JPH0680584A - Medicine containing glycentin as active ingredient - Google Patents

Abstract

PURPOSE:To provide a medicine containing glycentin as an active ingredient, stimulating the secretion of insulin without causing lower blood glucose, and useful as an insulin secretion-stimulating and diabete-treating agent. CONSTITUTION:The objective medicine contains preferably human type glycentin (especially preferably natural type human glycentin without methionine at its N-end, e.g. the amino acid sequence of the formula) as an active ingredient, and is administered so as to give a blood concentration of 100-10000pM [e.g. 0.01-10mg (preferably 0.1-1mg) as an oral administratior tablets, etc.].

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an insulin secretagogue and an antidiabetic agent containing glicentin as an active ingredient, and an antidiabetic agent which promotes insulin secretion by administration of glicentin to increase the blood level of the hormone. Concerning the use of glicentin as.

[0002]

2. Description of the Related Art Glicentin is one of the major components of intestinal GLI among glucagon-like immunoreactivity (GLI), which does not bind to glucagon-specific antibody but binds to non-specific antibody,
It is a peptide having 69 amino acid residues.

The glicentin contains a structure of glucagon, which is a peptide hormone consisting of 29 amino acids, from the 33rd position to the 61st position in the structure, and the Nth terminal side of the structure is from the 1st position to the 30th position via Lys-Arg. GRP
P (glucagon-related pancreatic peptide or gli
It has a structure called a centin-related pancreatic peptide) and a structure having a hexapeptide from the 64th position to the 69th position via Lys-Arg on its C-terminal side (N. Yanaihara and C. Yanaihara, Gastrointestinal.
Hormones, 141-162, edited by V. Mutt, Academic Press Inc.
(1988)).

Since the amino acid sequence of porcine glicentin has been clarified, glicentin (L. Thim and AJ Mood
y, Regulatory Peptides, 2, 139-150 (1981)),
Nucleotide sequence of preproglucagon, a glucagon precursor (S. Seino et. Al., FEBS LTTERS, 203, 25-30 (198
The amino acid sequence of glicentin of human, bovine, hamster, rat, guinea pig, etc. has been proposed by (6) etc.).

Glicentin and glucagon are tissue-specifically processed from the same precursor, preproglucagon, and are produced as glucagon with 29 amino acid residues in the pancreas and as glicentin in the intestine. Among these peptides, glucagon is known to promote gluconeogenesis and glycolysis to raise blood glucose level (R.
Ebert et. Al., Diabetes Metabolism Review, Vol.3,
No. 1, 1-26 (1987)].

[0006] Glucagon-like peptide-1 or GLP-1 (1-37), which is formed by tissue-specific processing from the same preproglucagon, is a polypeptide hormone consisting of 37 amino acids. -1 (1-37) derived from the above-mentioned glicentin amino acid sequence 7th to 36th
It is known that it has an action of promoting insulin secretion together with GLP-1 (7-36 amide), which is a polypeptide hormone consisting of up to the thirty amino acids [T.
Matsuyama et. Al., Diabetes Res. And Clinical Pra
ctice, Vol.5, 281-284 (1988), DA D'Alessio et. a
l., Diabetes, Vol. 38, 1534-1538 (1989), etc.].

[0007] Further, in the fraction of peak II obtained by fractionating the glucagon-like immunoreactant (GLI) extracted from the small intestine of dogs, promotion of insulin secretion was observed, and the fraction of peak II of GLI has a molecular weight. It is a fraction mainly composed of small glucagons. On the other hand, promotion of insulin secretion was not observed in the peak I fraction mainly containing glicentin [A. Ohneda et. Al., Horm. Metab. Res.
Vol.8, 170-174 (1976)].

However, the action of glicentin has not been clarified yet, and it is completely unknown as to which tissue or which cell glicentin directly acts. In addition, the glicentin isolated and purified so far is from mammals other than humans, and human glicentin is a purified product so far because of difficulty in obtaining raw materials for extraction, that is, human intestinal tract materials. Therefore, the physiological activity of human glicentin has not yet been elucidated.

