JPH0751592B2 - Cyclotriphosphazene derivative - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel cyclotriphosphazene derivative. More specifically, the present invention relates to a novel therapeutic physiologically active substance, cyclotriphosphazene derivative having high affinity with water.

<Prior Art> When a physiologically active substance that can be effective in treating a disease is administered to a living body (human or animal), the expected effect is often not observed due to various disorders.

For example, when orally administered, the absorption from the intestinal tract is poor and the expected effect may not be exhibited. Even if it is absorbed into the living body or is directly administered into the living body by a method such as injection, it may be rapidly decomposed by the action of the degrading enzyme in the living body and the effect may not be sustained. Furthermore, when the physiologically active substance is a peptide hormone or the like, an antibody is sometimes produced, which may cause serious side effects.

Although many methods for avoiding these disorders have been devised, it has recently been known that a method of binding a physiologically active substance for treatment to an appropriate water-affinity polymer is effective.

For example, it has been disclosed that hemoglobin bound to dextran using BrCN stays in the body as a blood substitute for an appropriate period (see Japanese Patent Laid-Open No. 52-51016). Further, it has been disclosed that peptide hormones directly modified with polyethylene glycol have reduced antigenicity (see Japanese Patent Laid-Open No. 61-178926).

However, the method of directly binding these therapeutic physiologically active substances to the water-affinity polymer is that a large amount of by-products are generated during the reaction, the reaction yield is low, and the binding of the bound physiologically active substances. It has drawbacks such as difficulty in controlling the release rate from the object.

Many methods have been proposed to solve these problems, but among them, it is said that the method of introducing cyanuric chloride between the water-affinitive polymer and the physiologically active substance for treatment is superior, and A reaction product of a therapeutic physiologically active substance and polyethylene glycol into which cyanuric chloride has been introduced (known as activated polyethylene glycol) is disclosed (for example, in Japanese Patent Publication No. 60-23084), activated polyethylene glycol-modified hemoglobin is disclosed. For example, see JP-A-62-62
-55079 discloses activated polyethylene glycol uricase, and JP-A-62-115280 discloses activated polyethylene glycol superoxide dismutase (SOD)).

<Problems to be Solved by the Invention> However, the method of modifying a therapeutic physiologically active substance with a water-affinity polymer such as polyethylene glycol activated with cyanuric chloride is firstly concerned with the safety of the cyanuric chloride group itself. Since its sex is unknown, its metabolism and degradation process in vivo are unknown, and the safety of degradation products has not been elucidated, it is necessary to use it with caution. Secondly, there is a problem that the activity of the physiologically active substance for treatment using cyanuric chloride, especially the activity of the enzyme, is significantly reduced.

<Means for Solving the Problem> The inventors of the present invention have made earnest studies in view of the above-mentioned problems of the prior art, and as a result, modify the physiologically active substance for treatment with a water-affinity polymer such as polyethylene glycol. In this case, by introducing cyclotriphosphazene instead of introducing cyanuric chloride between the water-affinity polymer and the therapeutic bioactive substance, the safety is high and the activity of the therapeutic bioactive substance does not decrease. Further, they have found that a water-affinity polymer derivative of a therapeutic physiologically active substance capable of controlling the kinetics of the therapeutic physiologically active substance in the body can be produced, and arrived at the present invention.

INDUSTRIAL APPLICABILITY The present invention provides cyclotriphosphazenes, which are highly safe physiologically active substances and / or novel physiologically active substances for treatment, whose kinetics in vivo can be controlled, and water-affinity polymer derivatives.

Cyclotriphosphazene is the minimum structural unit of the inorganic polymeric substance polyphosphazene. Since this cyclotriphosphazene is composed of nitrogen and phosphorus and is highly safe in vivo, it has already been known to attempt to bind a physiologically active substance for treatment thereto.

For example, an azirizene derivative of cyclotriphosphazene is known as cyclotriphosphazene bound to an alkylating agent (Labrre, JF et al., Eur. J. Cancer 1979,
15 (5) 637-43). However, derivatives in which a water-affinity polymer is bound to the phosphorus atom of these compounds are not known.

Further, for example, in the specification of USP 4,239,755, the following formula [II]
The steroidalized cyclotriphosphazenes represented by are disclosed.

In the above formula [II], R ₁ is a 3 or 17-hydroxysteroid residue and R ₂ is an alkyl group. However, this specification does not disclose the concept of binding a water-affinity polymer as a substituent of a phosphorus atom other than the phosphorus atom to which a steroid residue is bound.

US Pat. No. 4,495,174 discloses cyclotriphosphazene or polyphosphazene bound with a local anesthetic. However, although the specification discloses a method of substituting the chlorine group of cyclotriphosphazene with a local anesthetic having an amino group, there is no description about the handling of the unsubstituted chlorine group, Furthermore, there is no description or suggestion of binding a water-affinity polymer thereto.

Further, JP-A-56-30432 discloses an etophyllinated phosphasphazene polymer and a method for producing the same. The specification only describes an alkoxy group having 1 to 3 carbon atoms, a propylamino group or a phenoxy group as a substituent other than etophylline, and does not describe any water-affinity polymer. .

That is, all the physiologically active substances for treatment described in these prior arts are low-molecular weight substances, no disclosure is made about enzymes, polypeptides and proteins, and water-affinity polymers are There is no mention or suggestion of binding.

On the other hand, USP 3,674,509 discloses a protein crosslinked with cyclotriphosphazene as a protein feed for ruminants. The protein cyclotriphosphazene derivative of the same specification is a protein that crosslinks proteins with cyclotriphosphazene to make it resistant to digestion in the rumen of the ruminant animal, and alters the biokinetics of the protein. However, it is completely different from the derivative in which the protein of the present invention is bound to a water-affinity polymer through cyclotriphosphazenes.

