sequences can be synthesized and purchased from suppliers such as American Peptide Company, Bachem (USA), or Anygen (Korea).

To enhance the activity of wild-type oxyntomodulatory peptide at the glucagon receptor and GLP-1 receptor, the peptide of the present invention may be substituted with the following: 4-imidazolylacetyl, wherein the α carbon of the histidine at position 1 of the amino acid sequence shown in SEQ ID NO. 1 is deleted; deamino-histidyl, wherein the N-terminal amino group is deleted; dimethyl-histidyl (N-dimethyl-histidyl), wherein the N-terminal amino group is modified with two methyl groups; β-hydroxyimidazolylpropionyl, wherein the N-terminal amino group is replaced by a hydroxyl group; or β-carboxyimidazolylpropionyl, wherein the N-terminal amino group is replaced by a carboxyl group. In addition, the GLP-1 receptor binding region may be substituted with amino acids that enhance hydrophobic bonds, ionic bonds, or a combination thereof. Part of the oxyntomodulatory peptide sequence may be substituted with the amino acid sequence of GLP-1 or exendin-4 to enhance activity at the GLP-1 receptor.

Furthermore, some secretory acid-regulating peptide sequences can be replaced by sequences that stabilize the α-helix. Preferably, the amino acids at positions 10, 14, 16, 20, 24, and 28 of the amino acid sequence of Formula 1 can be replaced by amino acids or amino acid derivatives known to stabilize the α-helix, including: Tyr(4-Me), Phe, Phe(4-Me), Phe(4-Cl), Phe(4-CN), Phe(4-NO ₂ ), Phe(4-NH ₂ ), Phg, Pal, Nal, Ala(2-thienyl), and Ala(benzothienyl), and there is no limit on the type and number of α-helix-stabilizing amino acids or amino acid derivatives to be inserted. Preferably, the amino acids at positions 10 and 14, 12 and 16, 16 and 20, 20 and 24, and 24 and 28 can also be replaced by glutamic acid or lysine, respectively, to form a loop, and there is no limit on the number of loops to be inserted. Most preferably, the peptide can be a peptide having an amino acid sequence selected from the following formulas 2 to 6.

In one embodiment, the peptide of the present invention is an oxyntomodulin derivative comprising an amino acid sequence of the following formula 2, wherein the amino acid sequence of oxyntomodulin is replaced by the amino acid sequence of exenatide or GLP-1.

R1-A-R3 (Formula 2)

In another embodiment, the peptide of the present invention is an oxyntomodulin derivative comprising an amino acid sequence of Formula 3, which is prepared by linking a partial amino acid sequence of oxyntomodulin with a partial amino acid sequence of exenatide or GLP-1 via an appropriate amino acid linker.

R1-B-C-R4 (Formula 3)

In another specific embodiment, the peptide of the present invention is an oxyntomodulin peptide derivative comprising an amino acid sequence of the following formula 4, wherein part of the amino acid sequence of the oxyntomodulin peptide is replaced by an amino acid capable of enhancing the binding affinity to the GLP-1 receptor, for example, Leu at position 26, which binds to the GLP-1 receptor through hydrophobic interactions, is replaced by a hydrophobic residue Ile or Val.

R1-SQGTFTSDYSKYLD-D1-D2-D3-D4-D5-LFVQW-D6-D7-N-D8-R3 (Formula 4)

In another embodiment, the peptide of the present invention is an oxyntomodulin peptide derivative comprising the following formula 5, wherein part of the amino acid sequence is deleted, added, or replaced by other amino acids to enhance the activity of the natural oxyntomodulin peptide on the GLP-1 receptor and the glucagon receptor.

R1-E1-QGTFTSDYSKYLD-E2-E3-RA-E4-E5-FV-E6-WLMNT-E7-R5 (Formula 5)

In Formulas 2 to 5, R1 is the same as described in Formula 1;

A is selected from SQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO.38), SQGTFTSDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO.39), SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQID NO.40), GQGTFTSDYSRYLEEEAVRLFIEWLKNG (SEQ ID NO.41), GQGTFTSDYSRQMEEEAVRLFIEWLKNG (SEQ ID NO.42), GEGFTTSDLSRQMEEEAVRLFIEWAA (SEQ ID NO.43), and SQGTFTSDYSRQMEEEAVRLFIEWLMNG (SEQ ID NO.44);

