JPH05331071A - Lyophilized composition of calcitonin gene-related peptide and stabilization thereof - Google Patents

Abstract

PURPOSE:To provide a stabilizer capable of giving the CGRP lyophilized composition of high safety, stable for a long period of time. CONSTITUTION:The lyophilized composition characterized by containing (A) as active ingredient, a calcitonin gene-related peptide(CGRP) and (B) as stabilizer, either one of the following ingredients: (1) an effective amount of cyclodextrin, L-lysine, L-glutamine or L-methionine, or (2) an effective amount of sodium chloride and each effective amounts of at least one kind of saccharide selected from among mannitol, sucrose, inositol, sorbitol, maltase, trehalose, dextran, fructose and galactose, or amino acid(s) selected from among glycine, L-alanine, L-cysteine, L-leucine, L-arginine and L-histidine. The other objective stabilization method characterized by dissolving the above composition in an aqueous medium followed by lyophilization.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a freeze-dried composition containing calcitonin gene-related peptides (hereinafter abbreviated as CGRP) as an active ingredient and a method for stabilizing CGRPs.

[0002]

CGRPs are a group of peptides derived from the same gene as calcitonin, although they are structurally different from calcitonin. They are human, chicken, rat,
CGPRs derived from pigs and the like and their derivatives are known.

Human CGRP (h-CGRP) is a bone metabolism,
Known as a peptide that acts on the central nervous system [N
Nature, 308 (19), 746-748 (198).
4), FEBS Letters, 183 (2), 40.
3 (1985), Neuropeptides, 4, 4
25-434 (1984), Nature, 313.
(3), 54-56 (1984)].

Porcine CGRP (p-CGRP) is known as a peptide having a heart rate increasing action [Neur
optides, 9, 75-82 (1987)].
Rat CGRP (r-CGRP) is known as a peptide having vasodilatory action, gastric acid secretion inhibitory action, etc. [British J. Pharmacol. , 86,
544 (1985), Regulatory Pept.
des, 12, 81-89 (1985)].

Further, h-CGRP derivative, chicken CG
RP (c-CGRP) and c-CGRP derivatives are known as peptides having serum calcium and phosphorus lowering activity (JP-A-62-129297, JP-A-63-126894, 63-63). -25849
No. 0, JP 64-26598 A). CGRP
As described above, since various kinds have various physiological actions, a wide range of clinical applications are expected.

[0006]

When a small amount of peptides, proteins and the like are freeze-dried to prepare a preparation, it is essential to add an excipient. Mannitol is most frequently used as this excipient, but when mannitol is used as an excipient for freeze-drying CGRPs, its stabilizing effect is not so recognized. On the other hand, high molecular substances such as gelatin and human serum albumin have been used as stabilizers in freeze-drying. These have a high stabilizing effect but often have problems in immunological safety. Further, it has been reported for some proteins and the like that the stability is improved by adding an amino acid as a stabilizer, but it was not clear about CGRPs.

[0007]

[Means for Solving the Problems] To solve such problems, in order to obtain a safe and stable pharmaceutical formulation, as a result of various studies, surprisingly, it was surprisingly found that saccharides alone were used against CGRPs. Among the uses, only cyclodextrin showed an excellent stabilizing effect.

However, the stabilizing effects of mannitol, maltose and the like, which are commonly used, were not recognized. Also, when glucose, galactose, mannose, etc. are used, a hard and good freeze-dried product cannot be obtained, and it becomes a rugged or unevenly fragile solid substance such as a partly pulled string, and the ampule branches. The cake may have a bad shape. When sorbitol is used, small round solids are dispersed and cling to the bottom, and a uniform cake state is not obtained. When fructose is used, it does not become a powder but is liquefied and a freeze-dried product cannot be obtained. The use of these saccharides alone was not suitable as a stabilizing agent for CGRPs other than cyclodextrin.

