WO2008056726A1

WO2008056726A1 - Glp-1 derivative and use thereof

Info

Publication number: WO2008056726A1
Application number: PCT/JP2007/071687
Authority: WO
Inventors: Takahito Jomori; Yuji Hayashi; Mitsuhiro Makino
Original assignee: Sanwa Kagaku Kenkyusho Co., Ltd.
Priority date: 2006-11-09
Filing date: 2007-11-08
Publication date: 2008-05-15
Also published as: JP2010043001A

Abstract

[PROBLEMS] To provide a novel GLP-1 derivative having a largely improved capability of being absorbed through a mucous membrane. [MEANS FOR SOLVING PROBLEMS] Disclosed is a peptide which has (Lys)n-Arg [wherein n represents an integer of 4 to 8, and Arg is in the form of a carboxylic acid] added to the C-terminus of a peptide comprising an amino acid sequence corresponding to GLP-1 (7-35) or GLP-1 (7-36) or an amino acid sequence having the deletion, substitution and/or addition of one or several amino acid residues in the aforementioned amino acid sequence, having at least 85% homology to the aforementioned amino acid sequence and having a GLP-1 activity. The amino acid residue at position-8 in the amino acid sequence for GLP-1 is preferably serine or glycine, and the integer “n” is preferably 5.

Description

Specification

GLP-1 derivatives and their uses

Technical field

[0001] The present invention relates to a human glucagon-like peptide-1 (GL P-1; Glucagon like peptide-1) known as an incretin hormone. Specifically, the present invention has a high absorbability from mucous membranes. It is an invention of a novel derivative of GLP-1.

Background art

[0002] GLP-1 is known as an incretin hormone that is secreted from the gastrointestinal tract by food intake and works on the viscera to stimulate insulin secretion. GIP (Gas trie lnhioitory polypeptide or 7 lucose-depenaent insulinotropic polypeptide) has the same effect: Does the type 2 diabetes patient lack this incretin effect compared to healthy individuals? It is also suggested that the disorder is one of the causes of hyperglycemia, for example, blood GLP-1 levels are decreased in patients with type 2 diabetes and GIP is different from that of healthy individuals. Incretin hormone administration test for type 2 diabetic patients showed that GLP-1 administration did not show any difference in insulin secretion promoting response compared to healthy subjects, but GIP administration As a result, it has been reported that the response to GLP-1 is maintained in diabetic patients. Application Expected to be have! /, Ru.

[0003] The insulin secretion action of GLP-1 is characterized by glucose level dependency that insulin secretion is not stimulated when blood glucose level is 110 mg / dl or less, and insulin is secreted only when blood glucose level is higher than that. Is to represent. That is, administration of GLP-1 promotes insulin secretion according to blood glucose level, and insulin secretion does not occur when blood glucose level is below normal. Therefore, when GLP-1 is used, there are no significant concerns about hypoglycemia, and the great clinical merit is not to exhaust the viscera due to excessive insulin secretion. On the other hand, sulfonylurea, which has been used mainly for the treatment of type 2 diabetes, continuously closes ATP-sensitive K + channels and promotes insulin secretion. In other words, since it works on the presumptive insulin-secreting cells regardless of the blood sugar level, hypoglycemia, exhaustion of premature vigor due to excessive stimulation of β cells, Secondary ineffectiveness has been reported with long-term administration. Therefore, the pharmacological properties of GLP-1 are useful, different from conventional diabetes drugs.

[0004] However, the active body of GLP-1 is a polypeptide of GLP-1 (7-36) amide or GLP_1 (7_37). When GLP-1 is taken orally, it is digested and degraded by digestive enzymes in the digestive tract. Is not absorbed. Therefore, in clinical practice, intravenous injection or subcutaneous injection by infusion is being attempted. GLP-1 is also degraded by dipeptidyl peptidase IV (DPPIV) present in blood and tissues, and the in vivo half-life is known to be very short; 2 to 2 minutes. These are the bottlenecks for clinical application. Dipeptidyl peptidase IV, known as an enzyme that degrades GLP-1, is widely distributed in the kidney, liver, small intestine, salivary gland, various connective tissues, and other body fluids such as blood, urine, saliva, and nasal passages. It is becoming clear that it also exists in the mucosa.

[0005] In order to solve this problem, some research and development have been conducted. For example, there are reports of 8-position amino acid-substituted derivatives that are difficult to degrade and have a long half-life (Diabetologia 41: 271-278 (1998), Biochem 40: 2860-2869 (2001). Type GLP-1 injection (development name Liraglutide), or GLP-1 derivative with long-lived half-life that binds albumin in vivo (development number CJC-1131; Diabetes 52 (3) : 751_759 (2003)) In addition, the development of a synthetic exendin-4 injection (product name) derived from a lizard with GLP-ligand activity and long half-life in blood However, these are all injections, and given the burden of patients who have to be injected every day, GLP-1 is widely used as a treatment for diabetes. For administration, administration routes other than injection are desirable.