Accordingly, the present invention provides the following formula [I] It is a cyclotriphosphazene derivative represented by.

The water-affinity polymer of the present invention may be any as long as it is capable of binding to a cyclotriphosphazene ring, and among them, cyclotriphos may be directly bonded via a functional group existing in the molecule, for example, a hydroxyl group or an amino group. A water-affinity polymer residue capable of binding to the phosphorus atom of the sphazene ring is preferable, and a derivative thereof may have such a group even if it does not have such a group, for example, by amidation. A derivative of a water-affinity polymer which is bound to a cyclotriphosphazene ring by binding a cross-linking agent to the water-affinity polymer.

Such water-affinity polymers include polyethylene glycol, monomethoxy polyethylene glycol, and dextran.

In addition, examples of the water-affinity polymer for the above-mentioned water-affinity polymer derivative include water-affinity polymers such as styrene-maleic acid copolymers and divinyl ether-maleic anhydride copolymers. These water-affinity polymers may be used alone or in combination of two or more.

These water-affinity polymers and cyclotriphosphazene rings are cyclotriphosphazene rings in the case of water-affinity polymer compounds having a hydroxyl group such as polyethylene glycol, monomethoxypolyethylene glycol and dextran (general formula X-OH). And an ether bond P-O-X.

The molecular weights of these water-affinity polymers differ depending on the type of water-affinity polymer and cannot be unconditionally specified.

300 to 30,00 for polyethylene glycol, monomethoxy polyethylene glycol, polypropylene glycol, etc.
0, dextran, inulin, chondroitin, hyaluronic acid, etc. are usually used in the range of 1,000 to 3,000,000, and polylactic acid, polyglycolic acid, etc., in the range of 1,000 to 50,000.

Among these, polyethylene glycol, monomethoxy polyethylene glycol and polypropylene glycol having a molecular weight of 300 to 10,000 are preferable. Dextran preferably has a molecular weight of 5,000 to 100,000.

The therapeutic physiologically active substance of the present invention is a physiologically active substance that is administered for the purpose of treating human or animal diseases, and does not include substances such as amino acids and sugars that are used for nutritional supplementation. The therapeutic physiologically active substance of the present invention has one or more groups selected from the group consisting of a hydroxyl group, an amino group, an imino group, and a thiol group in the molecule, and peptide hormones, enzymes, proteins It refers to a therapeutic physiologically active substance or its derivative selected from the group consisting of drugs, anticancer agents and steroids. Peptide hormones are those in which two or more amino acids are linked by a peptide bond, and may be derived from various animals including humans, synthetic products, genetically engineered products, and the like, and have similar structures to these. It also includes substances having the physiological activity of. Specifically, insulin, ACTH, gastrin, calcitonin, endorphin, glucagon, somatostatin, urogastrone, growth hormone releasing factor (GRF),
Conticotropin-releasing factor (CRF), natriuretic peptide, FSH, LHRH, vasopressin, enkephalin, interferon, interleukins, TNF, protein C
Etc.

Enzymes may be any of those derived from animals including humans, synthetic products, genetically engineered products, and the like, and further include substances having a similar structure to these and having the same physiological activity. Specifically, superoxide dismutase, peroxidase, asparaginase, glutamase, arginase, arginine deaminase, RNase, hexosaminidase A, hexosaminidase B, alpha glucosidase, sphingomyelinase, allylsulfatase, uricase, batroxobin, streptokinase, Examples include elastase, β-glucuronidase, β-glucosidase, adenosine deaminase, urokinase and bilirubin oxidase.

Proteins may be derived from various animals including humans, synthetic products, genetically engineered products, and the like, and further include substances having a similar structure to these and having the same physiological activity. Specific examples include albumin, hemoglobin, fibrinogen, plasminogen, TPA (tissue type plasminogen activator), immunoglobulin G, α ₂ -macroglobulin, lactoferrin, ragweed pollen and the like.

Anticancer agents include melphalan, p-phenylenediamine mustard, chlorambucil, Ara-C, methotrexate, mercaptopurine, mitomycin C, pepleomycin, daunorubicin, doxorubicin, 5-FU and the like.

As the steroid agent, a steroid having a hydroxyl group at the 3- or 17-position, such as desoxoestrone, estrone, pregnanolone, estradiol-3-methyl ether, testosterone, ethinyl estradiol,
Prednisolone, triamcinolone, triamcinolone acetonide and the like can be mentioned.

Of the cyclotriphosphazene ring used in the present invention,
The portion excluding the water-affinitive polymers and the physiologically active substance for treatment is -OR ₇ (R ₇ is an alkoxy group which is a C _{1 to} C ₂₄ alkyl group) and -NHR ₈ (R ₈ is a glycine ethyl ester group). may be at least one selected from amino acid residues) it is, but specifically a methoxy group (-OCH _3), glycine ethyl ester residue (-NHCH ₂ -COOC ₂ H ₅₎ etc. Among them, glycine ethyl ester residue is preferable.

The compound of the present invention can be produced, for example, by the following method. First, a water-affinity polymer and hexahalogenated cyclotriphosphazene are reacted in a solvent, recrystallized and then filtered to produce water-affinity polymer-substituted cyclotriphosphazenes. The amount ratio of the water-affinity polymer and the hexahalogenated cyclotriphosphazene used depends on the amount of the water-affinity polymer to be introduced into the cyclotriphosphazene ring. Usually, a sufficient amount of water-affinity polymer is used to replace 1 to 3 of the 6 halogen atoms of the cyclotriphosphazene ring. Due to the difference in reactivity, a large excess of water-affinity polymer than the equivalent of the number of substitutions may be used.
Examples of the water-affinity polymers include hydroxyl group-containing water-affinity polymers such as polyethylene glycol, monomethoxy polyethylene glycol, dextran and derivatives thereof.

