JP3323305B2 - Elcatonin stabilizer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a stabilized peptide of an aqueous solution of elcatonin.

[0002]

2. Description of the Related Art Elcatonin has a chemical name of 1-butyl-7-.
(L-2-aminobutyric acid) -26-L-aspartic acid-27-L-valine-29-L-alanine calcitonin (salmon) [1-butyric acid-7- (L-2-aminob
utyric acid) -26-L-aspartic acid -27-L
-Valine-29-L-alaninecalcitonin (salmon)]
It is a kind of calcitonin used for hypercalcemia, Paget disease of bone or osteoporosis. At present, elcatonin is used as an aqueous solution for injection. However, like other calcitonins, when a peptide drug such as elcatonin is used as an aqueous solution, heat stability (long-term storage stability), light stability, In addition, it is necessary to formulate the preparation sufficiently considering the stability against shaking during transportation and the like. Conventionally, in the preparation of an aqueous solution of elcatonin, strict selection has been made with respect to the pH of the solution, the type of buffer used, and the ionic strength, and strict selection has also been made regarding the physical properties of the glass container such as an ampule used. Was needed.

[0003]

As described above, aqueous solutions of peptidic drugs such as elcatonin are extremely delicate, and effective stabilizers thereof have been long sought.

[0004]

Means for Solving the Problems The inventors of the present invention have screened a large number of substances for an elcatonin stabilizer in the form of an aqueous solution. In some of the compounds, it was found that an extremely small amount of the compound had a stabilizing effect on elcatonin, and the present invention was reached. That is, the present invention relates to the following formulas A to H

[0005]

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[0006]

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[0007]

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A stabilizer for an aqueous solution of elcatonin comprising one or more active ingredients selected from the group consisting of peptides represented by the following formula: The stabilizer for an injectable aqueous solution of elcatonin of the present invention can be synthesized by a known method for peptide synthesis. (1) In the case of production by a liquid phase method For example, the carboxyl group of the C-terminal proline group is converted into an amide group, and the amino acids represented by the formulas A to H are individually unprotected or preferably protected. Amino acids and / or lower peptides are condensed and subjected to a cyclization reaction in any process, and the condensation reaction is carried out. If there is a protecting group, for example, the protecting group of the active group is eliminated by acid decomposition in the final step It is obtained by doing.

The condensation reaction and the cyclization reaction are carried out by repeating the deprotection and deprotection of the protecting group and the condensation reaction in accordance with conventional methods for peptide synthesis. That is, various protecting groups used in the production of the present compound can be easily removed by known means such as hydrolysis, acid decomposition, reduction, aminolysis, hydrazinolysis and the like in peptide synthesis. Possible protecting groups are used. Such protecting groups are described in the literature of peptide synthetic chemistry as well as in reference books. In the present invention, for example, α-
A t-butyloxycarbonyl group, a benzyloxycarbonyl group or a p-methoxybenzyloxycarbonyl group is used for protecting the amino group, and the amino group in the side chain, that is, ε of lysine is used.
A benzyloxycarbonyl group, p-
Using a chlorobenzyloxycarbonyl group, a methyl ester group and a benzyl ester group for protecting the α-carboxyl group, and a benzyl ester group for protecting the side chain carboxyl group, that is, the carboxyl group of the side chain of aspartic acid or glutamic acid. A cyclohexyl ester group is used, a t-butyl ester group is used to protect the carboxyl group on the side chain of α-aminosuberic acid, a benzyl group is used to protect the hydroxyl groups of serine and threonine, and 2,6 is used to protect the hydroxyl group of tyrosine. It is preferable to use a dichlorobenzyl group and a mesitylene-2-sulfonyl group or a tosyl group for protecting the guanidino group of arginine.

In the synthesis of the present compounds, the condensation of individual amino acids and / or lower peptides can be carried out, for example, by condensing an amino acid or a lower peptide having a protected α-amino group and an activated terminal α-carboxyl group with a free α-amino group. And an amino acid or a lower peptide having a protected terminal carboxyl group, or an activated α
-It can be carried out by reacting an amino acid or a lower peptide having an amino group and a protected terminal carboxyl group with an amino acid or a lower peptide having a free terminal carboxyl group.

