DD267498A1 - PROCESS FOR BINDING METHOTREXATE TO MALEINSAEUREANHYDRIDE COPOLYMERS - Google Patents

Abstract

The invention relates to a process for the synthesis of methotrexate bound to maleic anhydride copolymers having improved performance properties. The aim of the invention is to provide a method for binding methotrexate to maleic anhydride copolymers whose products are water-soluble non-toxic polymeric cytostatics. According to the invention, the object is achieved by reacting a water-soluble alternating maleic anhydride-propene copolymer in a methotrexate-suitable solvent with methotrexate, if appropriate with addition of an acylation catalyst. The polymer-active ingredient conjugate is precipitated in a suitable precipitating agent, converted into the acid form after drying in the sodium salt and then by neutralization and p H value adjustment to 7.4. The polymers have good cytotoxic activity in vivo against the melanoma B16 tumor.

Description

Field of application of the invention

The invention relates to a method for binding methotroxate to maleic anhydride copolymers, which can be used for the synthesis of novel cytostatics which have improved applicability and can be used in human medicine.

Characteristic of the known technical solutions

It is known that the cytostatic methotrexate (formula I) can be covalently bound to various natural or synthetic polymer carriers, resulting in various appükative advantages.

COOH

The attachment to the macromolecule is in principle possible both via the NH ₂ groups of the pteridine ring and via the COOH groups of glutamic acid methotrexate.

While a depot effect can be achieved by the use of dextrans and polypeptides, poly (L-lysine) and immunoglobulins that are resistant to methotrexate, or specific tissues, become specifically accessible to a bookstore. J.M. WHITLEY; B.C.F.CHU and J.GALIVAN: Biomedical and dental applications of polymers p.41. Polymor Science and Technology Vol.14 Ed, by G.G. GEBELEIN ur.ci K. h COOUTZ; Plenum Press, New York, London 1981 However, the binding of methotrexate to monoclonal antibody allows the selective treatment of certain cancer cells R.W.BALDWIN and M.GARNETT: UCLASymp.Mol.Cell.Biol.NewSer.27,215 (1985).

The disadvantage of the previously known solutions is that they have a significant loss of activity in in vitro tests with good in vitro Ercjebnissen. In addition, the natural polymer carrier used are usually costly in the extraction and thus partly expensive.

Attempts have also been made to circumvent this deficiency by using more readily available synthetic polymers. Polyvinyl alcohol and polyethylenimine did not provide satisfactory efficacy of the polymer-drug conjugates. Divinyl ethorrelalic anhydride copolymers have been shown to be too toxic, although the latter polymer carrier itself has antineoplastic activity LGROS; H.RINGSDORF and H. SCHUPP: Angew. Chem. 93,311 (1981).

In addition to the loss of activity in vivo and toxic side effects, the achievement of a water solubility of the polymer-drug conjugates is another not yet completely dominated problem. The Wasserlöslichkeii the polymer carrier is significantly reduced by the connection of the water-insoluble methotrexate, whereby the application of the substances is difficult.

Object of the invention

The object of the invention is to provide a method for binding methotrexate to maleic anhydride copolymers whose products are said to be water-soluble, non-toxic polymeric cytostatics having improved performance properties.

-2- 267 498 Presentation of the Essence of the Invention

The object of the invention is to develop a synthesis process in which methotrexate is bound to maleic anhydride copolymers directly via the NHj groups of the pteridine ring and whose point is to be a water-soluble polymer with antineoplastic action.

According to the invention, this object is achieved in that water-soluble maleic anhydride propene copolymer in a suitable methotrexate solvent, preferably dimethylacetamide or dimethylformamide dissolved and methotroxate, optionally with the addition of 10 ~ ⁴ to 10 ~ ^e moles of an acylation catalyst per mole of methotrexate, to Reaction is brought. The polymer-drug conjugate is precipitated in a suitable precipitant, preferably chloroform or ether or mixtures of chloroform and ether, filtered off with suction and dried. After drying, the product is dissolved in an aqueous sodium bicarbonate solution, adjusted to pH 7.4 with dilute hydrochloric acid and the water and unreacted methotrexate are removed by conventional methods.

The result is a white to yellow powder, which is readily soluble in water and has in vivo antineoplastic activity.

The content of the product of methotrexate is determined by the molar ratio of anhydride to methotrexate in the reaction solution by the reaction temperature and time and can be adjusted in a customary manner on these variables.

It is for the work-up and application of the product advantageous if the reaction is carried out so that the degree of conversion of the methotrexate is over 50%, preferably more than 80%. Thus, for the process according to the invention at anhydride-methotrexate molar ratios of 20: 1 to 1: 1 reaction temperatures between 70 and 100 ⁰ C and reaction times of 8 to 20h proved to be favorable for the achievement of drug levels of Produktos between 5 and 45Ma. -%. The concentration of methotrexate in the reaction solution is determined by its solubility in the solvent used and can be varied within wide limits. When using dimethylacetamide good results are achieved with concentrations of 1 × 10 ^"J to 3 · 10"^"2 mol / l.

The molecular weights of the maleic anhydride propene copolymers may be between 20,000 and 10,000,000 g / mol, their methotrexate content may be between 5 and 45% by mass, but preferably 20 to 30% by mass.

