JPH1192405A - Medicinal complex - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject complex capable of site-specifically delivering a medicinal complex, staying in blood for a long time, and useful for anti-tumor medicines, anti-inflammatory medicines and the like by binding a carboxyalkylpullulan polyalcohol to the residue of a medicinal compound through a specific spacer. SOLUTION: This medicinal complex is obtained by binding a carboxy 1-4C alkylpullulan polyalcohol (especially a carboxymethylpullulan polyalcohol having a mol.wt. of 5,000-500,000 and a carboxymethylation degree of 0.01-2.66) to the residue of a medicinal compound (especially doxorubicin or the like) through a spacer comprising an amino acid or a spacer comprising 2-8 amino acids bound to each other through peptide bonds [especially (N-terminal)-Gly-Gly-Phe- Gly].

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a drug conjugate useful as a medicine. More specifically,
The present invention relates to a drug conjugate in which a carboxy C _1-4 alkyl pullulan polyalcohol, which is a polysaccharide derivative, and a pharmaceutical compound such as an antitumor agent or an anti-inflammatory agent are bound via a spacer.

[0002]

2. Description of the Related Art Antitumor agents used in the treatment of solid cancers such as lung cancer and gastrointestinal cancer and blood cancers such as leukemia are administered systemically by intravenous or oral administration and then specified. To a tumor site to inhibit or suppress the growth of cancer cells, thereby exerting a therapeutic effect. However, systemically administered antitumor agents are rapidly taken up from the blood into organs of the liver and retina or rapidly excreted in urine, resulting in a decrease in blood concentration and transfer to tumor sites. May not be enough. In addition, since ordinary antitumor agents themselves have low selectivity to transfer to tumor sites (tumor selectivity), the antitumor agents are distributed evenly to various cells and tissues throughout the body, and are distributed to normal cells and tissues. On the other hand, since it acts as a cytotoxin, there is a problem that side effects such as vomiting, fever, or hair loss occur at an extremely high rate. Therefore, there is a need to develop a means for efficiently and selectively transferring an antitumor agent to a tumor site.

As one of such means, a method has been proposed in which an antitumor agent is bound to a polysaccharide polymer to delay the disappearance of the antitumor agent from the blood and to enhance the directivity to cancer tissues. Have been. For example, Japanese Patent Publication No. 7-84481 discloses a drug conjugate in which daunorubicin, doxorubicin, mitomycin C, bleomycin, or the like is introduced into a carboxymethylated mannoglucan derivative via a Schiff base or an acid amide bond. ing. As the mannoglucan derivative in the present invention, a carboxymethylated mannoglucan polyalcohol is also used. However, the mannoglucan derivative has many branches and therefore has a complicated structure, and it has been difficult to obtain a product of uniform quality suitable for the production of pharmaceuticals.

[0004] WO94 / 19376 discloses that a carboxyl group of a polysaccharide having a carboxyl group is linked to a peptide chain (having 1 to 8 amino acids), and that the doxorubicin, daunorubicin, and mitomycin are further linked via the peptide chain. A drug conjugate bound with C or bleomycin is disclosed. Examples of the polysaccharide having a carboxyl group include polysaccharides originally having a carboxyl group in the structure thereof (eg, hyaluronic acid), and polysaccharides originally having no carboxyl group in the structure thereof (eg, pullulan, dextran, Examples thereof include polysaccharides in which a carboxy C _1-4 alkyl group is introduced into a hydroxyl group of chitin or the like, or a carboxyl group is introduced by ester bonding of a polybasic acid such as malonic acid or succinic acid. This drug conjugate has a structural feature that a drug such as doxorubicin and the above-mentioned polysaccharide moiety are bonded via a spacer, and provides an antitumor effect superior to doxorubicin and reduces toxicity and side effects. Have been.

[0005] In addition, regarding a technique relating to a drug conjugate using a polyalcoholated polysaccharide derivative as a drug delivery carrier, see "Study on polysaccharide-peptide-doxorubicin conjugate, relationship between blood stability of polysaccharide carrier and antitumor effect". (The 10th Annual Meeting of the DDS Society of Japan, 279, 1
994); “Study on polysaccharide-peptide-doxorubicin conjugate, pharmacokinetics and antitumor effect” (Proceedings of the 9th Annual Meeting of the Pharmacokinetic Society of Japan, 292, 1994); 19th Research and Development Trend Seminar (Pharmaceuticals Agency) Organizers) Abstracts, D-9, 1995; and "Study on Drug Delivery to Tumors by Polysaccharide Carriers" (12th Symposium on Colloid and Interface Technology, Chemical Society of Japan, Abstracts, 51, 1995) is there.

[0006]

An object of the present invention is to provide a drug conjugate capable of site-selectively transferring an active ingredient such as an antitumor agent or an anti-inflammatory agent to a tumor site or the like. is there. More specifically, it is a drug conjugate containing a pharmaceutical compound such as an anti-tumor agent or an anti-inflammatory agent as a partial structure, can stay in the blood for a long time, and It is an object of the present invention to provide a drug conjugate capable of site-selectively delivering the pharmaceutical compound. Another object of the present invention is to provide a method for producing a drug conjugate having the above characteristics.

[0007]

Means for Solving the Problems The present inventors have attempted to improve the drug conjugate disclosed in International Publication WO94 / 19376 in order to solve the above-mentioned problems, and as a result, have replaced polysaccharides having a carboxyl group. By using a dextran derivative obtained by polyalcoholizing dextran and carboxyl C _1-4 alkylated as a polysaccharide moiety, the drug after administration can be maintained at a high concentration for a long time, and site selection for tumor site and inflammation site can be achieved. Has been found to be able to significantly improve the performance. In addition, it has been found that such compounds significantly reduce the toxicity while significantly enhancing the main drug effects such as antitumor effects (PCT / JP97 / 01914). As a result of further research, the present inventors have found that a drug conjugate containing carboxyalkyl pullulan polyalcohol as a polysaccharide moiety has the same properties as the above-mentioned drug conjugate containing carboxyalkyldextran polyalcohol. Was. The present invention has been completed based on these findings.

