JP2874309B2 - Sugar-responsive polymer complex - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a saccharide-responsive polymer complex.

The sugar-responsive polymer complex can be used as a diabetes treatment system, a sugar sensor, or the like, which controls the release of a drug according to the sugar concentration.

(Prior Art) Conventionally, the fact that polyvinyl alcohol causes gelation by adding boric acid to an aqueous solution of polyvinyl alcohol is known.

In addition, as a product obtained by introducing a boronic acid group into agarose gel, Matrex (trademark) manufactured by Amicon Co., Ltd.
There is PBA-30 (benzeneboronic acid-crosslinked agarose gel), which contains boronic acid groups and dis-
It is used as a gel carrier for affinity chromatography utilizing the fact that a diol group-containing saccharide forms a complex.

However, up to now, there has been no report on the formation of a complex between a polymer having a boronic acid group and a polymer having a polyvalent hydroxyl group, or sol-gel transition caused by dissociation.

In general, in a living body, in a healthy state, an in-vivo regulation mechanism (homeostasis) works sufficiently and, for example, various advanced feedbacks such that the ion concentration and the blood glucose level in various blood are always kept constant. Tightly adjusted by the system. However, when a problem occurs in this regulatory mechanism for some reason, for example, when a chronic illness such as diabetes or hypertension occurs, insulin or a drug as an external therapeutic agent is periodically administered according to the symptoms. Need to be administered. The amount and timing of the drug to be administered at that time should be carefully considered. In particular, a great change in the balance of the regulation mechanism in the living body has serious consequences, and thus requires special care. So far, the main treatments for diabetes are diet and self-injection of insulin, even with the advancement of medicine and the proliferation of various medical devices.

From such a viewpoint, there is an increasing need for a diabetes treatment system that incorporates an auto-feedback mechanism that releases a drug only when necessary and stops the release immediately when the drug becomes normal.

Elliott and his colleagues detect blood glucose with a glucose detector,
A small portable device that injects a corresponding amount of insulin into a vein by a pump has been reported (J. Am. M.
ed. Assoc., 241 , 223 (1979)).

S. W. Kim et al. Have proposed an insulin release system using a complex of concanavalin A and a sugar chain-modified insulin as a molecular device having both a glucose sensor function and a drug release mechanism (DI
ABETES, 32, 499 (1983) ).

In other fields, the need for measurement of glucose, which is a kind of sugar, has been increasing, and glucose sensors have been actively developed in fields such as medical treatment, food, and fermentation processes.

(Problems to be Solved by the Invention) However, in the method of adding boric acid to an aqueous solution of polyvinyl alcohol, since boric acid to be added has a low molecular weight, it is easily diffused or permeated in a substance and is toxic. However, it was not suitable for medical applications.

The method of self-injection of insulin has disadvantages such as a mismatch between the required amount of insulin to be injected and the required amount, troublesome operation, risk of accidents such as low blood glucose coma, and the need for strong self-control of the patient himself. There is a need for the development of a simpler and safer insulin release control device (artificial spleen).

In addition, the method of Elliott et al. Connects the glucose sensor to the patient's bloodstream for a long time through the skin, causing problems such as bacterial infection and thrombus formation from the connection port, and furthermore, crystallization of insulin. It is not sufficient in safety and reliability because troubles such as clogging of the injection needle due to the above and troubles due to mechanical or electronic circuit failures are expected. In addition, since the conventional glucose sensor uses an enzyme, it has a drawback that its life is about one week.

In the example of Kim et al., The above-mentioned composite was dispersed in a pouch made of a polymer film. If this is implanted in the abdominal cavity, an exchange reaction occurs between the sugar chain-modified insulin bound to concanavalin A and glucose as the glucose concentration increases outside the pouch, and the sugar chain-modified insulin is released . On the other hand, when the glucose concentration is reduced, the exchange reaction is reduced, and the insulin release is also reduced. However, this system uses concanavalin A, which is extremely toxic, and thus has a problem in safety as a drug.

So far, no report has been reported that utilizes the fact that a boronic acid group is introduced into a synthetic polymer to cause a swelling change of a crosslinkable polymer by forming a complex with a sugar.

In the present invention, excellent sugar responsiveness, low toxicity, moldability,
It is an object of the present invention to provide a sugar-responsive polymer complex that can be easily processed.

