DK157601B - Therapeutic platinum or palladium preparation, and process for preparing it - Google Patents

Description

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DK 157601 B

The present invention relates to novel compositions containing platinum or palladium which are useful for controlling cancer cell and trypanosomal infection in mammals.

5 The compound cis-dichlorodiamine-platinum (11) (cisplatin) has been well known in the treatment of various cancers in mammals, but it is toxic and can therefore only be used in a low dose. It would be desirable to provide an effective product which could be tolerated in the mammalian body at larger doses and which would have a longer half-life. It would also be desirable to provide an effective composition, with improved solubility in water and serum as compared to cisplatin.

GB Patent No. 1,541,436 has been proposed to bind certain specified organic cytotoxic drugs with free amino or carboxyl groups having a peptide bond or other covalent bond to polymeric carriers having a molecular weight of 5,000 to 500,000 and being present in capable of binding to immunoglobulin, so that the cytotoxic drug is enzymatically cleaved on the target cell surface.

It is furthermore by C.R. Morris and G.R. Gale in Chem.

Biol. Interactions ", 1973, pages 305-315, have suggested that the compounds cis-dichloro (dipyridine) platinum (11) 25 and cis-Pt (II) may associate with electron-rich regions of the nucleic acid and proteins and also with the body assembly of free nucleotides and amino acids, but no such preparations have been isolated or specifically identified.

The present invention relates to a novel group of 30 platinum and palladium compositions which exhibit such advantages and / or are effective against trypanosomal infections.

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The novel compositions are characterized in that they comprise water-soluble anionic organic macro-molecules, namely, anionic polysaccharides, anionic polyamino acids or enzymes with anionic functional groups (e.g., enzymes with anionic side chains), having a molecular weight of 5,000. to 60,000, combined with a quadratic plane coordination portion around the metal atom M, of the formula cis- [MLL1] ++, where M is divalent platinum or divalent palladium, and L and L 'are ammonia or 10 mono- or difunctional primary amines, so that at least part of the valence requirements for the anionic functional groups of the anionic macromolecules are met.

The preparations are not specific compounds with readily determinable structures, but rather mixtures of related polymeric materials in which a majority of the square planar platinum or palladium coordination moieties are either supported on an aryl ionic macromolecule or serve. as a bridge or as bridges between two anionic 20 macromolecules. Where multiple bridges occur, more than two macromolecules may be linked together. In the ordinary case, the compositions will contain at least 2% by weight of platinum or palladium attached to the macromolecule (based on the weight of metal plus macromolecule).

By a water-soluble organic macromolecule is meant one which exhibits solubility in water when combined with cations such as H +, NH2 +, alkali metals, calcium and magnesium.

Examples of anionic macromolecular materials which have a sufficiently low toxicity and which can be used as bases for the compositions of the invention include (but are not limited to) poly-L-glutamic acid (PGA), carboxymethyl cellulose, ferritin, chondroitin sulfate, 3 DK 1: -7001 b poly-L-aspartic acid, dextran sulfate, polyphdivinyl ether-co-maleic anhydride], poly [acrylic acid-co-maleic anhydride], polyacrylic acid, serum albumin and desulfated heparin.

5 Ammonia or substituted ammonium groups L and L 'in the coordinated platinum or palladium moiety may be identical or be two primary amine functional ion groups present on a single molecule. Representative primary amines which can be used in compositions of the invention include isoamylamine, pentylamine, cyclohexylamine, 1,2-diaminocyclohexane and ethylenediamine.

The novel compositions may be prepared by reacting an aqueous solution of the anionic macromolecule which may be in salt form, e.g. sodium salt form, with an aqueous solution of an amine cis-platinum or cis-palladium compound in nitrate form containing water, MLL1 (H2O) 2 (NO0) 2, i.e. in salt form with crystal water. The platinum or palladium compound can be prepared by the method of Lippert, Lock, Rosen-20 berg and Zvagulis, Inorganic Chemistry, Vol. 16, no.

6, 1977, pp. 1525-1529. The reaction mixture is stirred for a considerable period of time, e.g. 16 hours or more and the product is purified by dialysis or filtration, depending on whether or not the product is soluble in the reaction medium.

The invention also relates to a process for the preparation of a composition according to the invention, characterized in that in an aqueous solution the anionic macromolecules are reacted with the compound cis-MLL'-30 (H2 O) 2 (NO3) 2.