That is, the present invention relates to a method in which a carboxy C _1-4 alkyl pullulan polyalcohol is linked to a residue of a pharmaceutical compound via a spacer consisting of one amino acid or a peptide consisting of 2 to 8 amino acids. A drug conjugate is provided. According to another aspect of the present invention, a medicament comprising the above drug complex; and a pharmaceutical composition containing the above drug complex as an active ingredient, for example, for injection or infusion in the form of a lyophilized product filled in a vial. And the like are provided. According to still another aspect of the present invention, there is provided a method for producing the above-described drug conjugate.

In a preferred embodiment of the present invention, the pullulan polyalcohol constituting the carboxy C _1-4 alkyl pullulan polyalcohol is a pullulan obtained by treating pullulan under conditions under which polyalcoholization can be performed substantially completely. The above drug conjugate, wherein the carboxy C _1-4 alkyl pullulan polyalcohol is carboxymethyl pullulan polyalcohol; the above drug, wherein the pharmaceutical compound is an antitumor agent or an anti-inflammatory agent Complex; an antitumor agent in which the pharmaceutical compound exhibits a concentration-dependent antitumor effect (an antitumor agent exhibiting a stronger antitumor effect at a higher concentration: in this specification, a concentration-dependent antitumor agent may be referred to as a concentration-dependent antitumor agent). The above drug conjugate; an antitumor agent wherein the pharmaceutical compound exhibits a time-dependent antitumor effect (when the drug compound has a longer action The above-mentioned drug conjugate, which is an antitumor agent exhibiting a stronger antitumor effect between the drug conjugate and the drug conjugate, which is sometimes referred to as a time-dependent antitumor agent in the present specification; and doxorubicin or (1S, 9S) -1-amino-9-ethyl
-5-Fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H, 12H-benzo [de] pyrano [3 ', 4': 6,7] indolizino
[1,2-b] The drug conjugate is provided, which is quinoline-10,13 (9H, 15H) -dione.

[0010] Similarly, in a preferred embodiment, a dipeptide whose spacer is represented by -XZ- (-XZ- is a compound having a hydrophobic amino acid (X) and a hydrophilic amino acid (Z) at the N-terminal side and the C-terminal side, respectively) And a residue obtained by removing one hydrogen atom and one hydroxyl group from the N-terminal amino group and the C-terminal carboxyl group of a dipeptide formed by peptide bond), or The above drug complex, which is a spacer containing a dipeptide as a partial peptide sequence; the above drug complex, wherein the hydrophobic amino acid is phenylalanine and the hydrophilic amino acid is glycine; the spacer is (N-terminal) -Gly-Gly-Phe-Gly- The above drug conjugate, which is:
In addition, the present invention provides the above drug conjugate, in which the amount of the introduced residue of the antitumor agent is in the range of 1 to 15% by weight, preferably 3 to 10% by weight, and more preferably 4 to 7% by weight.

In a particularly preferred embodiment of the present invention, H ₂ N-Gl
The N-terminal of the peptide represented by y-Gly-Phe-Gly-COOH has an acid amide bond to the carboxyl group of carboxymethyl pullulan polyalcohol, and the C-terminal of the peptide has (1S, 9
S) -1-Amino-9-ethyl-5-fluoro-2,3-dihydro-9-
Hydroxy-4-methyl-1H, 12H-benzo [de] pyrano
[3 ', 4': 6,7] Indolizino [1,2-b] quinoline-10,13 (9H,
(1S, 9S) -1-amino-9-ethyl-5-fluoro-
2,3-dihydro-9-hydroxy-4-methyl-1H, 12H-benzo [de] pyrano [3 ', 4': 6,7] indolizino [1,2-b] quinoline-10,13 (9H, 15H)-The introduction amount of dione residue is 2 to 10% by weight
And a carboxy C
_1-4 alkyl pullulan polyalcohol has a molecular weight of 5,000
Carboxymethyl pullulan polyalcohol having a carboxymethylation degree ranging from 0.01 to 2.66 per constituent sugar residue, preferably from 0.1 to 2.000, preferably from 10,000 to 350,000, more preferably from 50,000 to 200,000. The drug conjugate is provided in the range of 1.0, more preferably in the range of 0.3 to 0.6.

According to another aspect of the present invention, carboxy C
Provided is a drug delivery carrier consisting of _1-4 alkyl pullulan polyalcohols. In a preferred embodiment of the invention, the carboxy C _1-4 alkyl pullulan polyalcohol has a molecular weight in the range 5,000 to 500,000, preferably 10,000
~ 350,000, more preferably 50,000-200,000
And the degree of carboxy C _1-4 alkylation is in the range of 0.01 to 2.66, preferably 0.1 to 1.0, more preferably 0.3 to 0.6 per constituent sugar residue. Carboxymethyl pullulan polyalcohol is provided as the most preferred carrier. In another aspect of the invention, there is provided the use of a carboxy C _1-4 alkyl pullulan polyalcohol for the manufacture of a drug conjugate comprising a carboxy C _1-4 alkyl pullulan polyalcohol conjugated to a residue of a pharmaceutical compound. You.

[0013] In a preferred embodiment of the invention, carboxy C _1-4 alkyl pullulan polyalcohol for the manufacture of a drug complex in which residues a and carboxy C _1-4 alkyl pullulan polyalcohol is linked via a spacer of a pharmaceutical compound And that the carboxy C _1-4 alkyl pullulan polyalcohol is linked to the residue of the pharmaceutical compound via a single amino acid spacer or a peptide-bonded 2 to 8 amino acid spacer. There is provided the use of a carboxy C _1-4 alkyl pullulan polyalcohol for the manufacture of a characterized drug conjugate.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The drug conjugate of the present invention has a spacer or peptide bond of 2 to 8 amino acids.
Carboxy C via a spacer consisting of
It is characterized in that _1-4 alkyl pullulan polyalcohol and a residue of a pharmaceutical compound are bonded.

