JPH05124969A - Production of water-soluble polymerized medicine - Google Patents

Abstract

PURPOSE:To obtain the subject medicine exhibiting a high antitumor effect in the range of low toxicity, having excellent water solubility and useful for injections, etc., by reacting a specific copolymer with a medicine under a specific condition when the copolymer is bonded to the medicine through an amide bond. CONSTITUTION:When a medicine such as adriamycin is bonded through an amid bond to a block copolymer having a hydrophilic polymer structure part of a polyethylene glycol structure and a carboxyl group-having polymer structure part of an acidic polyamino acid structure, the block copolymer is reacted with the medicine in a solution maintained at a pH of <=8 to obtain the medicine of formula I (R1 is lower alkyl; R2 is bond group; R3 is methylene, ethylene; R is OH, group of formula II; (n) is 5-1000; (m) is 1-300; (x) is 0-300; (x) is smaller than (m); at least one of R is the group of formula II). The medicine is preferably administered in a total dose of 100-1000mg/m<3> once to three times a week.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing a water-soluble polymerizing agent from a block copolymer having a hydrophilic polymer structural portion and a polymer structural portion having a carboxyl group.

[0002]

2. Description of the Related Art Although water-soluble high-molecular-weight drugs are known, in many cases, denaturation of the drugs has occurred, so that a sufficient pharmacological effect cannot be expected in some cases.

[0003]

Several attempts have been made to bind a low molecular weight drug to a high molecular weight substance. However, it is the current situation that no one with a high pharmacological effect has been obtained.

[0004]

Means for Solving the Problems As a result of diligent studies to solve the drawbacks of conventional polymer drugs, the present inventors have found that a hydrophilic polymer structure portion and a polymer structure portion having a carboxyl group are formed. The present invention was completed by finding that a water-soluble polymerized drug having a dramatically improved pharmacological effect can be obtained by carrying out a reaction under a specific reaction condition when a drug is bound to a block copolymer with an amide bond. ..

That is, the present invention provides (1) when a drug is bound to a block copolymer having a hydrophilic polymer structural portion and a polymer structural portion having a carboxyl group by an amide bond, A method for producing a water-soluble polymerized drug, characterized in that the reaction with the drug is carried out in a solution whose pH is maintained at 8 or less, (2) The hydrophilic polymer structure portion has a polyethylene glycol structure, 1) A method for producing a water-soluble polymerizing agent, (3) A method for producing a water-soluble polymerizing agent according to the above (1) or (2), wherein the polymer structural portion having a carboxyl group has an acidic polyamino acid structure. Method, (4) above, wherein the drug is adriamycin (1),
(2) A method for producing the water-soluble polymerizing agent according to (3), (5) A method for producing the water-soluble polymerizing agent according to (1), wherein the water-soluble polymerizing agent has the structure of formula 1. ，

[0006]

[Chemical 3] (In the formula, R ₁ represents a lower alkyl group, R ₂ represents a bonding group, R ₃ represents a methylene group or an ethylene group, and R
Each independently represent a hydroxyl group or a residue of a drug, n is 5 to 1,000, m is 1 to 300, and x is an integer of 0 to 300, x is smaller than m, and at least one of R is at least one. Shall represent the residue of the drug. ) (6) The drug residue has the formula 2

[0007]

[Chemical 4] (7) A method for producing a water-soluble polymerizing agent according to (5) above, (7) A method for producing a water-soluble polymerizing agent according to (5) or (6) above, wherein R ₁ is a methyl group. ) A method for producing a water-soluble polymerizing agent according to the above (5), (6) or (7), wherein R ₂ is an alkylene group having 2 to 4 carbon atoms.

According to the present invention, a water-soluble polymerized drug having a high pharmacological effect can be obtained.

The present invention will be described in detail below.

Examples of the structure of the hydrophilic polymer structure portion include structures such as polyethylene glycol, polysaccharides, polyacrylamide, polymethacrylamide, polyvinyl alcohol, polyvinylpyrrolidone and chitosan, but any hydrophilic polymer structure may be used. There is no particular limitation. A particularly preferred structure is a polyethylene glycol structure.

