JPH029563B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to an antitumor agent containing a polymer bound to a cytotoxic substance such as an anticancer agent as an active ingredient, and a method for producing the same. By binding various drugs to polymeric carriers to make polymeric drugs, the hydrophilicity or hydrophobicity of the drug can be changed to improve absorption into the body or transfer to target organs. It is conceivable that it will be improved. Furthermore, since the drug is gradually dissociated and released from the polymeric carrier in the body, sustained release of drug efficacy over a long period of time and reduction in side effects can be expected. The present inventors have conducted extensive research into polymerizing cytotoxic substances such as anticancer agents in order to develop polymeric drugs that can be used as antitumor agents, and have arrived at the present invention. That is, the present invention provides a structural unit represented by the formula [] In [Formula [], Z represents a hydrogen atom or a monovalent cation. m represents an integer from 1 to 4. ] The constituent unit represented by the formula [] and In [Formula []], X represents a reactive residue of an alkylamine compound substituted with one or more hydroxyl groups. m represents an integer from 1 to 4. ] The constituent unit expressed by the formula [] and In [formula], Y represents an amino group- or imino-reactive residue of an antitumor alkylating agent, antimetabolite, or antibiotic containing an amino group or imino group in the molecule. m represents an integer from 1 to 4. ] Consists of the constituent unit represented by the formula [ ], In [Formula []], R represents a hydrogen atom, a methyl group, a benzyl group, or a hydroxymethyl group. ] The ratio of constituent units [ ], [ ] and [ ] is [ ] / [ ] + [ ] + [ ] = 0.05 to 0.95, and the constituent units [ ] are less than 40 mol% of the total constituent units,
It is a polymer bound to a cytotoxic substance with a degree of polymerization of 10 to 2000. In formula [], Z is a hydrogen atom or a monovalent cation, such as Na ⁺ , K ⁺ , NH ₄ ⁺ . m is 1
It represents an integer of ~4, but m is preferably 1 or 2. The polymer of the present invention also includes, for example, a polymer in which m=1 and m=2. The preferred degree of polymerization is 10
~2000. The ratio of constituent units [ ], [ ] and [ ] is [ ] / [ ] + [ ] + [ ] = 0.05 to 0.95. In formula [], Y represents a cytotoxic substance containing an amino group or imino group in the molecule, ie, an amino group- or imino-reactive residue of an antitumor alkylating agent, antimetabolite, or antibiotic. A cytotoxic substance in the present invention refers to a substance that exhibits toxicity to cells as it is, or a substance that does not exhibit toxicity as it is but can be converted into a substance capable of exhibiting toxicity to cells in vivo. Examples of these are: p-[N,N-bis(2-chloroethyl)]phenylenediamine p-[bis(2-chloroethyl)amino]L-
Phenylalanine (Melphalan) 2-Amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxypropionamide 1-(β-D-arabinofuranosyl)cytosine or its monophosphate 1-[5'-(n-aminoalkylphosphoryl)
-β-D-arabinofuranosyl]cytosines (n
=2~12) 1-[5'-(n-aminoalkylphosphoryl)
-2′-deoxy-β-D-ribofuranosyl]-5
-Fluorouracils 2-Amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethylpropionamide etc. In the present invention, the alkylamine compound substituted with one or more hydroxyl groups includes 2
-ethanolamine, 3-propanolamine,
4-butanolamine, 2,3-dihydroxy-
Propylamine and the like are preferably used, but are not limited thereto. In the polymer of the present invention, 40 mol% of the total structural units
Constituent units other than those represented by the formulas [], [], and [] may be included within the range below. Examples of these include α-glycine, alanine, phenylalanine, serine, etc. that do not have a carboxyl group (or its salt) in the α-position side chain.
-There are amino acids. Such a structural unit consisting of an α-amino acid does not participate in any way in binding with a cytotoxic substance, but it regulates the water solubility of a hydrophilic polymer and the lipid solubility and water solubility of a polymer obtained by binding a cytotoxic substance. It may be helpful. Therefore, if there is no particular need to adjust the fat solubility or water solubility, it is practically advantageous to use a product that does not contain such α-amino acid constituent units. The carboxyl terminal of the polymer of the present invention varies depending on the polymerization method. For example, when polymerizing α-amino acid N-carboxylic anhydride, if the polymerization initiator used is water, -COOH, and if ammonia is used, -COOH is used.

