KR0169195B1 - PVMA-Ara-C conjugate - Google Patents

Abstract

The present invention relates to a PVMA-Ara-C conjugate, and more particularly, to a PVMA-Ara-C conjugate represented by the following structural formula (I), which maintains drug concentrations for a long time in a single administration to enhance an anticancer effect, and a method of preparing the same. And it relates to an anticancer agent containing it as an active ingredient.

In the above formula, the ratio of x and y is 0: 100 to 80:20.

Description

[Name of invention]

PVMA-Ara-C conjugate

Detailed description of the invention

[Purpose of invention]

[Technical field to which the invention belongs and the prior art in that field]

The present invention relates to a PVMA-Ara-C conjugate, and more particularly, to a PVMA-Ara-C conjugate represented by the following structural formula (I), which maintains drug concentrations for a long time in a single administration to enhance an anticancer effect, and a method of preparing the same. And it relates to an anticancer agent containing it as an active ingredient.

In the above formula, the ratio of x and y is 0: 100 to 80:20.

Arabinopranosylcytosine (1-β-D-Arabinofuranosylcytosine; hereinafter referred to as Ara-C) shows a mechanism for killing cancer cells by blocking DNA synthesis of cells and is used in the treatment of leukemia clinically. Deoxycytidine deaminase is converted into uracil analogues, which prevents anticancer activity, and the drug is released by renal filtration even after maximum drug injection. Injection over time is effective for treatment.

Since Ara-C is active against stage S cancer cells, it is important to maintain blood concentrations above effective levels for a long time [Cancer Chemotherapy Report, V51, 125, 1967]. However, the long-term drip injection method is a burden on the patient, so research has been conducted on a new drug administration method rather than an injection method. As a result of this study, a formulation obtained by incorporating Ara-C into liposomes has been developed. Liposomes are sustained-release drugs that protect Ara-C from enzyme inactivation and release the drug slowly. Improve [Int. J. Cancer, V20, 581, 1977], liposomes are unstable in the blood and easily broken.

Recently, drug delivery methods using synthetic polymers have been used to overcome the instability of anticancer drug carriers.In the case of polymer drugs obtained by combining anticancer drugs with synthetic polymers, the anticancer drugs are stably maintained, and the anticancer drugs are gradually released to give a single dose. The drug has an excellent anti-cancer effect because of the prolonged blood concentration in the drug. J. Controlled Release, V19, 331, 1992.

[Technical problem to be achieved]

An object of the present invention is to provide a novel PVMA-Ara-C conjugate that enhances anticancer effect by binding Ara-C having excellent anticancer effect to a copolymer of polypyrrolidone-maleic anhydride (hereinafter referred to as PVMA).

[Configuration and Function of Invention]

The present invention is characterized by the PVMA-Ara-C conjugate represented by the following structural formula (I).

In the above formula, the ratio of x and y is 0: 100 to 80:20.

The present invention also includes an anticancer agent containing the PVMA-Ara-C conjugate represented by the above formula (I) as an active ingredient.

In addition, the present invention by polymerizing the solution of the solution dissolved in PVMA represented by the following structural formula (A) in a hydrophilic solvent and the solution of Ara-C represented by the following structural formula (B) in bromic acid or phosphate buffer solution of the structural formula (I) It includes a method for producing a PVMA-Ara-C conjugate represented by).

Wherein n is an integer from 10 to 500.

Referring to the present invention in more detail as follows.

In the present invention, the PVMA-Ara-C conjugate produced by the combination of Ara-C and PVMA having excellent anticancer effect is composed of amide bonds, and is applied to enzymes in vivo by a back-attack effect by adjacent carboxyl groups. The present invention relates to a PVAM-Ara-C conjugate, which slowly releases the drug regardless of the effective blood concentration, thereby enhancing the anticancer effect.

PVMA used as the Ara-C carrier in the present invention is a polymer material prepared by copolymerizing vinylpyrrolidone monomer and maleic anhydride by a known method [Makromol, Chem., 187, 297, 1986], which is biocompatible. It is well known as a very promising drug for the development of polymer drugs because it is water soluble. In addition, since many maleic anhydrides have high chemical reactivity in PVMA, they can be easily combined with drugs having active hydrogen functional groups such as amino group (-NH ₂ ) or hydroxyl group (-OH), Since a large number of water-soluble carboxyl groups (-COOH) are formed in the copolymer after the reaction, the resulting PVMA-drug conjugate is very soluble in water. In the present invention, PVMA having a repeating unit (n) of 10 to 500 and a molecular weight in the range of 2,000 to 100,000 is used. PVMA may have an anticancer promoting effect by binding to an anticancer agent regardless of molecular weight, but excretion and In consideration of side effects, it is preferable to use a molecular weight of 10,000 or less.