In this case, the carboxyl group is converted to an active azide, an acid anhydride, an imidazolide or an active ester such as a cyanomethyl ester, a p-nitrophenyl ester or an N-hydroxysuccinimide ester. Can be changed. Also, carbodiimides such as N, N'-dicyclohexyl-carbodiimide (DCC), N-ethyl-N'-3
-Dimethylaminopropyl-carbodiimide, N, N '
Activated by reacting with a condensing agent such as -carbonyl-diimidazole.

The preferred condensation method and cyclization reaction in the present invention are the azide method, active ester method, mixed acid anhydride method and carbodiimide method. In each stage of the condensation, it is desirable to use a method in which racemization does not occur or a method in which racemization is minimized. Preferably, an azide method, an active ester method, a Wunsch method [Z. Naturforsc
h. , 21b, 426 (1966)] or Geige
r method (Chem. Ber., 10., 788 (197)
0)].

The cyclization reaction can be performed in the same manner as the above-mentioned condensation reaction. The condensation order and the cyclization position can be synthesized at any position as long as it is the amino acid order shown in the formula. However, the amino acid and / or lower peptide are sequentially linked from the C-terminal side, and the ω of aminosuberic acid can be synthesized. It is preferred that the bond between the carboxyl terminus and the predetermined N-terminal amino acid be the cyclization position.

A peptide having an ε-amino group, a side chain carboxyl group, a guanidino group and a hydroxyl group thus protected is obtained. These protecting groups are preferably acid-decomposed, for example, trifluoromethanesulfonic acid, hydrogen fluoride anhydride and the like. In one step by the method according to the above method to obtain the desired compound. (2) In the case of production by the solid phase method In the present invention, in addition to the peptide synthesis method by the liquid phase method described above, the present compound may be synthesized by using a part or all of the peptide synthesis method by the solid phase method. it can.

For example, a C-terminal peptide fragment is synthesized by a solid phase method, a cyclic peptide fragment containing N-terminal α-aminosuberic acid is synthesized by a liquid phase method, and then the above two peptide fragments are synthesized by a solid phase method. To obtain a protected peptide resin obtained by condensation. These protecting groups and resin are eliminated in a single step by a known method, for example, a method using trifluoromethanesulfonic acid, hydrogen fluoride or the like, to obtain a target compound.

The resins used in the above solid phase method include:
Resins usually used in the solid-phase method include, for example, benzhydrylamine resin, p-methylbenzhydrylamine resin and the like. As this resin, a resin having desired properties depending on a difference in functional group equivalent or a degree of crosslinking can be obtained, and a commercially available resin can also be purchased. In the above solid phase method,
The resin is condensed one by one from the C-terminal amino acid in the order of the amino acid shown by the formula. The functional group of the amino acid is protected with a protecting group by a known method. Examples of the above protecting groups are as described above.

In the above solid phase reaction, the resin is put into a reactor, and a solvent for swelling the resin such as dichloromethane, chloroform, dimethylformamide and benzene is added to the resin 1.
The solvent is added at a ratio of 2 to 20 ml per g. To this, in a separate reactor, add 1 equivalent to 1 equivalent of amino group in the resin.
-6 equivalents of Boc (t-butyloxycarbonyl) amino acid is reacted with DCC, and the resulting symmetrical anhydride is separated from by-product dicyclohexylurea (DCU) and added to the reactor containing the resin. The amount of the condensing agent (DCC) used is 0.5 to 3 equivalents to 1 equivalent of the Boc-amino acid. The reaction is usually performed for 5 to 60 minutes.

A portion of the Boc-amino acid-resin or Boc-peptide-resin obtained in each step may be collected and the amount of the Boc-amino acid reacted in a conventional manner. Next, Boc, which is a protecting group for the α-amino group, may be eliminated with an acid such as trifluoroacetic acid, and a condensation reaction may be sequentially performed. The peptide synthesis by the above solid phase method uses an automatic solid phase synthesizer, but may be performed by a manual method. It is desirable that all of these operations be performed under a stream of nitrogen gas.

In this way, a resin to which the peptide is bound is obtained. As described above, in the thus obtained protected peptide-bonded resin, the protective group and the resin are eliminated in one step by anhydrous hydrogen fluoride or the like. (3) Separation and Purification and Others The compound thus obtained can be separated and purified by a known means for purifying a peptide or protein. For example, a gel filtration method using a gel filtration agent such as Sephadex G-25, Sephadex G-50, and Sephadex LH-20, column chromatography using carboxymethylcellulose and other ion exchange resins, and a reversed-phase synthetic polymer It can be carried out by column chromatography or high performance liquid chromatography using a resin or a chemically modified silica gel carrier.