[Formula] In the case of metal alcoholate,

[Formula] etc., but any physiologically harmless one may be used. The amine terminus is usually an amino group. The cytotoxic substance-bound polymer of the present invention has at least 60 mol% of the total structural units of the formula [] [Definitions of Z and m are the same as in the previous case. ] A polymer obtained by reacting a polymer consisting of the structural unit represented by the above with a cytotoxic substance containing an amino group or an imino group in the molecule, which has a primary or secondary amino group in the molecule. It can be obtained by reacting a water-soluble low molecular weight compound containing one or more hydroxyl groups. The reaction between the polymer of the present invention and a cytotoxic substance containing an amino group or imino group in the molecule is usually carried out in a homogeneous reaction system using water or an organic solvent such as dimethylformamide or dimethyl sulfoxide as a reaction solvent. For example, 1-ethyl-3-
The carboxyl groups in the polymer may be activated with (3-dimethylaminopropyl)carbodiimide hydrochloride or dicyclohexylcarbodiimide, or the carboxyl groups may be activated in the form of a mixed acid anhydride. Appropriate reaction temperature is -40 to 100°C and reaction time is 10 minutes to 10 days. The ratio of polymer to cytotoxic substance used varies depending on the desired degree of polymerization, but usually -COOZ in the polymer
An amount corresponding to 5 to 200% of the group is suitable. The desired polymer is obtained by removing low-molecular substances from the reaction mixture by dialysis, gel permeation chromatography, etc., and removing water by freeze-drying. Next, the obtained polymer is made into a homogeneous solution in water or dimethylformamide, etc., and a water-soluble low-molecular compound containing one primary or secondary amide group and one or more hydroxyl groups in the molecule is added. , for example, in the presence of a condensing agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride or dicyclohexylcarbodiimide. Appropriate reaction temperature is -40 to 100°C and reaction time is 10 minutes to 10 days. The ratio of polymer to cytotoxic substance used is
Although it varies depending on the desired degree of polymerization, an amount corresponding to 5 to 200% of the -COOZ groups in the polymer is usually suitable. In the above sequential reactions, the intermediate product (polymer) does not necessarily need to be isolated. That is, after reacting a cytotoxic substance containing an amino group or an imino group, one molecule is added to the same reaction system.
The reaction can also proceed by adding a water-soluble low molecule having one primary or secondary amino group and one or more hydroxyl groups and a condensing agent. The thus obtained polymer of the present invention is effective in treating leukemia and various malignant tumors. The compounds of the present invention are administered to patients by oral, subcutaneous, intramuscular, intravenous injection, suppositories, and other administration methods. For oral administration, solid or liquid preparations can be used. Examples of the preparation include tablets, pills, powders, granules, solutions, suspensions, and capsules. When preparing tablets, excipients such as lactose, starch, calcium carbonate, crystalline cellulose, or silicic acid; binders such as carboxymethylcellulose, methylcellulose, calcium phosphate, or polyvinylpyrrolidone; sodium alginate, Disintegrants such as sodium chloride, sodium lauryl sulfate and stearic acid monoglyceride; wetting agents such as glycerin; absorbents such as kaolin and colloidal silica; and additives such as talc and granular boric acid, etc., are used in formulations. be converted into Pills, powders, and granules are also formulated using the same additives as described above according to conventional methods. Liquid preparations such as solutions and suspensions are also formulated according to conventional methods. Examples of carriers used include glycerol esters such as tricaprin, triacetin, and iodized poppy oil fatty acid esters; water; alcohols such as ethanol; and oily bases such as liquid paraffin, coconut oil, soybean oil, sesame oil, and corn oil. . The above powders, granules, liquid preparations, etc. can also be contained in capsules made of gelatin or the like. Pharmaceutically acceptable carriers herein include stabilizers or preservatives. Formulations for parenteral administration are presented as sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Non-aqueous solutions or suspensions include, for example, propylene glycol, polyethylene glycol or vegetable oils such as olive oil, ethyl oleate,
