JPH01156925A - Freeze-dried drug composition of neocartinostatin derivative - Google Patents

Abstract

PURPOSE:To obtain the title preparation such as carcinostatic agent suitable as an injection storable at room temperature, by adding sugars selected from monosaccharide, disaccharide and dextran as a thermal stabilizing agent to a neocartinostatin derivative having a styrene-maleic acid copolymer residue. CONSTITUTION:The objective freeze-dried drug composition having excellent thermal stability can be produced by adding one or more sugars selected from monosaccharide (e.g., glucose or galactose), disaccharide (e.g., lactose, saccharose or maltose) and dextran (especially preferably disaccharide) to a neocartionostatin derivative having styrene-maleic acid copolymer residue (abbreviated as SMANCS: producible by adding 2mol of partially half-esterified styrene-maleic acid copolymer to 1mol of neocartionostatin which is a carcinostatic protein through an acid amide bond: having excellent carcinostatic action, low toxicity and prolonged action). The amount of the sugar is preferably 10-50mg per 1mg (titer) of SMANCS.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a lyophilized pharmaceutical composition of a neocarzinostatin derivative (hereinafter abbreviated as SMANC8) having a styrene-maleic acid copolymer residue.

(Prior art and problems) SMANC8 (Smanx) is a protein anticancer substance neocarcinostatin (hereinafter abbreviated as NC3, Japanese Patent Publication No. 42-21752) that undergoes partial half-esterification of two molecules per one molecule. Styrene maleic acid copolymer (hereinafter SMA)
It is abbreviated as ) is an NC8 derivative synthesized by linking with an acid amide bond (JP-A-60-75432 and JP-A-60-75499), and it has the same excellent anticancer effect as NC8 and has a higher anticancer effect than NC3. It is an excellent anti-cancer substance with reduced toxicity and increased durability.

When applying this SMANCS to humans as an injection,
Since SMANCS itself is a type of peptide, it is preferably freeze-dried and prepared into a preparation that is dissolved in an aqueous solvent before use.

However, even in the freeze-dried state, SMANCi9 is stable in a cold place (below 5°C, stored in a refrigerator) but unstable at room temperature.
This has been a major obstacle in supplying C8 as a pharmaceutical product. Therefore, there has been a demand for the development of a freeze-dried formulation of SMANC8 that has high stability against heat and can be stored even at room temperature.

On the other hand, it is known that physiologically active peptides such as enzymes, hormones, and lymphoids are unstable even in an aqueous solution state or even in a freeze-dried state, and lose their physiological activity significantly. Preventing the loss of physiological activity of such peptides,
Addition of a stabilizer is widely used as a method of increasing its stability.

For example, as a stabilizer in operations such as preservation, separation and purification of tumor necrosis factor, and freeze-drying, D-glucose, D-glucose,
-1j Lactose, D-xylose, D-glucuronic acid, trehalose, textolan, hydroxyethyl starch, etc. (Japanese Unexamined Patent Publication No. 59-59625), and polysaccharides such as dextran are known as interferon stabilizers. (Japanese Unexamined Patent Publication No. 60-59000).

Various heat stabilizers have been studied for this interferon, such as glycerin and sucrose (Japanese Patent Application Laid-Open No. 59-25333).

Dextran, hydroxyethyl starch (JP-A-60-1
55136), glycine, α-alanine and its salts, and human serum albumin (Japanese Patent Application Laid-Open No. 58-1465)
No. 04), glutamic acid (JP-A-59-18122)
4), inorganic salts, especially sodium chloride (Japanese Unexamined Patent Publication No. 5
9. -25364) A4 is known. Furthermore, mannitol is also commonly used as a stabilizer and excipient for ordinary freeze-dried preparations.

However, as mentioned above (SMANCS is an NC3 derivative in which the polymer SMA is bonded to the macromolecular bioactive peptide NC3, and is a very special anticancer substance that has both peptide and polymer properties. Therefore.

