JPS59139396A - Neocarzinostatin complex and its preparation - Google Patents

Abstract

PURPOSE:The reaction of neocarzinostatin with a styrene-maleic anhydride polymer which contains maleic anhydride residue and half-ester groups and has a specific molecular weight gives the titled complex that is used as a slow- releasing oily carcinostatic preparation with less side-effect. CONSTITUTION:The objective complex of formula I [n is 1-35 integer; NCS is residue of neocarzinostatin; SMA is residue of half-esterified styrene-maleic acid copolymer containing the groups of formula II and formula III where the maleic group bonds with NCS (where the sum of formulas II and III is more than 1 per 1 molecule and less than 50mol% on the average) and groups of formula IVwhere the maleic acid is half-esterified] is obtained by reaction of neocarzinostatin with a half-esterified styrene-maleic anhydride copolymer of 1,000-10,000 average molecular weight which has 1 or more maleic anhydride groups per one molecule.

Description

DETAILED DESCRIPTION OF THE INVENTION This issue 8A relates to a novel neocarzinostatin complex and a method for producing the same. More specifically, the present invention relates to the formula () [where n means an integer of 1 to 35, 0 means a neocarzinostatin residue, and G is a maleic acid moiety of Bonded maleic acid residue C9 ■ −→ Maleic acid residue not bonded (however, the sum of ■ and ■ is on average 1 or more and 50 mol% or less per molecule), and ■ Half-esterification The average molecular weight of the half-esterified product consisting of maleic acid residues i, ooo to io,
It means the styrene maleic acid copolymer residue of ooo.

] The present invention relates to a neocarzinostatin complex shown in the following and a method for producing the same.

Neocarcinostatin is produced from Streptomyces carcinostatecus variant F-4 Tokuroya (Strepto
myces carzinostaticus vir
-F-41Kuroya) is a proteinaceous anticancer substance produced in the culture of Ashi (Special Publication No. 42-21,752,
U.S. Pat. No. 3,334,022).

Its secondary structure has been reported by one of the inventors mentioned above as having a total number of amino acid residues of 109 and an estimated molecular weight of 10°700 (5 oience, 178
vol. 875-876, 1972, and Arch-B.
iochenx-Biophys-, vol. 163, 379
In the treatment of ~385jt 8 muscles, metastasis of muscle cells is the most important problem, and especially metastasis to the 77th node is the biggest problem. Previously, the present inventor conducted various studies on reducing the toxicity of neocarzinostatin, improving the durability of its efficacy, and specifically transferring the drug to the lymphatic system.
We have discovered that a neocarzinostatin derivative obtained by reacting two free amine groups present in the molecule of neocarzinostatin with a partially melted product of a water-soluble styrene-maleic acid copolymer meets the above objectives, A patent application was filed (Japanese Unexamined Patent Publication No. 117095/1983).

However, in addition to the need for anticancer agents to have the property of transferring to the lymphatic system as described above in order to suppress cancer metastasis, it is desirable that they have high affinity for tumor pgI. When the tumor compatibility 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 it has a maleic anhydride residue different from that of the styrene maleic anhydride copolymer or its partially decomposed product, and that the average molecular weight of the half-esterified product is i, ooo ~ We discovered that a neocarzinostatin complex obtained by reacting io, ooo styrene maleic anhydride copolymer with neocarzinostatin surprisingly achieved the above object, and filed another patent application (
(Japanese Patent Application No. 57-31555).

In this invention, the key point of the technology is to use a styrene-maleic anhydride copolymer in which maleic acid residues are half-esterified and which has an average of one or less manic anhydride residues per molecule. there were. In particular, it was important to have an average of one or less maleic anhydride residue per molecule. This is because the reaction between neocarzinostatin (hereinafter abbreviated as NC5) and styrene maleic anhydride copolymer half-ester (hereinafter referred to as SMA) is a reaction between polymers, and both are polyfunctional. Since various reactions may occur between molecules, we selected SMA with a small number of reactive maleic anhydride residues in order to suppress reduction reactions as much as possible and obtain the desired reactant. be. The invention was effective in solving the above problems. However, other VAs have been found to improve the reaction yield and are not effective against this problem. In other words, it was necessary to conduct further research in order to increase the reaction yield while suppressing side reactions. The present invention was completed as a result of this research.