The residue of the pharmaceutical compound contained in the drug complex of the present invention is used as a drug such as an antitumor agent, an anti-inflammatory agent, an antibacterial agent and the like for the treatment and / or prevention of diseases of mammals including humans. It is derived from a pharmaceutical compound and is constituted by its partial structure. However, the pharmaceutical compound from which the residue is derived is not limited to the above. As the pharmaceutical compound, any compound having one or more reactive functional groups (for example, an amino group, a carboxyl group, a hydroxyl group, a mercapto group, an ester group, etc.) capable of participating in the binding to the spacer is used. You may. As used herein, the term "pharmaceutical compound" also includes a prodrug compound which contains the main structure of a compound which itself has a medicinal action as its partial structure and can regenerate the compound in vivo.

More specifically, in the present specification, the residue of the drug compound is defined as a bond between the spacer and the residue of the drug compound when the reactive functional group in the drug compound reacts with the reactive functional group in the spacer. (Eg, dehydration condensation) means the partial structure derived from the pharmaceutical compound present in the compound after binding. For example, if the pharmaceutical compound is D-NH ₂ , D-COOH, D-COOR, D-OH, DS
When represented by H, D-CONH ₂ , D-NH-COOR (R is a lower alkyl group or the like), the residues of the drug compound are D-NH- (D-NH-COOR
-Q-), D-CO- (D-CO-NH-Q, D-CO-OQ, D-CO-SQ, etc.), D-CO- (D-CO-NH-Q, D-CO-OQ , D-CO-SQ etc.), D
-O- (DO-CO-Q, DOQ, etc.), DS- (DS-CO-Q, DSQ, etc.), D-CONH- (D-CO-NH-CO-Q, etc.), D-NH-CO- (D-NH-CO
(-OQ, D-NH-CO-NH-Q, etc.) (The parentheses indicate the bond between the spacer and the drug compound residue, and Q indicates the remaining partial structure excluding the reactive functional group from the spacer. Shown).
However, the type of bond between the spacer and the drug compound residue is not limited to the above. The residue of the pharmaceutical compound may be bound to a reactive functional group present on the amino acid constituting the spacer, in addition to the N-terminal amino group or C-terminal carboxyl group of the spacer.

The residues of the pharmaceutical compound include, for example, doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum antitumor agent (cisplatin or a derivative thereof), taxol or a derivative thereof, camptothecin or Its derivatives (Japanese Unexamined Patent Publication
No. 87746, antitumor agents, preferably (1S, 9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4- described in claim 2 Methyl-1H, 12
H-benzo [de] pyrano [3 ', 4': 6,7] indolizino [1,2-b]
Residues of antitumor agents such as quinoline-10,13 (9H, 15H) -dione) can be suitably used. Also suitable are, for example, residues of steroidal anti-inflammatory drugs such as hydrocortisone succinate and prednisolone succinate, or non-steroidal anti-inflammatory drugs such as mefenamic acid, flufenamic acid, diclofenac, ibuprofen and tinolidine.

As the spacer that binds to the residue of the pharmaceutical compound, a spacer consisting of one amino acid or a spacer consisting of 2 to 8 amino acids bound with a peptide can be used. More specifically, the spacer is 1
An oligopeptide consisting of two to eight amino acid residues (residues obtained by removing one hydrogen atom and one hydroxyl group from the amino group and carboxyl group of the amino acid, respectively), or peptide-bonded 2 to 8 amino acids (Meaning a residue obtained by removing one hydrogen atom and one hydroxyl group from the N-terminal amino group and the C-terminal carboxyl group, respectively).

Preferred spacers are residues of oligopeptides consisting of 2 to 6 amino acids. The type of amino acid constituting the spacer is not particularly limited, for example,
L- or D-amino acids, preferably L-amino acids, can be used, and in addition to α-amino acids, β-alanine, ε-aminocaproic acid, γ-aminobutyric acid, and the like may be used. It is preferable that such an amino acid other than the α-amino acid is arranged at a position close to the polysaccharide derivative.

The bonding direction of the spacer is not particularly limited. Generally, the N-terminal of the spacer is bonded to the carboxyl group of carboxy C _1-4 alkyl pullulan polyalcohol by an acid amide bond, and the spacer is bonded to the amino group of the drug compound. At the C-terminus. Further, for example, when a lysine residue is included as a constituent unit of the peptide spacer, and the α-amino group and the ε-amino group of the lysine residue are each bonded to the carboxyl group of another amino acid by an acid amide bond, both the peptide spacer Since the terminal is N-terminal, the carboxyl group of the pharmaceutical compound can be bound. Further, one or two or more residues of a diamine compound or a dicarboxylic acid compound (for example, a residue of a diamine such as ethylenediamine or a residue of a dicarboxylic acid such as succinic acid) are included in the spacer as constituent units. A spacer having both ends N-terminal and a spacer having both ends C-terminal may be used.

The amino acid sequence of the spacer is not particularly limited. For example, a residue of a dipeptide in which the spacer is represented by -XZ- (X represents a residue of a hydrophobic amino acid, and Z represents a residue of a hydrophilic amino acid) , -XZ- is a hydrophobic amino acid
(X) and the hydrophilic amino acid (Z) at the N-terminal and C
(This means residues obtained by removing one hydrogen atom and one hydroxyl group from the amino group at the N-terminus and the carboxyl group at the C-terminus of a dipeptide peptide-bonded on the terminal side.)
Or a spacer containing the residue of the dipeptide as a partial peptide sequence can be suitably used. As the hydrophobic amino acid, for example, phenylalanine, tyrosine, leucine and the like can be used, and as the hydrophilic amino acid, for example, glycine, alanine and the like can be used. The spacer may have a repeating sequence of such dipeptide residues (eg, -XZXZ-, -XZXZXZ-, etc.).