B is selected from SQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO.38), SQGTFTSDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO.39), SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQID NO.40), GQGTFTSDYSRYLEEEAVRLFIEWLKNG (SEQ ID NO.41), GQGTFTSDYSRQMEEEAVRLFIEWLKNG (SEQ ID NO.42), GEGFTTSDLSRQMEEEAVRLFIEWAA (SEQ ID NO.43), SQGTFTSDYSRQMEEEAVRLFIEWLMNG (SEQ ID NO.44), GEGFTTSDLSRQMEEEAVRLFIEW (SEQ ID NO.45), and SQGTFTSDYSRYLD (SEQ ID NO.45) ID NO.46);

C is a peptide with 2 to 10 amino acids composed of a combination of alanine, glycine, and serine;

D1 is serine, glutamate, or arginine;

D2 is arginine, glutamate, or serine;

D3 is arginine, alanine, or valine;

D4 is arginine, valine, or serine;

D5 is glutamine, arginine, or lysine;

D6 is isoleucine, valine, or serine;

D7 is methionine, arginine, or glutamine;

D8 is threonine, glycine, or alanine;

E1 is serine, Aib, Sar, d -alanine, or d -serine;

E2 is serine or glutamate;

E3 is arginine or lysine;

E4 is glutamine or lysine;

E5 is aspartic acid or glutamic acid;

E6 is glutamine, cysteine, or lysine;

E7 is cysteine, lysine, or deleted;

R3 is KRNRNNIA (SEQ ID NO. 32), GPSSGAPPPS (SEQ ID NO. 33), or GPSSGAPPPSK (SEQ ID NO. 34);

R4 is HSQGTFTSDYSKYLD (SEQ ID NO. 35), HSQGTFTSDYSRYLDK (SEQ ID NO. 36) or HGEGTFTSDLSKQMEEEAVK (SEQ ID NO. 37); and,

R5 is KRNRNNIA (SEQ ID NO. 32), GPSSGAPPPS (SEQ ID NO. 33), GPSSGAPPPSK (SEQ ID NO. 34), or deleted (excluded if the amino acid sequence of Formulas 2 to 5 is identical to SEQ ID NO. 1).

Preferably, the novel peptide of the present invention may be a peptide of the following formula 6.

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 (Formula 6)

wherein R1 is histidine, desamino-histidyl, 4-imidazolylacetyl or tyrosine;

X1 is Aib (aminoisobutyric acid), glycine, or serine;

X2 is glutamate or glutamine;

X3 is leucine or tyrosine;

X4 is serine or alanine;

X5 is lysine or arginine;

X6 is glutamine or tyrosine;

X7 is leucine or methionine;

X8 is aspartic acid or glutamic acid;

X9 is glutamate, α-methyl-glutamate, or deleted;

X10 is glutamine, glutamic acid, lysine, arginine, or deleted;

X11 is alanine, arginine, or deleted;

X12 is alanine, valine, or deleted;

X13 is lysine, glutamine, arginine, α-methyl-glutamate, or deleted;

X14 is aspartic acid, glutamic acid, leucine, or deleted;

X15 is phenylalanine or deleted;

X16 is isoleucine, valine, or deleted;

X17 is alanine, cysteine, glutamic acid, glutamine, α-methyl-glutamate, or deleted;

X18 is tryptophan or is deleted;

X19 is alanine, isoleucine, leucine, valine, or deleted;

X20 is alanine, lysine, methionine, arginine, or deleted;

X21 is asparagine or deleted;

X22 is threonine or deleted;

X23 is cysteine, lysine, or deleted;

X24 is a peptide having 2 to 10 amino acids consisting of glycine or is deleted; and

R2 is KRNRNNIA (SEQ ID NO.32), GPSSGAPPPS (SEQ ID NO.33), GPSSGAPPPSK (SEQ ID NO.34), HSQGTFTSDYSKYLD (SEQ ID NO.35), HSQGTFTSDYSRYLDK (SEQ ID NO.36), HGEGTFTSDLSKQMEEEAVK (SEQ ID NO.37) or is deleted (if the amino acid sequence of Formula 6 is the same as SEQ ID NO.1, it is excluded).

