JP3041627B2 - Stable compositions of anthracycline antibiotics - Google Patents

Description

The present invention relates to a stabilized pharmaceutical composition of an anthracycline antibiotic.

2. Description of the Related Art Anthracycline antibiotics having an amino sugar are widely used as therapeutic agents for malignant tumors (for example, Overview of Antibiotics [Third Edition Supplement], PP 156-165, University of Tokyo Press, 198).
4 years), but their use is limited due to cumulative cardiotoxicity. In addition, since these anthracycline antibiotics have a small molecular weight, they disappear quickly even after administration into blood vessels, and there is a problem in that there is little chance of being taken into a tumor. To solve this problem, anthracycline antibiotics were used in microspheres,
Various formulations have been proposed which release these drugs over a period of one to two days, one week, one month or several months by being encapsulated in microcapsules or liposomes. However, the stability of the encapsulated anthracycline antibiotics in the preparation and in vivo, or in the formulation process, is not always good.
There are few reports that have been studied in detail, but there is a sufficient concern that the degradation of drug efficacy due to degradation.

JP-A-63-215633 discloses a solution of doxorubicin or 4'-epi-doxorubicin in which the pH is adjusted.
It is disclosed that it can be stabilized by adjusting to 2.5 to 4.0. However, long-term pH control with various release controlling agents is difficult, and stabilization at a pH close to the in vivo pH is desired in consideration of administration to living organisms.

Problems to be Solved by the Invention When anthracycline antibiotics are administered to living organisms as microspheres, microcapsules, and liposomes, there are many factors that depend on interactions with the natural functions of living organisms. It is multifaceted.
In addition, since it relates to pharmaceuticals, it is required to provide a formulation that satisfies these various requirements as much as possible.

Considering the above situation, it is thought that the stability of the encapsulated anthracycline antibiotics is not ensured in some known anthracycline antibiotics, and a satisfactory effect is technically achieved. Is not good.

For example, in the case of microsphere (microcapsule) preparations that release doxorubicin continuously for about one month, the release is determined by a quantitative method that measures the fluorescence as the sum of doxorubicin itself and doxorubicin-derived products And does not necessarily mean that unmodified doxorubicin is released continuously. In other words, there may be no continuous release of effective drug over the required period. Furthermore, in the case of heating or stabilizing the wall substance of the microcapsule in the preparation step, when the reaction is carried out with a crosslinking agent or the like, there is a concern that such anthracycline antibiotics may also react and change. In addition, during storage of a solution of a doxorubicin preparation, doxorubicin is decomposed, and its amount may be significantly different from that in the initial stage, which may cause practical difficulties as a pharmaceutical.

Means for Solving the Problems In view of such circumstances, the present inventors have conducted intensive studies on stabilization of an anthracycline antibiotic having an amino sugar. It has been found that the stability of anthracycline antibiotics can be improved by adding a water-soluble polyanion to form an ion complex, and based on this, the present inventors have further studied and completed the present invention.

That is, the present invention provides a composition comprising an anthracycline antibiotic having an amino sugar and a water-soluble polyanion having an interaction of 40% or more with the anthracycline antibiotic.

The anthracycline antibiotic having an amino sugar used in the present invention refers to an anthracycline skeleton having a basic amino sugar moiety exhibiting a positive charge on the anthracycline skeleton.

Examples of the anthracycline antibiotics include doxorubicin (adriamycin), 4'-epi-doxorubicin, 4'-desoxy-doxorubicin, 4'-desoxy-4'-iodo-doxorubicin, daunorubicin (daunomycin) and 4-demethoxy. -Daunorubicin,
Aclarubicin and the like.

The anthracycline antibiotic may form a salt, and examples of the salt include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, succinic acid, tartaric acid, ascorbic acid, and citric acid. And physiologically acceptable salts with organic acids such as glutamic acid, benzoic acid, methanesulfonic acid and ethanesulfonic acid.

The water-soluble polyanion used in the present invention refers to a natural or synthetic polymer having a molecular weight of 5 to 2,000,000 and having an acidic residue such as sulfuric acid or sulfonic acid exhibiting a negative charge in the molecule.

Examples of natural polymers include polysaccharides, and those having 1.5 to 3 sulfuric acid residues per repeating unit sugar are preferable. Examples of synthetic polymers include polystyrene and polyvinyl alcohol, and 0.2 to 1.0 sulfuric acid per unit monomer. Those having a residue or a sulfonic acid residue are preferred.