The novel compounds of the present invention can be obtained in the form of a base or a salt thereof depending on the conditions of the method. For example, a salt with a known organic acid such as acetic acid can be formed. still,
Abbreviations described herein have the following meanings. Asu: L-α-aminosuberic acid Asn: L-asparagine Asp: L-aspartic acid Ala: L-alanine Thr: L-threonine Val: L-valine His: L-histidine Arg: L-arginine Leu: L-leucine Tyr: L-tyrosine Ser: L-serine Gly: glycine Lys: L-lysine Pro: L-proline Glu: L-glutamic acid Gln: L-glutamine D-Leu: D-leucine Boc-: t-butyloxycarbonyl Z- : Carbobenzoxy Fmoc-: Fluorenylmethyl Cl-Z: p-chlorobenzyloxycarbonyl Cl2 Bzl: 2,6-dichlorobenzyl Ac: acetyl Bzl: benzyl OBzl: benzyl ester OMe: methyl ester Tos: tosyl TFA: tri Fluoroacetic acid -Ether: diethyl ether DMF: N, N'-dimethylformamide MeOH: methanol DCM: dichloromethane DIEA: diisopropylethylamine HOBt: 1-hydroxybenzotriazole MBHA resin: p-methylbenzhydrylamine resin OSu: N-hydroxysuccinimide ester- OtBu: t-butyl ester WSCD.HCl: 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride The elcatonin stabilizer thus synthesized and purified is one or two of More than one species are mixed in advance with the bulk elcatonin powder, and then an elcatonin aqueous solution is prepared, or directly added and dissolved in the elcatonin aqueous solution, or a solution of the elcatonin stabilizer previously dissolved separately is used. And elcatonin aqueous solution. The effective amount is usually 0.5 to 10% by weight, more preferably 1 to 5% by weight based on the weight of elcatonin.
% By weight.

The aqueous solution of elcatonin is not particularly limited as long as it is commonly used, and includes physiological saline and various buffers. Examples of the buffer include, for example, citric acid, acetic acid and the like, and aqueous solutions of these acids. A salt is exemplified. Usually, their concentration is preferably about 0.05 to 20 mM,
The preferred pH is usually 5 to 7. Non-toxic strong electrolyte inorganic salts such as sodium chloride and potassium chloride may be added to this liquid as an osmotic pressure adjusting agent. Elcatonin, the active ingredient, depends on the indication and usage, etc.
Usually, an aqueous solution of 1 to 40 micrograms per container and a concentration of 1 to 40 micrograms per container is exemplified.

The aqueous solution of elcatonin for injection containing the above-mentioned elcatonin stabilizer can be filled into a medical glass container such as an ampoule or a vial or a plastic container to give an aqueous solution injection by a conventional method. In particular, an injection having excellent shaking stability can be obtained.

[0023]

EXAMPLES The present invention will be specifically described below with reference to production examples and examples, but the present invention is not limited to these. The amino acid analysis in each production example was performed by adding 6N hydrochloric acid to the sample, hydrolyzing the sample at 110 ° C. for 24 hours, drying it under reduced pressure, and analyzing it with an amino acid analyzer. Also,
The conditions of high performance liquid chromatography indicated by physical property values are as follows:
It is as follows. (High performance liquid chromatography conditions) Column: ODS column Inner diameter 4 mm, length 150 mm Eluent: Gradient A solution: 0.1% TFA water B solution: Acetonitrile Linear concentration from initial solution B concentration 15% to solution B concentration 45% Gradient elution (30 minutes) Flow rate 1 ml / min Detection wavelength 225 nm

[0024]

[Production Example 1]

[0025]

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Production of the compound represented by the above formula A (may be abbreviated as F04 compound) (1)

[0027]

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The title compound (hereinafter sometimes abbreviated as fragment (1)) was produced by the method of Examples 2 (1) to (14) of JP-B-53-41677. (2)

[0029]

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Production of Applied BiosystemS 430-A as a solid phase synthesizer
Solid phase synthesis was performed using a peptide synthesizer. M
BHA resin (Applied Biosystems, amino group 0.61m
0.8 mol (g / mol) in a reaction vessel for peptide solid phase synthesis, 8 ml of DCM (4 times, 1 minute each), 8 ml of a DCM solution containing 60% TFA (20 minutes), 4 ml of DCM (3 times, 15 seconds each), 3 ml of DMF solution containing 1 ml of DIEA (2
Each time, 1 minute each) and 8 ml of DMF (six times, 40 seconds each) in the order of stirring in a stream of nitrogen gas, followed by filtration after each treatment.