Using a natural or synthetic macro-molecular moiety that is water-soluble as a base, it is possible to produce both water-soluble and water-insoluble platinum or palladium-containing preparations. Som 4

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a general rule it can be said that if more than approx.

80% of the free anionic functional groups (e.g. carboxylic acid groups) on the macromolecule are complexed with the [cis-MLL '] ++ moiety, the resultant composition 5 of the invention will be insoluble in water or sera due to both the lower solubilization properties of the [cis-MLL '] ++ part compared to e.g. the sodium ion, and also the fact that at this high concentration of precious metal, the presence of cross-linking 10 of macromolecules to form even larger macromolecules is high in itself. However, there are a number of exceptions to this rule on an upper limit of 80% co-MP laxity for water or sera solubility.

First, a very high molecular weight macromolecule 15 can only tolerate that at most e.g. 50-60% of its anionic functional units are complexed to provide a final product which is water or plasma soluble. Second, if the primary amines are RNH 1 or NH 2 R'NH 2 in the [cis-MLL '] ++ moiety is such that the R-20 or R' group is substantially hydrophobic, the macro molecule with anionic functionality tolerates less complex bonding with such a [cis-MLL '] ++ moiety while maintaining water solubility for the final product than would be tolerated where L and L' are NH 2 or groups 25 of NH 2 CH 2 CH 2 NH 2.

Thus, the compositions of the invention are divided according to their properties into water-soluble and water-insoluble compositions. The effects of these types of preparations differ substantially when the preparations are used as drugs for the treatment of cancers or tumors. The water-soluble straight types are adapted for injection into mammalian body fluids, e.g. blood, and as they are soluble in the plasma, they pass through the mammalian body where the fluid flows, without a biofunctional barrier to the preparation.

Water solubility is also advantageous for the treatment of trypanosomal infections. Water-insoluble preparations

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on the other hand, according to the invention are readily adaptable for introduction into tumor tissue. Such introduced or implanted drug preparations can be expected to release relatively high concentrations of therapeutically effective substances by hydrolysis, by minimal solubility, by enzymatic attack or by a combination of these three ways in a very local volume, directly attacking the cancer mass.

Some examples will be given below using the following abbreviations: DACH = 1,2-diaminocyclohexane PGA = polyglutamic acid one = ethylenediamine.

The 1,2-diaminocyclohexane used in the compositions was obtained from Aldrich Chemical Co., as an 85 ° technical grade, and included a mixture of cis and trans isomers where the transisomer contained both plus and minusoptic enantiomers.

EXAMPLE 1 20 A. Preparation of cis-Pt [(DACH) (HnO) J (NO-)] In a 250 ml round-bottom flask 16.4 g (0.0431 mol) of pure cis-Pt (DACH) was introduced. (Cl) + · A silver nitrate solution was prepared by dissolving 14.3 g (0.0844 mol) of AgNO4 in 100 ml of distilled water. The solution was then added to the flask and stirred dark at 60 ° C for 30 minutes. The suspension was then cooled to room temperature with stirring for an additional 16 hours.

Next, the reaction mixture was filtered, resulting in a clear pale yellow aqueous solution containing Pt [(DACH) (h 2 O) 2] (NO 2) 2 ~ solution. Other nitrates

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6 platinum (II) and palladium (II) solutions as described below were prepared in a similar manner.

B. Preparation of cis-Pt (DACH) complexed with dextran sulphate In a 50 ml round bottom flask, 0.5 g of dextran sulphate sodium salt (medium molecular weight 5000) was dissolved in 20 ml of distilled water. Next, 2.0 ml of a 0.2615 M R-tLCDACB.M 2 O 2] (NO 2) 2 aqueous solution was prepared as in step (A) above, slowly added to the polymer by a syringe. resolution.

The reaction mixture was stirred for 16 hours.

The sample was then purified by dialysis over a 6 hour period (dialysis tube with a molecular weight cutoff of 5,000 -6,000). Analysis of the purified sample gave a 0.19 wt-ft of platinum content in solution. The dialysis product was used for the test as described in Table I.

A portion of the dialysis product was lyophilized to produce a chunky crystalline golden solid appearance. Lyophilization was performed over a week at 20 -60 ° C with a vacuum of 10 microns. The solid product could be reconstituted in distilled water.