Examples of natural polymer polyanions include dextran sulfate, keratan sulfate, heparan sulfate, pullulan sulfate, fucoidan, heparin, and sulfates of curdlan or its partially hydrolyzed products (see Japanese Patent Application No. 63-182188). Can be

The polyanion preferably forms a salt,
Examples include physiologically acceptable inorganic salts with sodium, potassium, lithium, ammonium and calcium.

The interaction of a polyanion with an anthracycline antibiotic is the ratio of the weight of an anthracycline antibiotic at which a polyanion can form an ion complex per unit weight (see Example 1). Preferably it is ~ 80%.

These polyanions may be used alone or as a mixture of two or more. The amount depends on the type of combination of the compound and the anthracycline antibiotic. 1 /
100-1000 times (weight ratio), more preferably 1/5
The amount is selected from the range of 0 to 200 times (weight ratio), especially the range of 1/1 to 10 times (weight ratio).

The composition of the present invention may further contain an organic acid or a salt thereof, such as citric acid, phosphoric acid, or acetic acid, which is commonly used for adjusting pH.

The composition of the present invention is produced, for example, by dissolving an anthracycline antibiotic in a buffer near neutral (pH 6 to 8) and adding a polyanion thereto.

The composition of the present invention can be directly administered to muscles, subcutaneous, blood vessels, organs, or lesions such as tumors as usual injections. Further, it can be formed into various controlled-release preparations and targeted preparations and administered, and can be used as a raw material when producing such preparations.

The microspheres, microcapsules, and liposomes containing the composition of the present invention can be formulated by a method known per se using various natural or synthetic polymers or various phospholipids exemplified below. it can.
Examples of the polymer include gelatin, collagen, albumin, hemoglobin, transferrin, globulin, fibrin, dextran, pullulan, chitosan,
Mannan, carrageenan, amylopectin, pectin, lentinan, polyfatty acid esters (lactic acid, glycolic acid,
Homopolymers and copolymers such as malic acid and citric acid), poly-β-hydroxybutyric acid, polyamino acids (poly-γ-benzyl-L-glutamic acid, poly-γ-methyl-L-glutamic acid, etc.), poly Aldehydes, polyvinyl polymers (ethylene, propylene, butadiene, acrylic acid, acrylic acid ester, methacrylic acid, methacrylic acid ester, vinyl acetate, vinyl chloride, vinyl alcohol, vinyl pyrrolidone, vinyl ether, vinyl carbazole, styrene, styrene derivative, α Homopolymers and copolymers of cyanoacrylate, acrylamide, divinylbenzene, etc.), maleic anhydride copolymers, ether cellulose (methylcellulose, ethylcellulose, carboxymethylcellulose, carboxyethylcellulose, B carboxymethyl cellulose, hydroxypropyl cellulose). Examples of the phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, dicetyl phosphate, phosphatidyl glycerol, phosphatidic acid, phosphatidylinositol and the like, respectively.

Preservatives, stabilizers, isotonic agents, solubilizing agents, and the like that are commonly used in injections may be added to the composition of the present invention.

In addition, preparations other than injections such as preparations for mucosal administration to the nose, mouth, rectum, vagina, uterus, etc., preparations for percutaneous administration, and preparations for administration directly to the tumor or tumor resection may be used.

 The composition of the present invention has, for example, the following characteristics.

(1) Good stability is ensured even in the neutral pH range where anthracycline antibiotics are unstable and decompose (degrade), and conventional anthracycline antibiotics secured by making the pH extremely acidic. A formulation that is less irritating to the living body than a solution of the substance is obtained.

(2) Changes in anthracycline antibiotic reaction and denaturation in a formulation step such as heating or reaction with a crosslinking agent can be suppressed.

(3) The stability is improved even in the neutral pH range, and various controlled release preparations and targeted preparations of anthracycline antibiotics can be prepared. In this way, when long-term administration is required, the preparation can be more stably provided with a predetermined pharmacological effect by administering once a week or once a month.

Examples Hereinafter, the present invention will be described more specifically with reference to Examples.

Example 1 10 ml of polyanion was added to 2 ml of a pH6.86 buffer in which 0.2 mg of doxorubicin (hereinafter abbreviated as DOX) hydrochloride was dissolved.
g was added and the mixture was stirred for 1 hour at room temperature. After that, 2 ml of 2.5 M sodium chloride pH6.86 buffer was added to each sample to dissociate DOX from the formed ion complex, and to the other sample, a filter with a pore size of 0.45 μm was used. After removing the ion complex, DOX was quantified respectively. D remaining in the latter sample
The former was subtracted from the amount of OX, and this was defined as the amount of DOX that had ionically interacted with each polyanion.