Injectable organic esters such as iodized poppy oil fatty acid esters are pharmaceutically acceptable carriers. Such formulations may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, dispersing agents, and stabilizing agents. These solutions, suspensions, and emulsifiers can be sterilized by, for example, filtration through a bacteria retention filter, addition of a sterilizing agent, or irradiation. Alternatively, a sterile solid preparation can be prepared and used by dissolving it in sterile water or a sterile injection solvent immediately before use. The effective dosage of the antitumor agent of the present invention is determined based on age, gender,
It varies depending on the patient's condition, but generally 10 ² ~
It is recommended to administer around 10 ⁵ μg/person/day. The toxicity of the cancer therapeutic agent of the present invention to experimental animals is as follows:
Although it depends on the type of cytotoxin it binds to, it is generally less toxic than the cytotoxin itself. For example, the _LD50 value for mice of a polymer conjugated with AraC is 250
mg/Kg or more. The present invention will be explained in detail below with reference to Examples. Example 1 This example demonstrates the antitumor effect of the following reaction and product (polymer conjugated with a cytotoxic substance). (1) 705 mg of poly-L-glutamic acid sodium salt (molecular weight 21000, degree of polymerization 140) and 1-[5'-
(2-aminoethylphosphoryl)-β-D-arabinofuranomyl]cytosine (hereinafter AraCMP
(referred to as derivative) 502 mg (1.40 mmole) in 50 ml of water
and add 1N NaOH to bring the pH of the solution to 7.50.
And so. Next, 2.683g (14.0/mmole) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (condensing agent) was added, and 2.75g (14.0/mmole) was added.
Stir for hours. During this time, the pH of the solution tends to rise, so add 1NHCl dropwise to reach a pH of 7 to 7.6.
I continued to make adjustments. After the reaction was completed, the mixture was concentrated under reduced pressure at 40° to a volume of about 40 ml, and dialyzed in a cellophane tube at 4°C against pure water for 48 hours. The recovered liquid was freeze-dried to obtain 1114 g of flocculent white solid. bound in the resulting polymer
For convenience, the amount of AraCMP residues is calculated using the molecular extinction coefficient ε ^273nm = 9000 (the Merck Index, 9th ed.
When the above sample was quantified, it was found that it contained 1.046 mmole (see page 2778). The reaction rate of Ara CMP was 74.7%. (2) Dissolve this substance in 50ml of water and add 503.4mg (8.25mmole) of 2-hydroxyethylamine to 1N.
Dissolve in 8.25ml of HCl and add, EDCI・HCl3.16g
(16.5 mmole) and adjust the pH of the solution to 7.0 with 1NHCl.
The mixture was stirred for 4 hours. During this period, the PH tends to rise, so
1NHCl was added to react at pH 7.0 to 7.5. After the reaction was completed, the volume was concentrated under reduced pressure to 20 ml, and dialyzed against pure water at 4°C for 48 hours. The recovered liquid was freeze-dried to obtain 742 mg of flocculent solid. The amount of AraCMP residues contained in the obtained polymer was 0.654 mmole as determined by absorbance measurement at 273 nm. Poly-L-glutamate sodium salt has a pH of
At pH=4.0, precipitation occurred from aqueous solution, but on the other hand, the obtained polymer was water-soluble even at pH=4.0. This shows that many side chain carboxyl groups of poly-L-glutamic acid were hydroxyethylated. Furthermore, the binding rate of AraCMP residues in the obtained polymer was calculated as follows. Since AraCMP residue amount = 0.654 mmole (determined by ultraviolet absorbance measurement) and polymer weight = 742 mg, the molecular weight of the AraCMP-bound unit (formula []) is 469, and the 2-hydroxyethylamine-bound unit ( The molecular weight of formula []) is 172, so 469×0.654+172x=742
becomes. From this, x = 2.53 mmole (amount of hydroxyethylated units). Therefore, _the binding rate ^of AraCMP = 0.654/2.53 + 0.654
=5), and 24 hours later, the test drug was administered once via IP, and the number of survival days was measured.

[Table] It can be seen that the polymer of the present invention has a greater life prolonging effect than AraC. (ii) 1 × ¹⁰⁵ L1210 cells were transferred to _CDF1 mice (N
= 5), and 24 hours later, the test drug was administered.
It was administered once iv and the number of days of survival was measured.