That is, the present invention is based on the formula (1) %Formula % (1) [where n means an integer of 1 to 35, ◎■ means a neocarzinostatin residue, [phase]■ means its maleic acid A maleic acid residue whose moiety is bonded to a maleic acid residue bonded to a maleic acid residue bonded to an
The sum of (1) and (2) is on average 1 to 50 moles per molecule. ) and ■ an average half-esterified molecule composed of half-esterified maleic acid residues 1 tl, 000 to 10
,000 styrene maleic acid copolymer residues. A neocarzinostatin complex represented by ``Neocarzinostatin, which has an average of 91 or more maleic anhydride residues per molecule, and has a half-esterified average molecule i11,000~ 10,000 styrene-maleic anhydride copolymer [wherein n means an integer of 1 to 35 and 柕 means a neocarzinostatin residue] , ω base) is a maleic acid residue whose maleic acid moiety is bonded to a maleic acid residue that is not bonded to a maleic acid moiety (however, the sum of ■ and ■ is on average 1 or more per molecule of 50 mol % or less) and (1) average half-esterified content composed of half-esterified maleic acid residues - 1 tl, 000 to 10
,000 styrene maleic acid copolymer residues. ] This is a method for producing a neocarzinostatin complex.

The complex (I) of the present invention has the same useful properties as the above-mentioned neocarzinostatin derivatives, and has excellent fat solubility, allowing it to be applied as a skin or oil-based preparation. When the complex (I) of the present invention is administered as an oily preparation, the drug can be concentrated at one ulcer site. and the complex of the present invention (
I) also has an excellent affinity for tumors, and can remain at the tumor site and exert a strong anticancer effect.

On the other hand, since the complex (I) of the present invention has water-soluble properties in addition to fat-solubility, it can also be administered systemically through a water-soluble preparation, such as intravenous injection.

A preferred modification of neocarzinostatin with the complex (I) of the present invention uses a semi-esterified styrene-maleic anhydride copolymer having one or more maleic anhydride residues. This is thought to be due to the fact that neocarzinostatin has both water-soluble properties and fat-soluble properties.

A particular object of the present invention is that when an anticancer drug is used as an intravenous injection, the anticancer drug is released into the capillaries and tissue, and then specifically transferred to the lymphatic system; When used, anticancer drugs are more likely to be in the blood than oil-based preparations.

It is possible that the anticancer drug is slowly released to the necessary site such as tissue fluid or lymph fluid, and regardless of the administration form, the anticancer drug can be easily accumulated in the tumor tissue (site) either as it is or after being degraded. It is to be safely excreted from the body.

For this purpose, since the complex (I) of the present invention leaks into tissues from capillaries, it is preferable that the molecular value is below the name, solubility in oily bases, and specificity for the lymphatic system. For good migration, the molecular weight is preferably 10,000 or more.

It is presumed that after the complex (I) of the present invention reaches a predetermined site in the living body, it is hydrolyzed as it is or partially, and neocarzinostatin is released and exhibits antitumor properties. The complex (I) of the present invention forms a polyanion and is also expected to have the effect of activating the immune system in vivo.

Complex of the invention (i) 1 to 35 molecules per qNcsi molecule, usually 2 to 15 molecules of SMA. NC8
The details of the bonding state between this and SMA are unknown. N
The structure of C8 has already been explained in 5 published in 1972.
Science 17, Vol.
Since there is only lysine at position 0, the two free amino groups of NC3 can react with the maleic anhydride residue of FisMA in the reaction between NC5 and SMA. Also, since NC8 has many secondary amine groups and hydroxyl groups, these functional groups and SM
It is also possible that A forms a secondary bond, and the formula (I)
The complex of is given.