By the way, the present inventors have previously described GI Bell.
Have been elucidated from the sequence of the human preproglucagon gene [see Nature, Vol. 304, 368-371 (1983)].
A DNA corresponding to the amino acid sequence of human glicentin was synthesized, a recombinant DNA vector was prepared using this DNA, and human glicentin was successfully produced from the transformed host cell (JP-A-4-36419).
(See No. 9). Thus, human-type glicentin can be easily obtained as a purified product in large quantities.

[0010]

By the way, as the standard of living has improved, the number of patients with diabetes, also known as civilization, has been increasing year by year, and the prevalence rate over the past 30 years has rapidly increased to 30 times or more. Shows a trend. It is said that the pathological condition is caused by an absolute or relative lack of insulin action, which is a hormone that plays a central role in blood sugar regulation. The main treatment methods are diet and insulin administration.

Conventionally, a sulfonylurea agent has been known as a drug for promoting insulin secretion, but there is a risk of causing hypoglycemia due to overdose or continuous administration, and it is sufficient to maintain a normal blood glucose level. There was a drawback that required great care. In addition, in patients with liver / kidney disorders,
Since drug or its metabolites accumulate, special caution is required during administration. In addition, it is not recommended for administration to pregnant women due to its ability to pass through the placenta, and cannot be administered to breast-feeding women due to its easy transfer to milk. Therefore, it is desired to develop an insulin secretagogue that is safe and has few side effects.

[0012]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to solve such problems and found that glicentin has a strong insulin secretory action,
The present invention has been completed.

[0013] According to the present invention, there is provided an insulin secretagogue, which comprises glicentin as an active ingredient. The glicentin in the present invention promotes insulin secretion from the pancreas without increasing blood sugar, and thus can be used for treatment and prevention of diabetes. Therefore, the present invention also provides a therapeutic agent for diabetes, which comprises glicentin as an active ingredient.

That is, the administration of glicentin promotes insulin secretion, and as a result, glicentin is used for the treatment and prevention of diabetes caused by a physical condition in which insulin secretion is insufficient.

The glicentin used in the present invention includes glicentin derived from any animal (eg, human, pig, cow, hamster, rat, guinea pig, etc.), or the N-terminal obtained when they are produced by a general genetic engineering method. Contains glycentin added with methionine (Met). However, it is preferable to use human glicentin considering the antigenicity and the like when administered to humans. Furthermore, it is more preferable to use a natural human glicentin in which methionine (Met) is not added to the N-terminus even if it is human.

Human glicentin (natural type) is represented by the following amino acid sequence. Arg-Ser-Leu-Gln-Asp-Thr-Glu-Glu-Lys-Ser-Arg-Ser-Ph
e-Ser-Ala-Ser-Gln-Ala-Asp-Pro-Leu-Ser-Asp-Pro-Asp-
Gln-Met-Asn-Glu-Asp-Lys-Arg-His-Ser-Gln-Gly-Thr-Ph
e-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-
Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Ar
g-Asn-Arg-Asn-Asn-Ile-Ala

Further, human glicentin with methionine added to the N-terminus is shown by the following amino acid sequence. Met-Arg-Ser-Leu-Gln-Arg-Thr-Gln-Glu-Lys-Ser-Arg-Se
r-Phe-Ser-Ala-Ser-Gln-Ala-Asp-Pro-Leu-Ser-Asp-Pro-
Asp-Gln-Met-Asn-Glu-Asp-Lys-Arg-His-Ser-Gln-Gly-Th
r-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-
Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Ly
s-Arg-Asn-Arg-Asn-Asn-Ile-Ala

These human glicentins have the corresponding D
By using a gene of NA sequence by a genetic engineering method,
Alternatively, it can be produced by a synthetic method. As an example of this genetic engineering method, the following DNA sequence proposed by the present inventors in Japanese Patent Application Laid-Open No. 4-364199 was previously mentioned.