EXAMPLE 2

Preparation of cis-Pd (DACH) complex bound with PGA

In a 50 ml round bottom flask, 1.0 g (0.0066 mol of 25 polymeric units) of poly-L-glutamic acid sodium salt was dissolved in 20 ml of distilled water. With stirring, 14.0 ml of a 0.1447 M cis-Pd [(DACH) (F₂O ^] (NO₂) aqueous solution was slowly added to the polymer solution through a separatory funnel. The reaction mixture was stirred for 16 hours.

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The sample was then purified by dialysis for 16 hours (dialysis tube with a molecular weight cut-off of 6,000). Analysis of the purified sample yielded a 0.22% by weight of palladium in solution.

EXAMPLE 3

Preparation of cis-Pt (DACH) complexed with PGA

In a 50 ml round bottom flask, 0.5 g (0.0033 mol based on polymer units) of poly-L-glutamic acid sodium salt (mean molecular weight 60,000) was dissolved in 20 ml of distilled water.

With stirring, 2.0 ml of a 0.2516 M

cis-Pt [(DACH) (h ^ O ^ l (NO) 2 aqueous solution slowly by means of a syringe added to the polymer solution. The reaction mixture was stirred for 16 hours.

The sample was then purified by dialysis for 6 hours (dialysis tube with a molecular weight cut-off of 6000). Analysis of the purified sample yielded a 0.24% by weight of platinum in solution. Part of the dialysis product was lyophilized as in Example 1, step (B) to produce a fluffy crystalline appearance of light golden solid.

Dissolve the solid in distilled water.

EXAMPLE 4

Preparation of cis-Pt (NH ^) kompleks complexed with PGA

In a 50 ml round bottom flask, 1.0 g (0.0066 mol based on polymer units) of poly-L-glutamic acid sodium salt (medium molecular weight 60000) was dissolved in 30 ml of distilled water.

Next, 7.8 ml of a 0.2537 M

cis-Pt [NH-j) 2 (H 2 O) 2] (NO 2) 2 aqueous solution slowly using a syringe added to the polymer solution.

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The reaction mixture was stirred for 16 hours.

The sample was then purified by dialysis for 60 hours (dialysis tube with a molecular weight cutoff of 5,000 - 6,000). Analysis of the purified sample gave a 0.61% by weight content of 5 platinum in solution (24.2% of the free carboxylic acid side chains on the PGA were thus complexed to platinum).

EXAMPLE 5

Cis-Pt (DACH) complexed with chondroitin sulfate In a 250 ml round bottom flask, 2.0 g of chondroitin sulfate sodium salt was dissolved in 80 ml of distilled water. With stirring, remained 8.0. ml 0.3405 M cis-Pt [(DACH) (H 2 O) 2] (NO 3) 2 aqueous solution slowly added to the polymeric solution. The reaction mixture was stirred for 16 hours. Overnight, the reaction product partially became a gel.

The sample was then purified by dialysis for 6 hours (dialysis tube with a molecular weight cut-off of 6000 - 8000). Analysis of the purified sample showed a 0.455 wt% platinum content in solution.

EXAMPLE 6 Preparation of cis-PtiNH 3) complexed with bovine serum albumin In a 50 ml round-bottom flask, 1.0 g of bovine serum albumin (prepared using Method IV by Cohn, EG et al., J. Am. Chem. Soc. ., 1947 69 (1953)) dissolved in 25 ml of distilled water. Next, 7.8 ml of 0.2537 M cis-Ptt (NH 2 ^ Ct ^ O ^) (^ 0 ^ 2 aqueous solution was slowly added by means of a syringe to the solution of serum albumin. The reaction mixture was stirred for 16 hours.

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The sample was then purified by dialysis for 6 hours (dialysis tube with a molecular weight cut-off of 6,000). Analysis of the purified sample showed a 0.51 wt-ft of platinum in solution.

EXAMPLE 7

In vivo experiments against trypanosomes

In vitro experiments against T. Brucei indicated that the cis-Pt (DACH) complexed with (PGA) and cis-Pt (NH4) 2 complexed with (PGA) preparations were immediately effective at low doses. Accordingly, in vivo testing was performed on mice.