Ara-C represented by the structural formula (A) has two nucleophilic reaction sites, and these show different tendencies depending on the reaction solvent. That is, in the aqueous solution, the nucleophilic reaction occurs at the primary amine group portion, and in the presence of an organic solvent, the nucleophilic reaction occurs at the primary hydroxyl group portion. Therefore, when Ara-C represented by Structural Formula (A) and PVMA represented by Structural Formula (B) react, as well as PVMA-Ara-C conjugate represented by Structural Formula (I) as an object of the present invention, the following Structural Formula (II) The by-products represented by) may be produced, but the by-products represented by the formula (II) are very low in yield since the Ara-C is separated during the amicon filtration process.

In the above formula, n is an integer of 10 to 500, and the ratio of x and y is 0: 100 to 80:20.

In the present invention, since the amide bond represented by Structural Formula (I) must be obtained by bonding the amine group of Ara-C to PVMA, the reaction should be carried out under aqueous solution if possible.

Therefore, in the present invention, a condition close to an aqueous solution is made by using a minimum amount of hydrophilic solvent in bromic acid or phosphate buffer. At this time, a single or two or more mixed solvents selected from water, N-methylformamide, N, N-dimethylformamide, acetone and alcohol are used as the hydrophilic solvent.

In other words, according to the present invention, the PVMA-Ara-C conjugate is prepared under aqueous solution conditions, and for this purpose, water must be contained in at least 50% by weight of the total solvent.

In the present invention, Amicon filtration is carried out to remove unreacted low molecular weight Ara-C in the reaction product, and it is filtered until the ultraviolet spectral value of the filtered solution becomes 0.01 or less. The remaining solution after filtration is lyophilized to obtain a solid target product.

The PVMA-Ara-C conjugate represented by Structural Formula (I) obtained by the above-described manufacturing process shows very good solubility in water due to the water solubility of the carboxyl group (COOH) and Ara-C. In addition, in order to further enhance the water solubility of the resulting PVMA-Ara-C conjugate, it can be used by converting it into a salt of the PVMA-Ara-C conjugate by treating with sodium bicarbonate or potassium bicarbonate.

According to the result of quantitative analysis by 272 mm ultraviolet spectrum, the Ara-C content rate contained in the PVMA-Ara-C conjugate represented by the structural formula (I) and salts thereof according to the present invention is 5 to 40%. As a result, Ara-C is associated with PVMA, which does not readily exit the kidney's glomerulus and circulates in the blood for a long time.

As described above, the PVMA-Ara-C conjugate represented by Structural Formula (I) according to the present invention has a property of slowly releasing Ara-C to maintain an effective blood concentration for a long time. It can avoid the nucleotide enzymatic disruption and overcome the short-term blood retention problem, showing a markedly increased anticancer effect.

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

Example 1

[Preparation of PVMA-Ara-C Conjugate]

Ara-C (200 mg) was dissolved in 0.1 M boric acid buffer (pH 8.6; 20 mL). A solution of PVMA (100 mg) having an average molecular weight of 43,000 in N-methylformamide (2 mL) was added to the solution and stirred at room temperature for 1 hour. The reaction mixture was transferred to a 200 ml amicon container, and the unreacted material and boric acid solution were filtered using PM30 (molecular weight 30,000), and the filtrate was filtered until the UV absorbance of the filtrate was 272 mm to 0.01. After the filtration step, the filtrate was ultrafiltered with a Millipore filter having a pore size of 0.2 µm and lyophilized to obtain 136 mg of white powder PVMA-Ara-C conjugate. Quantitative analysis by ultraviolet spectrum at 272 mm showed Ara-C content in the PVMA-Ara-C conjugate was 20% by weight.

Example 2

[Salt preparation of PVMA-Ara-C conjugate]

The PVMA-Ara-C conjugate (50 mg) obtained in Example 1 was dissolved in distilled water, adjusted to pH 7.8 with 0.1 M aqueous sodium bicarbonate solution, and stirred for 30 minutes. After ultrafiltration with distilled water using Amicon ultrafiltration membrane PM30 (separated molecular weight 30,000), freeze-dried to obtain 52 mg of sodium salt of PVMA-Ara-C conjugate of white powder. Quantitative analysis by ultraviolet spectrum at 272 mm showed that the Ara-C content in the sodium salt of the PVMA-Ara-C conjugate was 20% by weight. Soluble in water or acetic acid saline compared to the binder obtained in Example 1 above.

Example 3

[Preparation of PVMA-Ara-C Conjugate]

Ara-C (100 mg) was dissolved in 0.1 M phosphate buffer (pH 8,6; 20 mL). A solution of PVMA (100 mg) having an average molecular weight of 20,000 in ethanol (2 ml) was added dropwise to the solution and stirred at room temperature for 1 hour. The reaction mixture was transferred to a 200 ml amicon container, and the unreacted material and phosphate were filtered using PM30 (molecular weight 30,000), and the filtrate was filtered until the UV absorbance of the filtrate was 272 mm to 0.01. After the filtration step, the filtrate was ultrafiltered with a Millipore filter having a pore size of 0.2 μm and lyophilized to obtain 120 mg of white powder PVMA-Ara-C conjugate.