When a spacer containing such a dipeptide structure is used, the spacer is hydrolyzed at a tumor site or an inflammatory site considered to be rich in peptidase, and a pharmaceutical compound is liberated at a high concentration at the site. The partial structure formed by combining the spacer containing the dipeptide with the pharmaceutical compound is a preferred partial structure of the drug conjugate of the present invention. When a concentration-dependent antitumor agent (for example, doxorubicin) or the like is used as the residue of the pharmaceutical compound, a spacer consisting of the dipeptide residue represented by -XZ- or the dipeptide residue is a partial peptide It is particularly preferable to use a spacer containing the sequence.

Also, when a time-dependent antitumor agent requiring a sustained action time at a certain concentration or more is used as a residue of a pharmaceutical compound, a high antitumor effect can be obtained by using the above-mentioned spacer. May be achievable. Examples of such antitumor agents include the antitumor agents described in JP-A-6-87746, and preferably the antitumor agents described in claim 2. In general, it is necessary to select a preferable spacer from the viewpoint of the mechanism of action of the antitumor agent, characteristics of pharmacokinetics and toxicity expression, release of the antitumor agent in the body, etc., without being limited to the above spacers. There is. In general, it is preferable to select the above-mentioned spacer that can release a high-concentration pharmaceutical compound in a short time for a cancer type that grows rapidly.

Specific examples of the spacer are shown in the following table.
The spacer used in the drug conjugate of the present invention is not limited to the following, and it is needless to say that those skilled in the art can appropriately select a spacer so as to give an optimal release rate of the pharmaceutical compound. In the table, the left side of the peptide sequence is the N-terminal, and the residue of the pharmaceutical compound is bonded to the C-terminal side. D-Phe is D
-Phenylalanine residue, other amino acids are L-
Indicates an amino acid. The magnitude of the release rate was determined based on the degree of onset of drug effect of the drug complex bound to doxorubicin on Walker 256 tumor-bearing rats, or the concentration of free doxorubicin at the tumor site of Walker 256 tumor-bearing rats. Among these spacers, it is preferable to use a spacer capable of releasing a high-concentration drug compound in a short time, such as (N-terminal) -Gly-Gly-Phe-Gly-, for doxorubicin.

[0025]

[Table 1] (a) Spacer with high release rate -Leu-Gly- -Tyr-Gly- -Phe-Gly- -Gly -Phe-Gly- -Gly-Gly-Phe-Gly- -Gly-Phe-Gly-Gly- -Phe-Gly-Gly-Gly- -Phe-Phe-Gly-Gly- -Gly-Gly-Gly-Phe- Gly- (b) Spacer with relatively high release rate -Gly-Gly-Phe-Phe- -Gly-Gly-Gly-Gly-Gly-Gly- (c) Spacer with relatively low release rate -Phe-Phe-- Ala-Gly- -Pro-Gly- -Gly-Gly-Gly-Phe- (d) Spacer with low release rate -Gly- -D-Phe-Gly- -Gly-Phe- -Ser-Gly- -Gly-Gly --Gly-Gly-Gly- -Gly-Gly-Gly-Gly- ─────────────────────

Pullulan has a maltotriose residue of α1 →
It is a water-soluble α-glucan linked by six bonds and generally produced as an extracellular polysaccharide from sucrose or the like by Aureobasidium pullulans can be obtained as a commercial product. The origin of pullulan for use in production is not limited to the above. The molecular weight of pullulan is not particularly limited, and for example, those having a molecular weight of about 10,000 to about 2,000,000, preferably about 100,000 to about 1,000,000 can be used.

The polyalcohol degree of carboxy C _1-4 alkyl pullulan polyalcohol that constitutes the polysaccharide derivative moiety of the drug complex of the present invention is not particularly limited, Pururanpori constituting the carboxy C _1-4 alkyl pullulan polyalcohol It is preferred that the alcohol is obtained by treating pullulan under conditions that allow for substantially complete polyalcoholization. For example, pullulan polyalcohol in which pullulan is subjected to a large excess of sodium periodate and sodium borohydride in order to substantially completely convert pullulan into polyalcohol is suitable as a starting compound of the drug complex of the present invention. is there. However, the method of polyalcoholization of pullulan is not limited to the above method, and any method may be employed as long as it can be used by those skilled in the art.

[0028] C _1-4 configuring the carboxy C _1-4 alkyl group
As the alkyl, a linear or branched C _1-4 alkyl, specifically, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, or the like can be used. However, preferably, a methyl group can be used.
Carboxy C _1-4 alkylation is, for example, chloroacetic acid, bromoacetic acid, α
-Chloropropionic acid, α-methyl-α-chloropropionic acid, β-chloropropionic acid, α-methyl-β-chloropropionic acid, α-chlorobutyric acid, β-chlorobutyric acid,
The reaction can be carried out by reacting a halogenated C _1-4 alkylcarboxylic acid such as γ-chlorobutyric acid, preferably chloroacetic acid, to partially or completely carboxy C _1-4 alkylate the hydroxyl group.

For example, pullulan polyalcohol is dissolved in an inert solvent not involved in the reaction (eg, water, N, N-dimethylformamide, dimethyl sulfoxide, etc.), and a base (eg, sodium hydroxide, potassium hydroxide, etc.) is dissolved. A halogenated C _1-4 alkylcarboxylic acid or a salt thereof may be added in the presence thereof, and the reaction may be carried out at a temperature in a range of from about 100 ° C. to about 100 ° C. for several minutes to several days. The degree of introduction of the carboxy C _1-4 alkyl group can be easily determined, for example, by appropriately selecting the reaction temperature of the carboxy C _1-4 alkylation and the amounts of the halogenated C _1-4 alkyl carboxylic acid and the base used as the reagent. Adjustable, such means are well known to those skilled in the art. Although the degree of carboxy C _1-4 alkylation to the hydroxyl group of pullulan polyalcohol is not particularly limited, for example, in the range of 0.01 to 2.66 per constituent sugar residue, preferably
It is in the range of 0.1 to 1.0, more preferably 0.3 to 0.6. The molecular weight of the carboxy C _1-4 alkyl pullulan polyalcohol is about 5,000 to 500,000, preferably about 10,000 to 350,000, more preferably about 50,000 to 200,000, as measured by gel filtration.