[0006] As a non-invasive administration method that replaces injections and oral administration agents, administration by mucosal absorption such as in the lung, oral cavity, nasal cavity, vagina, eyes, and rectum is conceivable. However, since peptides such as GLP-1 are generally macromolecules, the absorption rate from the mucosa alone is low. For this reason, in general, a polymer such as a peptide is formulated together with an excipient such as an absorption enhancer. For example, a buccal tablet containing GLP-1 has been studied, and nasal formulation of GLP-1 by formulation technology using a polyvalent metal compound carrier is under development S, satisfactory bioavailability (biological (Utilization rate) is obtained! / ,! [0007] Thus, in WO2004 / 037859, the present inventors increase GLP-1 mucosal absorption including nasal mucosa by directly adding several arginine or lysine to the C-terminus of GLP-1. I found out. As a GLP-1 derivative similar to the GLP-1 derivative of WO2004 / 037859, WO2001 / 004156 discloses [Gly ⁸ ] -GLP-1 (7_36)-(Lys) -NH.

6 2

Yes. Here, there is no description of force mucosal absorption, which discloses that oral administration is possible by imparting stability and hydrophilicity to the GLP-1 derivative.

[0008] Patent Literature l: WO2004 / 037859

Patent Document 2: WO2001 / 004156

Non-patent literature l: Diabetologia 41: 271-278 (1998)

Non-Patent Document 2: Biochem 40: 2860-2869 (2001)

Non-Patent Document 3: Diabetes 52 (3): 751_759 (2003)

Disclosure of the invention

Problems to be solved by the invention

[0009] Absorption of GLP-1 from the mucosa is much less efficient than injection due to low membrane permeability and degradation at the site of absorption. For example, it is possible to administer GLP-1 intranasally, and the absorption yield is low, so a very high dose is required to obtain a sufficient pharmacological effect. Therefore, it is unrealistic to develop a pharmaceutical product using natural GLP-1 as a nasal agent in terms of the cost of producing the active ingredient of the peptide. In order to apply GLP-1 clinically, it is necessary to develop a GLP-1 derivative whose absorption rate from mucous membranes is comparable to that of injections. That is, an object of the present invention is to devise a novel GLP-1 derivative with improved absorption of mucosal force and to provide a mucosal administration agent in place of injection.

Means for solving the problem

[0010] In WO2004 / 037859, the present inventors increased mucosal absorption of GLP-1 derivatives by directly adding several arginine (A rg) or lysine (Lys) to the C-terminus of GLP-1. I found out. As a result of further investigations on the improvement of mucosal absorbability, the sequence added to the C-terminal side is a structure in which lysine is continuous. The mucosal absorbability of [0011] That is, in the present invention, GLP-7-35) or GLP-7-36) amino acid sequence IJ, or one or several amino acids in the amino acid sequence thereof is deleted, substituted and / or added. (Lys) n-Arg (n is an integer of 4 to 8, Arg is a carboxylic acid form) is added to the C-terminus of a peptide having a GLP-1 activity. In other words, the present invention comprises GLP-7-35) or GLP-7-36) amino acid sequences, or a sequence having 85% or more homology with those amino acid sequences, and A peptide in which (Lys) n-Arg (n is an integer of 4 to 8, Arg is a carboxylic acid form) is added to the C-terminus of the peptide having GLP-1 activity.

In the GLP-1 derivative of the present invention, in order to add resistance to dipeptidyl peptidase IV, alanine (Ala) at position 8 of natural GLP-1 is converted to serine (Ser) or glycine (G1 y ) Is preferred. Similarly, trypsin resistance can be achieved by substituting lysine at position 26 with glutamine (Gin) and lysine at position 34 with asparagine (Asn) or aspartate (Asp). Further, the number n of lysine residues added to the C-terminal is preferably 4 to 6, and most preferably 5. The most preferred GLP-1 derivative of the present invention is represented by [Ser ⁸ ] -GLP-l (7-35)-(Lys) -Arg (Arg is a carboxylic acid form).

Five

[0013] The present invention also relates to a pharmaceutical composition comprising the GLP-1 derivative of the present invention as an active ingredient. The pharmaceutical composition of the present invention is suitable for transmucosal administration, particularly nasal administration. In addition, the pharmaceutical composition of the present invention can be used for the treatment of diabetes, the treatment of obesity, the suppression of appetite, or the suppression of heart disease. In other words, the peptide of the present invention can be used for producing a therapeutic agent for diabetes, obesity or eating disorders.