In some cases, it is preferable that the water-affinity polymers having a hydroxyl group are alcoholates in which the hydrogen atom of the hydroxyl group is replaced with an alkali metal atom.

As hexahalogenated cyclotriphosphazene,
Hexachlorocyclotriphosphazene, hexafluorotriphosphazene, hexabromocyclotriphosphazene and the like can be mentioned. Among them, hexachlorocyclotriphosphazene is preferable.

Examples of the organic solvent used include non-aqueous solvents such as benzene, toluene, tetrahydrofuran, dioxane, and dimethylformamide, or a dimethylformamide-water mixed solvent. When the water-affinity polymer used has a high lipophilicity, a non-aqueous solvent is used, and when the water-affinity polymer has a low lipophilicity, dimethylformamide-
A water mixed solvent is used.

The reaction temperature is 0 ° C. to reflux temperature, and the reaction time is usually 1 to 24 hr, varying depending on the reactants. When the reaction is a dehydrochlorination reaction, addition of a base is desirable. Examples of the base used include sodium carbonate, triethylamine, pyridine, dimethylaminopyridine and the like.

For gel filtration, commonly used gels such as Sephadex LH-20, LH-60, G-100 are used. The gelation separates the water-affinity polymer-substituted cyclotriphosphazene in which the cyclotriphosphazene ring is replaced with a desired number of water-affinity polymer. The number of substitutions of the water-affinity polymer is preferably 1 to 3.

Next, the water-affinity polymer-substituted cyclotriphosphazenes thus produced are reacted with a therapeutic physiologically active substance in a solvent, and the water-affinity polymer and therapeutic physiologically active substance both-substituted cyclotriphosphazenes are reacted. To manufacture.

The amount ratio of the water-affinity polymer-substituted cyclotriphosphazenes to be used and the therapeutic physiologically active substance varies greatly depending on the therapeutic physiologically active substance. When the therapeutic physiologically active substance is high molecular weight peptide hormones, enzymes and proteins,
Since it has a plurality of amino groups that react with the cyclotriphosphazene ring in one molecule, those therapeutically active substances 1
A plurality of water-affinity polymer-substituted cyclotriphosphazenes can be bound per molecule. Therefore, in such a case, 1 to 1, for each free amino group of the physiologically active substance for therapeutic use.
000-fold, preferably 5-200-fold equivalents of water-affinity polymer-substituted cyclotriphosphazene are used.

On the other hand, when the therapeutic physiologically active substance is a low molecular weight peptide hormone or other low molecular weight compound, a plurality of therapeutic physiologically active substances can bind to one water-affinity polymer-substituted cyclotriphosphazene molecule. . Therefore, in such a case, the physiologically active substance for treatment is usually used in an amount of 1 to 100 times, preferably 1 to 50 times the equivalent of the water-affinity polymer-substituted cyclotriphosphazene.

The reaction is basically the same as the production method of the water-affinity polymer-substituted cyclotriphosphazenes in the previous period. When the therapeutic bioactive substance has a high lipophilicity, the reaction is performed with a non-aqueous solvent, and when the therapeutic bioactive substance has a low lipophilicity, the reaction is performed with a water-containing solvent. Examples of the water-containing solvent used include water, 0.1 M borate buffer solution (pH 10.0), and dimethylformamide-0.1 M borate buffer solution mixed solution.

The reaction temperature is 0 ° C. to the reflux temperature, but when the therapeutic physiologically active substance is a peptide hormone, enzyme, or protein, it is preferably carried out at a temperature as low as possible, preferably 0 to 40 ° C. to prevent inactivation. The reaction time is 1 to 24 hours, depending on the reactants.

After the reaction, gel filtration and ultrafiltration are performed to isolate the desired number of reaction products, water-affinity polymers into which therapeutic bioactive substances have been introduced, and cyclobiphosphazenes substituted with therapeutic bioactive substances. To do.

Preferably, the water-affinity polymer thus produced, the physiologically active substance for therapeutic use, and the unsubstituted halogen atom of the bisubstituted cyclotrophosphazene ring are alkoxy groups, secondary amino groups, secondary amino groups, alkyl groups, etc. Replace with.

When substituting with an alkoxy group, the both-substituted cyclotriphosphazenes are reacted with a sodium alkoxide such as sodium methylate in an organic solvent. Further, when substituting with a secondary amino group, the both-substituted cyclotriphosphazene is reacted with a primary amine such as methylamine, ethylamine, glycine ethyl ester in an organic solvent in the presence of triethylamine. When substituted with a tertiary amino group, the both-substituted cyclotriphosphazene is reacted with a secondary amine such as dimethylamine in an organic solvent in the presence of triethylamine. When substituting with an alkyl group, it is reacted with a Grignard reagent.

After the reaction, gel filtration and ultrafiltration are carried out to obtain a cyclotriphosphazene derivative which is a physiologically active therapeutic substance of the present invention.

The above-mentioned method is a method in which a water-affinity polymer is first introduced into the cyclotriphosphazene ring, then a therapeutic physiologically active substance is introduced into the cyclotriphosphazene ring, and finally an alkoxy group or the like is introduced.
It may be a method of introducing the therapeutic physiologically active substance and the other such as an alkoxy group in a different order.

