KR920003166B1 - Process for preparing granulation of drug using covalent bond between drug and polymer - Google Patents

Abstract

A process for preparing controlled release microsphere comprises: (a) adding drug into the polymer soln.; (b) reacting it in the presence of chemical catalyst to form the drug-polymer covalent complex (I); (c) suspending the obtd. (I) in the organic solvent to obtain the emulsified soln.; (d) hardening the (I) by heating at 80-170 deg.C or reacting the emusified soln. chemically; (e) replacing the surface of (I) with polr radical and centrifuging to obtain the precipitate; (f) washing the precipitate and freeze-drying to obtain the final product. The polymer is protein (e.g. serum albumin), sythetic polypeptide (e.g. poly-L-glutamic acid) or polysaccaride (e.g. dextran). The obtd. microsphere is useful as medicine, food additive etc.

Description

Method for preparing microspheres using drug-polymer covalent conjugate

1 shows a time-relative capillary elevation graph for determining the hydrophilicity of hydrophilic and nonpolar microspheres,

2 shows a graph of time-sedimentation volume of microspheres for the evaluation of physical stability of the microspheres.

Figure 3 shows a graph showing the time-drug release rate (%) of the microspheres made of drug-embedded microspheres and drug-albumin covalent conjugates by conventional physical methods to measure the release rate of microspheres,

Figure 4 shows a graph showing the time-drug release rate (%) of the microspheres made of drug-enclosed microspheres and drug-albumin covalent conjugates in order to measure the rate of drug release by the proteinase.

The present invention relates to microspheres prepared using drug-polymer covalent conjugates. Natural polymers such as serum albumin form complexes with radioisotopes and have been used as diagnostic tools for blood flow, blood distribution, and vascular diseases in humans and animals.Also, proteins such as albumin, gelatin, hemoglobin, starch, and dextran Polysaccharide polymers have been prepared in the form of microspheres containing radioactive elements, and have been used to diagnose and evaluate the distribution of reticular cells in vivo and the normal function of phagocytosis (US Pat. No. 3,663,685).

In addition, since these polymer microspheres are selective in organs distributed mainly in the body depending on their particle size, they are known to be used as drug transporters for the purpose of treating bacterial infection, tumor development, or other diseases in specific organs (US Pat. No. 3,663,687). Polymeric microspheres, such as albumin microspheres, which are less toxic during in vivo administration, have good adaptability in vivo and are gradually degraded by enzymes in vivo, have been developed as long-term sustained drug transporters.

Polymeric microspheres are generally dispersed in an aqueous solution of polymer or monoclonal drug in which the drug is dissolved in a physical method (eg, stirring) in an organic layer such as vegetable oil or liquid hydrocarbon to form an emulsion, which is then treated by heating or chemical method for a certain time. The formed microspheres were prepared through a process of separation, washing and drying in a liquid.

Until now, as a method of controlling drug release from the microspheres, a method of changing the degree of curing of the microspheres by adjusting the concentration in the solution of the drug during the manufacturing process or by increasing or decreasing the heating temperature, time, or chemical treatment strength has been attempted (US Pat. No. 4,147,767). ).

However, the following problems have been pointed out as a limiting factor in controlling drug release from microspheres by this physical method alone.

First, the decomposition of the drug is accelerated at a high temperature, the loss of drug efficacy.

Second, the problem of vascular occlusion may occur when the microspheres are modified or aggregates are formed according to the concentration of chemical hardener used.

Third, when the degree of hardening of microspheres is increased only by physical methods, the drug is slowly released from the microspheres in small amounts to reach the effective concentration required for pharmacological activity. If the concentration is increased, a large amount of drug is released at the beginning of administration, and the effective dose of the drug effectively released in the microspheres is often not greatly improved, and there are limitations depending on physicochemical properties such as solubility of the drug.

SUMMARY OF THE INVENTION An object of the present invention is to develop a method for effectively controlling the drug release from the microspheres by preparing the microspheres with the drug-polymer covalent conjugate in which the drug is covalently bonded to the polymer in view of the above problems.

In addition, due to the manufacturing process in which the microspheres are produced in the oil-in-water emulsion form, the hydrocarbon components remain on the surface to form aggregates during the storage period and have hydrophobicity to overcome the disadvantage of low dispersibility in the injection solution. The surface of the microspheres was substituted with a polar group to increase the hydrophilicity of the microspheres and provide physical stability suitable for injectable preparations.