Furthermore, it was found that among the amino acids, only L-lysine, L-glutamine and L-methionine have an excellent stabilizing effect. However, almost no stabilizing effect was observed with amino acids such as glycine, L-alanine, and L-leucine used in some protein preparations. Similarly, polymeric substances such as human serum albumin, which are often used in protein and peptide preparations, showed no stabilizing effect only for CGRPs.

Thus, cyclodextrin, L-
Lysine, L-glutamine and L-methionine are CG
It was found that the compound has a specific stabilizing effect on RPs, and by using these compounds as stabilizers for CGRPs, it is safe and excellent in thermal stability.
It has become possible to provide freeze-dried preparations of the same class.

Further, as a result of continuing the research, saccharides such as mannitol, sucrose, inositol, sorbitol, maltose, dextran, fructose, glucose, mannose and galactose alone or glycine, L-alanine, L-leucine as stabilizers were obtained. Amino acids such as L-cysteine, L-arginine and L-histidine alone have a low stabilizing effect, but surprisingly, a certain amount of sodium chloride is mixed with the above-mentioned various sugars or amino acids and freeze-dried. It was found that the stability of CGRPs is remarkably improved. As a result, CG
By using the above saccharides or amino acids in combination with sodium chloride as a stabilizer for the freeze-dried preparation of RPs,
It has become possible to provide a freeze-dried preparation of CGRP which is safe and excellent in heat stability.

That is, the object of the present invention is to use CGRPs as an active ingredient and as a stabilizer. 1) An effective amount of cyclodextrin, L-lysine and L-
Glutamine or L-methionine, or 2) consisting of an effective amount of sodium chloride and an effective amount of one or more of mannitol, sucrose, inositol, sorbitol, maltose, trehalose, dextran, fructose, glucose, mannose and galactose. A saccharide selected from the group or any one of glycine, amino acids selected from the group consisting of L-alanine, L-cysteine, L-leucine, L-arginine and L-histidine, To provide a lyophilized composition.

Further, the objects of the present invention are as CDRPs and stabilizers: 1) Effective amounts of cyclodextrin, L-lysine and L-
Glutamine or L-methionine, or 2) consisting of an effective amount of sodium chloride and an effective amount of one or more of mannitol, sucrose, inositol, sorbitol, maltose, trehalose, dextran, fructose, glucose, mannose and galactose. After dissolving one of a sugar selected from the group or an amino acid selected from the group consisting of glycine, L-alanine, L-cysteine, L-leucine, L-arginine and L-histidine in an aqueous medium And a method for stabilizing calcitonin gene-related peptides, which is characterized by freeze-drying.

First, the CGRPs, which are the active ingredients of the present invention, are CGRP, its derivatives or salts thereof.
CGRP and its derivatives are produced by a known peptide synthesis method, for example, a liquid phase method or a solid phase method. Examples of CGRP include h-α-CGRP, h-β-CGRP, c-
CGRP, r-α-CGRP, r-β-CGRP, p-
CGRP etc. are mentioned.

Examples of the above-mentioned derivatives include desalanyl
-Deamino-h-α-CGRP, desalanyl-deami
No-h-β-CGRP, desalanyl- [Asu^2,7]
-H-α-CGRP, desalanyl- [Asu^2,7] −
h-β-CGRP, [Asn ³, Phe¹⁵, Gly^{twenty three}]
-H-α-CGRP, desalanyl-deamino- [As
n³, Phe¹⁵, Gly^{twenty three}] -H-α-CGRP, [A
sn³, Asp¹⁴, Gly^{twenty three}] -H-α-CGRP, de
Sulanyl-deamino- [Asn³, Asp¹⁴, Gly
^{twenty three}] -H-α-CGRP, [Asn³, Asp¹⁴, Ph
e¹⁵] -H-α-CGRP, desalanyl-deamino-
[Asn³, Asp¹⁴, Phe¹⁵] -H-α-CGR
P, [Asp¹⁴] -H-α-CGRP, desalanyl-
Deamino- [Asp¹⁴] -H-α-CGRP, [Asn
³, Glu¹⁴, Gly^{twenty three}] -H-α-CGRP, Desua
Ranyl-deamino- [Asn³, Glu¹⁴, Gly^{twenty three}]
-H-α-CGRP, [Asn³, Glu¹⁴, Ph
e¹⁵] -H-α-CGRP, desalanyl-deamino-
[Asn³, Glu¹⁴, Phe¹⁵] -H-α-CGR
P, [Glu¹⁴] -H-α-CGRP, desalanyl-
Deamino- [Glu¹⁴] -H-α-CGRP, death ara
Nil- [Asu^2,7] -C-CGRP, desalanyl-
[Asp³, Asu^2,7] -C-CGRP, Death Alani
Ru-deamino-c-CGRP, desalanyl-deamino
-[(4-F-Phe)³⁷] -C-CGRP and the like
Be done.