The invention's effect

[0014] The GLP-1 derivative of the present invention has a continuous structure of lysine and / or arginine on the C-terminal side described in WO2004 / 037859, which is not limited to natural GLP-1. It has a much higher mucosal absorption rate than some GLP-1 derivatives. Therefore, the GLP-1 derivative of the present invention greatly enhances the possibility of clinical application of a mucosal absorption type GLP-1 preparation that is easy to administer and is not painful in place of conventional injections. It is considered to be very useful for improving the quality of life of patients, obese patients, and patients with heart disease.

[0015] Further, in the GLP-1 derivative of the present invention, since C-terminal arginine is a carboxylic acid form, When manufacturing, it is not necessary to carry out the amidation process like other GLP-1 derivatives, especially for industrial production!

[0016] It should be noted that the GLP-1 derivative of the present invention has an added lysine moiety that is readily cleaved in blood immediately after being decomposed by a protease and is present in a form close to that of natural GLP-1. The sex is also considered extremely low.

BEST MODE FOR CARRYING OUT THE INVENTION

[0017] Hereinafter, the present invention will be described in more detail. GLP-1 (7-36) is His-Ala-Glu-Gly-Thr-Phe_Thr_Ser_Asp_Val_Ser_Ser_Tyr_Leu_Glu_Gly_Gln_Ala_Ala_Lys_Glu_Phe_Ile_Ala-Trp-Leu-Va 卜 Lys-Gly-Aly Lys-Gly-Aly [Ser ⁸ ] indicates that the second, that is, 8-position alanine in the sequence is converted to serine, and is synonymous with 8S. In the GLP-1 derivative, it is possible to take the form of either an amide form in which the C-terminus of the amino acid is amidated, an amidated form! /, Na! /, Or a carboxylic acid form! / is there. The power of natural GLP-1 is an amide. The GLP-1 derivative of the present invention is a carboxylic acid. In the present specification, -NH represents an amide form, and -COOH represents a carboxylic acid form. In addition, the present invention is the first example showing that the effect of transmucosal administration is higher when the carboxylate of the amino acid of the GLP-1 derivative is carboxylic acid than when it is amide.

Yes

[0018] As described above, the GLP-1 derivative of the present invention lacks GLP-K7-35) or GLP-K7-36) amino acid sequence, or one or several amino acids thereof. (Lys) n — Arg (n is an integer from 4 to 8, Arg is a carboxylic acid form) is added to the C-terminus of a peptide having a lost, substituted and / or added sequence and having GLP-1 activity. Peptide. Here, GLP-K7-35) or GLP-K7-36) and a peptide having a GLP-1 activity consisting of a sequence in which one or several amino acids are deleted, substituted and / or added. Means that GLP-1 mutants are not lost even if some amino acids of the native GLP-1 peptide are deleted, substituted and / or added. is there. Such a GLP-1 variant can be expressed in another way! /, From a sequence having 85% or more homology with the amino acid sequence of GLP-K7-35) or GLP-K7-36). And can be expressed as a peptide having GLP-1 activity. Many of these natural GLP-1 peptide variants are It is technical common sense that there are numbers. Specifically, alanine at position 8 may be substituted with serine or glycine, which adds resistance to dipeptidyl peptidase IV. According to WO2004 / 037859 or WO2004 / 087910, lysine at position 26 may be substituted with glutamine, and lysine at position 34 may be substituted with asparagine or aspartic acid. Added. In addition, parin at position 16, serine at position 17, serine at position 18, glycine at position 22, glutamic acid at position 27, lysine at position 34, arginine at position 36 can each be substituted with alanine, and at the same time at least two can be substituted with alanine. It has been.

[0019] All of the GLP-1 mutants can be exemplified as follows based on the sequence of GLP-K7-35) or GLP-K7-36). GLP-K7-35) or GLP-K7-36) substitute for serine or glycine at position 8, substitute for glutamine at position 26, substitute for asparagine or aspartate at position 34, and these 8 A combination of two or more of the substitutions at positions 26, 34, and further, a substitute for alanine at position 16, a substitute for alanine at position 17, a substitute for alanine at position 18 , Substitution to alanine at position 22, substitution to alanine at position 27, substitution to alanine at position 34, substitution to alanine at position 36, and substitution from alanine at positions 16 to 36 A combination of two replacements.

[0020] The GLP-1 derivative of the present invention can be produced in the same manner as in WO2004 / 037859 by chemical synthesis or gene recombination techniques. The principles of chemical synthesis of polypeptides are well known in the art, and general texts in this area can be consulted as follows: Dugas Η · and Penney C, Bioorganic (chemistry (1981) springer- Verlag, New York, 54-92 shells, f row ;, m odd 430A heptad synthesizer (Applied Biosystems Inc, 850 Lincoln Center Drive, Foster City CA 94404) and a solid phase using a synthesis cycle supplied by PE-Applied Biosystems. The peptides of the present invention can be synthesized by methods Boc amino acids and other reagents can be purchased from Applied Biosystems and other drug suppliers.