The mice selected were NCS male mice weighing 20 - 25 g. Five animals were used for each dosing amount. Each mouse was injected interperitoneally with a fluid containing 50,000 Trypanosoma Conglolense. At this dose, control animals, without any anti-trypanosomal treatment, died in 5 - 8 days. One day after the injection of trypanosomes, the platinum drug of the invention was administered. In one experiment with 15 20 mg Pt / kg, cis-Pt (DACH) complexed with (PGA) gave a cure rate of 20 µg, and in another experiment with 6.25 mg Pt / kg the same compound gave a cure rate of 40 ° measured such as survival of the animals for 60 days. Other experiments with cis-Pt (NH 2) 2 complexed with (PGA) using 4 animals per day. the dose gave a cure rate of 75¾ at a dose of 20 mg Pt / kg and a cure rate of 50¾ at 15 mg Pt / kg. Mice (4 animals per dose level) treated with either 10 mg Pt / kg or 20 mg Pt / kg cis-Pt (NH 2) 2 complexed with (PGA) exhibited at day 30 Nos. 1, 3 and 6 cures of 100¾. After 60 days, blood from surviving animals was administered to other uninfected animals and no infection occurred.

In the test, cisplatin was ineffective at all doses.

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EXAMPLE 8

Various water and plasma soluble compounds of the invention were screened for anticancer activity in mice using ascites P388 leukemia cells.

The mice were CDF1, female, weighing approx. 20 g

each test animal was implanted approx. 4.8 x 10 5 cells interperitoneally. The test drugs were administered interperitoneally 1 day after infection. The results are shown in the following Table I. In Table I, the column entitled "Logg change in tumor burden at end Rx" means the logarithmic net change in viable tumor cell population at the end of the procedure as compared to the onset, forming an appropriate measure of the size of the cell population. amendment 15 due to treatment. A "-6 log change" means a 99.9999% reduction.

The results obtained as described appear to show that in many cases, compounds prepared in accordance with the invention are more tolerable in mammalian animal experiments than the standard cisplatin preparation. It was observed that the composition of cis-PtCcyclohexylamine with ferritin prepared in a manner similar to the procedure described in Example 6 was highly toxic in mice by administration as described above.

The therapeutic value of some of the water-soluble drugs in trypanosomal treatment is high. It is seen that the increased solubility properties of the compounds are a great advantage. The degree of solubility and biological parameters can be controlled by varying the total concentration of aminated platinum or palladium complex formed for the anionic macromolecules.

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TABLE I

Mean Log1Q change in Number of Dose mg Death Tumor burden by _Treatment_. animals ; PI.Ag_day ...% IL5 f 5 U nln ^ ''

Negative control (no 20 - 10 treatment), γ (3 96 16 + 60 -4.1 c / s-Pt (DACH) (PGA) complex <3 ^^ (3 12 14 + 40 -2.7 {3 96 14 +40 -2.7 3 48 11 +10 -0.7 3 24 11 +10 -0.7 3 12 11 +10 -0.7 {3 96 -— 13 + 30- -2.1 3 48 12 + 20 -1.4 3 24 11 +10 -0.7 3 12 11 +10 -0.7 i 3 96 Toxic - c / s-Pd (en) (PGA) complex j ^ 48 Toxic ^ i 3 12 10 0 - f 3 96 Toxic

c / s-Pd (NH 3) 2 (PGA) complex <(3 ^ Yy + 4yJ)

(3 12 9 -10 +0.7 {3 96 Toxic 3 S Toxic _0? 3 12 10 0 0 • r, „!. C 3 6 22 +100 -6.7 as-Pt (DACH) (dextran- ) 3 3 20 +81 -6 ^ 2 sul fat) complex \ 3 1> 5 18 +63 -4.5

Negative control (no treatment) (20 - 11 (10 56 Toxic j 10 38 17 + 54 -4.5 c / s-Pt (NH 3) 2 (PGA) complex {10 25 16.5 + 50 - 4.2 j 10 17 14 +27 -2.3 l10 11 13 + 18 _ -1.5 {10 5.2

Positive Control ^ 10 3.5 18.5 +68 -5.7 (cisplatin) 10 2> 3 16> 5 + 5 ° -4 »2

Negative control (no ^ 20 ~ 11 treatment)

Note: Each sample row is separated by a line.

% ILS = increase in life span.

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The following examples relate to the water-insoluble compositions of the invention.

