JPS6089433A - Preparation of carcinostatic substance composite - Google Patents

Abstract

PURPOSE:To prepare the titled composite, by opening the aldohexopyranose ring of dextran, reacting the aldehyde group of the dextran with a carcinostatic substance, masking the unreacted aldehyde group, and bonding with the antibody against the surface antigen of cancer cell. CONSTITUTION:The dextran having opened aldohexopyranose ring (the formula) is added preferably in a phosphate buffer solution at a concentration of 10- 30W/V%. A carcinostatic substance (e.g. daunomycin) is added to the above solution at a concentration of 0.5-5W/V% to effect the bonding of the substance to the aldehyde group of the opened dextrin, and then 85-95% of the free aldehyde group in the dextran-carcinostatic substance composite is masked e.g. by reduction. The objective composite composed of 5-30mol of dextran and 20-70mol of the carcinostatic substance bonded to 1mol of the antibody can be produced by bonding the masked composite with an antibody. The polymerization of the composite can be prevented by masking, and the objective product having excellent stability and carcinostatic activity can be produced.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a method for producing a newly regulated cancer active substance complex and a method for stabilizing an anticancer active substance complex.

[Prior art]

Today, a large number of anticancer substances have been developed as anticancer agents and are being applied clinically, but all of these are nonspecific to cancer cells and are also toxic to Seirei cells. Not much therapeutic effect has been obtained.

Based on this, treatment aims to combine an anticancer substance with a substance that has an affinity for cancer cells to form a complex, and to have this complex specifically reach cancer cells to exert its anticancer effect. The idea of law was born. As one of these, E, Hur
Wi et al. (Europe.

J, Cancer, Vol, 14, 1213 1220
．． (1978) developed a complex in which adriamycin, daunomycin, etc., which have anticancer effects, were bound to an antibody against a layer surface antigen via dextran. The method for producing this complex involves bonding the amino group of the antibody and the amino group of the anticancer substance to the aldehyde group of dextran whose aldohexopyranose ring has been opened. However, since the aldehyde group of oxidized dextran non-specifically binds to the easily bound amino group of the antibody, a complex is prepared in which 6 to 100 moles of antibody are bound per mole of dextran. This complex has a molecular weight of 1 million to 700
Many macromolecules have been found to have decreased stability during long-term storage, decreased antibody titers, and decreased anticancer activity, and their effective pharmacological effects have not been changed.

[Disclosure of the present invention]

Under these circumstances, the present inventors have conducted research on the production of anticancer drugs that are more stable and act more specifically against cancer cells, namely antibody-dextran-anticancer active substance complexes, and found that the conventional We found that masking the unreacted aldehyde groups in the complex stabilizes the complex, suppresses the decrease in antibody titer, and allows the complex to act more specifically on cancer cells. As a result, the present invention was completed.

That is, the present invention provides a step (Step A) of reacting an anticancer substance reactive with the aldehyde group with an aldehyde group of desquitran (hereinafter referred to as cleaved dextran) in which the aldohexopyranose ring is cleaved. Masking step (Step B) ■Antibodies against cancer surface antigens (hereinafter referred to as
(simply referred to as an antibody) (step C), and in the complex in which the anticancer substance and the antibody are bound via the cleaved dextran, the aldehyde of the cleaved dextran is The present invention relates to a method for improving the stability, antibody titer, and anticancer activity of the complex by masking groups.

The antibody used in the present invention may be any antibody that has been purified as a medicine, such as an animal-derived specific antibody obtained by immunizing an animal with an antigen, or a monoclonal antibody obtained by genetic engineering or cell fusion.

Dextran is not particularly limited as long as it can be used medicinally, and preferably has a molecular weight of 11,000 to
A range of 2 million is used.

The anticancer substance may be one that has a group reactive with an aldehyde group (for example, an amino group, a hydroxyl group), and even those that do not have such a reactive group may be used as an anticancer substance. The compounds may be used in the present invention by reacting them to make them reactive with aldehyde groups. Suitable anticancer agents include, for example, daunomycin,
Examples include mitomycin C and adriamycin.

Masking of the aldehyde group may be any method as long as it inactivates the aldehyde group, particularly to antibodies.

Examples of masking include treatment with a reducing agent such as metal borohydride or cyanogen metal salt borohydride, generation of acecool with alcohols, and treatment with amine compounds (particularly amino acids).

Examples of amino acids include the following.