Examples of the polymer structure portion having a carboxyl group include the structures of polymer carboxylic acids such as polyamino acid, polyacrylic acid, polymethacrylic acid and polymaleic acid. Particularly preferred structures are structures of acidic polyamino acids such as polyglutamic acid and polyaspartic acid.

Examples of the drug to be bound to the polymer structure having a carboxyl group include anticancer agents and antibacterial agents such as adriamycin, daunomycin, pinorbin, methotrexate, mitomycin C, etoposide and cisplatin,
Examples thereof include antiviral agents and the like, but are not limited to these.

In the above formula 1, R ₂ is not particularly limited as long as it does not impair the water solubility of the water-soluble polymerizing agent (preferably, further does not impair the micelle forming ability), and R ₂ of the hydrophilic polymer structural portion is Corresponding to the method and compound used for converting the terminal of the compound which will constitute the hydrophilic polymer structural part into a structure suitable for the formation when forming the polymer structural part having a carboxyl group at the terminal takes a structure, for example, an ethylene group (-CH ₂ CH ₂ -), propylene group _{(-CH (CH 3) CH 2} -), trimethylene group _{(-CH 2 CH 2 CH 2 -} ), butylene (-CH ₂ C
H (CH ₃ ) CH ₂ — and the like) include alkylene groups having 2 to 8 carbon atoms, preferably 2 to 4 carbon atoms, and the like, but are not particularly limited.

The molecular weight of the water-soluble polymerizing agent is not particularly limited as long as it is water-soluble, but it is preferably 1,00.
0 to 100,000, particularly preferably 5,000 to 5
It is 10,000. The ratio of the hydrophilic polymer structure part and the polymer structure part having a carboxyl group having a drug bound to the side chain in the water-soluble polymerized drug is not particularly limited as long as the water solubility of the polymerized drug is maintained. But preferably 1:
0.1-10 (weight ratio), particularly preferably 1: 0.2-
5 (weight ratio).

In the water-soluble polymerizing agent of the above formula 1,
R ₁ represents a lower alkyl group, preferably a methyl group. Further, n is 5 to 1,000, preferably 15 to 250, m is 1 to 300, preferably 10 to 100, and x is 0 to 300, preferably 0. ~ 100. The amount of the drug bound to the side chain of the polymer structure having a carboxyl group is not particularly limited, and it can be set to any binding amount, but it is bound to the side chain contained in the water-soluble polymerized drug. The amount of the added drug is usually 3 to 80% by weight, preferably 5 to 60% by weight. However, there is no problem in binding as many as possible unless the water solubility of the polymerizing agent is impaired.

The block copolymer can be produced by various methods. For example, a compound (for example, polyethylene glycol, polysaccharide, polyacrylamide, polymethacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, chitosan or a derivative thereof) which constitutes the hydrophilic polymer structural portion, or one whose terminal is modified A compound which reacts with a polymer compound having a carboxyl group or a carboxyl group protected by a protecting group, and then removes the protecting group in the case of containing a protecting group, or forms a hydrophilic polymer structural part. Alternatively, a modified carboxylic acid or its derivative monomer is reacted with a modified end thereof,
For those containing a protecting group, a block copolymer can be obtained by removing the protecting group.

The modification of the terminal of the compound that constitutes the hydrophilic polymer structural portion can be carried out by a known method. For example, as a method of converting a hydroxyl group into an amino group, a method of reacting ethyleneimine or the like, acrylonitrile. Method of converting nitrile group to amino group after Michael addition to or methacrylonitrile, etc., method of reacting alcohol amine such as ethanolamine after replacing hydroxyl group with halogen group, directly converting hydroxyl group to nitrile group and reducing It can be performed by a method of converting to an amino group. The method of removing the protecting group can be an alkali method, an acid method or a reduction method. As the alkaline substance used in the alkaline method, normal alkaline substances such as caustic soda, caustic potash, hydrazine and ammonia can be used. As the acidic substance used in the acid method, a usual acidic substance such as trifluoromethanesulfonic acid, methanesulfonic acid, trifluoroacetic acid, acetic acid, formic acid, hydrofluoric acid, hydrobromic acid, and hydrochloric acid can be used. .. Further, in order to prevent side reactions, anisole, thioanisole, m-cresol, o-cresol and the like can be added. As the reduction method, a general method such as a catalytic reduction method or a catalytic hydrogen transfer reduction method can be used.