The carboxy C _1-4 alkyl pullulan polyalcohols described above are useful as carriers for drug delivery. A drug conjugate in which a drug compound and a carboxy C _1-4 alkyl pullulan polyalcohol are bound has, for example, excellent selectivity such as tumor selectivity and can maintain a high blood concentration for a long time. There are features. As the bond between the pharmaceutical compound and the carboxy C _1-4 alkyl pullulan polyalcohol, for example, a method of directly linking both with an ester bond or the like, a method of linking both via an appropriate spacer such as the spacer described above, and the like Can be adopted.

With regard to the drug conjugate bonded via a spacer, the carboxy C _1-4 alkyl pullulan polyalcohol obtained as described above is bonded to the carboxyl group of the carboxy C _1-4 alkyl pullulan polyalcohol by the spacer bonded to the residue of the drug compound. By doing so, the drug conjugate of the present invention can be produced. The bond between the spacer and the carboxyl group of the carboxy C _1-4 alkyl pullulan polyalcohol is generally
It can be formed by bonding the N-terminal amino group of the spacer and the carboxyl group of the carboxy C _1-4 alkyl pullulan polyalcohol by an acid amide bond. However, the bond between the spacer and the carboxyl group of the carboxy C _1-4 alkyl pullulan polyalcohol is not limited to the above, and may be another chemical bond or a bond using one or more spacers. Good. For example, an acid anhydride may be formed by the C-terminal carboxyl group of the spacer and the carboxyl group of the carboxy C _1-4 alkyl pullulan polyalcohol, and each carboxyl group may be formed by using a diamine compound such as ethylenediamine as the spacer. An acid amide bond may be bonded to each amino group of the diamine.

N-terminal amino group of the spacer and carboxy
When the carboxyl group of the C _1-4 alkyl pullulan polyalcohol is bonded by an acid amide bond, a general dehydration condensing agent used for peptide chain synthesis, for example, N, N′-dicyclohexylcarbodiimide (DCC) N, N'-dicycloalkylcarbodiimides, carbodiimide derivatives such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAPC), 1-hydroxybenzotriazole (H
In addition to benzotriazole derivatives such as OBT), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (EEDQ) and the like can be used. Further, the reaction may be performed by an active ester method, an acid halide method, or the like.

The amount of the drug compound residue to be introduced into the carboxy C _1-4 alkyl pullulan polyalcohol is not particularly limited, but the physicochemical properties of the drug compound residue, the pharmacokinetics, drug efficacy, Should be selected appropriately from the viewpoint of toxicity and the like. Generally, 0.1 to 30% by weight, preferably 1 to 15% by weight, more preferably 3 to 10% by weight.
% By weight, particularly preferably about 4 to 7% by weight. The ratio of the drug compound residue introduced into the carboxy C _1-4 alkyl pullulan polyalcohol can be easily determined by, for example, absorbance analysis.

As an example of the method for producing the drug conjugate of the present invention, the case where a residue of a pharmaceutical compound which is an antitumor agent described in claim 2 of JP-A-6-87746 is introduced.
The production method is shown in the following scheme, but the drug conjugate of the present invention and the production method thereof are not limited to those shown in this scheme. In the following scheme, the introduced amount of the drug compound residue is, for example, 1 to 15% by weight, preferably 2 to 15% by weight.
About 10% by weight. Also, in the following scheme, only the structural unit in which one or two carboxymethyl groups are introduced among the structural units of polyalcohols are exemplarily described, but the polysaccharide derivative portion of the drug conjugate of the present invention is It should be understood that is not constituted by repeating the above constitutional unit.

[0035]

Embedded image

[0036]

Embedded image

The above-mentioned pharmaceutical compound is biased toward an equilibrium with a compound having a lactone ring (ring-closed form) in an acidic aqueous medium (eg, about pH 3), while being concentrated in a basic aqueous medium (eg, pH 3).
Lactone ring-opened compound (about 10)
It is known that the equilibrium is biased, but such a drug conjugate having a residue corresponding to a closed body and an open body has an equivalent antitumor effect, and both are within the scope of the present invention. It goes without saying that it is included in. In addition, if there is a reactive species in which the lactone ring is opened in the reaction system, the condensation reaction proceeds between the carboxyl group derived from the lactone ring and the amino group derived from the spacer, and the reaction yield is significantly reduced. In some cases, a desired uniform drug complex cannot be obtained. Such side reactions can be avoided by selectively using a ring-closure as a reactive species.

That is, after converting the sodium salt of carboxy C _1-4 alkyl pullulan polyalcohol to triethylammonium salt, the carboxyl group of carboxy C _1-4 alkyl pullulan polyalcohol is nonaqueous (in an organic solvent not containing water). On the other hand, by condensing the N-terminal amino group of the spacer to which the residue of the above-mentioned pharmaceutical compound is bonded, side reactions can be suppressed, and the desired product can be efficiently produced. Examples of the salt of carboxy C _1-4 alkyl pullulan polyalcohol soluble in an organic solvent include, for example, trialkylammonium salts such as triethylammonium salt and trimethylammonium salt, N-methylpyrrolidine, N-methylmorpholine, dimethylamino It is possible to use salts of organic bases such as pyridine (DMAP). As the organic solvent, N, N-dimethylformamide, dimethylsulfoxide and the like can be used.

The drug conjugate of the present invention provides desired pharmaceutical activity to a tumor site or an inflammatory site depending on the type of the residue of the pharmaceutical compound (for example, the residue of a pharmaceutical compound such as an antitumor agent or an anti-inflammatory agent). For example, and can be specifically expressed locally, and the toxicity of the pharmaceutical compound itself can be reduced. Without being bound by any particular theory, the polysaccharide derivative portion (eg, carboxymethyl pullulan polyalcohol) of the drug conjugate of the present invention has extremely excellent blood retention and accumulation at tumor / inflammation sites. Useful as a carrier for drug delivery, the drug conjugates of the present invention have tumor selectivity and inflammatory site selectivity. In addition, protease (peptidase) is considered to be expressed in tumor sites and inflammation sites.
The spacer of the drug conjugate of the present invention is easily hydrolyzed, and the released pharmaceutical compound exerts its medicinal effect.