Neutral amino acids; aliphatic amino acids [glycine, alanine, valine, leucine, isoleucine, etc.], oxyamino acids [serine, threonine, etc.], sulfur-containing amino acids [cystine, cystine, methionine, etc.], aromatic amino acids [phenylalanine, tyrosine, tri-amino acids, etc.] Acidic amino acids: aspartic acid, glutamic acid, etc. Basic amino acids: histidine, lysine, arginine, etc., imino acid residues such as niglycine, oxyproline, etc. Amino acids other than α-amino acids: β-alanine, etc. The present invention uses cleaved dextran, That is, bonding an anticancer substance reactive with the aldehyde group to the aldehyde group of the formula () (with a molecular weight of 10 to 2 million), masking the aldehyde group of the dextran, and the force produced through the binding of antibodies (, Kuwazi Tong is processed in the above order).

The above-mentioned cleaved dextran is known, and the method for producing it is Pr
oc, Natl, Acad, Sci, USA, 18.2
128 (1976), etc.

The composition ratio of each component in the complex obtained by the production method of the present invention is preferably 1 molecule of antibody G to 5 to 30 molecules of dextran, 20 to 70 molecules of the anticancer active substance, and the remaining aldehyde It is preferable that as many of the aldehyde groups as possible are inactivated, and it is preferable that as many as 80% of the aldehyde groups are masked.

An outline of an example of the manufacturing method of the present invention is as follows.

Cleavage of the aldohexopyranose ring of dextran is usually performed by oxidation. As the oxidizing agent, it is preferable to use sodium periodate). For example, about 100 to 700 μ of an oxidizing agent is added to 500 μ of dextran and the reaction is carried out for 5 to 24 hours. After the reaction is completed, the reaction solution is dialyzed, for example, against distilled water for 10 to 30 hours, and preferably freeze-dried to obtain oxidized (cleaved) dextran.

Considering the reaction between the cleaved dextran and the anticancer active substance, one molecule of the cleaved dextran is combined with the anticancer active substance 2 to I.
It is preferable to bond O, and in order to obtain a ratio similar to that of A, for example, an aqueous solvent (pH 6 to 9G) is used.
Preferably, cleaved dextran is added to a final concentration of about lθ to 30 W/V% and an anticancer substance is added to a final concentration of about 0.5 to 5 W/V% in a phosphate buffer solution, and the mixture is reacted for about 5 to 15 hours. It is preferable to let Desquitran - an anticancer active substance is thus obtained. Unreacted anticancer substances are removed by adsorption chromatography using Toyovar HW40 or other methods known per se.

When masking the aldehyde groups in the complex thus obtained, conditions may be selected that are sufficient to mask about 85 to 95% of the free aldehyde groups in the complex.

For example, reduction treatment is carried out using 1 to 10 moles of reducing agent.
Preferably, the treatment is carried out at 10°C for 15 to 20 hours. In addition, when using α-amino acids, the reaction time is usually 12 to
It is preferable that the time is 30 hours, and the PTL is about 5 to 8.

Thus, an anticancer active substance-masking dextran complex is obtained.

When binding an antibody to the complex, the antibody is added to a reaction solution containing 5 to 30 W/V% of the complex to a final concentration of, for example, 0.1 to 3 W/V%, and cooled. Let the reaction take place for 12 to 40 hours.

The complex related to the present invention thus obtained can be purified by applying it to a gel filtration ion exchange resin, antigen-Sepharose, or the like.

The thus obtained complex IJ is, for example, subjected to sterilization filtration, dispensed, and freeze-dried to form a dry preparation.

The present invention aims to prevent cancer by masking the aldehyde groups of the cleaved dextran, which has the disadvantage that the conventional complexes become macromolecules due to the binding of many antibodies to the aldehyde groups of the cleaved dextran.

That is, while the conventional complex has a molecular weight of 1 million to 7 million, the complex of the present invention has a molecular weight of 150,000 to 250,000, and contains 5 to 30 molecules of dextran per antibody molecule. It is a combination of a large number (20 to 70 molecules) of anticancer substances. As described below, this complex is superior in stability, properties, and cancer cell proliferation suppressive effect compared to macromolecules.

Regarding liquid storage stability, in the case of conventional macromolecular complexes, precipitation is observed after one month even when stored in a cold place, and a decrease in antibody activity is observed.

In addition, freeze-drying tends to produce insoluble matter.

However, in the complex obtained according to the present invention, no precipitation is observed even after one month of storage in liquid form, and no decrease in antibody activity is observed regarding storage stability after lyophilization.

Regarding antibody activity, when comparing the complex obtained by the present invention with the conventional complex, the conventional macromolecular complex shows only 40 to 60% of the activity of the antibody. In the complex obtained by the present invention, antibody activity is expressed at almost 100% without loss. This seems to be because, in the case of a macromolecular complex, multiple antibodies are bound to dextran, so that sufficient expression of activity is suppressed due to steric hindrance, etc.