The block copolymer thus obtained is reacted with a drug in a solution whose pH is kept at 8 or lower to obtain a water-soluble polymerized drug. For example, in order to obtain a water-soluble polymerizing agent of formula 1, all R
The drug may be reacted with the block copolymer in which is a hydroxyl group. When the drug is bound to the block copolymer with an amide bond, the reaction between the block copolymer and the drug is preferably performed by a coupling method using a condensing agent. On this occasion,
The pH of the reaction solution is adjusted to 8 or less, preferably 3 to 8.

As the condensing agent, 1-ethyl-3- (3-
Dimethylaminopropyl) carbodiimide (EDC),
1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl), dicyclohexylcarbodiimide (DCC), carbonyldiimidazole (CDI), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (EEDQ), diphenylphosphoryl azide (DPPA) and the like can be used. The condensing agent is preferably used in an amount of 1 to 10 times, more preferably 1 to 4 times the mol of the drug. At this time, in order to accelerate the reaction, N-hydroxysuccinimide (HONSu), 1-hydroxybenzotriazole (HOBt), N-hydroxy-5-norbornene-
2,3-dicarboxylic acid imide (HONB) and the like may be allowed to coexist, and in the case of coexisting with each other, it is preferable to use them in an amount of 0.2 to 10 times by mole, and particularly 0.5 to 2 times by mole. It is preferable to use mol.

The amount of the drug used is not particularly limited, and may be used in an amount desired to be bound to the block copolymer, but usually 0.1 to 1 equivalent of the carboxyl group of the block copolymer.
Use 2 moles.

The reaction is preferably carried out in a solvent, and various solvents such as dimethylformamide, dimethylsulfoxide, dioxane, tetrahydrofuran, water, and mixed solvents thereof can be used without any particular limitation. Although the amount of the solvent used is not particularly limited, it is usually used in an amount of 10 to 500 times by weight based on the block copolymer.

The reaction is preferably carried out at -10 to 40 ° C, particularly preferably -5 to 30 ° C. The reaction is 2
48 hours is enough.

Below, a water-soluble polymer in which adriamycin is bonded to the side chain of polyaspartic acid using a block copolymer consisting of a hydrophilic polymer structural part derived from a polyethylene glycol derivative and a polyaspartic acid structural part The synthetic method will be described in detail by taking a chemical agent as an example.

The water-soluble polymerizing agent can be synthesized as shown in the following reaction formula. That is, β-benzyl-L-aspartate-N-carboxylic acid anhydride (B
LA-NCA) has polyethylene glycol (PEG-NH ₂ ) having an alkoxy group such as a methoxy group at one end and a 3-aminopropyl group at the other end (preferably a molecular weight of 250 to 20,000). As an initiator, ring-opening polymerization is performed in a solvent such as dimethylformamide, dimethylsulfoxide, dioxane, chloroform, dichloromethane, tetrahydrofuran, acetonitrile, and the like to give a polyethylene glycol-poly (β-benzyl-L-aspartate) block copolymer (PEG -PBLA),
Then, the benzyl ester of PEG-PBLA is hydrolyzed to obtain a polyethylene glycol-polyaspartic acid block copolymer (PEG-P (Asp.)).
The anticancer agent adriamycin hydrochloride is added to this PEG-P (Asp.) To give a condensing agent or a condensing agent and HO at a pH of 8 or less.
By adding NSu or the like, the primary amino group of adriamycin and the side chain carboxyl group of polyaspartic acid are linked by an amide bond, and a water-soluble polymerizing agent (anticancer agent) (PEG-P (Asp.) ADR) is added. obtain.

