DK141773B - Process for preparing an anti-tumor agent. - Google Patents

Description

(11) PUBLICATION 141773 (§) DENMARK l '”ln, cl' A 61 K 39/395 • (21) Application No. 4224/75 (22) Filed in 19 * sep. 1975 (23) Race day 19 · Ββρ. 1975 (44) The application presented and the petition published on 16 Jun. 1 980 DIRECTORATE FOR __.

PATENT AND TRADE MARKET (30) Priority requested from dap

20 sep. 1974, 41037/74, GB

(7i) G.D. SEARLE & CO. LIMITED, Lane End Road, High Wycombe, Buckingham * Shir GB.

(72) Inventor: David Allen Lewis Davies, 10, Gillets Lane, High Wycombe, Buckinghamshire, GB.

(74) Clerk of the case;

The engineering company Budde, Schou & Co.

(54) Process for preparing an anti-thump agent.

The present invention relates to a process for the preparation of an antitumor agent characterized by reacting a cytotoxic agent, preferably p-di (2-chloroethyl) amino-L-phenylalanine or N, N-bis (2-chloroethyl). p-Phenylenediamine of the formula m 2 C-C of a peptide bond between the cytotoxic agent and the tumor specific antibody.

It is known to prepare antitumor agents in the form of specific antisera by conventional technique by immunization and then collecting sera. These antisera are then absorbed with e.g. normal cells to remove certain unwanted antibodies to cells other than the target cells, i.e. tumor cells. The remaining antibodies will then be directed predominantly to the selected target cells, ie. tumor cells.

It has now been found that such a tumor-specific antibody upon binding to a cytotoxic agent provides a significantly more effective, specific antitumor agent.

Namely, the drug-antibody complexes produced by the method of the invention are found to provide significantly greater protection against mice from tumor formation than either EL4 immunoglobulin administered alone or the cytotoxic agent associated with non-specific (antitumor) globulin. In addition, equivalent doses of the cytotoxic agent administered alone are lethal to mice; however, the maximum tolerable dose of the cytotoxic agent used alone is insufficient to substantially inhibit tumor formation under the conditions used in the biological assay below.

Tumor-specific antibodies are produced (by immunization) e.g. against prior art neoplastic tissue, as described by Ghose et al. in the British Medical Journal (1972) 3, pages 492-499. Thus, in a preferred embodiment of the method of the invention, antibodies against mouse EL4 lymphoma (EL4 immunoglobulin) are reacted with p-di (2-chloroethyl) amino-L-phenylalanine in the presence of 1-ethyl-3 (3-dimethylaminopropylene). pyl) -carbodiimide hydrochloride to provide an antitumor agent which per one tumor specific antibody contains approx. 13 molecules of cytotoxic agent chemically bound by peptide bonds.

Antibodies against neoplasm of lymphatic and hematopoietic tissues, including lymphosarcoma, chronic lymphatic leukemia,

Hodgkin's disease, ovarian cancer, breast and testes, and other epithelial tissue and melanoma, produced by techniques known in the art, are tumor-specific antibodies that can be used in the method of the invention. The immunoglobulin fraction which acts as an antibody to these tissues and is absorbed with normal tissue is bound particularly easily and without activity degradation by the method of the invention to a cytotoxic agent such as p-di (2-chloroethyl) amino-L-1. phenylanine in the presence of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide.

This gives a smooth running reaction to form the desired number of peptide bonds. Thus, it is convenient according to the invention that the carbodiimide be 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.

It is essential for the process of the invention that after the complex is formed, the tumor-specific antibody retains its specificity, just as the cytotoxic agent retains its ability to alkylate. Both of these essential features are accomplished by the easy-to-control peptide bond formation produced by carbodiimide, in which, according to the invention in the antitumor agent, 5 to 15 molecules of cytotoxic agent per molecule tumor specific antibody. This results in the tumor-specific antibody keeping the cytotoxic agent bound and delivering it first to the target cells, ie. at tumor sites where its high concentration release is desired. Presumably, this mechanism of action may account for the observation described above that greater administration of cytotoxic agent bound complex according to the invention is now possible in contrast to administration of the same cytotoxic agent in free form (which is otherwise completely insufficient at tolerable doses). This greatly increases the activity of the tumor-specific antibody.