Regarding the electrophoretic position, for antibodies with a hand at the γ position, a macromolecular complex has a band at the β position, and the complex obtained by the present invention has a band in the middle. Is recognized.

It can be seen that the charge changes due to the binding of dextran-anticancer active substance, but in the complex obtained by the present invention,
It can be seen that the antibody has properties more similar to the original antibody.

in vitro using α-fetoprotein-producing hepatoblastoma culture lines.
As shown in Figure 1, in vitro experiments have shown that the composite obtained by the present invention has a better cell proliferation inhibitory effect than the conventional composite in terms of cell number. .

As described above, compared to the conventional macromolecular complex, the complex obtained by the present invention is more effective than the antibody-based complex via dextran.
It is capable of binding a large number of anticancer active substances to its molecules, has excellent stability and antibody activity, and has properties close to the original properties of antibodies, such as ease of reaching target cells and good membrane permeability. There are advantages to this. In addition, its stability as a freeze-dried preparation is also recognized.

The dose and administration method for clinical use of the complex obtained in the present invention is to dissolve 10 to 300 mg of this product in 0.5 to 5 ml of distilled water for injection in the Japanese Pharmacopoeia, and to It is used by intravenous injection, intravenous drip injection, or drip injection with appropriate adjustment.

Experimental Example 1 When the complex obtained in the example described later and, for comparison, a conventional anti-α-fetoprotein-horse antibody-dextran-daunomycin complex (molecular weight 25 x 10'i) were stored at 4°C, Whereas precipitation was observed in the conventional product after one month, no precipitation was observed in the composite obtained by the present invention even after one month. In addition, R
When measured using the PHA method, a 40-60% decrease in activity was observed for the conventional product, whereas no decrease in activity was observed for the complex of Example 1 even after one month. .

Furthermore, when both complexes were freeze-dried, while the conventional product produced insoluble matter, the complex of Example 1 did not produce any insoluble matter, and no decrease in its activity was observed.

Experimental Example 2 α-fetoprotein-producing hepatoblastoma cells of the complex obtained in Example 1 and the conventional complex used in Experimental Example 1.
A comparative experiment was conducted on the growth inhibitory effect on 105 OX/ml. The results are shown in FIG.

The culture medium used was RPM1640 (Nissui Pharmaceutical Co., Ltd.).

Example 1 Dextran T-10, 100 mg, was dissolved in 20 ml of 0.015 M sodium periodate, treated at room temperature for 8 hours in the dark, and thoroughly dialyzed against distilled water overnight at 4°C. Thereafter, it was freeze-dried to obtain dry oxidized dextran loO+ng.

100 mg of this dried oxidized dextran was dissolved in 5 ml of a phosphate buffer solution containing sodium chloride, pH 9, 0, 0, 5 ml, and then 10 mg of daunomycin dissolved in the same amount was mixed and reacted at room temperature for 1 hour. Subsequently, the reaction solution was passed through a column packed with Toyopearl resin to remove free daunomycin, and 50 mg of glycine was added to the passed solution, followed by reaction at 4° C. for 24 hours. Next, 12.5 to 20 mg of anti-human alpha-fetoprotein horse antibody, which is a type of antibody against the surface antigen of hepatocellular liver cancer, was added to this, and the reaction solution was incubated at 4°C for 24 hours, and the reaction solution was transferred to Sephadex G200. (Figure 2) to obtain a fraction of the complex of the present invention by applying it to a gel filtration column.
. Furthermore, a stabilizer was added to this to obtain a freeze-drying agent.

The properties of the obtained complex were as follows: daunomycin/antibody molar ratio 40-50, dextran/antibody molar ratio 12, residual antibody activity, 100%, molecular weight 250,000, and electrophoretic position β2.
Example 2 Dextran T-40. and underwent dialysis. Thereafter, it was freeze-dried to obtain dry oxidized dextran.

50 mg of this dry oxidized dextran was added to 0.0 mg of physiological saline.
After dissolving in 5+Ill, 5 mg of adriamycin dissolved in the same volume was mixed and reacted at room temperature for 1 hour. The reaction solution was treated with a resin by the No\Fuchi method, and 20 mg of lysine was added to the treated solution, followed by reaction at 4°C for 24 hours.

Next, 2 mg of a monoclonal antibody (derived from mouse) against human melanoma was added to this, and the mixture was reacted at 4°C for 400 hours. The reaction solution was gel-filtered through Sephacryl 5300 to pool both parts of the complex of the present invention and stabilize it. A lyophilized preparation was obtained by adding the agent.