The microspheres made of drug-polymer covalent conjugates showed a significant increase in drug encapsulation efficiency, and the initial release of drug and the rate of degradation of microspheres were reduced compared to the microspheres in which the drug was physically enclosed.

Drug release pattern by proteolytic enzymes showed that the drug release from the microspheres was active from the early stage of enzyme action in the case of microspheres in which the drug was encapsulated by the conventional physical method, while in the microspheres in which the drug was enclosed in the form of drug-polymer covalent conjugate After this time, it was observed that the drug was gradually released.

Therefore, the microspheres using the drug-polymer covalent conjugate can increase the drug transport capacity of the microspheres to administer a large amount of the drug using a smaller amount of the microspheres, and the drug from the microspheres can be co-administered at an appropriate ratio of the existing microspheres. It can be said to be a formulation that can more effectively control the release pattern.

The microspheres developed in the present invention were prepared in the following steps.

(1) After the drug is added to the aqueous solution of the polymer, a chemical catalyst for promoting the covalent reaction is added and the reaction is carried out for a predetermined time to form a covalent compound between the polymer and the drug, which is separated and purified using Sephadex gel, and the drug is freeze-dried. Polymer covalent conjugate was prepared.

Drugs that can be used in this step possess a polar group (e.g., amide group, carboxyl group) capable of covalent bonding with the polymer in the molecular structure itself to form a covalent bond directly with the polymer (e.g., bleomycin, Reaction-inducing intermediate groups (e.g., tripeptides, tetrapeptides) for the purpose of forming covalents or increasing the reactivity with cytoribin, daunorubicin, adriamycin, melphalan, mitomycin C, methotrexate, or polymers ) Is a combined form of drug.

As the polymer, proteins (eg, albumin), synthetic polypeptides (eg, poly-L-glutamic acid, etc.), polysaccharides (eg, dextran polymers, etc.) may be used.

(2) The aqueous solution of the drug-polymer covalent conjugate is dispersed in an organic solvent layer to form an emulsion, and then heated to 90-200 ° C. or treated by a chemical method to cure the microspheres. Substitute the microspheres in the emulsion, centrifuge and wash with ether several times, then wash with a solvent several times to achieve a uniform dispersion in aqueous solution without addition of dispersant and then freeze-dried.

In this step, formaldehyde or glutaraldehyde is used as the chemical curing agent so that a highly reactive aldehyde group can be present on the surface of the microspheres. A polar group such as a carboxyl group was provided on the surface to increase the stability of the microspheres in the receiving medium.

In addition, the microspheres prepared in the present invention may be prepared as a suspension for injection by a conventional method, or may be mixed with conventional albumin microspheres and prepared as a sustained preparation.

The present invention is described below by way of examples, which do not limit the present invention.

Example 1

1) Preparation of serum serum albumin- methotrexate covalent conjugate

The pH of 20 ml of 5% albumin aqueous solution was adjusted to 4.75 with dilute hydrochloric acid. Then, 45 mg of methotrexate, an anticancer drug, was added with 150 mg of ethyl dimethyl aminopropyl carbodiimide as a carbodiimide catalyst and reacted for 6 hours. The polymer covalent binder was separated using a Sephadex column and lyophilized.

The concentration of albumin in the covalent conjugate was quantified by the burette reaction at 540 nm, and the concentration of methotrexate was also measured by spectroscopic method at 370 nm.

2) Preparation of microspheres using covalent binder

Among covalent binders prepared in 1), 1 ml of an aqueous solution of albumin containing the drug in a covalently bonded state was added to 20 ml of cottonseed oil using a covalent binder having a drug content of 13.3%, followed by stirring for 2 minutes to form a crude dispersion emulsion. This solution was added dropwise to 30 ml of cottonseed oil that was previously stirred.

The solution was stirred at 1700 rpm for 15 minutes and then dispersed for 5 minutes using an ultrasonic mill to form an emulsion of finely dispersed state, and then 2 ml of 25% glutaraldehyde solution was added dropwise. After 30 minutes, 1.5 ml of glycine aqueous solution (200mg / ml) was added to the suspension of the microspheres in water, and reacted for one hour.The aldehyde group on the surface of the microspheres was replaced with a carboxyl group to increase the hydrophilicity of the microspheres. After washing four times with ether, three times with ethanol, three times with distilled water, and freeze-dried for 2 days to obtain a powdery brown microspheres. The average particle diameter of this microsphere was 1.61 micrometer (standard deviation = ± 0.790).