[Chemical 1] A method for producing a desired human-type glicentin can be mentioned by preparing the human-type glicentin-synthesizing gene of 1., introducing this into a plasmid, and culturing transformed E. coli using the obtained plasmid. However, this method for producing human-type glicentin is only one example for producing human-type glicentin, and other methods,
It goes without saying that human glicentin prepared by any means can be used in the present invention as long as it has the above-mentioned amino acid sequence. As a method for producing human glicentin, another DNA corresponding to the amino acid sequence of glicentin described above is used.
The gene of the sequence is introduced into a plasmid, and Escherichia coli, Bacillus subtilis, yeast, and other microorganisms are transformed with the obtained plasmid, which is then cultured or produced by a method such as culturing human glicentin-producing cells. There is a method.

Usually, glicentin as an active ingredient can be administered orally or parenterally in the form of a suitable pharmaceutical preparation.

In the formulation of glicentin, it may be formulated alone or in admixture with pharmaceutical auxiliaries such as excipients, binders, stabilizers, buffers and the like.
The dosage form may be any of powders, granules, tablets, capsules, injections and the like depending on the dosage form of oral administration and parenteral administration. If necessary, it may be used in combination with other drugs such as a sulfonylurea drug or a biguanide drug.

An advantageous oral pharmaceutical formulation is in a form suitable for application in unit dosage form, eg an aqueous solution containing glicentin in an amount of 0.01 mg to 10 mg, preferably 0.1 mg to 1 mg per 5 ml. Alternatively, it may be an oily solution, or a suspension or emulsion preparation, or a tablet, capsule, granule or the like containing glicentin in an amount of 0.01 mg to 10 mg, preferably 0.1 mg to 1 mg.

When administered to the human body due to its chemical structure, glicentin is considered to be denatured by acid in the stomach by oral administration, and its activity may be reduced by digestion-induced degradation and modification. It is preferable to consider the release of the active ingredient within. Therefore, when glicentin is orally administered, it is coated with a known enteric coating agent. As such an enteric coating agent, a synthetic polymer compound such as Eudragit ^R (manufactured by Rohm Pharma Co.) containing polyacrylate as a main component, a semi-synthetic polymer compound such as cellulose acetate phthalate may be used.

However, the means of administration by parenteral administration is preferable as a means for administering glicentin to the human body without being denatured or decomposed. This parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection, enteral injection and the like, and glicentin is formulated in the form of an aqueous or oily solution or suspension or emulsion as described above. sell. It is also preferable to administer in the form of a depot preparation in order to maintain the effect of glicentin for a long time.

The dose depends on the symptoms, age, etc. of the patient to be administered.
It can be appropriately increased or decreased according to sex and body weight, and it is naturally changed depending on the administration form, that is, oral administration or parenteral administration. Regardless of which dosage form is used, it is appropriate that the blood concentration is in the range of 100 pM to 10,000 pM. In the case of parenteral administration per adult as a usual dose, it is used in the range of 0.5 to 500 μg / kg. However, smaller or larger doses may be administered within the safe range. Six-week-old male BALB / c mice were intraperitoneally administered with 10 mg / kg of human natural glicentin, but no apparent change occurred. Next, the present invention will be further described with reference to examples.

[0025]

EXAMPLES The present invention is explained in more detail by the pharmacological tests of the examples described below. For this test, mixed-breed dogs (weight: 13 to 17 kg) were used, and an experiment was performed after an overnight fast [A.Ohneda et al., Horm. Metab. Res., 9, 447-.
452 (1977)].

Nembutal anesthesia was performed to open the abdomen, and a cannula for injecting the solution was inserted into the upper pancreaticoduodenal artery, and a cannula for collecting blood used for hormone measurement was inserted into the upper pancreaticoduodenal vein. A 0.5% arginine solution prepared in physiological saline was injected at a flow rate of 2 ml / min. Twenty minutes after the start of injection, 100 pmol and 400 pmol glicentin solutions prepared with 0.2% bovine albumin and then 400 pmol glucagon solution were each added 4 times.
The injection was performed for 10 minutes at intervals of 0 minutes. Blood used for hormone measurement is 1,000 KIU aprotinin and 10 blood.
4 ml of blood was collected over time into a glass tube containing mg of EDTA.