The compound of the present invention is orally or parenterally administered to a mammal (monkey,
Dog, pig, rabbit, mouse, human etc.).

When administered orally, it is formulated into usual dosage forms such as tablets, granules, hard capsules, soft capsules, liquids and powders. In that case, it is formulated with additives such as an excipient, a binder, a disintegrant, and a lubricant.

For parenteral administration, injections, external preparations (ointments,
It is formulated into usual dosage forms such as creams, tapes, suppositories, nasal agents, oral agents). In that case, excipients, solvents,
It is formulated with additives such as dispersants and lubricants.

<Effects of the Invention> According to the present invention, first, the absorption rate of a physiologically active substance for treatment into a living body is increased, secondly the half-life in vivo is extended, and thirdly, side effects such as antigenicity are reduced. The fourth is a therapeutic bioactive substance having excellent characteristics such as enhanced retention in the diseased site, fifth is high safety, and sixth is retained activity. Triphosphazene derivatives are provided.

The characteristics as described above improve the efficacy and safety of the therapeutic physiologically active substance, and providing such a cyclotriphosphazene derivative of the therapeutic physiologically active substance is of great medical significance. .

The features of the present invention will be specifically described.

First, regarding the safety of the compound of the present invention, it goes without saying that among the parts constituting the compound of the present invention, the water-affinity polymer and the therapeutic physiologically active substance are safe.
Residues that replace cyclotriphosphazene are selected from ordinary alkoxy groups, amino groups, amino groups, imino groups, and alkyl groups, and it is safe to use amino acid residues. There is no end. Therefore, it is the cyclotriphosphazene ring itself that has a problem of safety. However, this has been shown by HR Allcock et al. (Inorg. Chem. 1982, 21, 515-521) to be decomposed into ammonia and phosphoric acid in an aqueous solvent. Therefore, it is clear that the cyclotriphosphazene ring is finally hydrolyzed into ammonia and phosphoric acid during the process of metabolism of the compound of the present invention in vivo. And it goes without saying that ammonia and phosphoric acid are safe at normal concentrations. Although the metabolic process of cyanuric chloride is completely unknown in conventional therapeutic bioactive substance derivatives of water-affinity polymer using cyanuric chloride, the safety of this compound is Easy to understand.

Secondly, the physiological activity of the therapeutic physiologically active substance derivative of the present invention is higher than the physiological activity of the conventional physiologically active substance derivative using cyanuric chloride, as is clear from Examples described below. As a result of comparing the SOD activity of the SOD derivative according to the present invention with the conventional method, it can be understood from the fact that the activity was higher than that of the conventional method.

Thirdly, as described in detail in Examples, mitomycin C is used for controlling in vivo kinetics of the present invention.
I investigated about the case. That is, the cyclotriphosphazene derivative in which 6 substituents of cyclotriphosphazene are substituted with monomethoxypolyethylene glycol 1, glycine ethyl ester 3, mitomycin C1 and the remaining one is substituted with an alkoxy group having a different alkyl group length Was prepared and the release rate of mitomycin C released therefrom in water was measured. As a result, it became clear that the longer the length of the alkyl group, the smaller the release rate of mitomycin C. This result indicates that the release rate of the therapeutically active physiologically active substance when the compound of the present invention is administered in vivo can be freely controlled.

<Examples> The present invention will be described in more detail with reference to Examples below, but the present invention is not limited thereto.

Example 1 Production of monomethoxypolyethylene glycol (PEG), glycine ethyl esterified cyclotriphosphazene-SOD (i) Production of sodium alcoholate of monomethoxyPEG 50 g of monomethoxyPEG (average molecular weight 5,000) was added to tetrahydrofuran (THF). Dissolved in 300 ml, sodium hydride
Add 0.24 g and reflux. The reaction was continued until the evolution of hydrogen stopped.

(Ii) Production of monomethoxy PEG-cyclotriphosphazene Subsequently, a solution prepared by dissolving 3.5 g of hexachlorocyclotriphosphazene in 100 ml of THF was added to the above reaction solution and reacted at room temperature for 2 hours. After the reaction was completed, NaCl formed was removed by passing.

(Iii) Monomethoxy PEG, glycine ethyl ester-
Preparation of cyclotriphosphazene Subsequently, the above liquid was cooled to 5 ° C, and a solution of 6.18 g of glycine ethyl ester (NH ₂ —CH ₂ —COOC ₂ H ₅ ) dissolved in 100 ml of THF was added thereto, and 6.00 g of triethylamine was added. Was added and reacted for 3 hours. The precipitate (triethylamine hydrochloride) formed after the reaction was removed by filtration, the liquid was concentrated, and the residue was dissolved in 200 ml of a chloroform-hexane (1: 1) solution. The solution was purified by gel filtration through a column (5 × 73 cm) of Sephadex LH-20 (Pharmacia) to separate the main components and distill off the solvent to obtain about 35 g of a dry solid. When the amount of Cl in this product was quantified, the ratio was 1 atom to 1 cyclotriphosphazene ring.

(Iv) Production of SOD derivative of monomethoxy PEG, glycine ethyl ester-cyclotriphosphazene 1.5 g of the dried product obtained above and 15 mg of SOD (manufactured by Nippon Chemical Research Co., Ltd.) were added to 0.1 M borate buffer solution. , pH 9.0 5.0 ml
After reacting at 4 ℃ for 2 hours in 0.1M phosphate buffer, pH 7.0
Was added to stop the reaction, and the ultrafiltration (Amicon,
Unreacted PEGylated cyclotriphosphazenes were removed with membrane YM-10), and 2.0 ml of the concentrate was purified by gel filtration through Sephacryl S-100 (Pharmacia) column (5 × 73 cm) to obtain monomethoxy. PEG, glycine ethyl esterification-
About 15 mg was obtained as cyclotriphosphazene-SOD.
The obtained product was analyzed by elemental analysis, nmr spectrum, and mass spectrum, and one of the six substituents on the cyclotriphosphazene ring was monomethoxy PEG, one was SOD, and the remaining four were glycine ethyl ester. It was confirmed. The obtained monomethoxy PEG-cyclotriphosphazene-SOD had the amino group at residue 7-12 of SOD modified, and the SOD activity was about 70% of the original value (for details, see the following study. reference).