The medicament containing the drug conjugate of the present invention is usually
It can be filled in vials or the like in the form of a lyophilized product, and is provided clinically as a parenteral administration preparation such as an injectable or infusion preparation which is dissolved at the time of use. It is not limited to the above embodiment. For the preparation of the above-mentioned preparations, for example, preparation additives available in the art such as a solubilizing agent, a pH adjuster, and a stabilizer can be used. Although the dose of the drug of the present invention is not particularly limited, usually, the dose of the drug compound constituting the drug compound residue, the amount of the drug compound residue introduced into the drug conjugate of the present invention, The decision should be based on the condition and the type of disease. For example, when the drug conjugate of the present invention in which the residue of the antitumor agent described in claim 2 of JP-A-6-87746 is introduced at a ratio of about 6% by weight is administered parenterally,
Generally day per body surface area 1 m ² per about 1 to 500 mg, preferably about administered once in a range of about 10 to 100 mg,. 3 to
It is preferable to repeat every four weeks.

[0041]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to the following Examples. In the examples, `` A-NH- '' refers to JP-A-6-87746.
Patent No. 2) The pharmaceutical compound described in claim 2 (in the examples,
It is called "DX-8951". ) Represents a drug compound residue in the case where a drug compound having a closed lactone ring is represented by A-NH ₂ (a group represented by A-NH- in the above-mentioned scheme: a lactone ring is formed). A'-NH- indicates that the lactone ring in the residue of the drug compound represented by A-NH- is either a closed or open ring or a mixed form thereof.

The degree of carboxymethylation of carboxymethyl pullulan polyalcohol (degree of substitution of carboxymethyl group per constituent sugar residue) was determined by converting sodium salt of carboxymethyl pullulan polyalcohol into a free acid form. 0.1N dissolved in aqueous sodium hydroxide
It was determined by titration with hydrochloric acid. Aqueous solution of sodium salt of carboxymethyl pullulan polyalcohol is Bio-R
The solution passed through an ad AG50W-x 2 (H ⁺ ) column was lyophilized and used as a sample. This sample was dissolved in a predetermined excess amount of a 0.1N aqueous sodium hydroxide solution, and titrated with 0.1N hydrochloric acid using phenolphthalene as an indicator. S
(mg), a predetermined excess amount of 0.1N aqueous sodium hydroxide
(ml) and the titration amount of 0.1N hydrochloric acid as b (ml), and the degree of carboxymethylation was determined by the formula of 15.4 (ab) / [s-5.8 (ab)]. The amount (% by weight) of the drug introduced was determined by absorbance analysis (around 362 nm) using characteristic absorption of the drug. Further, the gel filtration method was performed under the following conditions (column: TS
K gel G4000 PW _XL , eluent: 0.1 M NaCl, flow rate: 0.8 ml /
min, column temperature: 40 ° C).

Example 1: 3'-N- (Boc-Gly-Gly-Phe-Gly) -NH-A

Embedded image Boc-Gly-Gly-Phe-Gly (600 mg) and N-hydroxysuccinimide (160 mg) were converted to N, N-dimethylformamide
(20 ml), cooled to 4 ° C., and N, N′-dicyclohexylcarbodiimide (280 mg) was added. To this solution was added DX-8951 methanesulfonate (600 mg:
A solution of N, N-dimethylformamide (30 ml) in which triethylamine (0.16 ml) was dissolved was mixed with a solution of triethylamine (0.16 ml) and reacted with stirring at room temperature for 16 hours under light shielding. The reaction solution was evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane: methanol = 10: 1 solution containing 0.5% acetic acid) to obtain the title compound (1.0 g).

¹ H-NMR (DMSO-d ₆ ) δ: 8.40 (d, 1H, J = 8.3 H
z), 8.10-8.17 (m, 2H), 7.91-8.01 (m, 1H), 7.78 (d, 1
H, J = 10.75 Hz), 7.32 (s, 1H), 6.94-6.96 (m, 1H), 6.50
(s, 1H), 5.57 (t, 1H, J = 4.5Hz), 5.43 (s, 2H), 5.23
(s, 2H), 3.77 (dd, 2H, J = 5.85Hz, J = 8.80Hz), 3.70 (d,
2H, J = 4.40Hz), 3.65 (d, 2H, J = 5.35Hz), 3.56 (d, 2H, J
= 5.85), 3.15-3.25 (m, 2H), 2.40 (s, 3H), 2.05-2.25
(m, 1H), 1.86 (m, 2H), 1.35 (s, 9H), 0.88 (t, 3H, J = 7.
35) .Mass (FAB); m / e 854 (M + 1)

Example 2: 3'-N- (Gly-Gly-Phe-Gly) -NH-A tri
3'-N- (Boc-Gly-Gly-Phe-Gly) -NH-A (79 mg) obtained in Synthesis Example 1 of fluoroacetate
Was dissolved in trifluoroacetic acid (3 ml) and left for 1 hour. The solvent was distilled off and azeotroped twice with methanol (30 ml).
After azeotropic distillation twice with ethanol (30 ml), the residue was washed with ether to obtain the title compound (80 mg). ¹ H-NMR (DMSO-d ₆ ) δ: 8.53 (d, 1H, J = 8.3Hz), 8.40-8.4
8 (m, 2H), 8.28 (d, 1H, J = 8.3Hz), 7.95-8.07 (br, 3H),
7.81 (d, 1H, J = 10.2Hz), 7.30-7.37 (m, 2H), 7.15-7.3
0 (m, 5H), 6.50-6.55 (br, 1H), 5.50-5.57 (m, 1H), 5.4
1 (d, 2H, J = 7.82Hz), 5.25 (s, 2H), 4.55-4.62 (m, 1H),
3.55-3.92 (m, 6H), 3.15-3.25 (br, 2H), 2.98-3.03 (m,
1H), 2.73-2.82 (m, 1H), 2.40 (s, 3H), 2.05-2.25 (m, 1
H), 1.84-1.92 (m, 2H), 0.88 (t, 3H, J = 7.35Hz).