On the other hand, 2 mmol of Boc-Pro was dissolved in 5 ml of DCM, and DCC (0.5 M-D
(CM solution) 2 ml was added and reacted for 5 minutes. The reaction solution was filtered and transferred to a concentration vessel, to which 3 ml of DMF was added.
DCM was distilled off under a stream of nitrogen gas. DMF 3ml
Was added and transferred to the above-mentioned reaction vessel and reacted for 25 minutes. Then, it was washed with 8 ml of DCM (6 times, 20 seconds each) and filtered to obtain Boc-Pro-MBHA resin.

Next, the above Boc-Pro-MBHA resin was washed in a reaction vessel with 8 ml of DCM (four times, 1 minute each, filtered). To this, 8 ml of a DCM solution containing 60% TFA was added and stirred for 20 minutes. Boc was desorbed, and the obtained resin was subjected to DCM 4m.
1 (3 times, 15 seconds each), 3 ml of DMF solution containing 1 ml of DIEA (2 times, 1 minute each), 8 ml of DMF (6 times, 40 minutes each)
Second) and filtered.

Further, 2 mmol of BocThr (Bzl) was added to DCM5
in an amino acid activation vessel.
(M-DCM solution) 2 ml was added and reacted for 5 minutes. Next, the same treatment as in the case of Boc-Pro was performed, DMF was added, and the mixture was concentrated under a nitrogen gas stream, and then transferred to a reaction vessel and reacted for 20 minutes. Then 8 ml of DCM (6 times, 20 times each
Second) and filtered to obtain Boc-Thr (Bzl) -Pro-MBHA resin.

In the following, in order from the C-terminal to the N-terminal, in accordance with the sequence of the above-mentioned target substance, the corresponding protected amino acids are successively coupled and the last protected amino acid is bound, and then the N-terminal Boc group is deprotected. Treatment and subsequent cleaning,
2.2 g of the above compound were obtained. In the solid phase synthesis, Ar
g, Gln, 2 mmol of the corresponding protected amino acid is added to DMF-DCM (3: 1) mixed solvent 4 ml
After adding 2 ml of CC solution and 2 ml of HOBt solution (0.5 M-DMF solution) and reacting for 1 minute, the same treatment as other amino acids is performed, transferred to a reaction vessel and subjected to coupling reaction, washed with DCM, filtered, and then again. DCM in 4 ml of DMF-DCM (3: 1) mixed solvent
Solution 2ml, HOBt solution (0.5M-DMF) 2ml
Was added, and the mixture was reacted for 25 minutes, and transferred to a reaction vessel for coupling, that is, a so-called double coupling method.

The amino acids used in the solid phase method and in the production examples are as follows.

[0036]

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(3) Bonding of the compound obtained in the above (1) and the compound obtained in the above (2) 700 mg of the compound obtained in the above (1) was combined with 4 ml of DMF and N
Methylpyrrolidone is dissolved in a mixed solvent of 2 ml.
1.6 g of the compound obtained in the above (2) was added, and HOBt was added.
0.135 g, and cooled to -15 ° C.
0.192 g of HCl was added and stirred overnight. After completion of the reaction, the mixture was filtered by suction, and 5 ml of DMF and 5 ml of N-methylpyrrolidone 5
mixed solvent 10 ml, DMF 10 ml, DCM 10
ml, and dried under reduced pressure.
I got (4) Production of F04 compound 1.0 g of the compound obtained in the above (3) was transferred to an HF reactor (manufactured by Peptide Research Laboratories) and 2 ml of anisole was added.
, And 20 ml of anhydrous hydrogen fluoride was added thereto.
For 1 hour. After the reaction, anhydrous hydrogen fluoride was distilled off under reduced pressure, and the residue was washed with ether.
0 ml was added to extract the peptide. Extract is Dowe
x 1 × 2 column (2.6 × 15 cm).
It was eluted with 60 ml of 1M acetic acid and freeze-dried. This was purified by reversed-phase high performance liquid chromatography, and gel-filtered with 0.1 M aqueous acetic acid using Sephadex G-25 resin to obtain 4.5 mg. Physical properties Amino acid analysis values Asp 1.97 (2), Thr 3.87 (4), Ser 2.83 (3), Glu 3.13 (3), Pr
o 2.20 (2), Gly 3.95 (4), Ala 1.00 (1), Val 1.91 (2), Leu
6.32 (6), Tyr 0.97 (1), Lys 2.18 (2), His 1.15 (1), Arg
1.12 (1), Asu 1.18 (1) High performance liquid chromatography Retention time 25.5 minutes