The above-mentioned CGRP derivative can be produced, for example, by
JP-A-62-129297, JP-A-63-12689
4, JP-A-63-258490, JP-A-64-26
No. 598 and the like. As the salt of CGRP or a derivative thereof, a pharmacologically non-toxic salt is appropriately used. Examples thereof include salts with inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid, and salts with organic acids such as acetic acid, tartaric acid, succinic acid, citric acid and malic acid.

The CGRPs used in the present invention may be used in such an amount that they exhibit a physiologically active action effective as a drug, and usually 0.1 to 500 μg per dose.
It may be prepared to a certain extent.

As a stabilizer for CGRPs, cyclodextrin, L-lysine, L-glutamine or L-
Methionine may be used alone, or two or more kinds may be used in combination, or sodium chloride and saccharide or amino acid may be used in combination.

Examples of the above-mentioned saccharides include mannitol, sucrose, inositol, sorbitol, maltose, trehalose, dextran, fructose,
Examples thereof include glucose, mannose, galactose, etc. The saccharides may be used alone or in combination of two or more kinds. Examples of the above amino acids include glycine, L-alanine, L-cysteine, L-leucine, L
-Arginine, L-histidine, etc. are mentioned, and these amino acids may be used alone or in combination of two or more kinds. Furthermore, the saccharide and the amino acid may be used in combination.

The amount of the above stabilizer added is preferably about 0.2 to 100,000 parts by weight, more preferably about 2 to 10,000 parts by weight, relative to 1 part by weight of CGRP. The amount of sodium chloride added is preferably about 0.001 to 5 parts by weight, more preferably about 0.01 to 1 part by weight, based on 1 part by weight of the saccharide or amino acid. Examples of the aqueous medium include distilled water for injection and physiological saline. Further, the above-mentioned aqueous solvent may contain a water-soluble organic solvent, for example, a small amount of ethanol etc., as long as it does not show toxicity.

To produce the freeze-dried composition of the present invention,
For example, if necessary, a known pH adjusting agent, isotonicity agent, stabilizer, bulking agent, preservative, etc. are mixed with the CGRPs and stabilizer having the above composition and dissolved in the above aqueous medium. Then, it is subjected to aseptic filtration and freeze-dried according to a conventional method. For this freeze-drying, known freeze-drying methods such as tray freeze-drying and vial freeze-drying can be adopted under the conditions usually used.

[0022]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. The CGRPs used in the examples were all synthesized by Asahi Kasei Co., Ltd. according to the above-mentioned known documents.

Example 1 1 mg of desalanyl-deamino-c-CGRP and the stabilizer shown in Table 1 below were taken, and 50 ml of sterile distilled water was added and dissolved. After aseptic filtration, 0.5 ml each was dispensed into a glass ampoule, freeze-dried, replaced with nitrogen, and then sealed to obtain a freeze-dried preparation for dissolution before use.