[0025]

[Chemical 5] (In the formula, R is a hydroxyl group or formula 2

[0026]

[Chemical 6] Represents, n is 5 to 1,000, m is 1 to 300, and x is 0.
It represents an integer of ˜300, x is smaller than m, and at least one of R represents the formula (2). ) The water-soluble polymerizing agent of the present invention has good water solubility despite a high adriamycin substitution ratio (ratio of carboxyl groups to which adriamycin is bound in the number of carboxyl groups of polyaspartic acid), It retains its water solubility even when freeze-dried or concentrated.

The anticancer activity of this water-soluble polymerized drug is higher than that of the original adriamycin hydrochloride itself, as shown in Table 1. Moreover, its high anticancer activity is achieved in the range of fewer side effects than adriamycin.

The water-soluble polymerized drug of the present invention can be used in various commonly used dosage forms, for example, solid dosage forms, ointments, liquid dosage forms, etc. The dose is 1 to 3 times a week, and the total amount is 100 to 1,
It is about 000 mg / m ² / week.

[0029]

EXAMPLES Next, the present invention will be described in detail with reference to Examples and Reference Examples.

Example 1 5.7 g of β-benzyl-L-aspartate-N-carboxylic acid anhydride (BLA-NCA) was dissolved in 20 ml of N, N'-dimethylformamide (DMF). One end methoxy group, one end 3-aminopropyl group polyethylene glycol (PEG-NH ₂ ) (molecular weight 5,100) D
It was dissolved in 40 ml of MF and the solution was added to the BLA-NCA solution. After 40 hours, the reaction mixture was added dropwise to 2 liters of isopropyl ether. The precipitated polymer was collected by filtration, washed with isopropyl ether and then vacuum dried to obtain polyethylene glycol-poly (β-benzyl-L-
Aspartate) block copolymer (PEG-PBL
A) 7.99 g (yield 92.1%) was obtained.

The benzyl ester was hydrolyzed at room temperature while suspending 7.0 g of PEG-PBLA in 0.5N sodium hydroxide. After the copolymer was dissolved, the pH was acidified with acetic acid and dialyzed in water using a dialysis membrane (molecular weight cut off = 1,000). The solution in the membrane was freeze-dried to give 4.44 g of polyethylene glycol-polyaspartic acid block copolymer (PEG-P (Asp.)) (Yield 79%).
Got

157 mg of this PEG-P (Asp.) Was dissolved in 2.4 ml of water. Adriamycin hydrochloride 300mg
Was suspended in 24 ml of DMF, and triethylamine 72 was added under ice cooling.
After adding μl, an aqueous PEG-P (Asp.) solution was added. To this mixed solution, 54 μl of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was added, and the mixture was reacted at pH 7 to 8 under ice cooling for 4 hours. Then p
While adjusting H to 7-8, 54 μl of EDC was added and reacted at room temperature for 18 hours. The reaction mixture was dialyzed for 3 hours in 0.1 M sodium acetate buffer (pH 4.5) using a dialysis membrane (molecular weight cut-off = 12,000). After dialysis, AD
VANTEC UK-50 (molecular weight cut off = 50000)
Ultrafiltration was performed with the ultrafiltration membrane of 0) to remove unreacted adriamycin and other low molecular weight substances. Repeated washing with water and concentration, 21.2 ml of an aqueous solution of 10 mg / ml in terms of adriamycin (calculated from UV absorption at 485 nm)
Got

The obtained water-soluble polymerized anticancer agent (water-soluble polymerized drug) PEG-P (Asp.) ADR has the structure of the above formula 1, R ₁ is a methyl group, and R ₂ is trimethylene. The group R ₃ represents a methylene group. n = 115, m = 2
0, x = 4, part of R is a hydroxyl group and the rest is the above-mentioned residue (formula 2). Adriamycin content is 57.4% by weight
However, it showed good water solubility.