The reason that among the cytotoxic agents that can be reacted with tumor-specific antibody in the presence of water-soluble carbodiimide to form peptide bonds, thereby providing a predominantly targeted cytotoxic active (alkylating) agent, the two specifically mentioned are phenyl diamines, their known and proven cytotoxic activity and their easy accessibility, the first of which is available under the NFN name "Melphalan" and is commercially available under the registered trademark "Alkeran" ®, while the second is also a now well-known cytotoxic agent available in technical amounts.

The dosage will vary according to the nature and amount of the tumor-specific antibody and the type of cytotoxic agent bound to it, the extent of neoplastic growth (tumor size), and the individual's tolerance to the particular antitumor agent - (tumor specific antibody-cytotoxic agent complex). Since toxic side effects of the cytotoxic agent are greatly diminished by binding to the tumor-specific antibody, large doses of bound cytotoxic agent can be advantageously administered. Doses of p-di (2-chloroethyl) amino - L-phenylalanine can thus be increased 2-5 times compared to those described in Cuttings Handbook of Pharmacology, 4th edition 1969 published on Appelton Century Crafts, see in particular page 139th

The examples given below are intended to illustrate the method of the invention. In more detailed description of a preferred embodiment thereof.

Example_1_and_2 In rabbits, an antiserum is prepared against EL4 tumor cells (cells of transplantable lymphoma from C57B1 / 6 mouse strain). EL4 cells

Q

taken from mice are injected intravenously into rabbits at three doses of 10 cells per ml. dose at 10 day intervals. Ten days after the last dose, blood is drawn from the rabbits and the serum is collected. This serum is heated to 56 ° C for 30 min. to destroy complement activity.

The collected serum is absorbed with normal mouse cells, leaving tumor-specific antibodies. The serum is absorbed with a pool of mouse spleen cells, including at least some from mice of strain C57B1 / 6 or a similar strain. Repeated absorptions are made with a portion equal to 10 spleen to 1 milliliter serum for 16 hours at 4 ° C with stirring until no serum reactivity against normal cells of the strain C57B1 / 6 by conventional cytotoxicity tests. In the same assay and in addition, by indirect immunofluorescence or agglutination assays, the reactivity of the same serum is tested with EL4 tumor cells to demonstrate that it is tumor specific.

The globulin fraction is precipitated from the serum by the addition of saturated ammonium sulfate until the final concentration is 40%. The precipitated EL4 globulin (Ig) is redissolved in phosphate buffered saline so as to eventually provide a protein concentration of 30-50 g / ml as measured by the biuret reaction.

A peptide bond between an amino or a carboxylic acid group present on certain cytotoxic drugs, and corresponding respective groups on the antibody molecules, is produced in the presence of carbodiimide as described below.

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The following reagents are prepared (Al 10 ml of the above EL4 in immunoglobulin solution of 57 mg / ml adjusted to pH 6.5 with HCl, (B) either (1) 57 mg of N, N-bis (2-chloroethyl) p-phenylenediamine- -hydrochloride dissolved in 2 ml of dioxane or (2} 57 mg of p-di (2-chloroethyl) amino-L-phenylalanine dissolved in 0.8 ml of an acid: ethanol mixture (92% ethanol containing 2% HCl by weight / volume and then diluted with a buffer solution of 60% propylene glycol containing 1.2% (by weight / volume) of di-potassium hydrogen phosphate to reach a final pH of 6-6.5, (C) 114 mg 1-Ethyl-3 (3-dimethylaminopropyl) -carbodiimide HCl dissolved in 0.5 ml of physiological saline solution.

Reagents A, B and C are rapidly mixed for 5 seconds, after which the reaction is quenched by the addition of 6 ml of 1M sodium acetate at pH 6.5. The resulting solution is dialyzed against three portions of 0.3% sodium citrate solution over 24 hours at 4 ° C to remove unbound cytotoxic agent, i.e. (li respectively (2i.