SMANCS
It cannot be used as is.

In fact, the present inventors used glycine, which is known as a stabilizer for peptides, serum albumin, or mannitol, which is known as a stabilizer or excipient for lyophilized preparations, to
Attempts were made to prepare lyophilized formulations of MANCS, but no improvement in stability was observed in any of them.

(Another Means to Solve the Problem) The inventors of the present invention have conducted intensive research on a freeze-dried formulation composition of SMANCS that is stable against heat.

Conventional 1 For example, lactose, which is known as an excipient for tablets and capsules, is surprisingly effective in SMANCS in a freeze-dried state.
It has been found that this material has a remarkable effect on stabilizing the material against heat. Furthermore, the present inventors have found that stabilization of C8 to SM8 can be similarly achieved by other disaccharides, monosaccharides, dextran, etc. other than lactose! The present invention was completed.

That is, the two present inventions are (a) sMANes and (b)
This invention relates to a freeze-dried pharmaceutical composition comprising 1 fff1 or more of a saccharide selected from the group consisting of monosaccharides, disaccharides, and dextran.

Here, SMANCS refers to the following formula (N [wherein, (from the primary amino group in the alanine residue at the 1st position and the primary amino group in the norizine residue at the 20th position of neocarzinostatin) The bond of one remaining atom that has a bond with the neocarzinostatin residue in which the hydroxyl group has been removed from the neocarti of [i] is It means that it binds to carcinostatin residue); It means maleic acid residue and half-esterified male number] (-4 alkanol, the alkyl group has 1 or 2 carbon atoms) It is an alcohol residue obtained by removing the hydroxyl group from an ethylene glycol monoalkyl ether, or a glycerin dialkyl ether whose alkyl group has 1 or 2 carbon atoms. (Means an optional styrene-maleic acid copolymer residue.) This is a neocarzinostatin derivative represented by the following.

Details of SMANCS are described in JP-A No. 60-75432 and JP-A No. 60-75499, but a typical example is styrene which may be half-esterified in SMA. A compound in which the maleic acid copolymer residue is a half-butyl esterified styrene-maleic acid copolymer residue (hereinafter abbreviated as Bu-SMANC8).

).

In addition, monosaccharides include glucose, galactose, fructose, mannose, rhamnose, etc., trisaccharides include lactose, saccharose, maltose, etc., and dextran preferably has an average molecular weight of about 10,000 to 80,000, e.g. Dextran J0.40.70 (average molecular weights are] 10,000, 40,000, and 70,000, respectively). Among these, disaccharides such as lactose, saccharose, and maltose are preferably used. What is the amount of these sugars added?

1.0 mg or more per SMANCSI mg (potency),
Preferably it is about 1101T1 to 50111g.

Here, although the effect of sugars on the thermal stability of SMANCS in a freeze-dried state is not yet determined, it is speculated as follows.

In order to stably maintain the activity of SMANCS, the higher-order structure of the peptide moiety must be stably maintained.
Lyophilization of SMANCS results in the formation of hydrophobic bonds that are involved in maintaining the conformation of the peptide moiety.

Since the hydration water present around the peptide is removed, the structure of the peptide becomes unstable.

Sugar molecules have a strong ability to retain hydration water even during freeze-drying, and the sugar molecules themselves coordinate around peptide molecules instead of hydration water, forming a cohesive hydration layer. be able to. Due to such a mechanism, when saccharides coexist even in the freeze-dried state, the hydrophobic bond of the peptide portion of SMANCS is maintained, and its higher-order structure is thought to be stably maintained.

As is clear from the following experimental examples, among the monosaccharide, disaccharide and dextran of the present invention, disaccharide particularly showed a strong stabilizing effect. This is considered to be because, in the molecular structure of the trisaccharide domain, the conformation of the hydroxyl group is optimal for interaction through hydrogen bonding with the peptide portion of SMANCS.