Plasma insulin can be measured by a known method [CR Morgan and A. Lazarow, Diabetes, 12, 115-1.
26 (1963)]. Plasma glucagon was measured by the method of Ohneda et al. [A. Ohneda et al., Diabetes, 2
4, 811-819 (1975)], using an antiserum (G21) specific for the C-terminal portion of glucagon. Plasma glicentin was determined by the method of Ohneda et al. [A. Ohneda et al.,
Tohoku J. Exp. Med., 129, 207-217 (1979)], an antiserum (G25) that cross-reacts with glucagon-related substances.
Was used to measure the total glucagon-like immunoreactivity. Blood glucose was measured by a glucose oxidase method using a blood glucose level test reagent set (trade name: glucose B-test Wako, manufactured by Wako Pure Chemical Industries, Ltd.) for blood collected from the femoral artery.

The results of this experiment are shown in FIG. In the figure, the effects of glicentin and glucagon on blood glucose, blood insulin and blood glucagon in arginine-loaded dogs are shown.

The horizontal axis represents time, and the injection of arginine was started from -20 minutes. The time at which glicentin was first injected is indicated by A0 minutes. Human natural glicentin solution was injected for 10 minutes from A0, and human natural glicentin solution was injected for 10 minutes again at 40 minutes after B0.
At 40 minutes after C0, the glucagon solution was injected as above.

The vertical axis represents blood glucose level, insulin level (IRI), glucagon level [IRG (G21)],
The glucagon-like immunoreactivity value [IRG (G25)] is shown.

From these experimental results, insulin secretion is increased by administration of glicentin without changing blood glucose level. In addition, since the glucagon level does not increase with the administration of glicentin, it can be seen that glicentin is not immediately metabolized to glucagon. Further, it is shown that there is an insulin secretagogue effect as a unique effect of glicentin, which is different from the insulin secretagogue effect of glucagon, which is attributed to the increase in insulin level observed by administration of glucagon.

Next, formulation examples of glicentin of the present invention will be shown. Formulation Example 1 0.5 g of human glicentin, 2 kg of lactose, 20 g of magnesium stearate and 100 g of corn starch were mixed, the mixture was compressed, and the compressed mixture was ground to prepare a mixture granule. The obtained granules are placed in a tableting machine and 1
Tablets containing 5.0 μg of glicentin per tablet were obtained.
To make the tablets enteric, the tablets were enteric coated with cellulose acetate phthalate.

Formulation Example 2 Human glicentin 0.1 g, sucrose 30 g, 70% D-sorbitol 26 g, ethyl p-oxybenzoate 0.03
g and 0.015 g of propyl p-oxybenzoate were dissolved in 60 g of warm water. After cooling, a solution of the flavoring agent dissolved in 0.15 of glycerin and 0.5 g of 96% ethanol was added, and then water was added to this mixture to prepare a 100 ml syrup.

Formulation Example 3 1 g of human glicentin and 99 g of lactose were mixed and then dissolved in 1 liter of distilled water for injection, and this solution was aseptically filtered through a sterile filter, for example, a 0.22 μm membrane filter. Each 1 ml was dispensed into a vial bottle and freeze-dried to prepare a preparation for injection. This preparation for injection uses distilled water for injection 1
Dissolve in ml.

Formulation Example 4 Human glicentin 0.5 g, lactose 4 kg, crystallin cellulose 1.5 kg, calcium stearate 1.5 kg and talc 3 kg were thoroughly mixed, the mixture was compressed, and the compressed mixture was pulverized to obtain a mixture. Granules were prepared. The obtained granules were encapsulated in a two-piece capsule to obtain a capsule containing 10.0 μg of glicentin per capsule.

[0036]

[Effect] According to the present invention, there is provided insulin secretion promotion characterized by containing glicentin.

[Brief description of drawings]

FIG. 1 shows blood glucose levels, insulin levels (IRI) and glucagon levels [I] when glicentin and glucagon are administered.
RG (G21)] and glucagon-like immunoreactivity [IR
The figure which shows the transition of G (G25).

Claims (4)

[Claims] 1. An insulin secretagogue, comprising glicentin as an active ingredient. 2. The insulin secretagogue according to claim 1, wherein the glicentin is human glicentin. 3. A therapeutic agent for diabetes which comprises glicentin as an active ingredient. 4. The therapeutic agent for diabetes according to claim 3, wherein the glicentin is human glicentin.