Example 3: Carboxymethyl pullulan polyal
Synthesis of Cole-Gly-Gly-Phe-Gly-NH-A ' Pullulan 200 (20 g, Tokyo Kasei, molecular weight 200K) was 0.1 M
It was dissolved in acetate buffer (pH 5.5, 2000 ml), and an aqueous solution (2000 ml) of sodium periodate (44 g) was added. After stirring at 4 ° C. for 10 days while shielding from light, ethylene glycol (14.
0 ml) and stirred overnight. The reaction solution was adjusted to pH 6.5 using an 8 M aqueous sodium hydroxide solution. After adding and dissolving sodium borohydride (19 g), the mixture was stirred overnight. The reaction solution was adjusted to pH 5.5 with acetic acid, stirred at 4 ° C for 1 hour, and then adjusted to pH 7. using 8 M aqueous sodium hydroxide solution.
Adjusted to 5. The obtained aqueous solution was subjected to ultrafiltration using a Biomax-30 membrane to remove a low molecular fraction. The residual solution that did not pass through the membrane was passed through an ultrafiltration membrane 1000K (manufactured by Filtron), and then freeze-dried to obtain pullulan polyalcohol (7 g). The molecular weight (gel filtration, pullulan standard) of this material was 35K.

This pullulan polyalcohol (2 g) was added to an aqueous solution obtained by dissolving sodium hydroxide (8.4 g) in water (60 ml) and dissolved at room temperature. Monochloroacetic acid (12 g) was added to this solution under ice cooling to dissolve it, and then reacted at room temperature overnight. The reaction solution was adjusted to pH 10 with acetic acid, and then desalted by an ultrafiltration method using a Biomax-50 membrane. The remaining solution that does not pass through the membrane is lyophilized and the sodium salt of carboxymethyl pullulan polyalcohol (2.2
g) was obtained. The molecular weight (gel filtration, pullulan standard) of this material was 83K and the degree of carboxymethylation was 0.5. This sodium salt of carboxymethyl pullulan polyalcohol (1.0 g) was dissolved in water, and Bio-Rad AG 50W-X2
(200-400 mesh, Et ₃ NH ⁺ type) column and eluted with water. The eluate was freeze-dried to obtain carboxymethyl pullulan polyalcohol triethylammonium salt (1.0 g).

The triethylammonium salt of carboxymethyl pullulan polyalcohol (200 mg) was dissolved in N, N-dimethylformamide (12 ml). To this solution was added 3′-N- (Gly-Gly-Phe-Gly) -NH-A trifluoroacetate (42 mg) obtained in Example 2 and triethylamine (0.014 ml) of N, N-
A dimethylformamide (5 ml) solution and 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (200 mg) were sequentially added, and the reaction was allowed to proceed with stirring at room temperature overnight under light shielding. Water (300 ml) is added to the reaction solution, and the ultrafiltration membrane 10K
(Ultrafiltration using Filtron). Residual solution that does not pass through the membrane is adjusted to pH 10 with 0.1N aqueous sodium hydroxide solution.
And passed through a filtration membrane (0.16 μm, manufactured by Filtron). The solution passed through was desalted by ultrafiltration using a Biomax-50 membrane, followed by a Millipore filter.
(0.22 μm) and freeze-dried to give the title compound (210
mg).

After dissolving this compound in a 0.1 M aqueous sodium chloride solution, it was analyzed by GPC (column: Tosoh TSK Gel PW-4000XL, solvent: 0.1 M NaCl, flow rate: 0.8 ml / min).
1 and FIG. 2 show the ultraviolet absorption spectrum (0.1 M Tris buffer, pH 9.0, 0.1 mg / ml) of the present compound, respectively. The content of the drug compound residue in this compound was determined based on the absorbance at 362 nm in 0.1 M Tris buffer (pH 9.0), and was found to be 6.2% (W / W).

Example 4: Antitumor effect of the drug conjugate of the present invention 1 × 10 ⁶ mouse fibrosarcoma Meth A cells were transplanted subcutaneously into the right inguinal region of BALB / c male mice (7 weeks old). A
Tumor-bearing mice were created. On day 7, the drug conjugate of Example 3 dissolved in distilled water for injection was administered once into the tail vein of a Meth A tumor-bearing mouse. On day 21 after transplantation, the tumor was excised and weighed, and the tumor growth inhibition rate was calculated by the following formula: tumor growth inhibition rate (%) = [1
-(Average tumor weight of sample administration group / average tumor weight of control group)] × 100. As a result, the drug conjugate of Example 3 showed enhancement of the antitumor effect and expansion of the effective dose range as compared with the pharmaceutical compound itself.

[0051]

[Table 2] ¹⁾ Pharmaceutical compound equivalent

[0052]

EFFECT OF THE INVENTION The drug conjugate of the present invention into which an antitumor agent is introduced as a pharmaceutical compound is excellent in tumor site selectivity, can exhibit high antitumor activity, and has reduced toxicity. have.

[Brief description of the drawings]

FIG. 1 is a diagram showing a GPC chart of a drug conjugate of the present invention (Example 3).

FIG. 2 is a view showing an ultraviolet absorption spectrum of the drug conjugate of the present invention (Example 3).