(Means for Solving the Problems) The present invention is a sugar-responsive polymer composite comprising a boronic acid group-containing polymer and a polyvalent hydroxyl group-containing polymer.

 Hereinafter, the present invention will be described in more detail.

The boronic acid group-containing polymer in the polymer composite used in the present invention is a polymer of a boronic acid group-containing monomer, and a copolymer of this monomer and a copolymerizable monomer, and has a molecular weight of 1 to 10. It is about ten thousand.

Examples of the boronic acid group-containing monomer include 3-acryloylaminobenzeneboronic acid, 3-methacryloylaminobenzeneboronic acid, and 4-vinylbenzeneboronic acid.

Examples of the monomer copolymerizable with the boronic acid group-containing monomer include, for example, acrylamide, N-methylacrylamide, N, N-dimethylacrylamide, N, N-dimethylaminopropylacrylamide, N, N-dimethylaminopropylacrylamide various quaternary Salts, acryloyl morpholine, N, N-dimethylaminoethyl acrylate various quaternary salts, acrylic acid, various alkyl acrylates, methacrylic acid, various alkyl methacrylates, 2-hydroxyethyl methacrylate, N-vinyl pyrrolidone, monoglycerol methacrylate, acrylonitrile, Styrene and various macromonomers. The boronic acid group-containing monomer is used in the range of 0.1 to 90 mol%, preferably 0.5 to 30 mol%, based on all monomers.

Examples of the boronic acid group-containing polymer include, for example, condensation of a carboxyl group of an unsaturated carboxylic acid such as acrylic acid, methacrylic acid, itaconic acid, metaconic acid, fumaric acid, and maleic anhydride or a carboxyl group of a copolymer with carbodiimide or the like. A polymer obtained by an amide reaction with an amino group-containing boronic acid such as m-aminobenzeneboronic acid using an agent can also be used.

Examples of the polyhydric hydroxyl group-containing polymer that can be used in the present invention include polyvinyl alcohol having a polymerization degree of 100 to 10,000 or polysaccharides such as galactomannan, pullulan, dextran, and amylose. Further, a polymer obtained by hydrolyzing a polymer containing vinyl acetate, or a monomer having a polyvalent hydroxyl group such as monoglycerol methacrylate alone or with one or more other copolymerizable monomers may be used. A polymer obtained by polymerization can also be used. The amount of the monomer having a polyvalent hydroxyl group to be used is in the range of 0.1 to 90 mol% of all monomers.

Further, as the polyvalent hydroxyl group-containing polymer, acrylic acid,
Compounds having a carboxyl group of a homo- or copolymer of an unsaturated carboxylic acid such as methacrylic acid, itaconic acid, methaconic acid, fumaric acid, maleic anhydride or the like having a polyvalent hydroxyl group and a primary amino group, for example, tris (hydroxymethyl) amino A polymer reacted with methane or the like can also be used. Further, a polymer obtained by an amide reaction between a polymer containing a primary amino group and a carboxyl group-containing polyvalent hydroxyl compound may be used. Furthermore, examples of the polymer containing a primary amino group include a polymer or a protein obtained by homopolymerizing or copolymerizing a monomer such as aminostyrene or vinylbenzylamine. Examples of the carboxyl group-containing polyvalent hydroxyl group compound include protocatechuic acid, gallic acid, tricine and 2,2- (dihydroxymethyl) propionic acid.

In the polymer composite of the present invention, the mixing ratio of the boronic acid group-containing polymer and the polyvalent hydroxyl group-containing polymer varies depending on the molecular weight, but is preferably from 1:50 to 50: 1, and more preferably from 1:
10 to 10: 1. Particularly preferably, it is 3: 1 to 1: 1.

In the boronic acid group-containing polymer, the amount of the boronic acid group-containing monomer used is preferably 0.1 mol% or more of all monomers, and more preferably 0.5 to 30 mol%.