Example 2

The working time of the glutaraldehyde solution in Example 1-2) was 60 minutes, and the remainder was carried out in the same process to obtain brown microspheres. The average particle diameter of this microsphere was 1.59 m (standard deviation = ± 0.488).

Example 3

In Example 2, the action time of the glutaraldehyde solution was 90 minutes, and the remainder was performed in the same process to obtain brown microspheres. The average particle diameter of this microsphere was 1.46 μm (standard deviation = ± 0.455).

Example 4

1) Preparation of Cytaribin-Heraline Albumin Covalents

Dissolve 7.2 g of cytarabine hydrochloride (Cytarabine. HCℓ) and 650 mg of serum serum albumin in 20 ml of distilled water, then add 7.6 ml (40 mg EDC / ml) of ethyl dimethyl aminopropyl carbodiimide (EDC) solution slowly for 24 hours. After the reaction was carried out, the dialysis was carried out several times with 0.9% physiological saline solution to remove unreacted drug and carbodiimide catalyst from the reaction solution and freeze-dried at -40 ° C for 3 days to prepare the cytaribin-phosphate albumin covalent conjugate. Prepared.

2) Preparation of Microspheres Using Cytarabine-Albumin Covalent Conjugates

1 ml of 10 W / V% phosphorous albumin solution containing cytarabine-albumin covalent conjugate was added to 100 ml of cottonseed oil rotated at 1600 rpm and emulsified for 15 minutes to obtain an emulsion having a finely dispersed state using an ultrasonic mill. . The emulsion was spun at 100 rpm into 100 ml of cottonseed oil preheated to 100-105 ° C., and added dropwise to cure for 10 minutes.

After cooling the high temperature reaction solution in which the microspheres were suspended to room temperature, 1.5 ml of glutamine aqueous solution (200 mg / ml) was added and reacted for one hour, followed by centrifugation to separate the microspheres. Anhydrous ether was added, centrifuged at 4000 rpm for 15 minutes to remove the supernatant, and then washed four times with anhydrous ether to remove the cottonseed oil component on the surface of the microspheres and dried in a vacuum desiccator to obtain a microsphere.

Example 5

When preparing microspheres by the method of Example 4-2) using the cytarabine-human serum albumin covalent conjugate prepared in Example 4-1), cottonseed oil heated to 170-175 ° C. It was added dropwise to 100ml to cure for 10 minutes.

Example 6

1) Preparation of adriamycin-serum albumin covalent conjugate

a) Synthesis of Peptide Derivatives of Adriamycin

Manufactured by N-trityl Leu-Ala-Leu

1.3 g (0.01 M) of Leu-Ala-Leu was dissolved in 4 ml mol, 3 ml diethylamine, 8 ml isopropyl alcohol, and 3.6 g of trityl chloride was added in 12 portions for 12 hours (0.3 g / 1hr), followed by 30 ml The mixture was extracted with water and extracted twice with 30 ml of CHCl ₃ , washed with water, dried over sodium sulfate and vacuum distilled.

The residue was dissolved in anhydrous ethyl, and then diethylamine was added dropwise to cool the solution, washed with diethylammonium salt and transferred to the water layer.

Synthesis of Hydroxysuccinimide Ester of N-trityl Leu-Ala-Leu

Trityl-Leu-Ala-Leu and hydroxysuccinimide prepared above were dissolved in 125 ml anhydrous dimethoxyethane, cooled to 5 ° C., and dicyclohexyl carbodiimide was added thereto. After 20 hours the urea was removed by filtration at 5 ° C.

Binding with Adriamycin

0.15 mmol of hydroxysuccinamide ester of N-trityl Leu-Ala-Leu was added to 0.45 mmol adriamycin in 8 ml DMF, followed by 0.45 mmol triethylamine (Et ₃ N), followed by stirring at room temperature for 24 hours. It was dissolved in 5ml CHCl ₃ -MeOH (99: 1 V / V) and loaded into 60 columns of silica gel for purification.

b) succinate reaction of human serum albumin

Dissolve the serum serum albumin in water (100mg / ml) to pH 7.5 with 0.5M NaOH, add succinic anhydride (0.68mmol) stepwise, and then use phosphate buffer solution (NaCl 137mM, KCℓ 3mM, Na ₂ HPO). ₄ 8 mM) and then filtered using a Millipore GS filter and maintained at 4 ° C.

c) adriamycin and succinized serum serum albumin

20 mol of the adriamycin derivative prepared in a) and 50 mg of the serum serum albumin (5 ml of 10 mg / ml solution) prepared in b) were added to a beaker, and 7.5 mg of EDC was added thereto, and the mixture was left to stand at 4 ° C. without stirring. After 2 hours, another 3.75 mg of EDC was added and left overnight, followed by filtration over 50-80 of sephadex G to obtain a large amount of the title compound.

