JPH0244319B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for producing neocarzinostatin complexes. More specifically, the present invention is based on the formula () SMA _o - NCS () [wherein, NCS means a neocarzinostatin residue, SMA has an average of one or less maleic anhydride ring per molecule, and It means an alkyl esterified styrene-maleic acid copolymer residue having an average molecular weight of 1,000 to 10,000, and n means an integer of 1 to 35. ] The present invention relates to a method for producing a neocarzinostatin complex shown in the following. Neocarzinostatin is produced from Streptomyces carzinostaticus var. F-41.
41Kuroya) (see Japanese Patent Publication No. 42-21752 and US Pat. No. 3,334,022), and its primary structure was described by Maeda, one of the inventors of the present invention. It has been reported that the total number of amino acid residues is 109 and the estimated molecular weight is 10,700 [Science, vol. 178, 875-
876, 1972 and Archives of Biochemistry and Biophysics, Vol. 163, pp. 379-385). In the treatment of cancer, metastasis of cancer cells is the most important problem, and especially lymph node metastasis is the biggest problem. Previously, the present inventor conducted various studies on reducing the toxicity of neocarzinostatin, increasing the durability of its efficacy, and specifically transferring the drug to the lymphatic system, and found that it exists in the molecule of neocarzinostatin. It was discovered that a neocarzinostatin derivative obtained by reacting the two free amino groups of the polystyrene-maleic acid copolymer with a partial hydrolyzate of a water-soluble polystyrene-maleic acid copolymer met the above objectives, and a patent application was filed for the same. (See Publication No. 117095). However, in addition to the need for anticancer drugs to have the above-mentioned property of transferring to the lymphatic system in order to suppress cancer metastasis, it is also desirable that they have high tumor affinity. When the tumor affinity is high, the concentration of the anticancer drug in the tumor is selectively increased, and as a result, the occurrence of side effects is reduced, and the effects of the anticancer drug can be effectively exhibited. Therefore, as a result of further various studies, the present inventors found that the styrene-maleic anhydride copolymer or its partial hydrolyzate has a maleic anhydride ring different from that of the styrene-maleic anhydride copolymer or its partial hydrolyzate.
Half-alkyl esterified average molecular weight is 1000~
The neocarzinostatin complex represented by the formula () [hereinafter referred to as the complex ()] obtained by reacting 10,000 styrene maleic anhydride copolymer with neocarzinostatin is surprisingly It has been found that the above objectives can be achieved. That is, the complex (2) produced according to the present invention has the same useful properties as the neocarzinostatin derivatives described above, and has excellent fat solubility, so that it can be applied as an oil-based preparation. The complex () can be administered as an oil-based formulation to concentrate the drug at the tumor site. Furthermore, the complex () has excellent affinity for tumors, and can remain at the tumor site and exert a strong anticancer effect. On the other hand, since the complex () has water-soluble properties in addition to fat-solubility, it can be administered systemically through a water-soluble preparation, such as intravenous injection. The preferable properties of neocarzinostatin made of such a complex () include a semi-alkyl esterified styrene-maleic anhydride copolymer having one or less maleic anhydride rings (hereinafter referred to as this).
By using neocarzinostatin (hereinafter abbreviated as NCS),
This is thought to be due to the fact that it has both water-soluble properties and fat-soluble properties. What is required of an anticancer drug is that when the anticancer drug is used as an intravenous injection, it is released into the tissue through the capillaries and then specifically transferred to the lymphatic system; on the other hand, when the anticancer drug is used as an oil-based preparation, In such cases, anticancer drugs can be released in a sustained manner from oil-based preparations to the necessary sites such as blood, tissue fluid, or lymph. It must accumulate well in tissues (sites) and be safely excreted from the body. To meet this requirement, the complex () preferably has a molecular weight of 60,000 or less because it leaks into tissues from capillaries, and because of its solubility in oily bases and specific migration into the lymphatic system. It is desirable that the molecular weight is 20,000 or more. After the complex () reaches the designated site in the body, it undergoes hydrolysis as it is or in part.
It is presumed that neocarzinostatin is released and exhibits antitumor properties. The complex () forms a polyanion and is also expected to have the effect of activating the immune system in vivo. The complex () is a complex with 1 to 35 molecules, usually 5 to 15 molecules of SMA, per 1 molecule of NCS. NCS and
Although the details of the bonding state with SMA are unknown, NCS is not just a mixture, and the molecular weight of NCS is approximately 1.
