MXPA99010278A - Conjugate comprising a folic acid antagonist and a carrier - Google Patents

Abstract

The invention relates to conjugates comprising a D enantiomer of a folic acid antagonist and a carrier. The invention also relates to the production of such conjugates and the use thereof.

Description

COMPOSITE CONJUGATE THAT COMPRISES AN ANTAGONIST OF FOLIC ACID AND A CARRIER The invention relates to a conjugate compound comprising a folic acid antagonist and a carrier, the method of preparing this conjugate compound and its use. Folic acid is a natural compound that acts on cells for the transfer of methyl groups and that is therefore important for the growth of cells. Folic acid has the following formula: The CH group of glutamic acid is an asymmetric carbon atom. Folic acid therefore exists in two enantiomeric forms, namely, D enantiomer and L enantiomer. Of these enantiomeric forms, only the L-enantiomer is found in cells, so that only this form but not the D-enantiomer, is responsible for the action of folic acid. The reason for this is that folic acid is received in the cells through a folate receptor but that it receives only the L-enantiomer, but not the D-enantiomer. Folic acid antagonists are compounds that are derived from the acid Folic, but that counteract the action of the same in the place of action, that is to say in the cells. Therefore folic acid antagonists are presented as L-enantiomers, but not as D enantiomers. As examples of folic acid antagonists may be mentioned aminopterin and ametopterin which is also called methotrexate. Methotrexate, that is to say the L-enantiomer of ametopterin, is frequently used for the treatment of tumors and inflammations although it suffers from serious side effects because methotrexate is also absorbed by healthy tissues over which it has a toxic effect. Only DE-A-41 22 210.5- describes conjugated metatrexate and albumin compounds that are absorbed more intensely by tumors than by healthy tissue and therefore have less toxicity, but there is still a need to obtain products that still have fewer side effects The present invention therefore has the purpose of proposing a composition for the treatment of diseased tissues, particularly tumors, which has minimal side effects. This is achieved according to the invention with the objects defined in the claims. Accordingly, the object of the present invention is a conjugated compound comprising the D-enantiomer of a folic acid antagonist and a carrier. The present invention is based on the applicant's findings that the D-enantiomer of a folic acid antagonist in a conjugate compound comprising a carrier is preferentially absorbed by diseased tissues, particularly tumor cells, and develops in them an action against the disease. In addition, the Applicant has verified that the D-enantiomer of a folic acid antagonist by itself, that is to say that it is not found in a conjugate compound according to the present invention, lacks side effects on healthy tissue. The term "conjugate" means that the folic acid antagonist and the carrier are covalently linked, for example by amide and / or ester bonds or through an intermediate. The term "D-enantiomer of a folic acid antagonist" comprises compounds of any type derived from folic acid which act as folic acid antagonists and which are in the form of D-enantiomers. The D-enantiomer of a folic acid antagonist comprises as components the pteridine, particularly pterin, p-aminobenzoic acid and a D-amino acid, particularly D-glutamic acid, which are chemically modified in relation to the components that exist in folic acid . These modifications are for example substitutions such as the substitution of H atoms by C 1 -C 4 alkyl groups, particularly methyl group, halogen atoms such as F, Cl, Br, I, OH groups and NH 2. the replacement of OH groups by the alkyl groups mentioned above, NH2 groups, hydrogen and halogen atoms, as well as the substitution of NH2 groups by the alkyl groups mentioned above, OH groups and hydrogen and halogen atoms. In addition, one or both acid glutamic acid groups may exist in the form of acid derivatives, for example as esters or amides. Of these modifications there may be one or more in a folic acid antagonist used according to the invention. Preferably, the folic acid antagonists are D-ametopterin (hereinafter referred to as D-methotrexate), D-aminopterin and De, t-FMTX (a methotrexate analogue, in which glutamic acid (Glu) is substituted by D-erythro, threo-4-fluoro-Glu. Among the folic acid antagonists one or more can be found in the conjugated compound according to the invention If there are several, they may be the same or different from one another The term "carrier" comprises compounds of any type suitable for enrichment of the conjugated compound in the diseased tissue, for example the tumor or inflammation site. of these carriers can be mentioned proteins that can not be considered as foreign to the organism, and polyesters.The proteins are preferably found in native form.In the native form, the proteins do not have intermolecular and / or intramolecular crosslinking.Preferably the proteins have a molecular weight of up to 100,000 Dalton, particularly ,000 to 100,000 Dalton. It is also convenient that the proteins are of human origin. As an example of proteins can mention albumin, fibrinogen, transferrin, immunoglobulin and lipoproteins, with human albumin being preferred. Fragments of these proteins can also be used. In addition, the sequence of the proteins or their fragments can have modifications of one or several amino acids with respect to the known sequence of the proteins or their fragments. Examples of polyethers include polyethylene glycols, in particular those with a molecular weight of 100 to 20,000 Daltons. Polyethylene glycols which are esterified or etherified with a C 1 -C 2 alkyl group, particularly a methyl group, are preferred in their terminal hydroxyl group. The conjugate compound according to the invention can have one or more carriers of the type indicated above, particularly two of them. If you have several carriers, they may be the same or different from each other. As there are several polyethers, these are preferably chosen, so that the molecular weight of all the polyethers is approximately 20,000 Daltons or more. In the conjugate compound according to the invention, the folic acid antagonist can be combined with the carrier in a direct covalent form or through an intermediate, ie between the carrier and the folic acid antagonist, a linker is found. Suitable linkers are, for example, compounds which can couple the folic acid antagonist with the carrier. Preferably the linker is cleavable in a cell. The term "cell" comprises individual cells and set of cells. As an example of the former, cells of the organism itself can be cited, which are not found in a group. The sets of cells comprise tissues, organs and tumors. An intermediary or linker of the type mentioned above is known to experts in these arts, who also know factors, for example enzymes, which determine in the cells the division of certain chemical bonds. Therefore, it is in a position to construct a cleavable linker in a cell. With particular preference this linker comprises an azo group. It is particularly convenient if the linker has the following structure: -YRN = N-in which R represents an organic radical, preferably an aromatic radical, and with particular preference, phenylene or a derivative thereof, and Y is a radical chosen from C (0), S (0) 2, P (0) OH and As (0) 0H. The preceding structure of a preferred linker corresponds to that which exhibits the linker in a conjugated compound according to the invention. In addition, particularly when R comprises phenylene or one of its derivatives, the structure comprises an active compound, particularly suitable for the therapeutics of tumoral, inflammatory and autoimmune diseases. After cleavage of the linker, and eventually degradation of the protein still bound to the linker, the compound can develop its full efficiency. The conjugated compounds according to the invention can be prepared by covalently combining the folic acid antagonist with the carrier and optionally the linker. The procedures and known materials necessary for this purpose, are known to experts in these arts. When the folic acid antagonist has at least one carboxyl group, for example the one found in glutamic acid, the conjugated compounds can be prepared by reacting the folic acid antagonist with carbodiimides and hydroxysuccinimide to give reactive succinimidyl esters that are reacted with the carrier. In the case of conjugated compounds - with several folic acid antagonists, the preparation of the succinimidyl esters can be carried out together or separately. The reaction of the folic acid antagonist with carbodiimide and hydroxysuccinimide is carried out in an aprotic polar solvent, preferably dimethylformamide (DMF). The molar ratio of folic acid antagonist: carbodiimide: hydroxysuccinimide is about 1: 1.5: 10. The succinimidyl ester formed is then reacted in an aqueous buffer solution, preferably NaH03, with the carrier such as albumin. The carrier concentration is about 10 to 70 mg / ml. The carboxyl group thus activated can then react with the OH and NH groups of the carrier forming amide or ester bonds, whereby the conjugated compounds according to the invention are obtained. The conjugated compounds can be purified several times, for example by ultrafiltration, and then filtered in a sterile manner, whereby they are ready for administration.