Therefore, in the complex of the present invention, not only one or two of the two free amino groups of NC5 form an acid amide bond with SMA, but also the above functional group forms a secondary bond with SMA. Since there are a total of 35 functional groups, the result is that the SM for NC8 molecules in the complex is
The content ratio of A molecules can be 1 to 35. It is thought that normally 2 to 15 molecules of SMA react to form a complex.

The SMA used in the present invention has a semi-engineered maleic molecular weight of 1,000 to 10,000. Here R is methyl.

These are lower alkyl residues such as ethyl, globyl, and butyl, or polyhydric alcohol monoether residues such as ethylene glycol monoether and polyethylene glycol monoether. The number average molecular weight of these main chain units is i, ooo to io, ooo, preferably 1,500.
~5,000, and there are maleic anhydride residues. As the molecular weight increases, the number of maleic anhydride residues can increase, but if there is too much maleic anhydride residue, the reaction with NC8 will become complicated and a large number of by-products will be produced, so it is desirable to keep it in the above range.
Maleic anhydride residues in SMA that did not participate in the reaction with 8 are ring-opened by hydrolysis during the reaction process to become maleic acid residues. Therefore, when the reaction is carried out in an aqueous system, there is substantially no maleic anhydride residue in the SMA residue in the complex of formula (I). It is neglected that one of the two carboxyl groups of maleic acid is esterified,
It is preferable that all maleic acid residues other than maleic anhydride residues are half-esterified, but there may be some maleic acid residues that are not half-esterified.

The present inventors believe that in order to increase the reaction rate between NC8 and SMA, it is desirable to use SMA with a large number of maleic anhydride residues; I had a good guess that it would be possible. However, surprisingly, even when using SMA, 1===6
I remembered that if the reaction is carried out at a relatively low temperature below 0.degree. C., an SMA-based NC8 complex can be obtained in high yield without side reactions. NC3 and S
Since the reaction with MA is a reaction between polyfunctional polymers (or oligomers), it is impossible to specify the reaction position or molecular structure of the reaction products. However, the structure of the reaction product can be determined on average by analysis of molecular size using electrophoresis, gel ξ-mie chromatography, gel filtration, etc., and analysis of constituent components such as infrared absorption spectrum, ultraviolet absorption spectrum, and elemental analysis. It is possible to analyze

The neocarnanostatin complex (I) of the present invention comprises NC5
It is produced by reacting SMA with SMA. The reaction is usually carried out by dissolving NC8 in an aqueous solution of sodium charcoal, and
At room temperature or under cooling, preferably ÷4
It is added under stirring at a temperature below .degree. In addition, SMA is not reacted with ≠奔==x4-4 maleic anhydride and is a water-soluble organic solvent (eg, acetonitrile, dioxane).

It can also be produced by dissolving it in tetrahydrofuran), adding an aqueous solution of sodium bicarbonate (NC3) thereto, and then removing the solvent by drying under reduced pressure or the like.
The complex (I) of the present invention is thought to be generated by ring opening of the maleic anhydride residue of SMA and reaction with the functional group of NC8, and 1 to 35 molecules of SMA react with the NC3I molecule. may be used to form a complex, but usually 2 to 1
It is thought that five molecules of SMA react to form a complex.

Therefore, the resulting complex contains not only a reactant in which one or two of the two free amine groups of NC3 form an acid amide bond with SMA, but also a reactant in which SMA is secondary to other functional groups of NC3. At the same time, a complex with a bond formed is also generated. Note that the above reaction involves dissolving NC8 in an aqueous solution of sodium bicarbonate,
This is done by adding SMA to it and reacting it.
When such a system is used, the multi-reaction product contains yuzu salt derived from sodium bicarbonate, a portion of excess SMA, etc., and these are removed by various means such as dialysis.