Example 2 Polyethylene glycol-polyaspartic acid block copolymer (PEG-P (Asp.)) 2 obtained in Example 1
09 mg was dissolved in 3.2 ml water. 400 mg of adriamycin hydrochloride was suspended in 32 ml of DMF, 96 μl of triethylamine was added under ice cooling, and then an aqueous solution of PEG-P (Asp.) Was added. 279 mg of dicyclohexylcarbodiimide (DCC) was added to this mixed solution while adjusting the pH to 7-8, and the mixture was reacted for 4 hours under ice cooling and then for 18 hours at room temperature. The reaction mixture was passed through a dialysis membrane (molecular weight cut off = 12,
000) in 0.1M sodium acetate buffer (pH
It was dialyzed in 4.5) for 3 hours. The precipitate deposited after dialysis was filtered, and the filtrate was ultrafiltered with an ultrafiltration membrane of ADVANTEC UK-50 (molecular weight cut off = 50,000) to remove unreacted adriamycin and other low molecular weight substances.
Repeated washing with water and concentration, 10 mg in terms of adriamycin
20.0 ml of an aqueous solution / ml (calculated by absorption at 485 nm with an ultraviolet spectrophotometer) was obtained.

PE, the Water-Soluble Polymerized Anticancer Agent Obtained
G-P (Asp.) ADR has the structure of Formula 1 above and R
₁ represents a methyl group, R ₂ represents a trimethylene group, and R ₃ represents a methylene group. n = 115, m = 20, x = 4, part of R is a hydroxyl group, and the rest is the above-mentioned residue (formula 2). Although the adriamycin content was 48.8% by weight, it showed good water solubility.

Example 3 Polyethylene glycol-polyaspartic acid block copolymer (PEG-P (Asp.)) 3 obtained in Example 1
5.2 mg was dissolved in 0.4 ml water. 58 mg of adriamycin hydrochloride was suspended in 4 ml of DMF, 14 µl of triethylamine was added under ice cooling, and then an aqueous PEG-P (Asp.) Solution was added. To this mixed solution, 41.2 mg of dicyclohexylcarbodiimide (DCC) and 11.6 mg of N-hydroxysuccinimide (HONSu) were added while adjusting the pH to 7 to 8 and reacted for 4 hours under ice cooling, then at room temperature for 18 hours. It was made to react. The reaction mixture was dialyzed (molecular weight cut off = 1
2,000) for 3 hours in 0.1 M sodium acetate buffer (pH 4.5). The precipitate deposited after dialysis was filtered, and the filtrate was ultrafiltered with an ultrafiltration membrane of ADVANTEC UK-50 (fraction molecular weight = 50,000),
Unreacted adriamycin and other low molecular weight substances were removed. Repeated washing and concentration, 1 in terms of adriamycin
0 mg / ml (calculated from absorption at 485 nm with an ultraviolet spectrophotometer)
To obtain 4.3 ml of an aqueous solution of.

PE, which is the obtained water-soluble polymerized anticancer agent
G-P (Asp.) ADR has the structure of Formula 1 above and R
₁ represents a methyl group, R ₂ represents a trimethylene group, and R ₃ represents a methylene group. n = 115, m = 20, x = 4, part of R is a hydroxyl group, and the rest is the above-mentioned residue (formula 2). The adriamycin content was 58.0% by weight, but it showed good water solubility.

Reference Example 1 Mouse colon cancer Co was subcutaneously implanted on the dorsal part of a CDF1 female mouse.
lon26 cells were transplanted, and the water-soluble polymerized drug (anticancer drug) PEG-P (Asp.) ADR or adriamycin hydrochloride (ADR) obtained in Example 1 or 2 from the time when the tumor volume reached around 100 mm ³ . ) Was intravenously administered once every 4 days for a total of 3 times, and the effect on advanced cancer was examined.
Each drug was dissolved in physiological saline before use. Also PEG
-For P (Asp.) ADR, the dose converted to ADR was used. The antitumor effect of the drug was judged from the ratio T / C (%) of the median survival days of each group to the control and the tumor growth curve. The results are shown in Table 1 and FIG. As is clear from FIG. 1, when adriamycin hydrochloride (ADR) is administered, the growth inhibitory effect of the transplanted tumor is observed but the tumor shrinkage is hardly observed, whereas the water-soluble polymer anticancer agent of the present invention Was administered, the transplanted tumor disappeared.

