JPS6067433A - Complex of mitomycin, polysaccharide and antibody - Google Patents

Abstract

NEW MATERIAL:A complex of mitomycin, polysaccharide and antibody, consisting of the adduct of mitomycin and polysaccharide [bonded through the group of formula I (n is niteger of 3-7; the left-most N is N of mitomycin imino group; the right-most O is O of the polysaccharide OH), etc.] bonded with antibody through the group of formula II, formula III (R is lower alkylene; each left-most O is O of the polysaccharide OH; the right-most NH is the amino NH of the antibody), etc. EXAMPLE:The dextran.mitomycin C.anti-human erythrocyte Igg complex. USE:Agent for the missile therapy of cancer. The antibody is preferably an anti- tumor antibody. PREPARATION:The titled complex of formula VI can be prepared by (1) converting the mitomycin-polysaccharide adduct of formula P1-CO2H (CO2H is at the terminal of the spacer) to the mercapto-adduct of formula IV, and (2) reacting with the disulfide antibody of formula V (P2-NH2 is antibody) in an aqueous medium at 6-8pH.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a complex obtained by combining mitomycins, a polysaccharide, and an antibody. More specifically, the present invention provides that a conjugate of mitomycins and a polysaccharide and an antibody have the following formula: 0 represents O of the hydroxyl group of the polysaccharide, Nl at the right end (represents NH of the amino group of the antibody), ■ General formula % formula % (where n, R, 0 at the left end, 0 at the right end NH has the same meaning as above), or ① above ②.

Any hydrogen in the methylene chain represented by (CH2)n in the general formula of .

However, in the above, the conjugate of mitomycins and polysaccharides and r1 mitomycins and polysaccharides are expressed by the general formula % formula % (where n has the same meaning as above, and N at the left end is the imino group of mitomycins. , the rightmost 0 indicates the 0 of the hydroxyl group of the polysaccharide Ip + N-co-(cH2')n-o- (in the formula, 0 has the same meaning as above)
A group represented by ■′, or (cut)y in the general formulas of ■′ and ■′ above, in which one of the hydrogen atoms in the methylene chain is substituted with a lower alkyl group. shall be.

The complex of the present invention is characterized in that an antibody is further bound to the mitomycin-polysaccharide conjugate. When an antitumor antibody such as an anti-α-fetoprotein antibody is used as the antibody, there is an advantage that so-called missile therapy can be applied.

Conventionally, the present applicant has filed an application regarding a conjugate in which mitomycins are bound to a spacer bound to a hydroxyl group of a polysaccharide (% 97691/1989). In the JP-A show, the spacer part is "", N-C! 0(OH2)n-
Nl(-0-0-1, etc.). Also, regarding the above conjugate in which the spacer portion is >N-co-(CH2)n-o-, Honda published the same date as this application; ri
There is an application filed by a person (title of invention: conjugate of mitomycins and polysaccharide).

Next, the present invention will be explained in more detail.

The mitomycins used in the present invention include Iminohana (
Any mitomycin may be used as long as it has an aziridine ring imino group (aziridine ring imino group), and mitomycin A1 mitomycin C1 derivatives of these sites other than aziridine H are used.

Dextran, cellulose, agarose, alginic acid, etc. are used as the polysaccharide.

The preparation of conjugates of mitomycins and polysaccharides is described in Japanese Unexamined Patent Publication No. 54-97691, the above-mentioned patent application filed on the same date.

Next, as an antibody, any antibody may be used as long as the opposing antigen exists in the body and it does not cause serious symptoms by itself or by interaction with the antigen, but for the purpose of the present invention, anti-tumor antibodies is preferred. Various known anti-tumor antibodies, such as anti-α-fetoprotein, 0EA
(CarcinoEmbryotechnique Antidiene), HOG
(Human Choriogenic Gonadotropin) etc. can be used.

The complex of the present invention can be obtained by the following method.

(1) The conjugate of mitomycins and polysaccharides contains 18H.
Introduction of groups (1) Introduction of amino groups into the -1 conjugate In the conjugate, there is a portion of the spacer that is not used for binding to the mitomycins, i.e. - General formula % (wherein, 0 at the left end of n1 is ), or ■ - a group represented by the general formula % (where n1 is the same meaning as above), or ■ (CH2 in the general formulas of ■ and ■ above); ) There is a group in which any hydrogen in the methylene chain represented by n is substituted with a lower argyl group. First, a lower alkylene diamine is applied to this group to convert the carboxyl group at the right end of each formula of the above N1 to -coNH-R-NH2 (in the formula, R has the same meaning as above).