The neocarzinostatin complex (I) of the present invention can be administered to humans by local intratissue administration methods such as the primary site of cancer, the site of cancer removal after surgery, subcutaneous administration, intramuscular administration, intravenous administration, or intraarterial administration. Administrative methods such as internal administration, oral administration, and external administration methods such as topical application, spraying, herbal medicine, and intravesical injection are suitable. The dosage depends on the administration method and the malignancy of the cancer.

It is not fixed depending on the type, the patient's medical condition and membrane condition, the degree of cancer progression, etc., and 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. .01-10 Misaki/Same, mainly 1-2 times a week,
Alternatively, it is preferable to administer it on consecutive days. It is also possible to further increase the dosage for topical application and oral administration.

The complex (I) of the present invention is an X-ray contrast agent Biodol (manufactured by Laboratoire Guerbet, France, Libiodol Ultrafluid - Iodized poppy oil fatty acid ethyl ester).
Solubilize the suspension by ultrasonication.

Complex of the present invention (I) 1-'29/Ribiodol l rI
When an oil-based preparation of 1.0 ml is administered intraarterially, Lipiodol and the drug remain in the tumor blood vessels for a long period of time, exerting a strong antitumor effect. Furthermore, by dissolving in Libiodol, the state in which the complex (I) of the present invention stays locally can be observed by X-rays.

The present invention complex (
This is one of the usages that takes advantage of the properties of I).

Moreover, the complex (I) of the present invention is dissolved in a 1-9% aqueous sodium bicarbonate solution. When this aqueous solution is administered intravenously,
Since this drug is widely distributed in lymph vessels, it exerts a strong anticancer effect.

Using it as such an aqueous salt solution is also one of the ways to utilize the properties of the complex (I) of the present invention.

EXAMPLES The present invention will be explained in detail with reference to Examples below.

Reference example Number average molecule m2,1 determined by vapor osmotic pressure method in a test tube
00 styrene maleic anhydride copolymer 10y (0,
0048 mol), 0.1 g of lithium acetate, and predetermined amounts of alcohol and dioxane were charged, the upper part was melt-sealed, and the mixture was shaken in an enclosed room for 24 hours to uniformly dissolve. The resulting solution was then heated for 15 hours, then cooled to room temperature, the reaction solution was taken out, diluted approximately twice with dioxane, freeze-dried, and then further dried under reduced pressure at 60°C for 24 hours. A pale yellow flaky product (SMA) with some maleic anhydride rings remaining was obtained.

All the products obtained were semi-esterified SMAs.

The amount of residual anhydride rings in the obtained SMA was quantified by the ratio of the optical density at wave numbers 1780 and 7ooaa-' using infrared absorption spectroscopy. Table 1 summarizes the average molecular weight and residual anhydride ring content of the SMA obtained by vapor osmotic pressure method.

Table 1 Example 1-4 0.5 g of neocarzinostatin was dissolved in 0.5 M sodium bicarbonate aqueous solution 5 Qad under water cooling and shielded from light, and powdered SMA shown in Table 1 was added while stirring. 3-Of S
MA was added in several portions and thoroughly stirred until completely dissolved. After dissolution, the mixture was allowed to stand at 4-6°C for 16 hours. The pH of the reaction system was maintained between 8.3 and 867 during stirring.

Next, transfer the reaction solution into a cellophane tube, and perform 10.a.
M3! 4 under pressure against 11 ammonium carbonate aqueous solution
Dialysis was performed at ~6°C for 3 days with frequent changes of the dialysate. After the dialysis, the reaction solution was freeze-dried to obtain the desired SMANC8 complex as a white cotton-like solid. □When their molecular weights were determined by electrophoresis, they were approximately 57,000 each.
They were 0°56.000.40,000 and 65,000.

Table 2 summarizes the free amino group reaction rate (mol %) of this complex and the yield of the complex.