It is thought to be a polypeptide of 1,000,000,000, and has two primary amino groups at both ends, as well as a number of functional groups such as secondary amino groups and hydroxyl groups, and these functional groups are responsible for the reaction with SMA. It is thought that this may be involved.
However, since the reaction between NCS and SMA is a reaction between polyfunctional polymers (or oligomers), it is impossible to specify the reaction position or molecular structure of each reaction product. However, the structure of the reaction product can be determined by molecular size analysis such as electrophoresis, gel permeation chromatography, and gel filtration, and by component analysis such as infrared absorption spectrum, ultraviolet absorption spectrum, and elemental analysis. Therefore, it is possible to solve it on average. The complex () is produced by reacting NCS and SMA. The reaction is usually performed by dissolving NCS in an aqueous sodium bicarbonate solution, and adding one or more molecules of SMA, preferably three or more molecules of SMA, to one molecule of NCS.
powder is added under stirring at room temperature. Alternatively, it can be produced by dissolving SMA in an organic solvent, adding an aqueous sodium bicarbonate solution of NCS to the solution, and then removing the solvent by drying under reduced pressure or the like. The complex () is thought to be formed when the maleic anhydride ring of SMA opens and reacts with the functional group of NCS, and 1 to 35 molecules of SMA react with one molecule of NCS to form a complex. It is thought that normally 5 to 15 molecules of SMA react to form a complex. Therefore,
There is no maleic anhydride ring in the SMA residues contained in the complex (). The SMA used in the present invention has an average of 1 molecule per molecule.
or less, preferably 0.1 to 0.8 maleic anhydride rings, and the remaining maleic anhydride is ring-opened and semi-alkyl esterified, and has an average molecular weight of
It is a styrene-maleic anhydride copolymer having a molecular weight of 1,000 to 10,000, preferably 1,500 to 2,500. The SMA used in the present invention has one or less maleic anhydride,
In addition, since it is semi-alkyl esterified, it is possible to avoid the formation of high molecular weight by-products due to cross-linking reactions, and the reaction with NCS can be easily controlled. Here, the term "half-alkyl ester" means that half of the two carboxylic acids possessed by maleic acid, which is ring-opened from maleic anhydride, is converted into alkyl ester, and this half-alkyl ester is completely converted into half-alkyl ester. It is preferable that the maleic acid be esterified, but even if some of the maleic acid is not semi-alkyl esterified, there is no problem as long as most of the maleic acid is semi-alkyl esterified.
Moreover, half-alkyl ester usually means lower alkyl ester, and includes, for example, methyl ester, ethyl ester, propyl ester, butyl ester, or a mixed ester thereof, with half-butyl ester being particularly preferred. In addition, (poly)
It may also be an ester of a polyhydric alcohol such as an ester of ethylene glycol monoether, and in the present invention, these polyhydric alcohol esters are also included in the semi-alkyl ester. To administer the complex () to humans, there are several ways to administer the complex (), including intradermal, subcutaneous, intramuscular, intravenous, intraarterial, transverse administration, etc., at the primary site of cancer, the site of cancer removal after surgery, etc. and external methods of administration such as topical application, spraying, suppositories, and intravesical injection are preferred. The dosage varies depending on the administration method, the malignancy of the cancer, the type of cancer, the patient's medical condition and general condition, the progress of the cancer, etc.
It also depends on whether the purpose is to prevent lymph node metastasis after surgery, etc., or for therapeutic purposes, but for example, once a day.
It is preferable to administer 0.1 to 10 mg/Kg once or twice a week or every day. It is also possible to further increase the dosage for topical application and oral administration. The complex () is dissolved in the X-ray contrast agent Lipiodol (Lipiodol Ultrafluid iodinated poppy oil fatty acid ethyl ester, manufactured by Laboratoire Guerbet, France). Complex () 1-2 mg/
When 1 ml of Lipiodol is administered intraarterially, Lipiodol and the drug remain in the tumor blood vessels for a long period of time, exerting a strong antitumor effect. In addition, by dissolving in Lipiodol,
The state in which the complex () remains locally can be observed using X-rays. Using it as such an oil-based preparation is one way of using it that takes advantage of the properties of the complex (). The complex () is also dissolved in a 1-9% aqueous sodium bicarbonate solution. When this aqueous solution is administered intravenously, the drug exhibits a strong anticancer effect because it is largely distributed in the lymph vessels. Using it as such an aqueous salt solution is also one of the ways to utilize the properties of the complex (). The present invention will be specifically described below with reference to Examples. Reference example (a) 176 ml of p-cymene was heated to 132-134°C with stirring, and 29.4 g of maleic anhydride and 30.9 g of styrene were added.