The polymer used in the composite of the present invention is a known solution by a radical polymerization method, bulk, emulsification, polymerization temperature is 0 in the suspension polymerization.
It can be obtained at 100 ° C and a polymerization time of 10 minutes to 48 hours. Examples of the polymerization initiator used here include benzoyl peroxide, diisopropyl peroxydicarbonate, tertiary butyl peroxy-2-ethylhexanoate, tertiary butyl peroxypivalate, tertiary butyl peroxydiisobutyrate, lauroyl peroxide, and azoyl peroxide. One or more selected from the group of bisisobutyronitrile, 2,2'-azobis (2,4-dimethylvaleronitrile), and various redox initiator systems can be used, and 0.01 to 5.0 weight Used in%.

When used as the polymer complex of the present invention, a boronic acid group-containing polymer solution and a polyvalent hydroxyl group-containing polymer solution are mixed and used. The boronic acid group-containing polymer solution is 0.01 to 50% by weight.
It is preferably 0.1 to 20% by weight. The polyvalent hydroxyl group-containing polymer solution is used at 0.01 to 50% by weight, preferably 0.1 to 20% by weight.

The complex of the present invention is used in an aqueous medium or an aqueous medium containing 50% or less of an organic solvent, and is preferably used using a buffer. As the buffer, a sodium phosphate buffer, a sodium acetate buffer, an o-chlorophenol buffer, a sodium arsenate buffer, a sodium carbonate buffer, a Hepes buffer, a chess buffer and the like can be used. In this case, the pH value is preferably 6.0 or more. The composite is used at a temperature of 1 to 50C.

Sugars to which the polymer complex of the present invention responds include, for example, glucose, galactose, fructose, and mannose.

When the polymer complex of the present invention is used in the aqueous medium, it is used at a sugar concentration of 0.1 mg / dl or more, and preferably at a sugar concentration of 1 to 10,000 mg / dl.

The polymer complex of the present invention is also used as a sugar-responsive drug releaser, for example, by putting the complex and a drug in a commercially available dialysis membrane or semipermeable membrane or by microencapsulation. As a drug applicable to the polymer complex of the present invention, all drugs soluble in an aqueous solution can be used, and particularly, insulin, glucagon, somatostatin,
Corticosteroids are effective. Also, two or more of these drugs can be used in combination.

(Effect of the Invention) In the sugar-responsive polymer complex of the present invention, as the sugar concentration increases, the boronic acid group-containing polymer chain and the polyvalent hydroxyl group-containing polymer chain dissociate, while the sugar concentration decreases. Then, the dissociation between the polymer chains decreases. In addition, a drug is added to the complex in which a boronic acid group-containing polymer solution and a polyvalent hydroxyl group-containing polymer solution are mixed and used, whereby a sugar-responsive drug-releasing complex is obtained.

That is, the polymer complex of the present invention can control the release of the drug in response to the sugar concentration, has low toxicity,
Moreover, since it can be prepared in various forms,
It can be excellent in reliability, safety, ease of handling, etc.

For example, by using insulin as a drug,
It can be used as a preparation for treating diabetes or an artificial spleen having an auto-feedback mechanism that releases insulin with an increase in blood sugar level and stops insulin release when the blood sugar level decreases. Further, by using the polymer complex of the present invention in combination with a dye, the dye released from the complex can be quantified and used as a sugar sensor.

(Examples) Next, the present invention will be described in more detail with reference to Examples.
The present invention is not limited to these examples.

Since the polymer complex of the present invention utilizes the complex formation and dissociation of the boronic acid group-containing polymer chain and the polyvalent hydroxyl group-containing polymer chain, it is necessary to examine the complex formation and dissociation of the polymer chain. . In the present invention, the evaluation was made by examining the change in viscosity accompanying the complex formation. A multifunctional blood coagulation time measurement device (BIOM) for measuring the change in viscosity with blood coagulation
Using ATIC B-10), evaluation was performed by converting the value obtained from this device into a relative viscosity. As a principle of the multifunctional blood coagulation time measuring device, a wing attached to the tip of an arm is vibrated and immersed in a solution to be measured. The viscosity of the solution and the presence of the gel suppress the vibration of the wing, and its strength can be recorded as a resistance value. The value obtained from the multifunctional blood coagulation time measuring device was evaluated by converting it into a viscosity from a calibration curve prepared using a rotational viscometer.

Turbidity was measured using a spectrophotometer (500 nm).

Reference Example 1 Synthesis of a boronic acid group-containing polymer.