[0038]

[Production Example 2]

[0039]

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The compound represented by the above formula B (hereinafter referred to as F05)
(1) Production of fragment (1) Fragment (1) was produced by the method described in Examples 2 (1) to (14) of JP-B-53-41677. (2)

[0041]

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Production Example 1 Production Example 1 as in (2). When binding the changed Leu to the sequence produced in (2), Boc-Le
Solid phase synthesis was performed using Boc-D-Leu.H2O instead of u.H2O to obtain 2.1 g. (3) Binding of the compound obtained in the above (1) and the compound obtained in the above (2) 500 mg of the compound obtained in the above (1) and 1.1 g of the compound obtained in the above (2) are used. Production Example 1. The same procedure as in (3) was carried out to obtain 1.4 g of the target product. (4) Production of F05 compound 1.0 g of the compound obtained in the above (3) was prepared.
Treated in the same manner as in (4), purified by reversed-phase high performance liquid chromatography, and purified using Sephadex G-25 resin.
The mixture was subjected to gel filtration with 1M-acetic acid aqueous solution to obtain 5.6 mg of the above-mentioned target substance. Physical properties Amino acid analysis values Asp 2.02 (2), Thr 3.91 (4), Ser 2.85 (3), Glu 3.13 (3), Pr
o 2.33 (2), Gly 3.09 (3), Ala 1.00 (1), Val 1.93 (2), Leu
5.38 (5), Tyr 0.98 (1), Lys 2.20 (2), His 1.17 (1), Arg
1.07 (1), Asu 1.20 (1) High performance liquid chromatography Retention time 23.5 minutes

[0043]

[Production Example 3]

[0044]

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The compound represented by the above formula C (hereinafter referred to as F08)
(1) Production of fragment (1) Fragment (1) was produced by the method described in Examples 2 (1) to (14) of JP-B-53-41677. (2)

[0046]

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Production of Production Example 1. After reacting and binding Boc-Pro to the MBHA resin in the same manner as in (2), the corresponding protected amino acids are successively coupled according to the sequence of the target product in the order from the C-terminal to the N-terminal. After the ring was coupled with the last protected amino acid, TFA treatment and subsequent washing were performed to deprotect the N-terminal Boc group, thereby obtaining 2.3 g of the above compound. (3) Combination of the compound obtained in the above (1) and the compound obtained in the above (2) 500 mg of the compound obtained in the above (1) and 1.2 g of the compound obtained in the above (2) are used. Production Example 1. The same procedure as in (3) was carried out to obtain 1.4 g of the target product. (4) Production of F08 compound 1.0 g of the compound obtained in the above (3) was prepared.
Treated in the same manner as in (4), purified by reversed-phase high performance liquid chromatography, and purified using Sephadex G-25 resin.
The mixture was subjected to gel filtration with 1M-acetic acid aqueous solution to obtain 8.5 mg of the above-mentioned target substance. Physical properties Amino acid analysis values Asp 1.93 (2), Thr 3.88 (4), Ser 3.61 (4), Glu 5.00 (5), Pr
o 2.02 (2), Gly 3.02 (3), Ala 1.00 (1), Val 1.93 (2), Leu
6.74 (7), Tyr 1.01 (1), Lys 3.33 (3), His 1.03 (1), Arg
1.14 (1), Asu 1.07 (1) High performance liquid chromatography Retention time 23.3 minutes

[0048]

[Production Example 4]

[0049]

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The compound represented by the formula D (hereinafter referred to as F17)
(1) Production of fragment (1) Fragment (1) was produced by the method described in Examples 2 (1) to (14) of JP-B-53-41677. (2)

[0051]