[0021] A method for producing the peptide of the present invention by a gene recombination technique is as follows. GLP-1 DNA can be obtained by total synthesis or by modification of DNA encoded by a larger natural glucagon. The DNA sequence encoding the prebloglucagon is shown in Lund et al. [Proc Natl Acad Sci USA 79: 345-349 (1982)]. And can be used for the production of the compounds of the present invention. Methods for constructing synthetic genes are well known in the art, see Brown et al., Methods in Enzymology, Academic Press, NY Volume 68, pages 109-151. A DNA sequence encoding the peptide of the present invention is designed based on the amino acid sequence, and Model 3400 DNA synthesizer or ABI3900 high throughput nucleus' acid synthesizer (Applied Biosystems Inc, 850 Lincoln Center Drive, Foster City CA 94 404) A DNA having the sequence itself can be produced using a normal DNA synthesizer such as.

[0022] In addition, the DNA used for the production of the GLP-1 derivative of the present invention has a device that increases the expression level and stably accumulates the product in the host, a device that facilitates purification after production, or a fusion tank. It can be produced as ^ and can be devised to easily cut out the GLP-1 derivative. For example, the method of ligating the GLP-1 derivative gene of the present invention in tandem to increase the expression level, or purification of β-galatatosidase, β-lactamase, protein A, TrpE, ubiquitin, continuous histidine residues, etc. It is possible to adopt a technique in which an amino acid sequence that is advantageous for excision of a GLP-1 derivative by treatment with a specific protease is linked to an advantageous protein gene and produced as a fusion protein. In these cases, for example, in order to obtain a GLP-1 derivative as a simple substance after production, the target GLP-1 derivative can be obtained by performing purification using a fused protein and then cleaving with a specific protease. .

[0023] Next, the GLP-1 derivative gene prepared as described above is inserted into an appropriate recombinant DNA expression vector using an appropriate restriction endonuclease according to a common sense technique in this field. . For the effective expression method of GLP-1 derivative peptides at that time and various expression vectors that can be used for transformation in prokaryotic cells and eukaryotic cells, general methods in this region can be referred to. (Maniatis et al. (1989) Molecular Cloning; A Laboratory Manual, Cold Springs Harbor Laboratory Press, NY Volumes 1-3 and The Promega Biological Research Products Catalog and The Stratagene Cloning Systems Catalog).

[0024] Furthermore, an appropriate host cell is transformed with the constructed GLP-1 derivative peptide expression vector. Host cells can be either eukaryotic cells or prokaryotic cells! /. Techniques for transforming cells are well known in the art and can be found in general citations such as Maniatis et al. Prokaryotic host cells are generally more Produces protein at a high rate and is easier to culture. Proteins expressed in high level bacterial expression systems characteristically aggregate into particles or inclusion bodies that contain high levels of overexpressed protein. Such typically aggregated proteins are solubilized, denatured and refolded using techniques well known in the art. See Protein Folding, Kreuger et al. (1990) pages 136-142, edited by Gierasch and King, American Association for Advancement of science Publication.

[0025] The compound of the present invention can be formulated into a pharmaceutical composition by combining with a pharmaceutically acceptable carrier, diluent, excipient or absorption enhancer. Absorption enhancers include, for example, chelating agents (eg, EDTA, citrate, salicylate), surfactants (eg, sodium dodecyl sulfate (SDS)), non-surfactants (eg, unsaturated cyclic urea), And bile acid salts (for example, sodium deoxycholate, sodium taurocholate). Such a pharmaceutical composition can be produced by a well-known method in the pharmaceutical field. In addition, these pharmaceutical compositions are suitable for mucosal administration such as nasal cavity and can be administered individually or in combination with other therapeutic agents.

[0026] The compositions of the invention can be formulated using methods well known in the art to provide the patient with a rapid, sustained or delayed release of the active ingredient after administration. For example, suitable macromolecules (eg, polyesters, polyamino acids, polybutyrolipidone, ethylene vinyl acetate, methylcellulose, carboxymethylcellulose and protamine sulfate), or polyesters, polyamino acids, hydrated gels, polylactic acid, polydaricholic acid, lactic acid Using a dalcol copolymer or a polymer substance such as an ethyl acetate vinyl copolymer, the peptide of the present invention is complexed or adsorbed with the peptide of the present invention to produce a controlled release formulation be able to. Instead of mixing peptides with these polymer particles, for example, microcapsules manufactured by coacervation technology or interfacial polymerization, microcapsules made of hydroxymethylcellulose or gelatin, colloidal drug delivery systems (for example, Ribosome, albumin microsphere, microemulsion, nanoparticle and nanocapsule) or the microemulsion can encapsulate the peptide of the present invention.