0.995 g of 3-acryloylaminobenzeneboronic acid (5.
0 mol%) and 11.0 g (95.0 mol) of N-vinylpyrrolidone
%) In ethanol as a solvent, and 0.298 g (0.01 m2) of 2,2'-azobis (2,4-dimethylvaleronitrile) as an initiator.
ol /), polymerized in a degassed sealed tube at 45 ° C. for 0.5 hour, purified by reprecipitation three times with an ethanol / diethyl ether system, and dried. The yield of the polymer thus obtained was 21.7%. From the results of the atomic absorption analysis, 14.5% was obtained.
It was found to contain mol% boron. The molecular weight of the obtained polymer was 88,000.

The obtained polymer was soluble in ethanol and dimethyl sulfoxide, and was insoluble in benzene, n-hexane, acetone, tetrahydrofuran, chloroform, dioxane, diethyl ether and the like. Although slightly soluble in distilled water, it was readily soluble in alkaline aqueous solutions. The obtained polymer was dissolved in a 0.05N aqueous solution of sodium hydroxide, and titrated with a 0.1N aqueous solution of hydrochloric acid to measure the pH at which turbidity occurred. As a result, turbidity occurred around pH 9.

Reference Example 2 Synthesis of a boronic acid group-containing polymer.

Synthesis and analysis were performed in exactly the same manner as in Reference Example 1 except that the composition ratio was changed to 2.5 mol% of 3-acryloylaminobenzeneboronic acid and 97.5 mol% of N-vinylpyrrolidone.

The yield of the obtained polymer was 19.2%, and it was found from the result of atomic absorption analysis that it contained 12.0 mol% of boron. The molecular weight was 80,000.

Reference Example 3 Synthesis of a polymer containing a boronic acid group.

Synthesis and analysis were performed in exactly the same manner as in Reference Example 1, except that the composition ratio was changed to 1.0 mol% of 3-acryloylaminobenzeneboronic acid and 99.0 mol% of N-vinylpyrrolidone.

The yield of the obtained polymer was 17.5%, and it was found from the results of atomic absorption analysis that it contained 9.8 mol% of boron. The molecular weight was 80,000.

Reference Example 4 Synthesis of a boronic acid group-containing polymer.

0.386 g of 3-acryloylaminobenzeneboronic acid (2.
5 mol%) and 5.612 g of acrylamide (97.5 mol%)
Was polymerized at 45 ° C. for 45 minutes in a degassed sealed tube using 114 ml of distilled water / ethanol (v / v = 3/1) as a solvent and ammonium persulfate (0.025 mol /) as an initiator. After re-precipitation for three times, purification was performed, followed by drying. The yield of the obtained polymer was 70%. The molecular weight was 70,000.

Example 1 The polymer solution concentrations are shown in Table 1 using the polymer of Reference Example 1 as the boronic acid group-containing polymer and polyvinyl alcohol having a molecular weight of 88000 (saponification degree 99.5%) as the polyvalent hydroxyl group-containing polymer. And a complex was formed at a mixing ratio of 1: 1 to obtain a complex. Table 1 shows the measurement results of the viscosity change at this time.

Example 2 Using the polymer of Reference Example 1 as the boronic acid group-containing polymer and the polysaccharide (galactose) as the polyvalent hydroxyl group-containing polymer, the polymer solution concentration was changed as shown in Table 1, and the mixing ratio was changed. A complex was formed at a ratio of 1: 1 to obtain a complex. Table 1 shows the measurement results of the viscosity change at this time.

Comparative Example 1 A polymer synthesized in exactly the same manner as in Reference Example 1 except that the composition in Reference Example 1 was changed to only N-vinylpyrrolidone without adding 3-acryloylaminobenzeneboronic acid,
Using polyvinyl alcohol having a molecular weight of 88000 (degree of saponification: 99.5%) as a polyhydric hydroxyl group-containing polymer, changing the polymer solution concentration as shown in Table 1 and forming a complex at a mixing ratio of 1: 1 to form a complex I got Table 1 shows the measurement results of the viscosity change at this time.

In Table 1, the viscosity when no boronic acid group is contained is:
Approx. 1.0 (cps), almost constant even when the polymer concentration is changed
On the other hand, the viscosity of the composite comprising the boronic acid group-containing polymer and the polyvalent hydroxyl group-containing polymer clearly increases with the increase in the polymer concentration.