[0024]

[Table 1]

Example 2 CGRPs and a stabilizer were mixed in the composition shown in Table 2 below, dissolved in 20 ml of sterile distilled water, and then treated in the same manner as in Example 1 to obtain freeze-dried preparations. ..

[0026]

[Table 2]

Example 3 Desalanyl-deamino-c-CGRP 1 mg, D-mannitol 500 mg and sodium chloride 100 mg
Then, 50 ml of sterile distilled water was added and dissolved. After aseptic filtration, 0.5 ml each was dispensed into a glass ampoule, freeze-dried, replaced with nitrogen, and then sealed to obtain a freeze-dried preparation that was dissolved before use (invention product 5).

Example 4 In Example 3, the sugars or amino acids shown in Table 3 below were used in place of D-mannitol, and sodium chloride was treated in the same manner using the amounts shown in Table 3 to obtain freeze-dried preparations. (Invention products 6 to 17).

[0029]

[Table 3]

Example 5 0.5 mg of h-α-CGRP and 250 mg of D-mannitol
mg and 50 mg of sodium chloride were taken, and 25 ml of sterile distilled water was added and dissolved. After aseptic filtration, 0.5 ml each was dispensed into glass ampoules, freeze-dried, replaced with nitrogen, and then sealed to obtain a soluble preparation at the time of use.

Example 6 CGRPs, sugars or amino acids and sodium chloride were mixed in the composition shown in Table 4 below, and sterile distilled water 20 was added.
After dissolving in ml, the same treatment as in Example 5 was carried out to obtain freeze-dried preparations.

[0032]

[Table 4]

Example 7 10 mg of desalanyl-deamino-c-CGRP and D-
1 g of mannitol and 100 mg of sodium chloride were taken, and 50 ml of sterile distilled water was added and dissolved. After aseptic filtration, 0.5 ml each was dispensed into a glass ampoule, freeze-dried, replaced with nitrogen, and then sealed to obtain a freeze-dried preparation that was dissolved before use.

Example 8 50 mg of desalanyl-deamino-c-CGRP and D-
1 g of mannitol and 100 mg of sodium chloride were taken, and 50 ml of sterile distilled water was added and dissolved. After aseptic filtration, 0.5 ml each was dispensed into a glass ampoule, freeze-dried, replaced with nitrogen, and then sealed to obtain a freeze-dried preparation that was dissolved before use.

[0035]

【The invention's effect】

CGRP Stabilization Test [Preparation of Control Formulation] (1) 1 mg of desalanyl-deamino-c-CGRP was taken and dissolved in 50 ml of sterile distilled water. After sterile filtration,
Dispense 0.5 ml each into a glass ampoule, lyophilize, replace with nitrogen, and close by melting to obtain a freeze-dried preparation for dissolution before use (control product 1). (2) Take 1 mg of desalanyl-deamino-c-CGRP and 1000 mg of D-mannitol, and use sterile distilled water 50
It was dissolved in ml. Thereafter, the same treatment was carried out to obtain a freeze-dried preparation that was dissolved before use (control product 2). (3) 1 mg of desalanyl-deamino-c-CGRP and 1000 mg of maltose were taken and dissolved in 50 ml of sterile distilled water. Thereafter, the same treatment was carried out to obtain a dissolution-type freeze-dried preparation at the time of use (control product 3).

(4) Desalanyl-deamino-c-CG
1 mg of RP and 1000 mg of glycine were taken and dissolved in 50 ml of sterile distilled water. Thereafter, the same treatment was carried out to obtain a dissolution-type freeze-dried preparation at the time of use (control product 4). (5) Take 1 mg of desalanyl-deamino-c-CGRP and 1000 mg of L-alanine, and use 50 ml of sterile distilled water.
Dissolved in. Thereafter, the same treatment was carried out to obtain a dissolution-type freeze-dried preparation before use (control product 5). (6) Take 1 mg of desalanyl-deamino-c-CGRP and 100 mg of human serum albumin, and use sterile distilled water 50
It was dissolved in ml. Thereafter, the same treatment was carried out to obtain a dissolution-type freeze-dried preparation at the time of use (control product 6).