[0027] The GLP-1 derivative of the present invention is effective for various diseases for which a GLP-1 preparation is effective. That is The GLP-1 derivative of the present invention can be used, for example, for the treatment of non-insulin dependent diabetes, the treatment of insulin dependent diabetes, the treatment of obesity, the suppression of appetite, or the suppression of heart disease. .

[0028] The dosage of the GLP-1 derivative of the present invention is desirably determined by those skilled in the art for individual patients with various diseases. In general, however, the dosage will be in the range of 1 μg to 1 mg per kg of body weight, preferably in the range of 10 μg to 100 μg per kg of body weight. It can be used immediately before meals and administered once to three or more times a day.

[0029] Hereinafter, the present invention will be further described with reference to Examples and Test Examples. These examples do not limit the technical scope of the present invention.

Example

[0030] m GLP- book composition

The GLP-1 derivative was synthesized by solid phase synthesis using a Model 433A peptide synthesizer (Applied Biosystems, Foster City, Calif.), Purified by HPLC, and the synthesized product was confirmed by mass spectrum. Purity was over 95% for the majority, and was tested in vitro and in vivo. The synthesized compounds and their abbreviations are shown below.

[0031] Production Example 1. [Ser ⁸ ] _GLP-l (7-35) -Lys-Lys-Lys-Lys-Lys-Arg-COOH (SEQ ID NO: 2)

• · -8S-GLP-l (7-35) +5 R-COOH

Production Example 2 [Gly ⁸ ] -GLP-1 (7-35)-Lys- Lys- Lys- Lys- Lys- Arg- COOH (SEQ ID NO: 3)

• -8G-GLP-l (7-35) +5 R-COOH

Comparative production example 1. GLP-1 (7-36) _NH ₂ (Natural type: SEQ ID NO: 1, provided that the C-terminus is amidated) GLP-l (7-36) -NH

Comparative Production Example 2 [Ser ⁸ ] -GLP-l (7-36) -NH (SEQ ID NO: 4, where the C-terminus is amidated) · -8S-GLP-l (7-36) -NH

Comparative Production Example 3 [Ser ⁸ ] -GLP-l (7-35) -Lys-Lys-Lys-Lys-Lys-Arg-NH (SEQ ID NO: 2, provided that the C-terminus is amidated) -8S-GLP-l (7-35) +5 R-NH

Comparative Production Example 4 [Gly ⁸ ] -GLP-l (7-35) -Lys-Lys-Lys-Lys-Lys-Arg-NH (SEQ ID NO: 3. However, the C-terminal is amidated.) -8G-GLP-l (7-35) +5 R-NH

Comparative Production Example 5 [Ser ⁸ ] -GLP-l (7-36) -Arg-Arg-Arg-Arg-COOH (SEQ ID NO: 5) • -8S-GLP-l (7-36) + 4R-COOH

Comparative Production Example 6 [Ser ⁸ ] -GLP-l (7-36) -Arg-Arg-Arg-Arg-NH (SEQ ID NO: 5, provided that the C-terminal is amidated) -8S -GLP-l (7-36) + 4R-NH

Comparative Production Example 7 · [Ser ⁸ ] -GLP-1 (7-36)-Lys- Lys- Lys- Lys- Lys- Lys- COOH (SEQ ID NO:

6)---8S-GLP-l (7-36) +6 -COOH

Comparative Production Example 8 [Ser ⁸ ] -GLP-1 (7-36)-Lys- Lys- Lys- Lys- Lys- Lys- ΝΗ (SEQ ID NO: 6, provided that the C-terminus is amidated) -8S-GLP-l (7-36) +6 -NH

Comparative Preparation Example 9 [Gly ⁸ ] -GLP-1 (7-36)-Lys- Lys- Lys- Lys- Lys- Lys- COOH (SEQ ID NO:

7)---8G-GLP-l (7-36) +6 -COOH

Comparative Production Example 10. [Gly ⁸ ] -GLP-1 (7-36) -Lys-Lys-Lys-Lys-Lys-Lys-NH (SEQ ID NO: 7, provided that the C-terminus is amidated). -8G-GLP-l (7-36) +6 -NH

[0032] As the GLP-1 derivative of the present invention, the peptides of Production Examples 1 and 2 were synthesized. In addition, [Gln ²⁶ , Asn ³⁴ ] -GLP-l (7-35) -Ly s- Lys- Lys- Lys- Lys- Arg- COOH (sequence) added with trypsin resistance based on W 02004/087910 No. 8), [Ser ⁸ , Gln ²⁶ , Asn ³⁴ ] -GLP-1 (7-35) -Lys-Lys-Lys-Lys-Lys-Arg-COOH (SEQ ID NO: 9) etc. It can be synthesized as one derivative. Further, the GLP-1 derivative of the present invention can be synthesized based on the GLP-1 mutant described in paragraphs 18 and 19. On the other hand, among the GLP-1 derivatives synthesized as a comparative control, Comparative Production Examples 3 to 6 are peptides and their substitutes described in WO2004 / 037859, and Comparative Production Examples 7 to 10 are described in WO2001 / 004156. Peptides and their substitutes

Yes

[0033] The excellent effect of the GLP-1 derivative of the present invention will be shown in a comparative test between the GLP-1 derivative of the present invention and the comparative GLP-1 derivative.