When the PVMA-Ara-C conjugate was dissolved in water or acetic acid saline, the absorption spectrum of Ara-C was assumed to be the same as that of Ara-C. -C content was 17% by weight.

Example 4

[Preparation of PVMA-Ara-C Conjugate]

Ara-C (100 mg) was dissolved in 0.1 M phosphate buffer (pH 8.6; 100 mL). A solution of PVMA (100 mg) having an average molecular weight of 100,000 in acetone (2 mL) was added dropwise to the solution and stirred at room temperature for 1 hour. The reaction mixture was transferred to a 200 ml amicon container, and the unreacted material and phosphate were filtered using PM30 (molecular weight 30,000), and the filtrate was filtered until the UV absorbance of the filtrate was 272 mm to 0.01. After the filtration step, the filtrate was ultrafiltered with a Millipore filter having a pore size of 0.2 µm and lyophilized to obtain 125 mg of white powder PVMA-Ara-C conjugate. When the UVMA absorption spectrum and Ara-C in the aqueous solution of PVMA-Ara-C conjugates dissolved in water or acetic acid saline are the same, quantitative analysis of the UV spectrum at 272 mm shows that Ara-C in the PVMA-Ara-C conjugate The content was 18% by weight.

The PVMA-Ara-C conjugate represented by the above formula (I) according to the present invention and salts thereof are very useful as anticancer agents. Therefore, the present invention includes a PVMA-Ara-C conjugate represented by the above formula (I) and an anticancer agent containing a salt thereof as an active ingredient, which is PVMA-Ara-C conjugate represented by the above formula (I) and To their salts are prepared the addition of customary non-toxic pharmaceutically acceptable carriers, adjuvants and excipients. Anticancer agent according to the present invention can be administered by parenteral administration method, parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. In order to formulate into a parenteral dosage form, the PVMA-Ara-C conjugate represented by Structural Formula (I) is mixed with water with a stabilizer or buffer used in the preparation of conventional injections to prepare a solution or suspension, which is then used as an ampoule or vial. Formulated in unit dosage form of.

[Production of Injection Solution]

20 mg, 12 mg, and 8 mg of PVMA-Ara-C conjugate represented by Structural Formula (I) and salts thereof were dissolved in 5 ml, 3 ml, and 2 ml of distilled water, respectively. Sterilized by heat treatment at 121 ° C. for 30 minutes in an autoclave.

[Anticancer effect]

After dissolving the PVMA-Ara-C conjugate obtained in Example 1 in distilled water and filtering with a 0.2 μm filter, P388 leukemia cells were injected intraperitoneally into rats to evaluate antitumor activity as a% of the elongation effect. Experiments were carried out using 6 C3H / He mice in one group (mg / kg), and 24 hours after inoculating 1 × 10 ⁶ P388 leukemia cells into these mice, the following drugs were administered as shown in Table 1 below. After 4 days, the weight loss degree, mean survival days (MST) and life extension rate of the mice were measured and shown in Table 1 below.

In the following Table 1, the AMA-C content of 60 mg / kg, 30 mg / kg, and 15 mg / kg of PVMA-Ara-C conjugate was administered.Ara-C 60 mg / kg alone, PVMA 240 mg / Kg alone and a mixture of 240 mg / kg PVMA and 60 mg / kg Ara-C, respectively.

According to the results of Table 1, the PVMA-Ara-C conjugate administration group according to the present invention increased the life extension rate by 55-101% compared to the control group. In addition, Ara-C alone or PVMA and Ara-C as a comparison group showed a 16% life extension rate compared to the control group, which was rapidly diffused by Ara-C and reduced kidney glomeruli. Because it is easily discharged through.

[Effects of the Invention]

The PVMA-Ara-C conjugate according to the present invention has a property of slowly releasing Ara-C, which is useful in drug systems that enhance the efficacy of Ara-C and reduce toxicity.

Claims (5)

PVMA-Ara-C conjugate represented by the following structural formula (I). In the above formula, the ratio of x and y is 0: 100 to 80:20. An anticancer agent containing the PVMA-Ara-C conjugate represented by the following structural formula (I) or a salt thereof as an active ingredient. In the above formula, the ratio of x and y is 0: 100 to 80:20. The method of claim 2, wherein the anticancer agent is an injection. A solution obtained by dissolving a copolymer of polyvinylpyrrolidone-maleic anhydride (PVMA) represented by the following structural formula (A) in a hydrophilic solvent and arabinofranosyl toxin (Ara-C) represented by the following structural formula (B) A method for producing a PVMA-Ara-C conjugate represented by the following structural formula (I), characterized by polymerizing a solution dissolved in bromic acid or phosphate buffer. Wherein n is an integer of 10 to 500, and the ratio of x and y is 0: 100 to 80:20. The method of claim 4, wherein the hydrophilic solvent PVMA-Ara- characterized in that a single or two or more mixed solvents selected from water, N-methylformamide, N, N- dimethylformamide, alcohol, acetone is used. Method for preparing the C conjugate.