(Method for producing the composition of the present invention) To produce the composition of the present invention, 1. For example, add SMA to the buffer solution.
After dissolving the NCS, adding saccharides and adjusting the pH, the solution is sterilized using a conventional method and then freeze-dried. Here, the saccharide may be added to the buffer solution in advance before dissolving SMANCS, or may be added at the same time as SMANCS. As a buffer, phosphate buffer (
For example, phosphoric acid-sodium hydrogen-dihydrogen phosphate) I
Jum solution), Tris-hydrochloric acid buffer, glycine-sodium hydroxide solution, ammonium carbonate buffer, etc., but phosphate buffer is preferably used. The pH of the buffer solution is between 7.5 and 95. Preferably it is about 8.5.

Compositions of the invention include physiologically compatible and SMANC
Conventional tonicity agents, soothing agents, excipients, etc. that do not inhibit the activity of S can also be added as appropriate. Sodium chloride is used as an isotonic agent, and benzyl alcohol is used as an analgesic agent.

Examples include xylocaine, procaine, etc., and excipients include polyvinylpyrrolidone.

Adjust the pH of the solution thus obtained using hydrochloric acid, sodium hydroxide, etc. (preferably p) 18
．． 0±0.1), filtered and sterilized, and then freeze-dried to obtain the composition of the present invention.

Usually, the above operation is performed under light-shielded conditions, and preferably at low temperatures (
below 5°C).

Next, when the lyophilized preparation of the present invention is used as an injection preparation, the lyophilized composition is dissolved in an isotonic agent such as physiological saline or glucose injection before use. The pH of the reconstituted composition ranges from about 7.5 to 9.5, preferably from about 7.5 to 8.5.

If p) of the solution is less than 7.0, S in the solution
The acid amide bond between NC8 and SMA in the MANCS molecule is susceptible to hydrolysis, and the pII of the solution is 9.5.
If it is larger than , the biological activity of SMANCS may decrease, which is not preferable.

(Effects of the Invention) The freeze-dried pharmaceutical composition of the present invention has excellent thermal stability. Therefore, the composition of the present invention is based on SMANCS, which is an anticancer drug.
Sterile solutions containing as an active ingredient.

In particular, it is of great significance in that it further increases the filli value for the use of SMANCS as an injection.

Next, experimental examples will be listed to specifically explain the effects of the composition of the present invention.

Note that all the experiments described below were conducted in a dark place with an illumination intensity of 200 Lux or less.

Experimental example 1 (SMANCS temperature stability test) nu-
3MANC8 solution M (contains 2.omg (potency) of 11u-3MANC3 in 1m9P, 1780M ammonium carbonate buffer i pH 8,5) in 1 vial. Dispense in Omt portions.

A lyophilized formulation was prepared. i.e. nu in one vial
-8MANC34, OIng C titer).

For this preparation, 5℃, 15℃, 25℃, 40"C
And storage stability at 50°C was investigated.

In addition, the titer of 13u-3MANC3 is Micr
The antibacterial activity against Ococcus luteus ATCC9341 was determined by measuring by the cylindrical plate method described in 1 Japan Antibiotic Pharmaceutical Standards.

The results were expressed in terms of residual activity rate (%) and are shown in Table 9.

The titer at the start of standing was defined as 100%.

Table I Stability of SMANC8 When stored at 5°C, residual activity was 97% after 12 weeks, and the activity remained almost stable.

On the other hand, when stored at 15°C, residual activity decreased to 87% after 12 weeks, and when stored at 25°C, residual activity decreased to 76% after 8 weeks.
63% after 4 weeks when stored at 40°C, 4% when stored at 50°C
It dropped to 39% in a week.

In other words, the activity of Bu-8MANC8 is
It remained stable when stored in the refrigerator (refrigerated), but the stability decreased markedly at high temperatures. Stores at room temperature for about 2 months.

Approximately 25% was shown to be inactivated.

Experimental Example 2 (Comparative test of various stabilizers on stabilization of SMANC8) The storage stability of the composition of the present invention was investigated in comparison with a control product.