Test Example 1 Effect of GLP-1 Derivative on Lowering Blood Glucose Level by Nasal Administration

Test Examples 1 In 1 to 16, various GLP-1 derivatives were administered nasally to mice, and oral glucose tolerance test (OGTT) was conducted to investigate changes in blood glucose levels after oral loading with Darcos. The efficacy was compared.

GLP-1 derivatives were prepared in ImM with distilled water and stocked at _80 ° C. At the time of the test, it was diluted to a predetermined concentration with physiological saline and used. Using a micropipette, 20〃 1 GLP-1 derivative solution is slowly released directly from the tip of the tip directly into the mouse's nose using a micropipette. It was aspirated from the nose. Five minutes after the nasal administration of the GLP-1 derivative, a 10% glucose solution was orally administered with a sonde at a rate of 10 ml / kg.

For blood glucose level, blood sample 1 was squeezed from the wound where the tip of the tail was excised over time immediately after the test and after glucose administration, using a small blood glucose meter (Daltest Neo, Sanwa Chemical Laboratory Co., Ltd.). Measured. The area under the curve (AUC 0-20 minutes or 0-120 minutes) from the blood glucose level before administration in each GLP-1 derivative administration group was calculated.

[0035] Test Example 1 1

Here, GLP-1 derivative 8S-GLP-l (7_35) + 5KR-COOH from Production Example 1 and GLP-1 derivative 8S-GLP-1 (7_35) + 5KR-NH from Comparative Production Example 3 were each 1 nmol / Administration by mouse and comparison of efficacy. As a control group, 1 and 10 nmol / mouse of GLP-K7-3 6) -NH, which is a natural GLP-1 of Comparative Production Example 1, were set. The GLP-1 derivative of Comparative Production Example 3 is one of the most active GLP-1 derivatives in W 02004/037859.

The results are shown in Figure 1. 8S-GLP-l (7_35) + 5KR-COOH and 8S-GLP-1 (7_35) + 5KR-NH are both naturally-occurring GLP-beam highly active C-terminal Arg Was found to be more active in the carboxylic acid form than in the amide form.

[0036] Test Example 1 2

Here, GLP-1 derivative 8S-GLP-l (7_36) + 4R-COOH from Comparative Production Example 5 and GLP-1 derivative 8S-GLP-1 (7_36) + 4R-NH from Comparative Production Example 6 are each 1 nmol. Administered with / mouse and compared efficacy. As a control group, 1 and 10 nmol / mouse of GLP-1 (7-36) _NH of Comparative Production Example 1 were set. The GLP-1 derivative of Comparative Production Example 6 is one of the most active GLP-1 derivatives in WO2004 / 037859, and Comparative Production Example 5 is its C-terminal carboxylic acid form.

The results are shown in Figure 2. 8S-GLP-l (7_36) + 4R-COOH and 8S-GLP-1 (7_36) + 4R-NH were both active in nasal administration. It was also found that the carboxylic acid form is more active than the amide form.

[0037] Test Example 1 3 Here, 8S-GLP-l (7-35) + 5KR-COOH and 8S-GLP-l (7-36) + 4R-COOH, which were highly active in Test Example 1 1 and Test Example 1 2, respectively Administration was performed at 0 · 3, 1 and 3 nmol / mouse to compare the efficacy. As a control group, 10 nmol / mouse of GLP-1 (7-36) _NH was set.

Note that the number of basic amino acids on the C-terminal side of these two GLP-1 derivatives has a difference of one residue because position 36 of GLP-1 is arginine. This is described in WO2004 / 037859 This is because 4R is more active in nasal administration than 5R at the C-terminal! /.

The results are shown in Figure 3. It was found that 8S-GLP-l (7_35) + 5KR-COOH was more active than nasal administration than 8S-GLP-l (7-36) + 4R_COOH. Since the former 1 nmol / mouse is more active than the latter 3 nmol / mouse, the difference in activity is thought to be more than 3 times.

[0038] Test Example 1 4

Here, GLP-1 derivative 8S-GLP-l (7_35) + 5KR-COOH of Production Example 1 and GLP-1 derivative 8S-GLP-1 (7_35) + 5KR-NH of Comparative Production Example 3, Comparative Production Example 7 GLP-1 derivative 8S_GLP_1 (7_36) + 6K-COOH and GLP-1 derivative 8S-GLP-1 (7_36) + 6K-NH of Comparative Production Example 8 were each administered at 1 nmol / mouse to compare the efficacy. It was. The GLP-1 derivative of Comparative Production Example 8 is the 8-position serine of the GLP-1 derivative described in WO2001 / 004156, and Comparative Production Example 7 is its C-terminal carboxylic acid form.