It is an essential feature of the invention that after the reaction with Müthotrexat the polymer must first be converted into the sodium salt and by subsequent neutralization in the free acid.

If salt formation does not occur, the solubility in water and the associated in vivo efficacy are insufficient. Although the sodium salt shows good antineoplastic activity, in all tests premature deaths of the experimental animals are observed which indicate some toxicity of the compound. This is avoided with the acid form of the polymers.

It must be regarded as surprising that the polymer-active agent conjugates prepared by the method according to the invention have a better action in vivo compared to free methotrexate compared to the solid model tumor B16 melanoma.

In contrast to the leukemia P388, an effect approaching the free drug is observed.

The effect of the methotroxate-containing maleic anhydride copolymers is totally unexpected since the compound having monomeric maleic anhydride has no antineoplastic activity. It is the particular advantage of the process according to the invention that other compounds which have suitable functional groups can be bound simultaneously with Methotre.RTM. Such compounds may be other drugs or search groups for increasing organ or tissue specificity.

The following embodiments illustrate the invention without limiting it.

embodiments

Carrying out the tests

As animal material MDF-1-Mäuseböckchen were used from the breeding of the central institute for cancer research of the AdW of the GDR, Berlin book. As test tumors the leukemia P388 and the Molanom B16 were chosen.

At P388, each animal received about 10 ^b cells ο. p. and B16 melanoma 0.1 ml of a homogenized tumor.

B16 melanoma also grows in i.p. applications in solid cell aggregates.

As a parameter for assessing the efficacy of the substances, the prolongation of the survival time against tumor carriers without substance administration was chosen. Methotrexate from Laemema, Brno, Czechoslovakia served as a comparison preparation.

Example 1:

0.7 g (5 m mol) of maleic anhydride-propylene copolymer, and 0.45 g (1 m mol) of methotrexate are dissolved in 50 ml of dimethylacetamide is added 0.1 g Dimothylaminopyridin as the acylation catalyst and the homogeneous solution stirred at 80 ^c C for 16 h.

The mixture is then precipitated in 500 ml of dry chloroform, filtered off with suction and dried at 50 ° C. in vacuo.

1.0p of the product is dissolved in 10% NaHCOj solution and added dropwise with 0.1 N HCl solution. the pH of the solution is adjusted to 7.4. The water is distilled off on a rotary evaporator and the residue is dried in vacuo.

The Wiricstoffgehalt of the product is 28Ma .-%.

Table 1 shows an effect similar to methotrexate in the leukemia P388. Table 2 shows the good effect of the product over Molanom B16, which is higher than that of methotrexate.

Example 2:

The reaction is carried out as in Example 1, but concentrated after dissolution in NaHCOy solution without neutralization and dried.

As can be seen in Tables 1 and 2, premature deaths during testing indicate that the toxicity is higher.

Example 3:

The reaction is carried out as in Example 1, but the product isolated after precipitation in chloroform is used for the test. It is not soluble in water and has clearly toxic side effects in the test.

Example 4:

The reaction is carried out as in Example 1, but instead of Copoiymeren the monomeric maleic anhydride used. As can be seen from Tables 1 and 2, this low molecular weight product has no antineoplastic effect.

Table 1 Mean survival time (in days) of BDFr mice after i.p. Injection of P 388 and treatment

dose 2.5 15 20 40 mg / MTX / kg / injection Day of the application 1-4 1 1 1 after tumor incubation sample control 9 10 10 10 MTX · ' 16 12 13 14 1 16 12 15 - 2 16 10 ^b 14 4 ^b 3 14 12 5 ^b 5 ^b 4 10 11 10 10

a) MTX: methotrexate

b) premature deaths

Table 2 Mean survival time (in days) of BDF, mice after B16 melanoma incubation and treatment

dose 2.5 10 10 20 mg MTX / KG / injection Day of the application 1,3,4,7-10 1,5,9 1 1 after tumor incubation sample control 25 18 21 21 MTX ' ¹ 20 21 24 22 1 26 29 42 29 2 28 32 '' 27 "» 3 5 '' 27 • 3 "» 4 - 18 - -

a) MTX: MothoUexat

b) premature deaths

Claims (4)

1. A method for binding methotrexate to maleic anhydride copolymers, characterized in that 1 to 20 moles of water-soluble maleic anhydride propene copolymer having molecular weights of 20,000 to 1000000g ΜοΓ ¹ in methotrexate suitable organic solvents with 1 mole of methotrexate, wherein the methotrexate concentration · 10 " ^{2 to} 3-10" ² MoIT ¹ , reacted at temperatures of 343K to 373K and in a time of 8 to 20 hours, isolated, converted to the sodium salt and then neutralized to the acid form. 2. The method according to claim 1, characterized in that the polymer after the reaction with methotrexate in a suitable precipitating agent, preferably chloroform and / or diethyl ether precipitated, dried, dissolved in aqueous NaHCOa solution, with 0.1 N aqueous hydrochloric acid of the pH Value is set to 7.4 and then the water is removed. 3. The method according to claim 1, characterized in that is used as a solvent for methotrexate dimethylacetamide and / or dimethylformamide. 4. The method according to claim 1, characterized in that is used as acylation dimethylaminopyridine in concentrations of 10 " ⁴ to 10 ~ ⁶ mol ΜοΓ ¹ methotrexate.