The conjugate of mitomycins and polysaccharides is prepared by ■-C02H (
However, Co2H indicates C02H at the end of the spacer part)
The above reaction can be expressed as follows.

■-co2a + R (NHz)z→■-0ONH-R
-NH211'r, V, Il, -y +, k +
−, + −S ”ir 5, / +y −h−1,
+7M-f'14'? As N-kylene, carbon number is 2~
A linear or branched version of 6 is preferably used. For example, ethylene diamine, propylene diamine, tetramethylene diamine, hexamethylene diamine, etc. are used.

The above reaction is usually carried out in an aqueous medium in the presence of a suitable condensing agent at room temperature and pH 6 to 8 for 5 to 50 hours.

The aqueous medium means water or a mixed solvent of water and a lower alcohol such as methanol or ethanol, acetone, tetrahydrofuran, etc., or a buffer solution. As the condensing agent, for example, 1-ethyl-3-C5-dimethylaminopropyl)carbodiimide or the like is used.

The above reaction may also be carried out in one step by allowing lower alkylene diamine to coexist when mitomycins are bonded to the polysaccharide and spacer bonded intermediate for producing the conjugate. Reaction condition a in this case: tl-i
i' Fu After 1 production〒 1--After the reaction is completed, unreacted substances are removed by ultrafiltration.

(1) Introduction of -2-5-s-group The aminated conjugate is reacted with N-succimidyl 3-(2-pyridyldithio)propionate (hereinafter referred to as 8PDp) to add an -8-B- group to the conjugate. Introduce. The reaction is carried out in an aqueous medium, usually at a pH of 6 to 8, for 10 minutes to 5 hours. As the aqueous medium, those mentioned above are used.

After the reaction is completed, the product is purified by dialysis or the like.

(1) -3-5-s-→-8R -÷■-Cot()(-R-NH-00-(, H20H
28N (a disulfidated conjugate is reduced to a mercapto conjugate. A known reduction method in which -5=S- is converted to -8H can be applied, but dithiothreitol (dithj, o
Particular preference is given to reduction with .threitol).

When dithiothreitol is used, the reaction is carried out by reacting the disulfide bond and dithionuretol in an aqueous medium, usually at room temperature and pH 6 to 8 for 5 minutes to 3 hours.

The reaction may be carried out by adding an aqueous dithiothreitol solution to the solution after dialysis in (1)-2.

After the reaction is completed, the mercapto conjugate is purified by dialysis or the like.

(2) Introduction of -5-s- group into antibody (In the formula, ■-NH2 represents an antibody) A -5-S- group is introduced by reacting the antibody with 5PDP.

The reaction conditions may be the same as in the case of (1)-2 above. After the reaction is completed, it is purified by dialysis or the like.

(3) Reaction between mercapto conjugate and 2-sulfide antibody (generation of target complex) ■-〇〇NH-R-NH-C! 0-01(2C!H2S
H+■-CONH-R-NH-CO-OH2CH2-E
3-8-(3H2CI12C,N'H-(9) Reaction C: Carry out in an aqueous system, usually at room temperature and pH 6-8 for 1-20 hours.

As the aqueous medium, the same ones as mentioned above can be used, but a buffer solution is particularly preferred. The mercapto conjugate solution after dialysis and the disulfide antibody solution are mixed, and after the reaction is completed, tiIl is produced by gel filtration or the like.

The pharmaceutical and pharmacological properties of the complex of mitomycins, polysaccharide, and antibody of the present invention are combined with the dextran-mitomycin C conjugate obtained using 6-aminohexanoic acid as a spacer and anti-human red blood cell Iff (fluorescence) as an antibody. The case of a complex (from Example 1) obtained from (labeled with helical isothiocyanate) is shown below.

(1) Release rate The release of mitomycin C from the complex in 67° C., pH 7, 41JL buffer is according to a first-order rate equation (i.e., a linear relationship), and the half-life is about 24 hours. This is almost the same as the case without antibody binding.

(2) 1n using antitumor active mass L1210 leukemia
The results of examining the antitumor activity in vitro were as follows, and are equivalent to mitomycin.

△ Sacrificial product concentration Growth inhibition μ9 shoulder, Mitomai (,,,,,710) (%) 0, 5 8 5. 1 0, 5 7 5. 7 (3) Antibody activity Using human red blood cells as the antigen and guinea pig complement as the complement, the antibody titer was assayed using hemolytic reaction as an indicator. 50%
Comparing the concentrations that cause hemolysis, it was found that the complex retained 85.6% of the activity of the original antibody, indicating that the activity involved in the complement system was almost maintained even after synthesis.