The antitumor agents thus prepared are assayed for protein content and alkylation activity. Protein content is measured using a conventional biuret bovine serum albumin assay as standard. The alkylation activity is tested using -Epstein's method (cf. J. Epstein, R. W. Rosenthai and R. J. Ess.

Anal. Chem. 27, 1434 (1955)).

The final preparation of N, N-bis (2-chloroethyl) p-phenylenediamine / tumor specific antibody complex prepared according to Example 1 contains 12.2 mg EL4 immunoglobulin / ml and 122 pg N, N-bis (2-chloroethyl) ) of p-phenylenediamine / ml. This represents a ratio of approx. 5.0 molecules of cytotoxic agent per molecule tumor specific ig.

Similarly, the prepared solution of p-di (2-chloroethyl) amino-L-phenylalanine / tumor specific antibody complex prepared according to Example 2 contains 13.9 mg of EL4 immunoglubulin / ml and 380 µg of p-di (2-chloroethyl) amino-L-phenylalanine / ml, representing a ratio of approx. 13.6 molecules of cytotoxic agent per molecule tumor specific antibody.

Thus, it is seen that the cytotoxic agents of this invention and their close analogs to tumor-specific antibodies can be covalently linked by a peptide bond. The activity of such cytotoxic agent / tumor specific antibody complexes is shown below.

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Biological Assay of Cytotoxic / Tumor-Specific Antibody - ^ __ in _ Complex as Antitumor Agent _____________; _ ¥ _______

The compositions of Examples 1 and 2 above are used to protect mice against the lethal effects of injecting EL4 tumor cells.

Groups of mice get intraperitoneally injected approx. 10,000 4 lethal doses (5 x 10 1 tumor cells collected from the abdomen of pre-infected mice. 24 hours after injection of the cells, the animals receive the first of 4 daily doses of therapeutic agent or control agent as described below.

1. Treatment with N, N-bis (2-chloroethyl) p-phenylene di-mln / EL4 immunoglobulin complexes ____ 50% of mice survive

Treatment with i (number of days)

Saline (control) 14.5

Normal rabbit globulin / N, N-bis (2-chloroethyl) p-phenylenediamine (1/2 ml doses containing 245 µg N, N-bis (2-chloroethyl) p-phenylenediamine and 7.0 mg of normal rabbit globulin) (Comparison! 14.5 EL4 immunoglobulin alone (25 mg) (Comparison! 24.5 N, N-bis (2-chloroethyl) p-phenylenediamine / -EL4 immunoglobulin (1.4 ml doses containing 170 pg N, N-bis (2-chloroethyl in p-phenylenediamine + 17 mg EL4 immunoglobulin), (prepared according to the invention 34.0 2, Treatment with p-di {2-chloroethyllamino-L - phenylalanine / EL4 immunoglobulin complexes 50% of mice survive

Treatment with i (number of days)

Saline (control) 15.5 p-di (2-chloroethyl) amino-L-phenylalanine mixed with normal rabbit globulin as in 1. other (comparison) 15.0 EL4 immunoglobulin alone (1 mg) (comparison) 26.6 p -di (2-chloroethylamino-L-phenylalanine / -EL4 immunoglobulin complex (760 µg β-di (2-chloroethylamino-L-phenylalanine + 28 mg EL4 immunoglobulin)) (predominantly 80% surviving mice according to the invention) 42 days 7 141773

It is seen from the experimental results that a pure physical mixture of the cytotoxic agents at tolerable doses of non-antitumor-specific immunoglobulin from the same test animal (rabbits) is as ineffective as saline injections (control).

A somewhat better activity is the known administration of EL4 immunoglobulin alone (comparison).

The sharp improvement is seen in the administration of the antitumor agent prepared according to the invention by reacting the same cytotoxic agent with the same tumor-specific antibody, i.e. EL4 immunoglobulin. The dose of cytotoxic agent is thereby raised above the limit of the toxicity of these compounds.