2) Preparation of microspheres using adriamycin-serum albumin covalent conjugate

The covalent conjugate prepared in 1) was prepared in the same manner as in Example 1-2) using an amount of 1 ml as an aqueous albumin solution.

Example 7

1) Preparation of Cytarabine-Poly-L-Glutamic Acid Covalent Bond

Dissolve 0.004 mole of poly-L-glutamic acid (PLGA) in dimethylformamide (DMF) and maintain isobutyloxycarbonyl chloride (IBC) and triethylamine (TEA) at -8-5 ° C. After adding 0.004 mol each, stirred for 1 hour at constant temperature, and then added 0.004 mol cytarabine-containing DMF solution and TEA, which was left for 3 days at 4 ℃ and again at room temperature for 4 hours.

The reaction solution was added to a fixed amount of cooled 0.4M phosphate buffer (pH 8.0), filtered, and the filtrate was dialyzed with a semipermeable membrane in the order of 3% NaCl solution and distilled water to prepare a covalent binder. Measuring the absorbance of the acrylic portion of the cytosine λ _max, 300nm (ε8,000) - Sy other Lavigne -PLGA covalent N ⁴ PSY content of other Lavigne weight Sy other Lavigne in 0.1M phosphate buffer (pH7.5) And analyzed.

2) Preparation of Microspheres Using Cytarabine-Poly-L-Glutamic Acid (PLGA) Covalent Conjugates

It was prepared in the same process as in Example 1-2).

Example 8

1) Method for preparing dextran T70-C ₁₀ -methotrexate covalent conjugate

Hiraku Onishi & Tsuneji Nagai, Chem, Pharm. Bull. 34, 2561]. Namely, 5.0 g of dextran T70 was dissolved in 100 ml of water and oxidized for 1 hour at room temperature using 1.139 g of sodium periodate. 3.83 g ethylene glycol was added to stop the reaction, followed by dialysis at 4-5 ° C. for 24 hours and 12 hours using water and 0.01 M carbonate buffer (pH9.5), respectively (cellulose tubing, pore diameter: 24 kPa).

1.063 g of diaminodecane was then added and after 4 hours 0.467 g of sodium borohydride was added. This was dialyzed against water at 4-5 ° C. for 24 hours to obtain dextran T-70-C ₁₀ . 20 ml of dextran T70-C ₁₀ was separated on a Sephadex G50 column, followed by 3 mg of methotrexate and 300 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). After adjusting its pH to 6.0, it stirred at the dark and room temperature for 24 hours.

2) Method for preparing microspheres using dextran T70-C ₁₀ -methotrexate covalent conjugate

It was prepared in the same manner as in Example 1-2) using dextran T70-C ₁₀ -methotrexate covalent conjugate.

Experimental Example 1

To compare the hydrophilicity of microspheres whose surface is substituted with carboxyl groups and microspheres not treated with polar groups, 10 mg of each microsphere is filled in capillaries, and distilled water is used as a developing solvent. The degree of capillary rise of the solvent was measured over time. The results are shown in FIG. In Figure 1 it can be seen that the hydrophilicity of the microspheres increased significantly with the presence of a polar group on the surface.

Experimental Example 2

In order to evaluate the physical stability of microspheres as an injectable preparation, the rate at which the evenly dispersed microsphere solution settled over time was compared by plotting the time zone sedimentation volume from 0 to 50 minutes. As the microsphere sample, microspheres whose surface was treated with a polar group and untreated microspheres were used. The above result is shown in FIG. In FIG. 2, the microspheres whose surface was treated with a polar group maintained a physically stable dispersion state for a long time.

Experimental Example 3

The drug loading amount of albumin microspheres physically encapsulated with the same amount of drug and albumin microspheres prepared with drug-albumin covalent conjugates was as follows.

TABLE 1

a) HAMF: albumin microspheres by conventional manufacturing method

b) HAMC: microspheres prepared by the method of Example 1 of the present invention

In the case of the microspheres prepared by the method of Example 1, the amount of drug loading was increased more than two times compared to the microspheres using the mixed solution of albumin and drug.