EXAMPLE 9 1 g of sodium salt of poly-L-glutamic acid was dissolved in 5 20 ml of distilled water in a round bottom flask equipped with a magnetic stirrer. To this flask was then slowly added 10.5 ml of an aqueous solution of cis-Pt (NH 3) 2 (NO 2) 2.2 H 2 O containing 0.495 g / ml platinum with stirring. A product from the stirred solution slowly precipitated, but it was redissolved after the addition of 10 ml of distilled water. An additional 2.1 ml of the platinum solution was added to provide a precipitated product of a crosslinked nature. After filtration, washing and drying, the preparation thus prepared contained 33.68¾ platinum, calculated that 86.0¾ of the carboxylic acid groups on the side chains of the poly-L-glutamic acid are complexed with cis- [Pt (NH -part.

EXAMPLE 10 In a preparation similar to that described in Example 9, 0.5 g of the sodium salt of poly-L-glutamic acid was reacted with 0.312 g of platinum in the form of the cis-PtCNH-CN of a product containing 38.55¾ platinum. This corresponds to a reaction of 95.1¾ of the carboxylic acid units to the poly-L-glytamic acid with the 25 cis- [Pt (NH₂) 2] ++ moiety.

Example 11 0.857 g of sodium carboxymethyl cellulose was dissolved in a mixture of 60 ml of water and 20 ml of dimethylformamide. The vessel was covered with aluminum foil and 9.6 ml of an aqueous solution of cis-Pt (NH

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13 added with stirring. After 1 hour, a product began to precipitate. Then the product was collected, vacuum filtered, washed with water and acetone and dried under vacuum. The product thus produced showed by analysis 24.8 24 platinum, which corresponds to the reaction of 88.2 88 of the free carboxylic acid groups in the carboxycellulose complex with the cis- [Pt (NH NH) ++ moiety.

EXAMPLE 12

The preparation as prepared in Example 9 was tested 10 for activity against ascites P388 leukemia in CDF1 female mice. At a non-toxic dose of 30 mg / kg of mouse weight, administered interperitoneally as a single dose the day after implantation of 10 6 tumor cells, the preparation of Example 9 gave a 36¾1 increase in lifespan 15 and a ca. -3.0 log ^ g change in tumor burden at end of trial. Toxicity data for the preparation of Example 9 derived from non-tumor bearing CDF1 female mice indicated LD ^ q, LD ^ g and LD ^ g of 68, 99 and 141 mg of platinum / kg mice, respectively.

Claims (10)

A platinum or palladium composition, characterized in that it comprises water-soluble anionic organic macromolecules, in particular anionic polyamino acids, anionic polysaccharides or enzymes with anionic functional groups, having a molecular weight of 5,000 to 60,000, combined with a part with square planar coordination around the metal ion M, of the formula cis- [MLL1] ++, wherein M is divalent platinum or divalent palladium and L and L 'are ammonia or mono- or di-functional primary amines, so that at least part of the valence requirements for the anionic functional groups of the anionic macromolecules is satisfied. Composition according to claim 1, characterized in that the valence requirements for at least 80% of the anionic functional groups in the anionic macromolecules are satisfied and the composition is insoluble in water. Composition according to claim 1 or 2, characterized in that the macromolecules are poly-L-glutamic acid, poly-L-aspartic acid, serum albumin, chondroitin sulfate, dextran sulfate, carboxymethylcellulose, poly [divinylated-maleic anhydride], poly [acrylic acid]. co-maleic anhydride], polyacrylic acid, ferritin or desulfated heparin. Composition according to any one of the preceding claims, characterized in that the groups L and L 'are ammonia, 1,2-diaminocyclohexane or ethylenediamine. Composition according to claim 1, characterized in that M is platinum, L and L 'are ammonia and the macromolecule is poly-L-glutamic acid. DK 157601 B Composition according to claim 1, characterized in that M is platinum, L and L 'are the functional amino groups of 1,2-diaminocyclohexane and the macromolecule is poly-L-glutamic acid or dextran sulfate. Composition according to claim 6, characterized in that it is lyophilized. Composition according to claim 2, characterized in that M is platinum, L and L1 are ammonia and the macromolecule is poly-L-glutamic acid or carboxymethyl cellulose. Process for the preparation of a composition according to claim 1, characterized in that the anionic macromolecules are reacted with the compound cis-MLL 1 (H 2 O) 2 (NO 2) 2 in aqueous solution. Process for the preparation of a composition according to claim 4, characterized in that in an aqueous solution the anionic macromolecules are reacted with the compound cis-MLL '(H2O) 2 (NO0) 2j wherein L and L' are ammonia, 1, 2-diaminocyclohexane or ethylenediamine.