More preferably, the peptide of the present invention may be selected from the peptides of SEQ ID NOs. 1 to 31. Still more preferably, the peptide of the present invention may be an Oxyntomodulin peptide derivative described in Table 1 of Example 2-1.

Oxyntomodulin has the activity of two peptides, GLP-1 and glucagon. GLP-1 lowers blood sugar, reduces food intake, and inhibits gastric emptying, while glucagon increases blood sugar, promotes lipolysis, and reduces body weight by increasing energy metabolism. The different biological actions of the two peptides can lead to undesirable effects, such as increasing blood sugar if glucagon shows a stronger dominant effect than GLP-1, or causing nausea and vomiting if GLP-1 shows a stronger dominant effect than glucagon. Therefore, the oxyntomodulin peptide derivatives of the present invention are not only intended to increase these activities, for example, the amino acids at positions 1 and 11 of the oxyntomodulin peptide that inhibit glucagon activity can be modified to balance the activity ratio of glucagon and GLP-1.

The inventors conducted in vitro experiments to demonstrate that the peptide of the present invention exhibits superior activity against the GLP-1 receptor and the glucagon receptor compared to oxyntomodulin. This indicates that the peptide of the present invention activates the GLP-1 receptor and the glucagon receptor, thereby exhibiting a superior therapeutic effect on obesity compared to conventional oxyntomodulin. Furthermore, the peptide's inhibitory effect on food intake in vivo was investigated, and it exhibited a superior inhibitory effect on food intake compared to conventional oxyntomodulin ( FIG1 ).

It is apparent to those skilled in the art that when the Oxintomodulin peptide derivatives of the present invention are modified using conventional techniques, including modifications such as PEG and sugar chains or fusions with albumin, transferrin, fatty acids, and immunoglobulins to enhance the therapeutic effects of the Oxintomodulin peptide derivatives, they exhibit therapeutic effects superior to those of native Oxintomodulin. Therefore, modified Oxintomodulin peptide derivatives are also encompassed within the scope of the present invention.

In another aspect, the present invention provides a polynucleotide encoding the peptide.

The term "homology", as used herein with respect to polynucleotides, refers to sequence similarity between wild-type amino acid sequences or wild-type nucleotide sequences, and includes gene sequences that have 75% or higher, preferably 85% or higher, more preferably 90% or higher, and even more preferably 95% or higher identity with the polynucleotide sequence encoding the peptide. Homology evaluation can be performed by the naked eye or using commercially available programs. Using commercially available computer programs, the homology between two or more sequences can be expressed as a percentage (%), and the homology (%) between adjacent sequences can be evaluated. The polynucleotide encoding the peptide is inserted into a vector and expressed, thereby obtaining a large amount of peptide.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating obesity comprising the peptide.

As used herein, the term "prevention" means all actions that limit or hinder the occurrence of obesity by administering a peptide or composition, and the term "treatment" means all actions that ameliorate or beneficially change the symptoms of obesity by administering a peptide or composition.

As used herein, the term "administer" means to introduce a predetermined amount of a substance into a patient by some appropriate method. The compositions of the present invention can be administered by any common route, as long as they can reach the intended tissue, such as, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary or rectal administration. However, since peptides are digested after oral administration, the active ingredient of the composition for oral administration should be coated or formulated to prevent degradation in the stomach.

As used herein, the term "obesity" means the accumulation of excess fat tissue in the body, and a body mass index (weight in kg divided by height squared in m) of 25 or above is considered obese. Obesity is generally caused by an energy imbalance—when dietary intake exceeds energy expenditure for a long time. Obesity is a metabolic disease that affects the entire body and increases the risk of diabetes, hyperlipidemia, sexual dysfunction, arthritis, and cardiovascular disease, and in some cases is associated with the development of cancer.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent. As used herein, the term "pharmaceutically acceptable" means that the composition is sufficient to achieve a therapeutic effect without harmful side effects, and can be easily determined based on the following: disease type, patient age, weight, health status, sex and drug sensitivity, administration route, administration mode, administration frequency, duration of treatment, drugs combined with or taken simultaneously with the composition of the present invention, and other factors known to the drug.

The pharmaceutical composition comprising the derivative of the present invention may further comprise a pharmaceutically acceptable carrier. For oral administration, the carrier may include, but is not limited to, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant, and a flavoring agent. For injectable formulations, the carrier may include a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, and a stabilizer. For topical formulations, the carrier may include a matrix, an excipient, a lubricant, and a preservative.