Table 2 Table 3 Elemental analysis results (1%) After the reaction, the remaining free amine groups of this complex were diluted with water and reacted with trinitrobenzene sulfonic acid, and the resulting nitrobenzene derivative was analyzed using visible light absorption spectroscopy. It was quantified spectroscopically using a meter. Table 3 summarizes the results of elemental analysis of each composite. The infrared absorption spectra of each composite obtained by the KBr tablet method are shown in Figure 1 (ta).
, (b), (C), and (d). Figure 2 (a), (b), (C), (d) shows Toyo Soda■&! A gel permeation chromatogram measured at pH 7.9 using a G-30005W column and using 105M ammonium bicarbonate as a mobile phase is shown. For comparison, FIG. 3 shows the Tagervermiasi = J/chromatogram obtained for NC8. Figure 4 (Otori), (b),
(C) and (d) show the ultraviolet and visible light absorption spectra of each complex in a 0-5M bicarbonate aqueous solution. Further, Table 4 summarizes the solubility of the complexes in various solvents.

Table 4 Solubility of composite (1 cape/1 scj) Comparative example 1 NC80.2f was dissolved in 0.8 M sodium bicarbonate aqueous solution 305E/ under ice cooling, and a number average molecular weight of 3.100 was dissolved in this.
, maleic anhydride ring inclusion average 0.40/molecular partial half-butyl cellosolve esterified styrene maleic anhydride 1
:1 Copolymer powder 1. Of was added in several portions as in the example. After the powder is completely dissolved, place it in the refrigerator for 1 hour.
After standing for 6 hours, the remaining amino groups were quantified and the reaction rate was 16.9. The reaction solution was cooled on ice in Seronave for 3 hours.
After dialysis while replacing the external dialysis solution (5mM ammonium bicarbonate aqueous solution) for several days, freeze-drying resulted in 0 white fluff-like solids.
．． I got 759. The analyte yield was 79.3%.

The elemental analysis values of the solid were as follows.

Ni2.32%, C; 63.31%, H; 6.69%

[Brief explanation of the drawing]

FIG. 1 shows an example of an infrared absorption spectrum of a complex according to the present invention, and FIG. 2 similarly shows an example of a gel permeation chromatogram (GPC). FIG. 3 shows an example of GPC of NC5, and FIG. 4 shows an example of the ultraviolet/visible light absorption spectrum of the conjugate of the present invention. Patent Applicant Kuraray Yamanouchi Pharmaceutical Co., Ltd. Pharmaceutical Antibiotic Research Institute Co., Ltd. Patent Attorney Ken Honda v+12I (d) (b) 3g, V1!4. ．． Dedication 1214-IG 19 '20!
2% IQ 11141G '920222φ
2625 籟? +X1”f) σ reaction 2
Good yield of 80 kuzuki L゛. . , No. ′+wa <b) Advancing 5th grade) Left throw (Qi) Song Gitai,)
Kumi Chogo,) 1-2 Minamiikebukuro, Toshima-ku, Tokyo
4 number 5

Claims (1)

[Claims] 19 Formula (1) means a neocarzinostatin residue, and ■ means a maleic acid residue whose maleic acid moiety is not bonded to ■ ■□□□ (however,
The sum of (1) and (2) is on average 1 to 50 moles per molecule. ) and ■ Half-esterified maleic acid residue m i,ooo
~10,000 styrene maleic acid copolymer residues. Neocarzinostatin complex designated J. 2. Ocarthinostatene, and a half-esterified styrene-maleic anhydride copolymer having an average molecular ffi of 1,000 to 10,000 and having an average of E or more maleic anhydride residues per molecule. The formula CI) means a neocarzinostatin residue, and the maleic acid moiety is bonded to the (However, the sum of ■ and ■ is on average 91 to 50 moles per molecule.) and ■ Half-esterified maleic acid residues composed of half-esterified maleic acid residues. average molecule m i,ooo~10
,000 styrene maleic acid copolymer residues. ” A method for producing a neocarzinostatin complex.