The results are shown in Figure 4. 8S-GLP-l (7_35) + 5KR-COOH was more active in nasal administration than other V and misaligned GLP-1 derivatives. From the comparison of 5KR-COOH and 6K-COOH on the C-terminal side, it can be seen that the amino acid at the C-terminal is more effective in nasal administration of arginine than lysine. In addition, in the GLP-1 derivative with 6K added to the C-terminal side, unlike the case of 5KR, it has been clarified that the effect of the amide form is higher than that of the carboxylic acid form.

[0039] Test Example 1 5

Here, the same test as in Test Example 1-4 was performed on the 8-position glycine body. That is, GLP-1 derivative 8G-GLP-l (7_35) + 5KR-COOH of Production Example 2 and GLP-1 derivative 8G-GLP of Comparative Production Example 4 -l (7-35) +5 R-NH, GLP-1 derivative 8G-GLP-1 in Comparative Preparation Example 8G-GLP-l (7_36) + 6K-COOH, and GLP-1 derivative 8G-GLP-1 in Comparative Preparation 10 (7_36) + 6K-NH was administered at 1 nmol / mouse, and the efficacy was compared. As a control group, 10 nmol / mouse of GLP-1 (7-36) _NH was set. The GLP-1 derivative of Comparative Production Example 10 is the GLP-1 derivative described in WO2001 / 004156, and Comparative Production Example 9 is its C-terminal carboxylic acid form.

The results are shown in FIG. The same results as in the 8-position serine were obtained in the 8-position glycine form. In the GLP-1 derivative of the present invention and the GLP-1 derivative described in WO2001 / 004156, the 8-position glycine body and the 8-position serine body show substantially the same activity.

[0040] Test Example 1 6

Here, GLP-1 derivative 8S-GLP-l (7_35) + 5KR-COOH from Production Example 1 and GLP-1 derivative 8G-GLP-1 (7_36) + 6K-NH from Comparative Production Example 10 are 0.3 and 1 respectively. And 3 nmol / mouse to compare the efficacy.

The results are shown in FIG. The force 8S-GLP-K7-3 5) + 5KR-COOH, which is comparable to the 8-position serine and the 8-position glycine, was more active. IC value is 8S-GLP-1 (7_35) + 5KR-C

OOH was 1.6 nmol / mouse, 8G-GLP-1 (7_36) + 6K-NH power 9 nmol / mouse, and the ratio was 3.1 times.

[0041] Trial, Trial 2 GLP- ί 本 FL grass

Here, GLP-1 derivatives were administered intranasally to mice, and their blood kinetics and absorption rate were examined. The administered GLP-1 derivative was 8S-GLP-l (7_35) + 5KR-COOH of Production Example 1, and was administered at 30 and lOOnmol / kg. As control drugs, natural GLP_1 (7_36) -NH from Comparative Production Example 1 and GLP-1 derivative 8S-GLP-1 (7-36) _NH from Comparative Production Example 2 were administered at 100 and 300 ηmol / kg, respectively. .

Mice were ddY mice (7 weeks, male), and 100 1 was collected from the orbital venous plexus using a heparin-treated glass capillary immediately before nasal administration of the GLP-1 derivative. The blood GLP-1 concentration was defined as the value at 0 minutes after administration. Next, the mice were lightly anesthetized with ether, and 20 1 GLP-1 derivative solution was slowly released from the tip of the pipette tip directly into the nose of the mouse using a micropipette. Thereafter, 100 blood samples were collected from the orbital venous plexus over time, and plasma was obtained by centrifuge separation. Plasma GLP-1 concentration was measured using the GLP-l (total) RIA kit (LINCO, Ca t No. GLP1T-36HK) to determine the absorption rate. The absorption rate indicates the absorption rate by nasal administration of each GLP-1 derivative when the absorption when each GLP-1 derivative was intravenously administered was taken as 100%.

The results are shown in Fig. 7 and the changes in blood concentration of GLP-1 derivatives. Table 1 shows the absorption rate of GLP-1 derivatives. The 30 nmol / kg group of 8S-GLP-l (7_35) + 5KR-COOH, which is the GLP-1 derivative of the present invention, is the control group of natural GLP-1 (7-36) _NH. It showed almost the same blood concentration transition of the nmol / kg administration group and the 100 nmol / kg administration group of 8S-GLP-1 (7-36) _NH. Similarly, the 100 nmol / kg administration group of the product of the present invention The blood concentration was almost the same as that of the 300 nmol / kg group of the control drug. Thus, 8S-GLP-K7-35) + 5KR-COOH, which is a GLP-1 derivative of the present invention, is a natural GLP-1 (7_36) -NH or 8S − It was found that the same blood concentration could be achieved with almost 1/3 dose of GLP-l (7-36) -NH. Comparing the absorption rate at the same dose of 100 nmol / kg, 8S-GLP-1 (7_35) + 5K R-COOH is the natural GLP-1 (7-36) _NH and 8S-GLP-1 (7_36) The absorption rate was almost 6 times higher than that of -NH.