The compositions of the present invention used in this experiment were those obtained in Examples 1 to 7. In addition, as a control and a comparative example, those obtained in the preparation examples described later were used. Each of these compositions contains nu-3MANC34 in one vial.
mg (potency) and 5.56 mg of sodium phosphate.

25°C and 40°C for these lyophilized formulations
The storage stability was investigated over a period of 12 weeks. Also,
The titer of SMANCS is Micrococcus
The antibacterial activity against 1 uteus ATCC 9341 was determined by measuring by the cylindrical plate method listed in the Japanese Antibiotic Pharmaceutical Standards.

The results were expressed as activity residual rate (%) and shown in Table 1 (25°C) and Table 3 (40°C). For each formulation, the titer at the start of leaving was set as 100%.

Table 2 Stabilizing effect of SMANCS by various additives (temperature 25°C) Table 3 Stabilizing effect of SMANCS by various additives (temperature 40°C) As a result, the residual activity of the control was 73% after being left at 25°C for 12 weeks However, the residual activity of the formulation containing monosaccharide, disaccharide, and dextran was maintained at a high value, and a remarkable stabilizing effect was observed.

The degree of stabilization increases in the order of dextran, monosaccharide, and disaccharide, and the residual activity of the formulation containing disaccharide is 25°C.
It was almost 100% when left for 12 weeks, and over 95% even when left at 40°C for 12 weeks.

In addition, if the decomposition reaction mechanism of the test substance is a pure first-order reaction of tb, the condition of leaving it at 40°C for 12 weeks is 2 weeks.
Since 48 weeks at 0° C. corresponds to the condition of being left for about 1 year, this was set as a standard for evaluating stability for about 1 year at room temperature.

From the above results, among the saccharides, polyhydric alcohols, neutral amino acids, and human serum albumin, which are generally known as stabilizers for peptides, only the monosaccharide, disaccharide, and dextran of the present invention specifically inhibit SMANCS. It was found that it stabilized.

The following solutions were dissolved in the control and comparative sodium salt buffers (pH 8.0) used in this experiment, adjusted to pH 8.0 ± 0.1 with 0.2N hydrochloric acid and 0.2N sodium hydroxide, and then further Add .01 MIJ acid sodium salt buffer (p Ll 8.0) and make @final WffL 100
mA. Filter sterilize this solution and add 4 m to 1 vial.
1 (Ilu-8 MANCS 34'11 g (titer)) was dispensed and lyophilized to obtain a lyophilized Bu-S MANCS formulation composition.

(2) Comparative Example I Ru-3MANC3 solution (contains z, omg (potency) of 13u-SMANC8 in 1mt.0.OIM sodium phosphate buffer it! pH8,0) in 50 ml,
Add 2.0 g of glycine, 0.2 N hydrochloric acid and 0.0 g.
After adjusting the pH to 8.0±0.1 with 2N sodium hydroxide, 0.01 M sodium phosphate buffer (pH 8.0) was added.

The final liquid volume was 100 mA. This solution was sterilized by FA,
4 mt (Bu-8MANC84mg) per vial
After dispensing (titer)), lyophilize +Bu-S M
A NCS-glycine lyophilized formulation composition was obtained.

(3), Comparative Example 2, Mannitol 2 instead of glycine used in Comparative Example 1
．． 0g was added and treated in the same manner as in Comparative Example 1.

A lyophilized Bu-13MANC3-mannitol formulation composition was obtained.

(4) Comparative Example 3 Instead of glycine used in Comparative Example 1, human serum Alp,
Add 0.2 g of mint and treat in the same manner as in Comparative Example 1,
A Bu-3MANC3-human serum albumin lyophilized formulation composition was obtained.

For clinical application of the composition of the present invention to humans, this composition can be dissolved at the time of use to form an injectable preparation.