The compositions of the present invention can be formulated into a variety of dosage forms in combination with the above-mentioned pharmaceutically acceptable carriers. For example, for oral administration, the pharmaceutical composition can be formulated into tablets, lozenges, capsules, elixirs, suspensions, syrups, or wafer-thin capsules. For injectable formulations, the pharmaceutical composition can be formulated into ampoules as single-dose forms or into multi-dose containers. The pharmaceutical composition can also be formulated into solutions, suspensions, tablets, pills, capsules, and long-acting preparations.

On the other hand, the example that is suitable for the carrier, excipient and diluent of pharmaceutical preparation comprises lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.In addition, pharmaceutical preparation can further comprise filler, anticoagulant, lubricant, wetting agent, flavoring agent and antibacterial agent.

Further, the pharmaceutical composition of the present invention may have any formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquids for internal use, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

Furthermore, the composition can be formulated into a single dosage form suitable for the patient's body, and is preferably formulated into a preparation that can be used for peptide drugs according to general methods in the pharmaceutical field, so as to be administered orally or parenterally, such as through the skin, intravenous, intramuscular, intraarterial, intramedullary, intramedullary, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intracolonic, topical, sublingual, vaginal or rectal administration, but not limited thereto.

The peptide can be applied by mixing with various pharmaceutically acceptable carriers such as physiological saline or organic solvents. To increase stability or absorbability, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers can be used.

The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined by the type of active ingredient together with various factors such as the disease to be treated, administration route, age, sex and weight of the patient and severity of the disease.

According to the graded treatment regimen, the effective total dose of the composition of the present invention can be administered to the patient in a single dose, or can be administered in multiple doses for a long time. In the pharmaceutical composition of the present invention, the content of the active ingredient can vary according to the severity of the disease. Preferably, the total daily dose of the peptide of the present invention can be about 0.0001 μg to 500 mg per 1 kg of patient body weight. However, in addition to the administration route and treatment frequency of the pharmaceutical composition, the effective dose of the peptide is also determined taking into account a variety of factors: including patient age, weight, health status, sex, severity of disease, diet and secretion rate. In view of this, those skilled in the art can easily determine the effective dose suitable for the specific application of the pharmaceutical composition of the present invention. The pharmaceutical composition according to the present invention is not specifically limited to the preparation and administration route and mode, as long as it shows the effect of the present invention.

The pharmaceutical composition of the present invention exhibits excellent duration of efficacy and titer in vivo, thereby significantly reducing the amount and frequency of administration thereof.

In addition, the pharmaceutical composition can be administered alone or in combination with other pharmaceutical preparations that show a preventive or therapeutic effect on obesity, or simultaneously. Pharmaceutical preparations that show a preventive or therapeutic effect on obesity are not particularly limited and may include GLP-1 receptor agonists, leptin receptor agonists, DPP-IV inhibitors, Y5 receptor antagonists, melanin concentrating hormone (MCH) receptor antagonists, Y2/3 receptor agonists, MC3/4 receptor agonists, gastric/pancreatic lipase inhibitors, 5HT2c agonists, β3A receptor agonists, pullulan receptor agonists, ghrelin antagonists and/or ghrelin receptor antagonists.

In still another aspect, the present invention provides a method for preventing or treating obesity, comprising the step of administering the peptide or a pharmaceutical composition comprising the peptide to a subject.

In the present invention, the term "subject" refers to a subject suspected of obesity, and refers to mammals that are obese or potentially obese, including humans, mice, and livestock. However, any subject treated with the peptides or pharmaceutical compositions of the present invention is included without limitation. A pharmaceutical composition comprising the peptides of the present invention is administered to a subject suspected of obesity, thereby effectively treating the subject. Obesity is as described above.

The treatment method of the present invention may include the step of administering a composition comprising the peptide in a pharmaceutically effective amount. The total daily dose should be determined by the appropriate medical judgment of a physician and administered once or several times. In terms of the objectives of the present invention, the specific therapeutically effective dose level for any particular patient may vary depending on various factors well known in the medical field, including the type and degree of response to be achieved, the specific composition - depending on whether other agents are used together with it, the patient's age, weight, health status, sex and diet, the number and route of administration, the secretion rate of the composition, the treatment period, other drugs used in combination or concurrently with the composition of the present invention, and similar factors well known in the art.