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Production Example 1 Production Example 1 as in (2). When combining the sequence produced in (2) with the changed predetermined Lys, Boc-Ly
Solid phase synthesis using Boc-Lys (Ac) instead of s (Cl-Z) gave 2.1 g. (3) Binding of the compound obtained in the above (1) and the compound obtained in the above (2) 500 mg of the compound obtained in the above (1) and 1.1 g of the compound obtained in the above (2) are used. Production Example 1. The same procedure as in (3) was carried out to obtain 1.4 g of the target product. (4) Production of F17 Compound Production Example 1 was prepared by using 1.0 g of the compound obtained in the above (3).
Treated in the same manner as in (4), purified by reversed-phase high performance liquid chromatography, and purified using Sephadex G-25 resin.
The mixture was subjected to gel filtration with 1M-acetic acid aqueous solution to obtain 4.9 mg of the above-mentioned target substance. Physical property value Amino acid analysis value Asp 1.94 (2), Thr 3.65 (4), Ser 2.56 (3), Glu 3.06 (3), Pr
o 1.96 (2), Gly 2.91 (3), Ala 1.00 (1), Val 1.96 (2), Leu
4.76 (5), Tyr 0.96 (1), Lys 1.95 (2), His 0.97 (1), Arg
0.98 (1), Asu 0.90 (1) High performance liquid chromatography Retention time 28.5 minutes

[0053]

[Production Example 5]

[0054]

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The compound represented by the above formula E (hereinafter referred to as F23)
(1) Production of fragment (1) Fragment (1) was produced by the method described in Examples 2 (1) to (14) of JP-B-53-41677. (2)

[0056]

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Production Example 1 After reacting and binding Boc-Pro to MBHA resin in the same manner as in (2), the same procedure was followed.
In accordance with the sequence of the target substance in the order of the terminal, the corresponding protected amino acids are sequentially coupled and the last protected amino acid is bound. After that, in order to deprotect the N-terminal Boc group, TFA treatment and subsequent washing are performed. 1.6 g of compound were obtained. (3) Bonding of the compound obtained in the above (1) with the compound obtained in the above (2) 500 mg of the compound obtained in the above (1) and 1.0 g of the compound obtained in the above (2) are used. Production Example 1. By carrying out in the same manner as in (3), 1.3 g of the above-mentioned target product was obtained. (4) Production of F23 compound 0.9 g of the compound obtained in the above (3) was produced.
Treated in the same manner as in (4), purified by reversed-phase high performance liquid chromatography, and purified using Sephadex G-25 resin.
The mixture was subjected to gel filtration with 1M-acetic acid aqueous solution to obtain 5.6 mg of the above-mentioned target substance. Physical properties Amino acid analysis values Asp 2.13 (2), Thr 3.88 (4), Ser 1.97 (2), Glu 1.07 (1), Pr
o 1.56 (2), Gly 3.06 (3), Ala 1.00 (1), Val 2.04 (2), Leu
3.10 (3), Tyr 0.92 (1), Lys 1.02 (1), Arg 0.94 (1), Asu
1.16 (1) High performance liquid chromatography Retention time 18.5 minutes

[0058]

[Production Example 6]

[0059]

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The compound represented by the above formula F (hereinafter referred to as F26
(1) Production of fragment (1) Fragment (1) was produced by the method described in Examples 2 (1) to (14) of JP-B-53-41677. (2)

[0061]

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Production Example 1 After reacting and binding Boc-Pro to the MBHA resin in the same manner as in (2), the corresponding protected amino acids are successively coupled according to the sequence of the target product in the order from the C-terminal to the N-terminal. After the ring was coupled with the last protected amino acid, treatment with TFA and subsequent washing were performed to deprotect the N-terminal Boc group, thereby obtaining 2.4 g of the above compound. (3) Binding of the compound obtained in the above (1) and the compound obtained in the above (2) 500 mg of the compound obtained in the above (1) and 1.3 g of the compound obtained in the above (2) are used. Production Example 1. By carrying out in the same manner as in (3), 1.6 g of the above-mentioned target product was obtained. (4) Production of F26 compound 1.0 g of the compound obtained in the above (3) was prepared.
Treated in the same manner as in (4), purified by reversed-phase high performance liquid chromatography, and purified using Sephadex G-25 resin.
The mixture was subjected to gel filtration with 1M-acetic acid aqueous solution to obtain 9.1 mg of the above-mentioned target substance. Physical properties Amino acid analysis values Asp 2.20 (2), Thr 4.94 (5), Ser 2.93 (3), Glu 4.41 (4), Pr
o 2.42 (2), Gly 3.05 (3), Ala 1.00 (1), Val 2.06 (2), Leu
6.19 (6), Tyr 1.66 (2), Lys 3.20 (3), His 2.05 (2), Arg
0.98 (1), Asu 1.15 (1) High performance liquid chromatography Retention time 24.5 minutes