The following experiment was conducted on the obtained SOD derivative.

Measurement of SOD activity Method of utilizing SOD activity of the obtained compound by reduction of cytochrome C (Seikagaku Corporation Enzyme Data Seet Lib)
rary No. 68) and compared with the activity of unmodified SOD.

At the same time, the SOD activity of PEG derivatives of SOD using cyanuric chloride was compared as a control. PE of SOD using cyanuric chloride
The G derivative was produced by the following method.

40 g of monomethoxy PEG (average molecular weight 5,000) and 0.73 g of cyanuric chloride in 150 ml of benzene molecular sieve 3A 7 g
And reacted at 80 ° C. for 20 hours in the presence of 15 g of sodium carbonate. After completion of the reaction, the precipitate was removed by filtration, and then petroleum ether was added to precipitate monomethoxyPEG-modified cyanuric chloride. This is washed with petroleum ether and then dried to give the product.
I got 10g.

1.5 g of the obtained product and 15 mg of SOD (manufactured by Nippon Chemical Research Co., Ltd.) in 4 ml of 5.0 ml of 0.1 M borate buffer pH 9.0.
After reacting for 2 hours at ℃, 30 ml of 0.1M phosphate buffer pH 7.0
The reaction is stopped by adding, and ultrafiltration (manufactured by Amicon, membrane 7M-1
Unreacted monomethoxy PEG-cyanuric chloride was removed in 0), and 2.0 ml of the concentrated solution was purified by gel filtration through a column (5 × 73 cm) of Sephacryl S-100 (Pharmacia) and freeze-dried to obtain monomethoxy. PEG-Cyanuric chloride-SOD
As a result, about 15 mg was obtained. In the obtained compound, the amino group of the residue was modified at 7- of SOD.

Table 1 shows the relative comparison results of the SOD activities of Example 1 obtained above and the target age group.

It can be seen from Table 1 that the SOD derivative of the present invention has higher SOD activity than the conventional derivative using cyanuric chloride.

Measurement of half-life in blood The half-life in blood of the compound of the present invention and unmodified SOD was intravenously injected into rats (3 rats each), and blood was sampled from the tail vein over time to determine the SOD activity in plasma as described above. Comparison was made by measuring by.

The results obtained are shown in Table 2.

As shown in Table 2, the blood half-life of the compound of the present invention is remarkably prolonged.

Immunogenicity The compound of the present invention (Example 1) and unmodified SOD were each added in an amount of 20 μg as Freund's complete supplement solution (Freund's comp).
The emulsion was emulsified with lete adjuvant) (FCA), and then intraperitoneally administered (0.2 ml) to A / J mice, and thereafter, additional administration was performed on the 14th and 28th days. Blood was collected from the orbital vein of the mouse every 7th day from the first administration day, and the serum was pre-diluted with the rat by intradermal injection (0.1 ml), and 4 hours later. SOD 0.5mg and Evans Bull-20
2 ml of a mixed solution of mg was intravenously injected, and passive skin anaphylaxis (Passive Anaphyla
The antibody production by the xis, PCA) reaction was evaluated.

The results are shown in Table 3. The numerical values in the table are shown as the maximum dilution factor of the serum in which the PCA reaction was positive.

From the results in Table 3, antibody production was observed for unmodified SOD but not for the compound of the present invention.

Absorbability from duodenum The compound of the present invention and unmodified SOD were each treated as 100m as SOD activity.
G / Kg was fasted for 20 hours in a rat (SD male, 6-week-old) by duodenal administration, and the SOD activity in plasma after the administration was measured.

The results are shown in Table 4.

The results in Table 4 suggest that SOD may be absorbed orally by the compounds of the present invention.

Example 2 Production of monomethoxy PEG, glycine ethyl ester-cyclotriphosphazene-SOD An example of producing the same compound as in Example 1 by another method is shown below.

(I) Preparation of monomethoxy PEG-cyclotriphosphazene 50 g of monomethoxy PEG (average molecular weight 5,000) and 3.5 g of hexachlorocyclotriphosphazene in 200 ml of benzene in the presence of 10 g of molecular sieve 3A and 20 g of sodium carbonate at 80 ° C. Reacted for hours. After completion of the reaction, the precipitate was removed by filtration, and then petroleum ether was added to add monomethoxy PEG.
Cyclotriphosphazene was precipitated. This was washed well with petroleum ether and then dried to obtain 52 g of a product. It was confirmed by Cl analysis that 5 Cl atoms were attached to 1 molecule of this compound.

(Ii) Preparation of monomethoxy PEG-cyclotriphosphazene-SOD 1.5 g of the product obtained above and 15 mg of SOD (Nippon Chemical Research Co., Ltd.) in 5.0 ml of 0.1 M borate buffer pH 9.0. so
After reacting at 37 ℃ for 1 hour, 30 ml of 0.1M phosphate buffer pH 7.0
In addition, the reaction was stopped and ultrafiltration (Amicon, membrane 7M-1
Unreacted monomethoxy PEG-cyclotriphosphazene was removed in 0), 2.0 ml of the concentrate was purified by gel filtration through a column (5 × 73 cm) of Sephacryl S-100 (Pharmacia), and freeze-dried. , About 15 mg was obtained as monomethoxy PEG-cyclotriphosphazene-SOD.