Next, the production of the complex of mitomycins, polysaccharide, and antibody of the present invention will be explained with reference to Examples.

Example 1 l-(1) Introduction of mercapto group into mitomycin C1 dextran conjugate with 6-aminehexanoic acid as a spacer Reaction intermediate between dextran and 6-aminehexanoic acid (
There is a production example in JP-A-54-97691) 100■, mitomycin G101'li7% ethylenediamine 0.
45■, 1-ethyl-3-(3-dimethylaminepropyl)carbodiimide hydrochloride! +00■ was dissolved in 10w1 of phosphate buffer at pH 7.5, and after reacting at room temperature for 24 hours,
Unreacted substances are removed by ultrafiltration with a pH 9 sodium carbonate aqueous solution using a 20,000-cut ultrafiltration membrane.

Next VC0,3ml x t/-tall (7) 5PDj
4.46 Add Tng and react at 25°C for 50 minutes.

After the reaction is complete, add 0.5 ml of 0.1M lithium-hydrochloric acid buffer.
and dialyzed against 0.1 M pH 7.5 phosphate buffer.

Next, 40 mM dithiothreitol was added, reacted at 23°C for 20 minutes, and dialyzed against an acetone buffer solution of pH 4.5.

1-(21 antibody with a -5-s- group introduced into it, use anti-human erythrocyte 7g labeled with FITC. 150 mg of the antibody was dissolved in 107 g of phosphate buffer, and 1.95 μg of 5PDP in ethanol was added. , 23
Incubate at ℃ for 30 minutes. Then, 0.5 ml of 0.1M Tris-HCl buffer is added, and the mixture is dialyzed against 0.1M pH 15 phosphate buffer.

1-(3) Formation of complex The product of 1-(i) + 1121 is reacted in a phosphate buffer at pH 7.5 at room temperature for about 10 hours.

1-(4) Purification (gel filtering) The reaction completed solution was fractionated using Sephadex G-200, unreacted antibodies were separated, and the complex fraction was purified by void □], ume
Obtained as a fraction (Figure 1). Furthermore, Sephadex 4
e) Fractionate in B and 1'r'r the three peaks (Figure 2) The first peak (void vci'lume) is the conjugate of mitomer f-sin C and dextran and the antibody. Covered with polymer. The second peak is the complex of interest. The third peak is a conjugate of unreacted mitomycin C and dextran. In addition, in the above, the conjugate of mitomycin C and dextran is 0)6. So, the antibody is 0~
Measure at 0.

The binding ratio of the complex was determined by the absorbance on the gel filtration chart, and the simitomycin-dextran conjugate was quantified, and LOWr7
When the amount of protein was quantified using a method, it was revealed that the antibody was bound at a ratio of conjugate:antibody=1:2.

[Brief explanation of drawings]

Figure 1 shows Sephadex G-2, the solution after completing the complex formation reaction.
The relationship between time and absorbance during 00 fractionation is shown. FIG. 2 shows the relationship between time and absorbance during Cefdex 4B fractionation of the solution after completion of the complex formation reaction. Patent applicant (102') Representative of Kyowa Hakko Kogyo Co., Ltd.
Book Celebration Part

Claims (2)

[Claims] (1) The conjugate of mitomycins and polysaccharide and the antibody have the general formula % formula % xylene group. Also, the 0 at the left end of each is the 0 of the hydroxyl group of the polysaccharide.
, the NH on the right side indicates the NH of the amino group of the antibody. ), ■ a group represented by the general formula % (in the formula, n, R, 0 at the left end and NH at the right end are the same as above), or ■ a group represented by the general formula (■, ■) above. A complex of mitomycins, a polysaccharide, and an antibody, in which any hydrogen of the methylene chain represented by (CH2)n is substituted with a lower alkyl group. However, in the above, the conjugate of mitomycins and polysaccharide is defined as the combination of mitomycins and polysaccharide. 1-NH helmet C. - (in the formula,
n has the same meaning as above, N at the left end represents N of the imino group of mitomycins, 0 at the right end represents 0 of the hydroxyl group of polysaccharides), ■' general formula % formula %) (in the formula , N1 at the left end and 0 at the right end have the same meaning as above)
A bale represented by or ■′ above ■′. It refers to a bond in which any hydrogen in the methylene chain represented by (OHg)n in the general formula (2) is bonded by a group substituted with a lower alkyl group. (2) The complex according to claim 1, wherein the polysaccharide is dextran, agarose, cellulose, or alginic acid.