[0063]

[Production Example 7]

[0064]

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The compound represented by the above formula G (hereinafter referred to as F32
(1) Production of fragment (1) Fragment (1) was produced by the method described in Examples 2 (1) to (14) of JP-B-53-41677. (2)

[0066]

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Production Example 1 After reacting and binding Boc-Pro to MBHA resin in the same manner as in (2), the same procedure was followed.
In accordance with the sequence of the target substance in the order of the terminal, the corresponding protected amino acids are sequentially coupled and the last protected amino acid is bound. After that, in order to deprotect the N-terminal Boc group, TFA treatment and subsequent washing are performed. 2.5 g of compound were obtained. (3) Binding of the compound obtained in the above (1) and the compound obtained in the above (2) 500 mg of the compound obtained in the above (1) and 1.1 g of the compound obtained in the above (2) are used. Production Example 1. The same procedure as in (3) was carried out to obtain 1.4 g of the target product. (4) Production of F32 compound 1.0 g of the compound obtained in the above (3) was prepared.
Treated in the same manner as in (4), purified by reversed-phase high performance liquid chromatography, and purified using Sephadex G-25 resin.
The mixture was subjected to gel filtration with 1M-acetic acid aqueous solution to obtain 11.7 mg of the above-mentioned target compound. Physical property value Amino acid analysis value Asp 2.04 (2), Thr 4.62 (5), Ser 3.42 (4), Glu 6.03 (6), Pr
o 3.05 (3), Gly 2.99 (3), Ala 1.00 (1), Val 2.14 (2), Leu
7.71 (8), Tyr 1.61 (2), Lys 3.82 (4), His 1.87 (2), Arg
1.16 (1), Asu 0.97 (1) High performance liquid chromatography Retention time 25.5 minutes

[0068]

[Production Example 8]

[0069]

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The compound represented by the formula H (hereinafter referred to as F48)
(1) Production of fragment (1) Fragment (1) was produced by the method described in Examples 2 (1) to (14) of JP-B-53-41677. (2)

[0071]

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Production Example 1 Production Example 1 as in (2). When combining the sequence produced in (2) with the changed predetermined Lys, Boc-Ly
Solid phase synthesis was performed using Boc-Lys (Ac) instead of s (Cl-Z) to obtain 2.0 g. (3) Binding of the compound obtained in the above (1) and the compound obtained in the above (2) 500 mg of the compound obtained in the above (1) and 1.1 g of the compound obtained in the above (2) are used. Production Example 1. The same procedure as in (3) was carried out to obtain 1.4 g of the target product. (4) Production of F48 compound 1.0 g of the compound obtained in the above (3) was prepared.
Treated in the same manner as in (4), purified by reversed-phase high performance liquid chromatography, and purified using Sephadex G-25 resin.
The mixture was subjected to gel filtration with 1M aqueous acetic acid to obtain 7.8 mg of the above-mentioned target substance. Physical properties Amino acid analysis values Asp 1.98 (2), Thr 3.63 (4), Ser 2.55 (3), Glu 3.09 (3), Pr
o 1.96 (2), Gly 2.89 (3), Ala 1.00 (1), Val 1.94 (2), Leu
4.71 (5), Tyr 0.80 (1), Lys 1.93 (2), His 0.96 (1), Arg
1.00 (1), Asu 0.88 (1) High performance liquid chromatography Retention time 27.7 minutes

[0073]