[Table] Also in this case, the polymer of the present invention shows an excellent life prolonging effect. Example 2 This example demonstrates the following reactions and products. 100 mg of sodium salt of poly-L-glutamic acid (molecular weight 31000; degree of polymerization 240) and mitomycin
33mg of C in 0.025M sodium phosphate buffer (PH
7.24) Dissolved in 20ml. Next, 254 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added and dissolved therein, and the mixture was stirred overnight in the dark. The reaction solution was then dialyzed against 0.9% NaCl for 24 hours and against pure water at 4°C for 24 hours.
By freeze-drying, 96.7 mg of a flocculent solid of mitomycin C-poly-L-glutamic acid conjugate, which is an intermediate product, was obtained. Dissolve this substance in 20ml of water, add 81mg of 2-hydroxyethylamine to 1N.
The mixture was dissolved in 1.33 ml of HCl, added with 509 mg of EDCI.HCl, and stirred for 4 hours. During this time, the pH tended to increase, so the pH was adjusted to 7.0 to 7.5 with 1NHCl. The reaction solution was then dialyzed against pure water at 4°C for 48 hours.
The recovered liquid was freeze-dried to obtain 105 mg of flocculent solid. Poly-L-glutamate sodium salt has PH=
On the other hand, the polymer obtained above was an aqueous solution even at PH=4.0. This indicates that many side chain carboxyl groups of poly-L-glutamic acid were hydroxyethylated. Further, the amount of mitomycin C residues bound to the obtained polymer was determined by measuring the ultraviolet absorption spectrum (in water) of the polymer. That is, the ultraviolet absorption spectrum has an absorption maximum at 360 nm based on the mitomycin C residue, confirming that the object of the present invention is formed, and the molecular extinction coefficient of the mitomycin C residue. For convenience, ε360nm=23000 (JSWebb et al., JACS,
84, p. 3185 (1962)), the amount of mitomycin and residues contained in 105 mg of the resulting mitomycin C-polymer conjugate was determined to be 29.7 μmole.
(equivalent to 9.93 mg). Also, the binding rate of MMC is
When calculated using the same method as in Example 1, it was 5.3%. Example 3 This example demonstrates the following reaction and product (cytotoxic substance conjugated polymer). Poly-L-glutamate sodium salt (molecular weight
21000, degree of polymerization 140) 100 mg and p-[N,N-bis(2-chloroethyl)]phenylenediamine hydrochloride
17.8 mg was dissolved in 20 ml of water, 130 mg of EDCI.HCl was added, and the mixture was stirred overnight. The reaction solution was then dialyzed against pure water at 4° C. for 48 hours and diaphragm-dried to yield 92 mg of the intermediate as a flocculent white solid. water this thing
Add 60 mg of 2,3-dihydroxypropylamine dissolved in 0.66 ml of 1N HCl,
Furthermore, 130 mg of EDCI/HCl was added and stirred overnight. The reaction solution was dialyzed against pure water at 4°C for 48 hours and then freeze-dried to obtain the target product, p-
122 mg of a polymer conjugate of [N,N-bis(2-chloroethyl)]phenylenediamine was obtained. Poly-L-glutamate sodium salt has PH=
The polymer obtained above was water-soluble even at pH=4.0, whereas it precipitated from an aqueous solution at pH=4.0. This shows that many side chain carboxyl groups of poly-L-glutamic acid were 2,3-dihydroxypropylaminated. Further, the amount of p-[N,N-bis(2-chloroethyl)]phenylenediamine residue bonded to the obtained polymer was determined by measuring the ultraviolet absorption spectrum of the polymer. That is, the ultraviolet absorption spectrum shows an absorption maximum based on the same residue at 275 nm, which confirms that the object of the present invention is formed, and the molecular extinction coefficient of the same residue is calculated as ε275 nm= 16200 (for convenience, the extinction coefficient of the conjugate of acetic acid and p-[N,N-bis(2-chloroethyl)]phenylenediamine was adopted), and the p contained in 122 mg of the obtained conjugate was
- The amount of [N,N-bis(2-chloroethyl)]phenylenediamine residue was determined to be 40 μmole.