10 mg of each polyanion was added to 2 ml of a pH6.86 buffer in which 0.2 mg of DOX was dissolved, and the mixture was stored with stirring at 37 ° C. for 7 days.
Thereafter, DOX was dissociated from the ion complex formed by adding 2 ml of a 2.5 M sodium chloride pH6.86 buffer solution to each, and the amount of remaining DOX was quantified by a fluorescence HPLC method. These residual amounts and the DO when no polyanion was added
The difference from the residual amount of X was defined as the stabilized DOX amount.

Table 1 shows that the polyanion interacted ionically.
The amount of DOX and the amount of stabilized DOX are shown for each anion.

Using daunorubicin hydrochloride, the interaction with each polyanion and the amount of daunorubicin stabilized thereby were examined in the same manner as described above.

Table 2 shows the amount of daunorubicin interacting ionically with the polyanion and the amount of stabilized daunorubicin for each anion.

Example 2 pH 6.86 buffer 2 in which 0.2 mg of doxorubicin hydrochloride was dissolved
50 μg or 200 μg of dextran sulfate sodium (hereinafter abbreviated as DS-Na) or heparin sodium (hereinafter abbreviated as Hep-Na) having an average molecular weight of 5000 per ml, respectively.
g was added and stored under stirring at 37 ° C for 6 hours. Thereafter, 2 ml of 2.5 M sodium chloride pH6.86 buffer solution was added to each to dissociate DOX from the formed ion complex, and the amount of remaining DOX was quantified by a fluorescence HPLC method. Table 3 shows the results. The residual% of each DOX is calculated by the above p
Using pH 4.01 buffer instead of H6.86 buffer and 2.5 M sodium chloride pH6.86 buffer solution, and storing at 0 ° C
It was expressed as a relative value with the residual amount of DOX taken as 100%.

When no polyanion was added, DOX was rapidly degraded and decreased to 38.4% after 6 hours. In contrast,
When 25 and 100% of each polyanion was added, the residual amount of DOX after 6 hours increased with the addition amount, and DS-N
a, In the case of Hep-Na, the degradation of DOX was reduced to about 1/3 of that without addition.

Example 3 pH6.86 buffer 2 in which 0.1 mg of doxorubicin hydrochloride was dissolved
Add 10 mg each of DS-Na with different average molecular weight to ml
It was stored under stirring at 37 ° C. for 7 days. Thereafter, the amount of residual DOX was quantified in the same manner as in Example 2, and the results are shown in Table-4. The residual% of each DOX was represented by the same relative value as in Example 2.

When DS-Na was not added, DOX was 2.4% after 7 days.
Down to. In contrast, each DS-Na with a different molecular weight
When DOX was added, 50-60% of DOX remained in each case. In addition, DS-Na having a larger molecular weight tended to have a stronger stabilizing effect.

Example 4 pH 6.86 buffer 2 in which 0.1 mg of doxorubicin hydrochloride was dissolved
10 mg of various salts of heparin was added to each ml and stored at 37 ° C. for 7 days with stirring. After that, the residual% of DOX was obtained in the same manner as in Example 2.
Is calculated, and the results are shown in Table-5.

Without the addition of each salt of heparin, DOX decreased to 2.4% after 7 days. On the other hand, in each case where each salt of heparin was added, 66.5 to 78.8% of DOX remained. The stabilizing effect was slightly different depending on the type of heparin salt, but not so large.

Example 5 Doxorubicin hydrochloride (20 mg) was added to 0.15 ml of water, and 10 mg of DS-Na having an average molecular weight of 5,000 was added and suspended therein. This was added to a solution prepared by dissolving 2 g of a copolymer of lactic acid and glycolic acid (lactic acid / glycolic acid = 75/25 molar ratio, weight average molecular weight: 10500, hereinafter abbreviated as PLGA) in 2.05 g of dichloromethane, and a probe-type sonicator was added. Disperse using for 30 seconds.
This emulsion was added to a 0.1% aqueous solution of polyvinyl alcohol 4
The mixture was poured into 00 ml and made into a three-phase emulsion using a small homogenizer. The dichloromethane in this emulsion was volatilized under stirring, and the obtained microspheres (hereinafter abbreviated as msp) were collected by a centrifuge.
This msp was dispersed again in distilled water and further centrifuged to remove free drug. After washing, the collected msps were more completely desolvated and dehydrated by freeze-drying.

Table 6 shows the results of the dissolution test of DOX from msp (A-102) prepared by the above method and msp (A-101) prepared without adding DS-Na. Dissolution test: pH 7.0, 1 / 30M at 37 ° C, containing 0.2% by weight of heparin calcium (added to suppress DOX degradation after dissolution) as dissolution test solution
A phosphate buffer was used. A certain amount of msp was dispersed, and on day 7, msp and DOX in the eluate were quantified respectively.