(7) Desalanyl-deamino-c-CG
1 mg of RP and 1000 mg of sodium chloride were taken and dissolved in 50 ml of sterile distilled water. Thereafter, the same treatment was carried out to obtain a dissolution-type freeze-dried preparation at the time of use (control product 7). (8) Take 1 mg of desalanyl-deamino-c-CGRP and 500 mg of D-mannitol, and use sterile distilled water 50 m.
It was dissolved in 1. Thereafter, the same treatment was performed to obtain a freeze-dried preparation that was dissolved before use (control product 8). (9) Desalanyl-deamino-c-CGRP 1 mg, D-mannitol 500 mg and glycine 200 mg
It was taken and dissolved in 50 ml of sterile distilled water. Thereafter, the same treatment was carried out to obtain a dissolution-type freeze-dried preparation before use (control product 9).

[Test Method] Each of the products 1 to 17 of the present invention and the products 1 to 9 of the controls obtained in the above examples were stored at 60 ° C. for 3 months, and then subjected to high performance liquid chromatography (HPLC) under the following conditions. Was used to measure the content of CGRPs, and the content immediately after the sealing was set to 100% to determine the residual rate.

HPLC measurement conditions Column: YMC AM-302 ODS S-5 12
0Å (manufactured by YMC) Inner diameter 4.6 × 150 mm Mobile phase: 0.1% TFA: acetonitrile = 69: 31 Flow rate: 0.7 ml / min Detection: 220 nm [Test result] Remaining rate of CGRPs of each sample is shown. 5 shows.

[0040]

[Table 5]

As shown in the results in Table 5, saccharides other than cyclodextrin, for example, mannitol, maltose, and the like, which were added alone as a control product, amino acids other than L-lysine, L-glutamine, and L-methionine, such as glycine and L, were used. -The stability of CGRP is remarkably reduced in the control product in which alanine and the like are added alone, the above-mentioned saccharides and amino acids in combination, for example, the control product in which mannitol and glycine are added in combination, and the control product in which sodium chloride is added alone. In contrast, cyclodextrin, L-lysine, L
-The product of the present invention added with glutamine and L-methionine alone exhibits excellent stability, and the product of the present invention in which the above saccharides or amino acids and sodium chloride are used in combination exhibits excellent stability. The combined effect of sodium chloride was observed.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display area A61K 9/14 L 7329-4C E 7329-4C B 7329-4C 47/02 J 7433-4C 47 / 18 J 7433-4C 47/26 J 7433-4C

Claims (2)

[Claims] 1. A calcitonin gene-related peptide as an active ingredient and as a stabilizer, 1) An effective amount of cyclodextrin, L-lysine, L-
Glutamine or L-methionine, or 2) a group consisting of an effective amount of sodium chloride and an effective amount of one or more kinds of mannitol, sucrose, inositol, sorbitol, maltose, trehalose, dextran, fructose, glucose, mannose and galactose. Sugars or glycine selected from
Lyophilization of calcitonin gene-related peptides, characterized in that it contains any one of amino acids selected from the group consisting of L-alanine, L-cysteine, L-leucine, L-arginine and L-histidine. Composition. 2. As a calcitonin gene-related peptide and a stabilizer, 1) an effective amount of cyclodextrin, L-lysine, L-
Glutamine or L-methionine, or 2) a group consisting of an effective amount of sodium chloride and an effective amount of one or more kinds of mannitol, sucrose, inositol, sorbitol, maltose, trehalose, dextran, fructose, glucose, mannose and galactose. Sugars or glycine selected from
After dissolving any one of amino acids selected from the group consisting of L-alanine, L-cysteine, L-leucine, L-arginine and L-histidine in an aqueous medium,
A method for stabilizing calcitonin gene-related peptides, which comprises freeze-drying.