Example 1 Eight types of elcatonin stabilizers obtained by synthesizing and purifying in Production Examples 1 to 8 were added to an aqueous solution for injection of elcatonin in order to examine the stabilizing effect thereof.
Elcatonin was tested for shaking stability. (1) Preparation of an aqueous solution of elcatonin for injection: distilled water 100
4.63 sodium citrate / 3H ₂ O per 0 ml
g, 0.37 g of anhydrous citric acid and 7.0 g of sodium chloride were added and dissolved to prepare an aqueous solution having a pH of 6.0. Elcatonin was dissolved in this aqueous solution to a concentration of about 10 μg / ml to prepare an aqueous solution of elcatonin for injection. (2) Preparation of injection: 4.63 g of sodium citrate / 3H ₂ O, 0.37 g of anhydrous citric acid, and 7.0 g of sodium chloride were added to and dissolved in 1000 ml of distilled water, and an aqueous solution having a pH of 6.0 was added. Prepared. Eight kinds of elcatonin stabilizers produced in Production Examples 1 to 8 were dissolved in this aqueous solution to a concentration of about 10 μg / ml or about 1.0 μg / ml, respectively, to prepare aqueous solutions of the elcatonin stabilizer. . This aqueous solution of elcatonin stabilizer and the aqueous solution for injection of elcatonin are mixed, and a test solution of an aqueous solution of elcatonin for injection containing 5% or 0.5% of the total amount of one or more stabilizers relative to the amount of elcatonin is prepared. Prepared.
Next, 1 ml of each test solution was filled into a normal glass ampule to prepare about 50 ampules each.

As a control, an ampoule of an aqueous solution of elcatonin for injection containing no stabilizer of the present invention was prepared in the same manner. (3) Stability test: The ampoule of the above test solution was placed in a thermostatic shaker, and the content of elcatonin was measured over time by liquid chromatography to examine the shaking stability. - shaking conditions [amplitude: 10 cm, shaking rate: 180 times / min, temperature: 25 ° C], liquid chromatography measurement conditions [column: ODS column (4.6 × 150 mm), mobile phase: CH ₃ CN @ -
0.1% TFA (1: 2), flow rate: 1 ml / min, detection: UV220 nm] Table 1 shows the residual ratio of each injection for 4 weeks with shaking.

[0075]

[Table 1]

As is evident from Table 1, the ampoule of an elcatonin aqueous solution for injection containing an elcatonin stabilizer of the present invention has improved shaking stability over the control ampoule containing no stabilizer. Was.

[0077]

Example 2 1) Preparation of injection: 4.63 g of sodium citrate · 3H ₂ O, 0.37 g of anhydrous citric acid, and 7.0 g of sodium chloride were added to and dissolved in 1000 ml of distilled water. 0 was prepared. F08 produced in Production Example 3 was dissolved in this aqueous solution to a concentration of about 10 μg / ml. This aqueous solution of F08 and the aqueous solution for injection of elcatonin prepared in Example 1 were mixed, and a test solution of an aqueous solution of elcatonin for injection containing 0%, 1%, 5%, and 10% of F08 relative to the amount of elcatonin was prepared. Prepared.
Next, 1 ml of each test solution was filled into a normal glass ampule to obtain about 100 ampules each. 2) Stability test: Example 1 using the ampoule of the above test solution
In the same manner as described above, the mixture was placed in a thermostatic shaker, and the content of elcatonin was measured over time by liquid chromatography, and the shaking stability was examined. Separately, the ampoule of the above test solution was placed in a thermostat at 40 ° C., and the content of elcatonin was measured over time by liquid chromatography to examine its storage stability. The shaking conditions and liquid chromatography measurement conditions were the same as in Example 1.

Table 2 shows the residual ratio of each injection for 4 weeks with shaking.
Table 3 shows the storage stability at 40 ° C for 6 months.

[0079]

[Table 2]

[0080]

[Table 3]

From the results shown in Tables 2 and 3, the elcatonin injection containing F08, which is the elcatonin stabilizer of the present invention, was excellent in shaking stability and storage stability at 40 ° C.

[0082]

Industrial Applicability The peptide of the present invention can improve the stability of elcatonin in an aqueous solution for injection in a very small amount, and is useful as a stabilizer for an aqueous solution of elcatonin.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-58-148827 (JP, A) JP-A-3-90033 (JP, A) JP-A-5-178894 (JP, A) (58) Field (Int.Cl. ⁷ , DB name) A61K 38/00-38/58 A61K 47/42 A61K 9/08 C07K 14/585 CA (STN) MEDLINE (STN) BIOSIS (STN) REGISTRY (STN) JICST file ( JOIS)

Claims (1)

(57) [Claims] 1. A compound represented by the following formulas A to H: Embedded image Embedded image DOO vibration of elcatonin solution of one or more in selected from the group consisting of peptides represented as an active ingredient
Cormorant stabilizer.