(Iii) Monomethoxy PEG, glycine ethyl ester-
Preparation of cyclotriphosphazene-SOD 15 mg of monomethoxy PEG-cyclotriphosphazene-SOD obtained above was dissolved in water, and 10 mg of glycine ethyl ester was added thereto together with 10 mg of triethylamine, and the mixture was reacted at 40 ° C for 2 hours. After the reaction is complete, dialyze to remove small molecules,
The reaction product obtained by freeze-drying was Sephacryl S-100.
The product was purified by gel filtration on a column (Pharmacia) (5 × 73 cm) and freeze-dried to obtain about 5 mg of the same compound as the final product of Example 1.

Example 3 Production of monomethoxy PEG, glycine ethyl esterified cyclotriphosphazene-insulin (i) Monomethoxy PEG, glycine ethyl esterified cyclotriphosphazene was prepared in the same manner as in (i) to (iii) of Example 1. Obtained.

(Ii) Production of insulin derivative of monomethoxy PEG, glycine ethyl esterified cyclotriphosphazene 1.5 g of the dried product obtained above and 150 mg of porcine insulin
Was reacted in 5.0 ml of 0.1 M borate buffer, pH 9.0 for 2 hours at 4 ° C., and then 30 ml of 0.1 M phosphate buffer pH 7.0 was added to stop the reaction, and the reaction was carried out by ultrafiltration (Amicon). , Membrane YM-10) to remove unreacted PEGylated cyclotriphosphazenes and concentrate
2.0 ml was purified by gel filtration through a Biogel P-30 column (5 × 73 cm) to obtain about 130 mg of freeze-dried monomethoxy PEG, glycine ethyl esterified cyclotriphosphazene-insulin as the main eluate.

Example 4 In the same manner as in Example 3, monomethoxy PEGs having an average molecular weight of 350,750,1,900 were also used.
EG, glycine ethyl esterified cyclotriphosphazene-insulin was produced (Example 4- (a)-
(C)). The glucose lowering effect of the compound of Example 3 and these insulin derivatives is described in the literature (Z. Anal. Chemie. 25
2 , 224 (1970)).

The results are shown in Table 5.

Example 5 Production of monomethoxy PEG, glycine ethyl ester-cyclotriphosphazene-alparaginase (i) Production of monomethoxy PEG-cyclotriphosphazene In the same manner as in Example 2, monomethoxy PEG-cyclotriphosphazene was obtained. .

(Ii) Production of monomethoxy PEG-cyclotriphosphazene-alparaginase 1.5 g of the product obtained above and asparaginase (EC
ol-derived, Kyowa Fermentation) 150 mg 0.1M borate buffer pH 9.0 5.
After reacting in 0 ml at 37 ℃ for 1 hour, 0.1M phosphate buffer pH
Unreacted monomethoxy PEG-cyclotriphosphazene was removed by ultrafiltration (Amicon, membrane 7M-10) by adding 30 ml of 7.0, and 2.0 ml of concentrate was added to Sephacryl S-100 (Pharmacia). Column) (5 × 73 cm) gel filtration, freeze-dried and monomethoxy PE
About 300 mg was obtained as G-cyclotriphosphazene-alparaginase. It was confirmed that these compounds had 4 Cl atoms.

(Iii) Production of monomethoxy PEG, methylamine-cyclotriphosphazene-alparaginase 100 mg of monomethoxyPEG-cyclotriphosphazene-asparaginase obtained above was dissolved in water and kept at 4 ° C, and then methylamine was added thereto. It was blown in as a gas. After 30 minutes, the blowing was stopped, the pressure was reduced to remove methylamine, the mixture was dialyzed to remove low molecules, and the reaction product obtained by freeze-drying was applied to a column of Sephacryl S-100 (manufactured by Farfacia) (5 × 7).
It was purified by gel filtration (3 cm) and freeze-dried to give about 70 mg of monomethoxy PEG-methylamine-cyclotriphosphazene-alparaginase. It was confirmed that there was no Cl atom in the obtained product.

Example 6 Production of Monomethoxy PEG, Glycine Ethyl Esterified Cyclotriphosphazene-Hemoglobin (i) Monomethoxy PEG, glycine ethyl esterified cyclotriphosphazene was prepared in the same manner as in (i) to (iii) of Example 1. Manufactured.

(Ii) Production of hemoglobin derivative of monomethoxy PEG, glycine ethyl esterified cyclotriphosphazene 1.8 g of the dried product obtained above and 0.5 mg of hemoglobin were added to 0.1
After reacting in 150 ml of M borate buffer, pH 9.0 under ice-cooling for 2 hours, 30 ml of 0.1 M phosphate buffer, pH 7.0 was added to stop the reaction, and the reaction was stopped by ultrafiltration (molecular weight 100,000 blocking membrane). Unreacted raw materials and the like were removed in step (1) and freeze-dried to obtain about 2 g of monomethoxy PEG, glycine ethyl esterified cyclotriphosphazene-hemoglobin.

Example 7 Production of monomethoxy PEG, glycine ethyl ester-cyclotriphosphazene-mitomycin C (i) Production of monomethoxy PEG-cyclotriphosphazene In the same manner as in Example 2, monomethoxy PEG-cyclotriphosphazene was obtained. It was

(Ii) Production of monomethoxy PEG-cyclotriphosphazene-mitomycin C 1.5 g of the product obtained above and 200 mg of mitomycin C were dissolved in acetone, 100 mg of potassium carbonate was added, and the mixture was refluxed for 5 hours. Acetone was distilled off after filtering the precipitate, and the residue was purified by Sephadex LH-20 gel filtration using monomethoxy PEG.
-Cyclotriphosphazene-mitomycin C 1.0 g
Obtained.