and 1 g of benzoyl peroxide to p-cymene.
The solution obtained by dissolving in 200 ml was continuously added dropwise over 3 hours. A polymerization reaction occurred simultaneously with the dropping, and a cloudy precipitate of styrene maleic anhydride copolymer was successively formed. After the polymerization reaction was completed, the precipitate was collected, washed, and dried to obtain 58 g of a white powder of styrene-maleic anhydride copolymer. This styrene-maleic anhydride copolymer is considered to be a 1:1 alternating copolymer based on the polymerizability of each monomer, and the results of elemental analysis also support this. By vapor pressure method using acetonitrile as a solvent
The number average molecular weight was determined to be 1760 using a KNAUER VPO device. (b) Dissolve 20 g of this styrene maleic anhydride copolymer in 40 ml of dioxane, and add n-
Add 12 ml of butanol and 0.2 g of lithium acetate,
The tube was sealed and reacted at 90°C for 25 hours. After the reaction was completed, the solvent was removed by evaporation from the reaction solution by drying under reduced pressure, and the resulting residue was washed with a mixture of n-hexane and acetone (volume ratio 9:1), and then dried under reduced pressure to obtain some pale yellow flakes. A half-butyl esterified styrene-maleic acid copolymer with maleic anhydride rings remaining was obtained. The molecular weight of this half-butyl esterified styrene-maleic acid copolymer was determined by vapor pressure method and was 2200, which was found in the infrared absorption spectrum.
Average maleic anhydride ring content from D ₁₇₈₀ / D ₇₀₀
It was calculated to be 0.75 pieces/molecule. Elemental analysis values (actual measurements) C: 67.29%, H: 6.83%, N: 0.19% Example: 0.2 g of neocarzinostatin was dissolved in 80 ml of 0.5 M sodium bicarbonate aqueous solution, and the mixture was stirred at room temperature according to the reference example. 0.9 g of the obtained semi-butyl esterified styrene maleic anhydride copolymer powder having a number average molecular weight of 2200 and an average maleic anhydride ring content of 0.75/molecule was added in four portions at 20 minute intervals. After the half-butyl esterified styrene maleic anhydride copolymer powder was completely dissolved, the mixture was further stirred for 40 minutes. The reaction solution was transferred to a cellophane tube and filtered under pressure in a 5 mM aqueous sodium bicarbonate solution. After changing the 5mM sodium bicarbonate aqueous solution several times and performing diafiltration at 4°C for 2 days, diafiltration was performed in a 5mM ammonium bicarbonate aqueous solution at 4°C for another 2 days, and then in a 1.25mM ammonium bicarbonate aqueous solution at 4°C for an additional 2 days. It was transparent for several days. After freeze-drying the diafiltration-purified reaction solution, suspending it in pure water, washing it, and collecting the insoluble part using a centrifuge, repeat the process three times, and then freeze-dry it as a white cotton-like solid. Neocarzinostatin-semi-butyl esterified styrene maleic acid copolymer complex (hereinafter abbreviated as Neo SMANCS)
0.15g was obtained. The elemental analysis values (actually measured values) of the Neo SMANCS were as follows. C: 60.52%, H: 6.48%, N: 3.33% From these elemental analysis values, it was estimated that in Neo SMANCS, about 5.6 molecules of SMA were bound on average per molecule of NCS. Neo SMANCS
The infrared absorption spectrum (KBr formulation method) of
The infrared absorption spectra (KBr tablet method) of the half-butyl esterified styrene maleic anhydride copolymer and neocarzinostatin (NCS) used in the reaction are shown in FIGS. 2 and 3, respectively. In addition, Neo SMANCS can be used on acrylamide gel,
The molecular weight was determined to be approximately 43,000 by electrophoresis with the addition of 0.1% sodium dodecyl sulfate. Neo SMANCS softens slightly at 220℃ and begins to decompose at 250℃. Neocarzinostatin (NCS) using the solubility of Neo SMANCS in solvents as a reaction raw material
Table 1 shows a comparison with the styrene maleic anhydride copolymer and the semi-butyl esterified styrene maleic anhydride copolymer.

[Table] As is clear from the table above, Neo SMANCS
It is clear that the NCS and the semi-butyl esterified styrene maleic anhydride copolymer form a complex, not just a mixture.

[Brief explanation of drawings]

Figure 1 shows the infrared absorption spectrum of Neo SMANCS, Figure 2 shows the infrared absorption spectrum of the half-butyl esterified styrene maleic anhydride copolymer, and Figure 3 shows the infrared absorption spectrum of NCS.

Claims (1)

[Claims] 1 neocarzinostatin and an average of 1 per molecule
Formula () SMA _o − NCS () [ In the formula, NCS means a neocarzinostatin residue, and SMA is a styrene-maleic acid copolymer having an average molecular weight of 1,000 to 10,000 and having an average molecular weight of 1,000 to 10,000 and having an average of one or less maleic anhydride rings per molecule. refers to a combined residue, and n refers to an integer from 1 to 35. ] A method for producing a neocarzinostatin complex.