The results of this test indicate that the GLP-1 derivative of the present invention showed high activity in the nasal administration evaluation system of Test Example 1, and that it has high mucosal absorbability.

[Table 1] Absorption rate of GL P-1 derivative by nasal administration to mice

Brief Description of Drawings

[Fig.1] Comparison of the blood glucose lowering effects of 8S-GLP-l (7_35) + 5KR-COOH and 8S-GLP-1 (7_35) + 5KR-NH by nasal administration in Test Example 1 1! It is a figure which shows the result. In the figure, the number in brackets after the compound abbreviation name indicates the dose (nmol / mouse).

[Figure 2] In Test Example 1-2, 8S-GLP-1 (7_36) + 4R-COOH and 8S-GLP-1 (7_36) + 4R-N It is a figure which shows the result of having compared the blood glucose lowering effect of nasal administration. In the figure, the number in brackets after the compound abbreviation name indicates the dose (nmol / mouse).

[Figure 3] In Test Example 1-3, 8S-GLP-l (7_35) + 5KR-COOH and 8S-GLP-1 (7_36) + 4R-

It is a figure which shows the result which compared the blood glucose lowering effect of COOH by nasal administration. In the figure

The number in brackets after the compound abbreviation name indicates the dose (nmol / mouse).

[Fig. 4] In Test Example 1-4, 8S-GLP-l (7_35) + 5KR-COOH, 8S-

GLP-l (7-35) +5 R-NH, 8S-GLP-l (7_36) + 6K-COOH, and 8S-GLP-1 (7_36) + 6K-

It is a figure which shows the result of having compared the blood glucose lowering effect of NH by nasal administration. In the figure, the number in brackets after the compound abbreviation name indicates the dose (nmol / mouse).

[Fig. 5] In Test Example 1-5, 8G-GLP-l (7_35) + 5KR-COOH, which is 8-position glycine, 8

G-GLP-l (7-35) +5 R-NH, 8G-GLP-l (7_36) + 6K-COOH, and 8G-GLP-l (7_36) + 6-NH It is a figure which shows the result compared by. In the figure, the number in brackets after the compound abbreviation name indicates the dose (nmol / mouse).

[Figure 6] In Test Example 1-6, 8S-GLP-l (7_35) + 5KR-COOH and 8G-GLP-1 (7_36) + 6K-

[Fig. 7] In Test Example 2, 8S-GLP-l (7_35) + 5KR-COOH, GLP-1 (7_36) -NH, and 8S

It is a figure which shows the blood concentration transition when nasally administering -GLP-l (7-36) -NH. In the figure, the number in parentheses after the compound abbreviation name indicates the dose (nmol / kg).

Claims

The scope of the claims

[I] The amino acid sequence of GLP-7-35) or GLP-7-36), or a sequence in which one or several amino acids in the amino acid sequence are deleted, substituted and / or added. A peptide in which (Lys) n-Arg (n is an integer of 4 to 8, Arg is a carboxylic acid) is added to the C-terminus of a peptide having GLP-1 activity.

[2] An amino acid sequence of GLP-7-35) or GLP-7-36), or a peptide having a GLP-1 activity consisting of a sequence having 85% or more homology with those amino acid sequences Peptides with (1 ^ ¾) 11-8 ( ₁ 1 is an integer of 4-8, Arg is a carboxylic acid) added to the C-terminus.

[3] The peptide according to claim 1 or 2, wherein position 8 of the GLP-1 amino acid sequence is Ser or Gly.

[4] The peptide according to claim 1 or 2, wherein position 26 of the GLP-1 amino acid sequence is Gln and position 34 is SAsn.

[5] The peptide according to claim 1 or 2, wherein n is 5.

[6] The peptide according to claim 1 or 2, represented by [Ser ⁸ ] -GLP-l (7-35)-(Lys) —Arg.

Five

[7] The peptide according to any one of claims 1 and 2, wherein the natural GLP-beam also has a high mucosal absorption rate.

[8] A pharmaceutical composition comprising the peptide according to claim 1 or 2 as an active ingredient.

9. The pharmaceutical composition according to claim 8, which is used for transmucosal administration.

10. The pharmaceutical composition according to claim 9, wherein the transmucosal administration is nasal administration.

[I I] The pharmaceutical composition according to claim 8, which is used for treating diabetes, treating obesity, or suppressing appetite.

[12] Use of the peptide according to claim 1 or 2 for producing a therapeutic agent for diabetes, obesity or eating disorders.