Example 3 Using the polymer of Reference Example 1 as a polymer containing a boronic acid group and polyvinyl alcohol having a molecular weight of 25,500 (a saponification degree of 99.6%) as a polymer containing a polyvalent hydroxyl group, a polymer solution concentration of 0.1 was used.
The complex was formed by changing the mixing ratio at 5% to obtain a complex. Table 2 shows the measurement results of the viscosity change at this time.

Example 4 Table 2 shows the measurement results of the change in viscosity when the composite was obtained by changing the polymer solution concentration in Example 3 to 0.75%.

Example 5 Table 2 shows the measurement results of the change in viscosity when the composite was obtained in the same manner as in Example 3 except that the polymer solution concentration was changed to 1.0%.

Example 6 Using a polymer of Reference Example 2 as a polymer having a boronic acid group and polyvinyl alcohol having a molecular weight of 25,500 (a saponification degree of 99.6%) as a polymer having a polyvalent hydroxyl group, a polymer solution concentration of 1.
A complex was formed by changing the mixing ratio at 0% to obtain a complex. Table 2 shows the measurement results of the viscosity change at this time.

Table 2 shows the results of Examples 3 to 6. The viscosity change accompanying the complex formation when the polymer concentration and the mixing ratio of the polymer containing a boronic acid group and the polymer containing a polyhydric hydroxyl group (polyvinyl alcohol) are changed is shown.

From this result, the complex formation of the complex is clearly recognized when the mixing ratio of the boronic acid group-containing polymer and the polyvalent hydroxyl group-containing polymer is in the range of 3: 1 to 1: 1.

Example 7 A 0.75% aqueous solution of the boronic acid group-containing polymer of Reference Example 1 and polyvinyl alcohol having a molecular weight of 25500 (degree of saponification 99.6%)
A 0.75% aqueous solution was mixed at a mixing ratio of 1: 1 to form a complex to form a complex, and then the sugar concentration was 0,100,200,500,1000.
FIG. 1 shows the results of examining the change over time in the decrease in viscosity (gel strength) due to complex dissociation with the addition of mg / dl glucose.

In the figure, △ is 0, black □ is 100, white □ is 200, ●
Indicates the case of 500 and ○ indicates the case of the sugar concentration of 1000 mg / dl.

From this figure, it can be seen that as the sugar concentration increases, the decrease in viscosity due to complex dissociation becomes faster. In particular, when the sugar concentration is 1000 mg / dl, the viscosity becomes constant after 20 minutes.

Example 8 As a boronic acid group-containing polymer, 2% of the polymer of Reference Example 4
Using a solution, polyvalent hydroxyl group-containing polymer with a molecular weight of 88000
Using a 2% solution of polyvinyl alcohol of the above, a complex was synthesized by mixing insulin and put into a dialysis membrane, and in a phosphate buffer (pH = 7.4) in the absence of glucose and in glucose 1000 mg / dl Insulin release was examined. In addition,
As insulin, bovine insulin manufactured by Sigma was used, and the amount of insulin was determined by UV measurement (274 nm). As the dialysis membrane, a dialysis tube manufactured by Spectra, Spectra / Bore 7 (fraction molecular weight: 25,000) was used. The concentration of insulin in the complex was 0.5 mg / ml, and 6 ml of the complex was placed in a dialysis tube, and the concentration of insulin released in 12 ml of a buffer solution or a buffer solution containing glucose was examined. The concentration of insulin released after 3 hours was 11 μg / ml when immersed in the buffer and 36 μg / ml when immersed in a buffer containing 1000 mg / dl glucose.

[Brief description of the drawings]

FIG. 1 is a graph showing a time-dependent change in viscosity decrease due to complex dissociation when glucose of each sugar concentration is added to the obtained complex in Example 7 of the present invention. The vertical axis is the gel strength (cps), the horizontal axis is the elapsed time (minutes), Δ is 0, black □ is 100, white □ is 200, ● is 500, ○
Indicates the case of a sugar concentration of 1000 mg / dl.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C08L 101/02 C08L 101/02 101/06 101/06

Claims (1)

(57) [Claims] 1. A saccharide-responsive polymer complex comprising a boronic acid group-containing polymer and a polyvalent hydroxyl group-containing polymer.