The release of msp prepared with the addition of DS-Na was faster than that without the addition, but the sum of the amount of DOX in the msp and that in the eluate was 93.4%, whereas the sum in the eluate was 93.4%. The sum in msp without added Na is 75.9%, about 18% less.
Heparin calcium was added to the eluate as a stabilizer, and this difference was considered to be due to decomposition in msp.
This shows that the stability of X in msp is significantly improved by the addition of DS-Na.

Example 6 8 mg of doxorubicin hydrochloride was dissolved in 4 ml of water, 16 mg of DS-Na having a molecular weight of 8000 was added thereto, 1.5 g of alkali-treated gelatin was added, and the mixture was heated to 70 ° C. To this, 30 ml of sesame oil heated to 70 ° C. and 1 ml of sorbitan sesquioleate were added to form a water / oil emulsion using a small homogenizer, which was immediately cooled to 0 ° C. and left for 20 minutes. Thereafter, centrifugation was performed, and the oil layer of the supernatant was replaced with sesame oil in which glutaraldehyde (GA) was saturated and dissolved, followed by stirring at room temperature for 3 hours to perform a crosslinking reaction. Thereafter, this emulsion was centrifuged, and the washing operation of replacing the supernatant with the same volume of acetone was repeated three times.
To obtain gelatin msp.

After the msp prepared by the above method was sufficiently degraded with collagenase, the unchanged encapsulated DOX was quantified to calculate the DOX encapsulation rate. The results are shown in Table-7.

While the inclusion ratio of DOX in G-101 was 69.3%,
In G-201 prepared by adding DS-Na, it was 87.0% and 1%.
It was 7.7% higher. This difference is due to the fact that water /
It is considered that the stability of DOX in the oil emulsification stage was improved by the addition of DS-Na.

Furthermore, the ratio of G-101 to that of D-OX in G-101 is extremely low at 4.8%. G-2
Its ratio in G-202 to DOX inclusion ratio in 01 was 4
2.3% and 37.5% were higher. This difference is considered to be due to the fact that the modification of DOX by the crosslinking agent, the reaction between DOX and the additive, etc. were protected by the addition of DS-Na in the crosslinking reaction step by GA.

These experiments show that the stability in processing steps such as heat and cross-linking to make DOX a more useful dosage form is DS.
It shows that it was obtained by the addition of -Na.

Effect of the Invention The composition of the present invention is stabilized with an anthracycline antibiotic and can be administered in various dosage forms.

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Claims (9)

(57) [Claims] 1. A stabilized pharmaceutical composition comprising an anthracycline antibiotic having an amino sugar and a water-soluble polyanion having an interaction of 40% or more with the anthracycline antibiotic. 2. The stabilized pharmaceutical composition according to claim 1, wherein the anthracycline antibiotic forms a physiologically acceptable salt with an inorganic or organic acid. 3. An anthracycline antibiotic comprising doxorubicin (adriamycin), 4'-epi-doxorubicin, 4'-desoxy-doxorubicin, 4'-desoxy-4'-iodo-doxorubicin, daunorubicin (daunomycin), 4- 2. The stabilized pharmaceutical composition according to claim 1, which is demethoxy-daunorubicin or aclarubicin. 4. The stabilized pharmaceutical composition according to claim 1, wherein the water-soluble polyanion is a natural or synthetic polymer having a molecular weight of 5 to 2,000,000 and having an acidic residue exhibiting a negative charge in the molecule. 5. A natural polymer comprising 1.5 to 3 per repeating unit sugar.
5. The stabilized pharmaceutical composition according to claim 4, which is a polysaccharide having a sulfuric acid residue. 6. The stabilized pharmaceutical composition according to claim 4, wherein the natural polymer is dextran sulfate, keratan sulfate, heparan sulfate, pullulan sulfate, fucoidan, heparin or a sulfate of curdlan or a partial hydrolyzate thereof. Stuff. 7. A synthetic polymer comprising 0.2 to 1.0 per unit monomer.
The stabilized pharmaceutical composition according to claim 4, which is polystyrene or polyvinyl alcohol having a sulfuric acid residue or a sulfonic acid residue. 8. The stabilized pharmaceutical composition according to claim 1, wherein the water-soluble polyanion forms a physiologically acceptable salt. 9. The stabilized pharmaceutical composition according to claim 1, wherein the content of the water-soluble polyanion is 1/100 to 1000 times (weight ratio) the amount of the anthracycline antibiotic.