Experimental Example 4

In order to compare the initial release rate of the drug from the microspheres obtained by the preparation of Example 1 with the conventional microspheres in which the drug was physically encapsulated, each microsphere was uniformly dispersed in a phosphate buffer solution and then subjected to 300 rpm in a water bath at 37 ° C. ± 2 ° C. The sample was collected by a certain amount of time with stirring to remove the microspheres in the liquid, and then the drug concentration in the filtrate was quantified by spectroscopic method at 370 nm.

This result is shown in FIG. From the microspheres according to the conventional manufacturing method, the drug is mainly released from the surface portion of the drug is released through the pores of the microspheres through a simple diffusion mechanism, but in the case of the microspheres according to the present invention, the drug is released as the hydrolyzate from the surface portion is gradually hydrolyzed. Initial release rate and release rate were reduced.

Experimental Example 5

Dispersion suspension of the microspheres using the covalent conjugate prepared by the method of Example 1 and the conventional microspheres in which the drug was encapsulated by the physical method in the albumin microspheres were added with proteolytic enzyme while stirring in a water bath at 37 ° C ± 2 ° C. As a result, a sample was collected and spectroscopically measured the decrease in turbidity in the solution, and the time required for 25% degradation of the microspheres was observed.

As a result, it was found that the decomposition rate of the microspheres according to the present invention was significantly reduced.

TABLE 2

* T0.25: Time taken for microspheres to degrade 25% by protease

HAMF: albumin microspheres by conventional manufacturing method

HAMC: Microspheres prepared by the method of Example 1 of the present invention

Experimental Example 6

In order to compare the drug release pattern by the proteolytic enzyme from the albumin microstructure prepared by the conventional method of the albumin microsphere prepared by the method of Example 1, each microsphere was dispersed evenly in 80ml of phosphate buffer solution of pH7.4 by 4mg The amount of drug released over time after the device was placed in a water bath at 37 ° C. ± 2 ° C. and acted as a prozyme as a protease was spectroscopically determined at 370 nm.

This result is shown in FIG. Drug release from the microspheres prepared by the conventional method is actively progressed from the beginning of the action of the enzyme, while the microspheres according to the present invention showed a positive release of the drug gradually after several hours.

Preparation Example 1

1.92 mg (50 μg as etotrexate) of the microspheres of methotrexate-serum albumin covalent conjugate prepared in Example 1 were dispersed in 2 ml of physiological saline to prepare a suspension injection.

Preparation Example 2

2.27 mg of human serum albumin microspheres of methotrexate prepared by the conventional method and 0.96 mg of microspheres (25 μg each as methotrexate) prepared in Example 1 were well mixed and dispersed in 5 ml of saline solution to prepare a suspension injection.

Claims (14)

A method for producing a polymer fine particle by heating in a dispersion solvent, wherein a covalent compound of a polymer and a drug is used as a raw material of the polymer microspheres, instead of the polymer in which the drug is dissolved by a physical method. A method for producing a polymeric microsphere by chemical curing in a dispersion solvent, wherein a covalent compound of a polymer and a drug is used as a raw material of the polymeric microsphere, instead of the polymer in which the drug is dissolved by a physical method. Method of increasing hydrophilicity by introducing a polar group on the surface of the microspheres made of a polymer-drug covalent bond. The method according to claim 1 or 2, wherein the drug possesses a reactor in which the drug can bind with the polymer, and uses a covalent bond in a form in which the drug and the polymer are directly connected. The method according to claim 1 or 2, wherein the drug uses a covalent compound in which the drug is bonded by an intermediate group to the polymer to introduce a highly reactive chemical group to the drug or to increase the reactivity with the polymer. The method of claim 1 or 2, wherein the polymer is a protein. The method of claim 1 or 2, wherein the polymer is a synthetic polypeptide. The method of claim 1 or 2, wherein the polymer is a polysaccharide. The method of claim 6, wherein the protein is serum albumin. The method of claim 1 or 2, wherein the drug is a cytotoxic substance. The method of claim 10, wherein the cytotoxic agent is an anticancer agent. The method of claim 1, wherein the microspheres of the polymer-drug covalent conjugate are prepared in a dispersion solvent heated to 80 ° C. to 170 ° C. 7. The method of claim 3, wherein the polar group is introduced by an aqueous amino acid solution. A composition in which microspheres made of a polymer-drug covalent conjugate are dispersed in a solvent for injection.

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