(Iii) Monomethoxy PEG, glycine ethyl ester-
Production of cyclotriphosphazene-mitomycin C 500 mg of monomethoxy PEG-cyclotriphosphazene-mitomycin C obtained above was dissolved in THF, and 100 mg of glycine ethyl ester was added thereto together with 100 mg of triethylamine, followed by reaction at 4 ° C. for 2 hours. After completion of the reaction, the precipitate was removed by filtration and THF was distilled off to obtain a reaction product, which was then used as a Sephadex LH-20 (Pharmacia) column (5 × 7).
The product was purified by gel filtration (3 cm) to obtain 320 mg of monomethoxy PEG, glycine ethyl ester-cyclotriphosphazene-mitomycin C. No Cl atom was detected in this product. Further, it was confirmed from nmr that four glycine ethyl esters were attached to the cyclotriphosphazene ring.

Example 8 Monomethoxy PEG, glycine ethyl ester alkoxy-
Preparation of Cyclotriphosphazene-Mitomycin C Using the same method as in Example 7, glycine ethyl ester was reacted in 3 equivalents instead of 4 equivalents of cyclotriphosphazene in the step (iii), and hexachlorocyclo A compound was prepared in which one Cl atom of triphosphazene remained. Using this as a raw material in THF, CH ₃ ONa,
C ₆ H ₁₃ ONa, C ₁₂ H ₂₅ ONa, C ₁₈ H ₃₇ ONa are reacted with each other at room temperature for 10 hours, and the remaining Cl atoms are respectively -OCH ₃ ,, -OC ₆ H ₁₃ ,, -OC ₁₂ H ₂₅ ,-.
A compound substituted with OC ₁₈ H ₃₇ was prepared. Table 6 shows the release rate of mitomycin C when these compounds were dissolved in water so as to be converted into mitomycin C in the same amount and stirred at 37 ° C.

These results show that the release of mitomycin C bound to the cyclotriphosphazene ring can be freely controlled by changing the nature of the group substituted on the same ring.

Example 9 Preparation of monomethoxy PEG, glycine ethyl ester-cyclotriphosphazene-doxorubicin Doxorubicin instead of mitomycin C of Example 7
The same reaction was performed using 200 mg to obtain monomethoxy PEG,
Glycine ethyl ester-cyclotriphosphazene-
290 mg of doxorubicin was obtained.

The nmr spectrum of the reaction product confirmed that the amino group of doxorubicin was bonded to the cyclotriphosphazene ring.

Example 10 Production of monomethoxy PEG, methoxy-cyclotriphosphazene-taunomycin (i) Production of monomethoxy PEG-cyclotriphosphazene In the same manner as in Example 2, monomethoxy PEG-cyclotriphosphazene was obtained.

(Ii) Production of monomethoxy PEG-cyclotriphosphazene-daunomycin 1.5 g of the product obtained above and 200 mg of daunomycin were added to acetone, 100 mg of potassium carbonate was added, and the mixture was refluxed for 5 hours. After removing the precipitate in excess, the acetone was distilled off,
The residue was purified by Sephadex LH-20 gel filtration to obtain 1.3 g of monomethoxy PEG-cyclotriphosphazene-daunomycin.

(Iii) Production of monomethoxy PEG, methoxy-cyclotriphosphazene-daunomycin 500 mg of monomethoxy PEG-cyclotriphosphazene-daunomycin obtained above was dissolved in methanol, and 200 mg of sodium methylate was added thereto at 4 ° C. The reaction was carried out for 2 hours. After completion of the reaction, the precipitate was removed by filtration and the reaction product obtained by distilling off methanol was separated by Sephadex LH-20.
The product was purified by gel filtration on a column (Pharmacia) (5 × 73 cm) to obtain 330 mg of monomethoxy PEG, methoxy-cyclotriphosphazene-daunomycin. No Cl atom was detected in this product. It was also confirmed from nmr that four methoxy groups were attached to the cyclotriphosphazene ring.

Example 11 Preparation of monomethoxy PEG, glycine ethyl esterified cyclotriphosphazene-estradiol 3-methyl ether (i) Monomethoxy PEG-cyclotriphosphazene was prepared in the same manner as in (i) to (ii) of Example 1. Obtained.

(Ii) Preparation of monomethoxy PEG-cyclotriphosphazene-estradiol 3-methyl ether 2.86 g of estradiol 3-methyl ether was added to 100 ml of THF.
It was dissolved therein, 0.24 g of sodium hydride was added, and the mixture was refluxed for 5 hours. After the reaction, the reaction solution was cooled to room temperature, and then (i)
The liquid obtained in step 1 was added and the reaction was carried out at room temperature for 5 hours. After the reaction was completed, NaCl formed was removed by passing. The THF was distilled off to obtain 52 g of monomethoxy PEG-cyclotriphosphazene-estradiol 3-methyl ether.

(Iii) Monomethoxy PEG, glycine ethyl ester-
Preparation of cyclotriphosphazene-estradiol 3-methyl ether 10 g of monomethoxy PEG-cyclotriphosphazene-estradiol 3-methyl ether obtained in (ii) was added to THF30.
1 ml of glycine ethyl ester in THF5
A solution dissolved in 0 ml was added, 1 g of triethylamine was further added, and the mixture was reacted for 5 hours. The precipitate (triethylamine hydrochloride) formed after the reaction was removed by filtration, and the solution was concentrated,
The residue was dissolved in 20 ml of chloroform-hexane (1: 1) solution. This solution was purified by gel filtration through a column (5 × 73 cm) of Sephadex LH-20 (Pharmacia), and the solvent was distilled off to remove monomethoxy PEG, glycine ethyl ester-cyclotriphosphazene-estradiol 3-methyl. 15 g of ether was obtained.