The conjugated compounds according to the invention are distinguished because they remain in the patient's circulatory system for a long time. They are also enriched in diseased tissues, particularly tumors and inflammatory foci. They are also distinguished because they have side effects even lower than those conjugated with L-folic acid antagonists as are known from DE-A-1 22 210.5 but retaining their effectiveness against diseased tissues, particularly against tumors and inflammatory foci. The conjugated compounds according to the present invention are therefore suitable for the treatment of tumors, such as hematological and solid tumors, inflammations, for example rheumatic diseases, such as chronic polyarthritis or psoriasis, and autoimmune diseases. The invention will be illustrated below by the following exemplary embodiments: Example 1: Preparation of a conjugated compound according to the present invention from D-methotrexate and human serum albumin. D-methotrexate (D-MTX) is dissolved in DMF at a concentration of 20 mg / ml. To the light yellow solution is added the 1.5 molar amount of di-cyclohexylcarbodiimide and a 10-fold molar amount of hydroxysuccinimide. After a reaction time of approximately 12 hours, the reaction is complete, having obtained succinimidyl ester (D-MTX-HSIE) which can be seen in the crystallized amount of di-cyclohexyl urea (DCH). The analytical control of the reaction was carried out by DC. Plates: Silica gel 60 with fluorescence indicator, Eluent: Ethyl acetate / MeOH: 75/25 Rx values: D-MTX 0.0 D-MTX-HSIE 0.35-0.38 The clear yellow solution of D-MTX -HSIE in DMF is added under continuous stirring and slowly to the protein solution (50-70 mg of human serum albumin in 0.17 M NaHCO 3, pH 8.5), forming after some time a turbidity consisting of unreacted cyclohexylcarbodiimide and DCH still dissolved in DMF. After a reaction time of at least 30 minutes, the turbidity is separated by means of a sterile filter (0.22 μm) and DMF by ultrafiltration with through-membrane filter (YM30; Amicon). The purity control is carried out by means of HPLC: Preliminary column: 50 x 4 mm Zorbax Diol Columns: 1. Zorbax GF 450 2. Zorbax GF 250 Eluent: 0.2 M phosphate buffer pH 7.4 Flow rate: 1.0 ml / min. Pressure: about 65 bar A conjugated compound according to the present invention is obtained from D-methotrexate and human serum albumin. Example 2: Comparison of the toxicity of a conjugated compound according to the invention of D-methotrexate and human serum albumin, with a conjugate of methotrexate and human serum albumin. For this test, the conjugate compound according to the present invention constituted by D-methotrexate and human serum albumin (D-MTX-HSA) of Example 1 was used. In addition, the methotrexate conjugate and human serum albumin (MTX-HSA) conjugate was used. from DE-A-41 22 210.5. Each of 5 healthy Sprague-Dawley rats was administered either D-MTX-HSA or MTX-HSA, respectively, injected each with 4 mg portions of conjugated compound per kg body weight (referred to the proportion of MTX or D-MTX). of the conjugated compounds) at 2-day intervals. The results are summarized in Table 1.