It is administered intravenously or subcutaneously to patients with cancer, the elderly, and patients with gastrointestinal abnormalities who are unsuitable for oral administration. Administration is 1-3m at a time
It is desirable to administer 1 to 3 times a day. Target cancer types can be used for solid tumors as well as tL cancers (leukemia). Ascites again.

It can also be used for tumors in prepared water.

(Example) The composition of the present invention will be specifically explained below with reference to Examples, but the composition of the present invention is not limited by this practical side chain.

All adjustments were made in a cool, dark place with a temperature of 5°C or less and an illuminance of 200 Lux or less.

Example 1 11u-SMANC8 solution Q (2.0 m in 1 ml)
g (titer) of Bu-3MANC3. 0. OI
M Sodium phosphate salt slow extraction solution, pH 8,0) 50m
Add 2.0 g of glucose to Z.

p with 0.2N hydrochloric acid and 0.2N sodium hydroxide
After adjusting H to 8.0±0.1, further adjust it to 0. Add OIM sodium phosphate buffer (p) (s, o) to a final volume of 1o.

ml. This solution was sterilized by filtration, and 4
ml (Bu-SMANCS4lTlg (titer)) was dispensed and lyophilized to obtain a lyophilized Bu-3MANC3-glucose preparation composition.

Examples 2 to 17 In Example 1, the following compounds were added in place of glucose, and treated in the same manner as in Example 1, containing monosaccharides, disaccharides, and dextran in the following composition ratios.
A lyophilized formulation composition of ANCS was obtained.

All of these compositions contain Bu-3 per vial.
Contains 34 mg (potency) of MANC.

Examples 18 to 22 A freeze-dried pharmaceutical composition containing Bu-8MANC8 with the following composition was obtained by processing in the same manner as in Example 1.

Patent applicant Yamanouchi Pharmaceutical Co., Ltd. Agent: Patent Attorney Kiyoya Fujino

Claims (5)

[Claims] (1) (a) Neocarzinostatin derivatives having styrene-maleic acid copolymer residues (hereinafter abbreviated as SMANCS); and (b) one or more types selected from the group consisting of monosaccharides, disaccharides, and dextran. A lyophilized pharmaceutical composition comprising a saccharide and a saccharide. (2) SMANCS has the following formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ is the primary amino group in the alanine residue at the 1st position of neocarzinostatin and Means a divalent neocarzinostatin residue obtained by removing one hydrogen atom from each primary amino group in the lysine residue at position 20, ▲ mathematical formula, chemical formula,
There are tables, etc. ▼ indicates the removal of the hydroxyl group from one carboxyl group in the styrene residue (▲ there are mathematical formulas, chemical formulas, tables, etc. ▼); maleic acid residues (▲ there are mathematical formulas, chemical formulas, tables, etc. ▼). One residue (▲There are mathematical formulas, chemical formulas, tables, etc.▼) that has a bond with the neocarzinostatin residue in the form of ); and maleic acid residues (▲mathematical formulas, chemical formulas, tables, etc.▼), or maleic acid residues and half-esterified maleic acid residues (▲ There are mathematical formulas, chemical formulas, tables, etc.▼,
In the formula, R is an alkanol having 1 to 4 carbon atoms, an ethylene glycol monoalkyl ether having an alkyl group having 1 or 2 carbon atoms, or an alcohol obtained by removing the hydroxyl group from glycericin alkyl ether having an alkyl group having 1 or 2 carbon atoms. It is a residue. ); means a monovalent styrene-maleic acid copolymer residue which may be partially half-esterified. ] The claim is a neocarzinostatin derivative represented by (
1) The composition described. (3) Claim (2) wherein the monovalent partially half-esterified styrene-maleic acid copolymer residue is a monovalent partially half-butyl-esterified styrene-maleic acid copolymer residue.
). (4) The composition according to claim (1), wherein the monosaccharide is glucose or galactose. (5) The composition according to claim (1), wherein the disaccharide is lactose, saccharose, or maltose.