Example 12 Preparation of dextran, glycine ethyl ester-cyclotriphosphazene-SOD (i) Dextran-cyclotriphosphazene-SO
Preparation of D Hexachlorocyclotriphosphazene 3.5 g in a solution of 10 g dextran (average molecular weight 40,000) dissolved in 100 ml H ₂ O.
Was dissolved in a mixed solvent of 10 ml of water and 40 ml of acetone and added dropwise. During this period, the pH was adjusted to 10-11 with 2N caustic soda solution.
Kept at. After the dropping was completed, the pH was adjusted to 3 with 2N hydrochloric acid. The dextranated cyclotriphosphazene formed was precipitated and purified by adding acetone.

(Ii) Dextran-cyclotriphosphazene-SO
Production of D 5.0 g of the product obtained above and 15 mg of SOD (Nippon Chemical Research Co., Ltd.) in 5.0 ml of 0.1 M borate buffer pH 9.0.
After reacting at 37 ℃ for 1 hour, add 0.1M phosphate buffer pH 7.0 to 30m.
l Add the reaction to stop the reaction,
Unreacted dextran-cyclotriphosphazene was removed with M-10) and 2.0 ml of the concentrate was purified by gel filtration through a column (5 × 73 cm) of Sephacryl S-100 (Pharmacia) and freeze-dried. , About 100 mg was obtained as dextran-cyclotriphosphazene-SOD.

(Iii) Production of dextran, glycine ethyl ester-cyclotriphosphazene-SOD Dextran-cyclotriphosphazene-obtained above
Dissolve 80 mg of SOD in water and add glycine ethyl ester 1
0 mg was added together with 10 mg triethylamine, and the mixture was reacted at 4 ° C. for 2 hours. After completion of the reaction, the reaction product obtained by dialysis to remove low molecular weight compounds and freeze-drying was purified by gel filtration through a column (5 × 73 cm) of Sephacryl S-100 (Pharmacia) and freeze-dried to give dextran, 60 mg of glycine ethyl ester-cyclotriphosphazene-SOD was obtained. The obtained SOD derivative had the amino group at residue 7-12 of SOD modified, and the SOD activity was about 60% of the original value.

Example 13 Production of dextran, glycine ethyl ester-cyclotriphosphazene-insulin (i) Production of dextran-cyclotriphosphazene In the same manner as in Example 12, dextran-cyclotriphosphazene was obtained.

(Ii) Production of dextran-cyclotriphosphazene-insulin 4.0 g of the product obtained above and 150 mg of porcine insulin
Was reacted in 5.0 ml of 0.1 M borate buffer pH 9.0 at 37 ° C. for 1 hour, then the reaction was stopped by adding 30 ml of 0.1 M phosphate buffer pH 7.0, and the reaction was carried out by an ultrafiltration (manufactured by Amicon). , Membrane 7M-10) to remove unreacted dextran-cyclotriphosphazene,
Biogel P-30 column (5 x 73 cm) with 2.0 ml of concentrate
Of the dextran-cyclotriphosphazene-insulin by lyophilization.
0 mg was obtained.

(Iii) Production of dextran, glycine ethyl ester-cyclotriphosphazene-insulin Dextran-cyclotriphosphazene-obtained above
Insulin (50 mg) was dissolved in water, and glycine ethyl ester (10 mg) was reacted with triethylamine (10 mg) at 4 ° C. for 2 hours. After the reaction is complete, dialyze to remove small molecules,
The reaction product obtained by freeze-drying was purified by gel filtration on Biogel P-30 column (5 × 73 cm) and freeze-dried to obtain 33 mg of dextran, glycine ethyl ester-cyclotriphosphazene-insulin.

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Claims (10)

[Claims] 1. The following formula [I]: A cyclotriphosphazene derivative represented by. 2. The cyclotriphosphazene derivative according to claim 1, wherein the molecular weight of monomethoxypolyethylene glycol is 300 to 30,000. 3. The molecular weight of dextran is 1,000 to 3,000,000.
The cyclotriphosphazene derivative according to claim 1, wherein 4. The cyclotriphosphazene derivative according to claim 1, wherein the peptide hormones are insulin, calcitonin, or natriuretic peptide. 5. The cyclotriphosphazene derivative according to claim 1, wherein the enzyme is superoxide dismutase, asparaginase, or bilirubin oxidase. 6. The cyclotriphosphazene derivative according to claim 1, wherein the protein is hemoglobin, TPA or interferon. 7. The cyclotriphosphazene derivative according to claim 1, wherein the anticancer agent is mitomycin C, daunorubicin, or doxorubicin. 8. The cyclotriphosphazene derivative according to claim 1, wherein the steroid is estradiol-3-methyl ether, testosterone or triamcinolone acetonide. 9. When the total of water-affinity polymer or its derivative and therapeutic physiologically active substance or its derivative among R _{1 to} R ₆ is 5 or less, the remaining cyclotriphosphazene substituents are glycine. The cyclotriphosphazene derivative according to any one of claims 1 to 8, which is an ethyl ester residue. 10. An effective amount of the following formula [I]: A pharmaceutical composition comprising the cyclotriphosphazene derivative represented by: and (b) a pharmaceutically acceptable carrier.