In another aspect, the present invention provides use of the peptide or a pharmaceutical composition comprising the peptide in preparing a medicament for preventing or treating obesity.

Embodiments of the invention

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only, and the present invention is not intended to be limited by these examples.

Example 1. Preparation of in vitro activated cell lines

Example 1-1: Preparation of a cell line showing cAMP response to GLP-1

PCR products were obtained by performing PCR using the ORF (open reading frame) region of cDNA corresponding to the human GLP-1 receptor gene (OriGene Technologies, Inc. USA) as a template and the following forward and reverse primers including HindIII and EcoRI restriction sites.

Forward primer: 5'-CCCGGCCCCCGCGGCCGCTATTCGAAATAC-3' (SEQ ID NO. 47)

Reverse primer: 5'-GAACGGTCCGGAGGACGTCGACTCTTAAGATAG-3' (SEQ ID NO. 48)

The PCR product was cloned into a known animal cell expression vector xOGC/dhfr to prepare a recombinant vector xOGC/GLP1R.

CHO DG44 cells cultured in DMEM/F12 (10% FBS) were transfected with the recombinant vector xOGC/GLP1R using Lipofectamine (Invitrogen, USA) and cultured in a selection medium containing 1 mg/mL G418 and 10 nM methotrexate. Monoclonal cell lines were selected from these cells using the limiting dilution technique, and ultimately, cell lines that exhibited a superior cAMP response to GLP-1 in a concentration-dependent manner were selected.

Example 1-2: Preparation of a cell line showing cAMP response to glucagon

PCR products were obtained by performing PCR using a region corresponding to the ORF of cDNA of the human glucagon receptor gene (OriGene Technologies, Inc. USA) as a template and the following forward and reverse primers including EcoRI and XhoI restriction sites, respectively.

Forward primer: 5'-CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC-3' (SEQ ID NO. 49)

Reverse primer: 5'-CTAACCGACTCTCGGGGAAGACTGAGCTCGCC-3' (SEQ ID NO. 50)

The PCR product was cloned into a known animal cell expression vector xOGC/dhfr to prepare a recombinant vector xOGC/GCGR.

The CHO DG44 cell line cultured in DMEM/F12 (10% FBS) medium was transfected with the recombinant vector xOGC/GCGR using Lipofectamine and cultured in a selection medium containing 1 mg/mL G418 and 10 nM methotrexate. Monoclonal cell lines were selected from these cells using the limiting dilution technique, and ultimately, a cell line that exhibited an excellent cAMP response to glucagon in a concentration-dependent manner was selected.

Example 2. Testing of in vitro activity of oxyntomodulin peptide derivatives

Example 2-1: Synthesis of Oxyntomodulin Derivatives

To measure the in vitro activities of the oxyntomodulin peptide derivatives, oxyntomodulin peptide derivatives having the following amino acid sequences were synthesized (Table 1).

【Table 1】

Oxyntomodulin and Oxyntomodulin derivatives

In Table 1, the bold and underlined amino acids in each of SEQ ID NOs.: 19, 20, 22, 25, 26, 27, 29, 30, and 31 form a ring together, and the amino acid represented by X means an unnatural amino acid, α-methyl-glutamic acid. In addition, CA represents 4-imidazolylacetyl, and DA represents desamino-histidyl.

Example 2-2: Testing of the in vitro activity of oxyntomodulating peptide derivatives

In order to measure the anti-obesity efficacy of the Oxyntomodulin derivative synthesized in Example 2-1, cell activity was measured in vitro using the cell lines prepared in Examples 1-1 and 1-2.

This cell line is a cell line produced by transfecting CHO (Chinese Hamster Ovary) cells to express the human GLP-1 receptor gene and the glucagon receptor gene, respectively. Therefore, it is suitable for measuring GLP-1 and glucagon activities. Therefore, the activity of each oxyntomodulin peptide derivative was measured using each transformed cell line.

Specifically, each cell line was subcultured two or three times a week and aliquoted at a density of 1×10 ⁵ in each well of a 96-well plate, followed by culture for 24 hours.