[Brief explanation of the drawing]

FIG. 1 shows the inhibition of proliferation of α-fetoprotein-producing hepatoblastoma cells by the complex of Example 1 and the conventional complex (macromolecule anti-α-feto1-protein horse antibody-dextran-daunomycin complex). FIG. 2 is a graph showing a comparison of effects, and FIG. 2 is a diagram showing separation of the complex of the present invention and unreacted dextran-glycine-downmycin complex. ■...Complex of the present invention (Example 1) 2...Conventional complex 3...Control 4...Unreacted dextran complex Figure 1 Figure 2 now Iil#o. March 15, 1980 Commissioner of the Japan Patent Office 1. Indication of the case 1988 Patent Application No. 198131 2. Name of the invention Method for manufacturing an anticancer active substance complex 3. Relationship with the person making the amendment Patent application Name of Person Midori Juji Co., Ltd. 4, Agent ■541 Address New Life Hirano-cho 4-53-3 Hirano-cho 4-chome, Higashi-ku, Osaka Telephone: (06) 227-1156 6. 1 patent of the specification subject to amendment "Scope of Claims" column, "Detailed Description of the Invention" column, and "Brief Description of Drawings" section 7, contents of amendment (1) Scope of Claims 1 in the Description are corrected as shown in the attached sheet. do. (2) rHurwiJ on page 2, line 15 of the same book [)1
Corrected by urwitzJ. (3) On page 2, line 17 of the same book, ``cancer surface antigen J'' is corrected to ``cancer antigen 1''. (4) Delete "Adriamycin," from lines 18 to 19 on page 2 of the same book. (5) Delete "etc." on page 2, line 19 of the same book. (6) On page 4, line 8 of the same book, "1 cancer surface" is corrected to "cancer cell surface." (7) Delete "If it has been purified as a medicine" on page 4, lines 17 to 18 of the same book. (8) Delete "If the material can be used for medicinal purposes" in the second and third lines of page 5 of the same book. (9) “Rysine” in the same book, page 6, line 14, is “lysine”
Correct. (10) On page 10 of the same book, after “gel filtration” in the first line, “
Add a carrier. (11) On page 10 of the same book, line 4, after “dispense,” “
Add ``liquid frozen crystals''. (12) On page 10 of the same book, line 4, “lyophilize” was replaced with “
Corrected to ``freeze-dried.'' (13) Correct rlnJ to rinJ on page 12, line 5 of the same book. (14) “Fetoprotein” on page 13, line 4 of the same book.
"Equine antibody" is corrected to "fetoprotein horse antibody." (15) On page 13 of the same book, line 5, replace “25” with “100”
Correct. (16) rRP on page 13, line 9 of the same book) TAJ as “P
Correction to HAJ. (17) “Fetoprotein” on page 13 of the same book, last line
is corrected to "fetoprotein." (18) rRPMJ on page 14, line 4 of the same book
Correct to IJ. (19) rG200J on page 15, line 2 of the same book [G-
Corrected to 200J. (20) On page 15 of the same book, line 5, "desiccant" is corrected to "dry preparation." (21) On page 15 of the same book, add ``Toyobal'' before ``resin'' in the last line. (22) "Fetoprotein" on page 16, line 10 of the same book is corrected to "fetoprotein." (23) "Fetoprotein" on page 16, line 12 of the same book is corrected to "fetoprotein." Above (Separate IE) Claims fil A step of reacting an anticancer substance reactive with the aldehyde group with the aldehyde group of desquitran whose aldohexopyranose ring has been opened (Step A) ■ Masking the aldehyde group Step (Step B) ① Step (Step C) of binding an antibody against a cancer cell surface antigen to the aldehyde group (Step C) A method for producing an anticancer active substance complex. (2) The method for producing an anticancer active substance complex according to claim (1), characterized in that Step A, Step B, and Step C are carried out in this order. (3) The control according to claim (1) or (2), characterized in that 5 to 30 molecules of dextran and 20 to 70 molecules of an anticancer substance are bound to one molecule of the antibody. Method for producing cancer agent conjugate.

Claims (1)

[Claims] (11) Step of reacting an anticancer substance reactive with the aldehyde group with the aldehyde group of desquitran whose fludohexopyranose ring has been cleaved (Step A); ■ Step of masking the aldehyde group ( Step B) ■ A method for producing an anticancer active substance complex, which comprises a step (Step C) of binding an antibody against the layer surface antigen to the aldehyde group (Step C). (2) Processing in the order of Step A, Step B, and Step C. (3) 5 to 30 molecules of dextran and 20 to 70 molecules of anticancer activity per antibody molecule; A method for producing an anticancer active substance complex according to claim 11 or claim 2, which comprises combining substances.