(equivalent to 10.8 mg). Further, the binding rate was calculated in the same manner as in Example 1 and was 6.0%. Example 4 (1) Poly-L-glutamic acid (molecular weight 30000) 129
mg was dissolved in 30 ml of dry dimethylformamide, 121 mg of pivaloyl chloride and 100 mg of triethylamine were added under ice cooling, and the mixture was stirred for 40 minutes. Then,
1-(β-D-arabinofuranosyl)cytosine
A solution of 243 mg dissolved in 3 mg of dimethylformamide and 100 mg of triethylamine were added and stirred. After 20 minutes, the ice bath was removed, and the reaction was allowed to proceed at room temperature for 17 hours. Then, 2-hydroxyethylamine
61 mg was added and reacted for 2 hours. Thereafter, the reaction solution was added dropwise to 30 ml of ice water, and the resulting aqueous solution was dialyzed against water at 4° C. for 24 hours through a cellophane membrane. The dialysate was freeze-dried to obtain 205 mg of a flocculent solid of the following formula. The above-obtained polymer was water-soluble even at pH=4.0. This shows that many side chain carboxyl groups of poly-L-glutamic acid were hydroxyethylated. 247nm in ultraviolet absorption spectrum (UV)
By showing the characteristic absorption maximum of 1N-acylated AraC at 298 nm, it was confirmed that AraC is polymer-bonded with the ^4N amino group. (M.
Akiyama et al., Chem.Pharm.Bull., vol. 26, 981
(1978)). The amount of AraC residues in the polymer is determined by setting the molecular extinction coefficient at 300 nm to 8000 for convenience (see the above literature).
When it was quantified, it was 0.18 mmole. Therefore,
The binding rate of AraC residues in the polymer was calculated as follows. Since AraC residue amount = 0.18 mmole (quantified by ultraviolet absorbance measurement) and polymer weight = 205 mg,
Furthermore, the molecular weight of the unit bound to AraC (formula []) is 354, and the molecular weight of the unit bound to 2-hydroxyethylamine (formula []) is 172, so 354×0.18+172x=205. From this, x = 0.82 mmole (amount of hydroxyethylated units). Therefore, the binding rate of AraC=0.18/0.82+0.18×100=18%. (2) Anti-tumor test 1× ¹⁰⁵ L1210 cells were injected into _CDF1 mice (N=
5), and 24 hours later, 1 dose of the study drug was administered ip.
The animals were administered twice, and the survival days were measured.

[Table] It can be seen that the polymer of the present invention has a greater life prolonging effect than AraC.

Claims (1)

[Claims] 1. A structural unit represented by the formula [], In [Formula [], Z represents a hydrogen atom or a monovalent cation. m represents an integer of 1 to 4. ] The constituent unit represented by the formula [ ] and In [Formula []], X represents an alkylamine residue substituted with one or more hydroxyl groups. m represents an integer of 1 to 4. ] The constituent unit represented by the formula [ ] and In the formula [], Y represents an amino group- or imino-reactive residue of an antitumor alkylating agent, antimetabolite, or antibiotic containing an amino group or imino group in the molecule. m represents an integer of 1 to 4. ] Consisting of the constituent units represented by the formula [ ], In [Formula []], R represents a hydrogen atom, a methyl group, a benzyl group, or a hydroxymethyl group. ] The ratio of constituent units [ ], [ ] and [ ] is [ ] / [ ] + [ ] + [ ] = 0.05 to 0.95, and the constituent units [ ] are less than 40 mol% of the total constituent units,
An antitumor agent whose active ingredient is a polymer bound to a cytotoxic substance with a degree of polymerization of 10 to 2000. 2 At least 60 mol% of all structural units are of the formula [] In [Formula [], Z represents a hydrogen atom or a monovalent cation. m represents an integer of 1 to 4. ] Consisting of the constituent units represented by [ ], where the constituent unit [ ] is
When it is not 100 mol%, the remaining structural units are of the formula [] In [Formula []], R represents a hydrogen atom, a methyl group, a benzyl group, or a hydroxymethyl group. ] One polymer is obtained by reacting a polymer consisting of the structural unit represented by the following with an antitumor alkylating agent, antimetabolite, or antibiotic containing an amino group or imino group in the molecule. Alternatively, a method for producing an antitumor agent containing a polymer bound to a cytotoxic substance as an active ingredient, which comprises reacting an alkylamine compound substituted with a plurality of hydroxyl groups.