Table 1: Results of determination of toxicity of MTX-HSA and D-MTX-HSA after the 3rd day.

Abbreviations: SE: Inflammation of mucous membranes; DU: Diarrhea; SF: Caked skin; GV: Weight loss.

As can be seen in Table 1, when administering MTX-HSA the rats suffer strong side effects already from the 3rd day. On the 4th day two dead rats were found in the cage. The other 3 rats had to be sacrificed because they suffered very strong side effects. In contrast, none of the rats that were treated with the conjugate compound according to the present invention D-MTX-HSA exhibited side effects. Consequently the compounds conjugates according to the invention have few side effects. Example 3: Therapeutics of tumors with a conjugated compound according to the present invention of D-methotrexate and human serum albumin in comparison with the conjugate of methotrexate and human serum albumin. For this test, the conjugated compounds indicated in Example 2 were used. As rats, rats affected by alker-256 carcinosarcoma were used. The start of the therapy was on the 6th day after tumor transplantation with tumor volumes between 1000 and 2500 mm3 (tumor diameter l x l up to 1 x 2 cm). Five rats were administered MTX-HSA (3 injections at 2-day intervals with a dose of 2 mg of MTX-HSA (referred to the MTX component) per kg of live weight). The conjugate compound according to the invention D-MTX-HSA was administered in the double dose according to the preceding protocol. With this dose in the case of MTX-HSA there were strong side effects, but not in the conjugate according to the invention D-MTX-HSA (see Example 2). The results are summarized in Table 2.

Table 2: Results of tumor therapy with the MTX-HSA conjugate and the conjugate according to the invention D-MTX-HSA.

Abbreviations: NW: Secondary effect; Death Death due to tumor recurrence. The results in Table 2 show that the tumor was absorbed (remission) in the MTX-HSA group in 3 of the rats. In this group, 2 rats suffered from recurrence and had to be sacrificed. In 3 rats side effects occurred. On the other hand, in the rats that were treated with the conjugate compound according to the invention D-MTX-HSA, all the tumors were cured in all the animals, without observing side effects, although double dose was used in comparison with MTX-HSA. Consequently, the conjugated compounds according to the invention have minimal side effects, and excellent conjugates can be obtained with these conjugates.

Claims (14)

Claims

1. - Conjugated compound comprising a D-enantiomer of a folic acid antagonist and a carrier.

2. Conjugated compound according to claim 1, characterized in that the folic acid antagonist comprises one or more derivatives of pteridine, p-aminobenzoic acid and a D-amino acid.

3. Conjugated compound according to claim 2, characterized in that the D-amino acid is glutamic acid.

4. Conjugated compound according to claim 3, characterized in that the folic acid antagonist is D-ametopterin or D-aminopterin.

5. Conjugated compound according to one of claims 1 to 4, characterized in that the conjugate comprises several antagonists of folic acid.

6. Conjugated compound according to one of claims 1 to 5, characterized in that the carrier is a native protein or a polyether not considered as foreign to the organism.

7. Conjugated compound according to claim 6, characterized in that the protein is serum albumin.

8. Conjugated compound according to claim 6, characterized in that the polyether is a polyethylene glycol.

9. Conjugated compound according to one of claims 1 to 8, characterized in that it comprises several carriers.

10. Conjugated compound according to one of claims 1 to 9, characterized in that a linker is found between folic acid antagonist and the carrier.

11. Conjugated compound according to claim 10, characterized in that the linker is cleavable in a cell.

12 - Conjugated compound according to claim 11, characterized in that the linker comprises an azo group.

13. Process for the preparation of conjugated compounds according to one of claims 1 to 12, characterized in that the folic acid antagonist, the carrier and, optionally, the linker are combined in a covalent manner.

14. Use of the conjugated compounds according to one of the claims. 1 to 12 for the treatment of tumoral, inflammatory and / or autoimmune diseases.