 ──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Masahiro Ikeda 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo Inside the Daiichi Pharmaceutical Co., Ltd. Tokyo R & D Center

Claims (26)

[Claims] 1. A carboxy C _1-4 alkyl pullulan polyalcohol is linked to a drug compound residue via a spacer consisting of one amino acid or a peptide-bonded spacer consisting of 2 to 8 amino acids. A drug conjugate comprising: 2. The method according to claim 1, wherein the pullulan polyalcohol constituting the carboxy C _1-4 alkyl pullulan polyalcohol is a pullulan polyalcohol obtained by treating pullulan under substantially completely polyalcoholizable conditions. The drug conjugate according to claim 1, wherein 3. The drug conjugate according to claim 1, wherein the carboxy C _1-4 alkyl pullulan polyalcohol is carboxymethyl pullulan polyalcohol. 4. The drug conjugate according to claim 1, wherein the pharmaceutical compound is an antitumor agent or an anti-inflammatory agent. 5. The drug conjugate according to claim 4, wherein the pharmaceutical compound is an antitumor agent exhibiting a concentration-dependent antitumor effect. 6. The drug conjugate according to claim 4, wherein the pharmaceutical compound is an antitumor agent that exhibits a time-dependent antitumor effect. 7. The antitumor agent is doxorubicin or (1S, 9S)
-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-
Hydroxy-4-methyl-1H, 12H-benzo [de] pyrano [3 ',
4 ': 6,7] Indolizino [1,2-b] quinoline-10,13 (9H, 15H)
-The drug conjugate of claim 4, which is a dione. 8. A dipeptide whose spacer is represented by -XZ- (-XZ- is a hydrophobic amino acid (X) and a hydrophilic amino acid (Z)
Are N-terminal and C-terminal, respectively, to form a dipeptide formed by peptide bonding.
A residue obtained by removing one hydrogen atom and one hydroxyl group from the terminal carboxyl group) or a spacer containing the dipeptide as a partial peptide sequence. Item 14. The drug conjugate according to Item. 9. The drug conjugate according to claim 8, wherein the hydrophobic amino acid is phenylalanine and the hydrophilic amino acid is glycine. 10. The method according to claim 10, wherein the spacer is (N-terminal) -Gly-Gly-Phe-
10. The drug conjugate according to claim 9, which is Gly-. 11. The introduction amount of a residue of an antitumor agent is 1 to 15% by weight.
The drug conjugate according to any one of claims 5 to 10, which is in the range of 10%. 12. The introduction amount of a residue of an antitumor agent is 3 to 10% by weight.
The drug conjugate according to any one of claims 5 to 10, which is in the range of 10%. 13. The introduction amount of a residue of an antitumor agent is 4 to 7% by weight.
The drug conjugate according to any one of claims 5 to 10, which is in the range of 10%. 14. The peptide represented by H ₂ N-Gly-Gly-Phe-Gly-COOH has an N-terminal acid amide bond to a carboxyl group of carboxymethyl pullulan polyalcohol, and the C-terminal of the peptide has (1S , 9S) -1-Amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,
12H-benzo [de] pyrano [3 ', 4': 6,7] indolizino [1,2-
b] The drug complex according to claim 1, wherein the 1-amino group of quinoline-10,13 (9H, 15H) -dione and an acid amide bond. 15. (1S, 9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,
12H-benzo [de] pyrano [3 ', 4': 6,7] indolizino [1,2-
b] The amount of quinoline-10,13 (9H, 15H) -dione residue introduced was 2
15. The drug conjugate of claim 14, which is in the range of ~ 10% by weight. 16. The carboxy C _1-4 alkyl pullulan polyalcohol is a carboxymethyl pullulan polyalcohol having a molecular weight of 5,000 to 500,000, and has a carboxymethylation degree of 0.01 to 2.66.
16. The drug conjugate according to 15. 17. The method according to claim 14, wherein the carboxy C _1-4 alkyl pullulan polyalcohol is a carboxymethyl pullulan polyalcohol having a molecular weight of 10,000 to 350,000 and a carboxymethylation degree of 0.1 to 1.0. Drug conjugate. 18. The method according to claim 14, wherein the carboxy C _1-4 alkyl pullulan polyalcohol is a carboxymethyl pullulan polyalcohol having a molecular weight of 50,000 to 200,000 and a carboxymethylation degree is in a range of 0.3 to 0.6. Drug conjugate. (19) (1S, 9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,
12H-benzo [de] pyrano [3 ', 4': 6,7] indolizino [1,2-
b] The amount of quinoline-10,13 (9H, 15H) -dione residue introduced was 5
15. The drug conjugate according to claim 14, wherein the carboxy C _1-4 alkyl pullulan polyalcohol has a molecular weight of about 83,000 and a degree of carboxymethylation of about 0.5. 20. A drug delivery carrier for binding a pharmaceutical compound, said drug delivery carrier comprising a carboxy C _1-4 alkyl pullulan polyalcohol. 21. A carboxy C _1-4 alkyl pullulan polyalcohol having a molecular weight in the range of 5,000 to 500,000,
21. The carrier for drug delivery according to claim 20, wherein the degree of carboxy C _1-4 alkylation ranges from 0.01 to 2.66. 22. The drug delivery system according to claim 20, wherein the molecular weight of the carboxy C _1-4 alkyl pullulan polyalcohol ranges from 10,000 to 350,000 and the degree of carboxy C _1-4 alkylation ranges from 0.1 to 1.0. carrier. 23. The drug delivery system according to claim 20, wherein the molecular weight of the carboxy C _1-4 alkyl pullulan polyalcohol ranges from 50,000 to 200,000 and the degree of carboxy C _1-4 alkylation ranges from 0.3 to 0.6. carrier. 24. The drug delivery carrier according to any one of claims 20 to 23, wherein the carboxy C _1-4 alkyl pullulan polyalcohol is carboxymethyl pullulan polyalcohol. 25. Use of a carboxy C _1-4 alkyl pullulan polyalcohol for the manufacture of a drug complex containing the carboxy C _1-4 alkyl pullulan polyalcohol bound to residues of pharmaceutical compounds. 26. A carboxy C _1-4 alkyl pullulan for the production of a drug conjugate in which a residue of a pharmaceutical compound and a carboxy C _1-4 alkyl pullulan polyalcohol are bound via a spacer or without a spacer. Use of polyalcohol.