The cells of culture were washed with KRB buffer and suspended in 40ml KRB buffer comprising 1mM IBMX and left at room temperature for 5 minutes. Oxyntomodulin (SEQ ID NO.1) and Oxyntomodulin derivatives (represented by SEQ ID NOs.2-6, 8, 10-13, 17, 18, 23-25, 27-30 and 31) were diluted from 1000nM to 0.02nM by 5 times of serial dilution, and each 40mL was added to the cells and incubated at 37°C in a CO2 incubator for 1 hour. Then, 20mL of cell lysis buffer was added to carry out cell lysis, and the cell lysate was used for a cAMP assay kit (Molecular Device, USA) to measure cAMP concentration. _EC50 values were calculated thus and compared with each other. _EC50 values are shown in Table 2 below.

【Table 2】

Comparison of in vitro activities of oxyntomodulin and its derivatives at GLP-1 and glucagon receptors

As shown in Table 2, compared with the natural oxyntomodulin peptide of SEQ ID NO. 1, the oxyntomodulin derivatives showed excellent in vitro activities and different activity ratios at the GLP-1 receptor and the glucagon receptor.

Oxyntomodulin is known to activate GLP-1 and glucagon receptors to suppress appetite, promote lipolysis, and promote satiety, thereby exhibiting anti-obesity effects. The oxyntomodulin derivatives according to the present invention exhibit higher in vitro activity at both the GLP-1 and glucagon receptors than wild-type oxyntomodulin, and thus can be used as obesity therapeutic agents with higher potency than known oxyntomodulin.

Example 3. In vivo activity test of oxyntomodulin peptide derivatives

To measure the in vivo therapeutic activity of the Oxyntomodulin derivatives, changes in food intake were examined in ob/ob mice administered with the Oxyntomodulin derivatives, using native Oxyntomodulin as a control.

Specifically, obese diabetic ob/ob mice, commonly used to test the efficacy of therapeutic agents for obesity and diabetes, were fasted for 16 hours and administered 1 or 10 mg/kg of oxyntomodulin or 0.02, 0.1, 1, or 10 mg/kg of an oxyntomodulin derivative of SEQ ID NO. 2. Food intake was then observed for 2 hours ( FIG. 1 ). FIG. 1 is a graph showing changes in food intake according to the dose of oxyntomodulin or an oxyntomodulin derivative administered. As shown in FIG. 1 , administration of 1 mg/kg of an oxyntomodulin derivative exhibited a superior inhibitory effect on food intake compared to administration of 10 mg/kg of oxyntomodulin.

In conclusion, the Oxyntomodulin derivatives of the present invention have much higher anti-obesity effects than wild-type Oxyntomodulin even when administered at lower doses, suggesting that the problem that wild-type Oxyntomodulin shows lower anti-obesity effects and should be administered at high doses three times a day is improved.

Claims (8)

1. A peptide comprising an amino acid sequence selected from SEQ ID NO. 23 to 31, wherein the peptide is not modified with a non-peptide polymer, or the peptide is modified with a non-peptide polymer selected from polyethylene glycol, sugars, albumin, transferrin, fatty acids, and immunoglobulins. 2. The peptide according to claim 1, wherein the peptide has an anti-obesity effect. 3. A polynucleotide encoding the peptide of claim 1. 4. A pharmaceutical composition for the prevention or treatment of obesity, comprising the peptide of claim 1 or 2 as an active ingredient. 5. The pharmaceutical composition according to claim 4, further comprising a pharmaceutically acceptable carrier. 6. The pharmaceutical composition according to claim 4, wherein the composition is administered alone or in combination with or simultaneously with other pharmaceutical formulations that have shown preventive or therapeutic effects on obesity. 7. The pharmaceutical composition of claim 6, wherein the pharmaceutical formulation exhibiting a preventive or therapeutic effect on obesity is selected from GLP-1 receptor agonists, leptin receptor agonists, DPP-IV inhibitors, Y5 receptor antagonists, melanin-concentrated hormone (MCH) receptor antagonists, Y2/3 receptor agonists, MC3/4 receptor agonists, gastric/pancreatic lipase inhibitors, 5HT2c agonists, β3A receptor agonists, amylopectin receptor agonists, auxin-releasing peptide antagonists, and auxin-releasing peptide receptor antagonists. 8. Use of the peptide of claim 1 or 2 or the composition of claim 4 in the preparation of a medicament for the prevention or treatment of obesity.