[0039]

[Table 1] Table 1 Anti-cancer activity sample for mouse colon cancer Colon26 Dose Mean survival days T / C Tumor disappeared mice (mg / kg) (%) Example 1 25 60.2 233 2/3 50 60.2 233 2/3 100 17.2 67 0/3 Example 2 25 60.0 233 2/3 50 22.5 87 0/3 ADR 10 60.0 233 0/3 Results up to 60 days Mean survival of untreated group. 25.8 days

[0040]

The water-soluble polymerized drug obtained by the method of the present invention has good water solubility even if the amount of the drug bound is increased. Moreover, the present invention can provide an extremely useful drug because it exhibits a high antitumor effect in a range of low toxicity as compared with an anticancer agent not bound to a block copolymer.

[Brief description of drawings]

FIG. 1 is a tumor growth curve of mouse colon cancer Colon26 when adriamycin hydrochloride is administered.

FIG. 2 shows a tumor growth curve of mouse colon cancer Colon26 when PEG-P (Asp.) ADR of Example 1 was administered.

FIG. 3 is a tumor growth curve of mouse colon cancer Colon26 when PEG-P (Asp.) ADR of Example 2 is administered.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kazunori Kataoka 1083-4 Omuro, Kashiwa City, Chiba Prefecture 141-9, Kashiwa Village, Mitsuo Okano 6-12-12 Kunifudai, Ichikawa City, Chiba (72) Invention Authors Takashi Fuji, 45-3 Shimokawa-cho, Maebashi City, Gunma Prefecture (72) Inventor Shigeto Fukushima 239 Iwahana-cho, Takasaki City, Gunma Prefecture (72) Inventor Hisao Yumoto 2-11-1-803 Shimo Kita-ku, Tokyo (72) Inventor Kazuya Okamoto 5-7-10-305 Higashiohisa, Arakawa-ku, Tokyo

Claims (8)

[Claims] 1. When a drug is bound to a block copolymer having a hydrophilic polymer structure part and a polymer structure part having a carboxyl group by an amide bond, the reaction between the block copolymer and the drug is conducted at pH. The method for producing a water-soluble polymerizing agent is characterized in that 2. The method for producing a water-soluble polymerizing agent according to claim 1, wherein the hydrophilic polymer structural portion has a polyethylene glycol structure. 3. The method for producing a water-soluble polymerizing agent according to claim 1, wherein the polymer structure portion having a carboxyl group has an acidic polyamino acid structure. 4. The method for producing a water-soluble polymerized drug according to claim 1, wherein the drug is adriamycin. 5. The method for producing a water-soluble polymerizing agent according to claim 1, wherein the water-soluble polymerizing agent has the structure of formula 1. [Chemical 1] (In the formula, R ₁ represents a lower alkyl group, R ₂ represents a bonding group, R ₃ represents a methylene group or an ethylene group, and R
Each independently represent a hydroxyl group or a residue of a drug, n is 5 to 1,000, m is 1 to 300, and x is an integer of 0 to 300, x is smaller than m, and at least one of R is at least one. Shall represent the residue of the drug. ) 6. The residue of the drug has the formula 2 6. The method for producing a water-soluble polymerized drug according to claim 5. 7. The method for producing a water-soluble polymerizing agent according to claim 5, wherein R ₁ is a methyl group. 8. The method for producing a water-soluble polymerizing agent according to claim 5, 6 or 7, wherein R ₂ is an alkylene group having 2 to 4 carbon atoms.