Organic Complex of Platinum, Its Preparation and Its Use for Treating Malignant Tumors

This invention relates to an organic complex of platinum, a process for preparing it and its therapeutic use for the treatment of malignant tumours.

According to one aspect of the invention, there is provided as a novel chemotherapeutic agent having anti-tumour activity cis-isocitrato (1,2-diaminocyclohexane) platinum (II), which may be abbreviated to PHIC. This organic complex of platinum can be prepared by reacting a molecular moiety in which the platinum atom is partially chelated with 1,2-diaminocyclohexane (trans-d and l isomers) represented by the general formula: ##STR1## with trisodium isocitrate (dl isomer), which has the formula: ##STR2## which acts as a labile ligand, and thereafter recovering the resulting platinum complex from the reaction mixture. Two bonds can be formed between molecule (1) and any pair of reactive positions 1, 2, 3 and 4 of the molecule (2). Chelation most likely occurs at the 1- and 2-positions and at the 1- and 3-positions. Interaction at the 1-4 3-4, 2-3 and 2-4 positions is also possible. A monodentate interaction between (1) and (2) is also quite possible: the first Pt bond being a Pt-carboxylato bond, the second being a Pt--OH bond. The invention includes all possible complexes as well as mixtures of these complexes in all proportions.

In a preferred embodiment of the invention, the organic complex of platinum is prepared by reacting cis-dichloro (1,2-diaminocyclohexane) platinum (II) with silver nitrate to form the corresponding dinitrato platinum compound which is thereafter reacted with trisodium isocitrate to form the required platinum complex.

The platinum complex of the invention, i.e. PHIC, has been found to have useful anti-tumor activity. It is useful in the treatment of malignant tumors in warm blooded animals including humans. Apart from its anti-tumor activity, PHIC has also been found to exhibit anti-microbial activity and to be useful as a disinfectant.

Thus in accordance with another aspect of the invention, there is provided a method of treating malignant tumours in a human subject comprising the administration to the subject of an effective dose of PHIC.

It will be appreciated that for chemotherapeutic use, PHIC will normally be administered in the form of a pharmaceutical composition comprising PHIC as an active ingredient in association with a pharmaceutically acceptable diluent or excipient therefor. For the treatment of malignant tumours, PHIC may be administered orally, intramuscularly or intravenously. Hence the pharmaceutical composition will normally be made up in a form appropriate to the desired mode of administration, preferably in a dosage unit form containing a discrete amount of the active ingredient PHIC. For oral administration the dosage unit may be, for example, a capsule, pellet or packaged powder, whereas for intravenous or intramuscular administration, the dosage unit may be, for example, a discrete quantity of an injectable solution or suspension packaged in a suitable sterile container, such as an ampoule.

For the treatment of malignant tumours, the daily dosage of PHIC will generally be from about 1 to about 400 mg per kilogram body weight of the treated subject. It may be administered in one or several administrations.

The inhibition of cellular proliferation by, and the anti-tumor activity of, platinum coordination complexes, in particular cis-dichlorodiammine platinum (II), which may be abbreviated to PDD, of the formula: ##STR3## were discovered some fifteen years ago. Since that time, much research has been devoted to this category of compound in the hope of first, identifying the most active members, and second, of overcoming the disadvantages which were rapidly found to present, these compounds as known to in particular, their low degree of solubility which renders administration difficult, their nephrotoxicity and ototoxicity. Research has also been done to elucidate their mechanism of action. Recent studies have shown that the solubility of these compounds, their anti-tumor activity and their nephrotoxic effects can be favourably influenced by causing various alterations into the molecule. For instance, the replacement of the two chlorine atoms in PDD by bidentate ligands such as oxalato and malonato, was proposed as early as 1973. Then, in 1974, the replacement of the two NH.sub.3 molecules in PDD by 1,2-diaminocyclohexane was suggested. The complex so obtained had the advantage of not causing any cross-resistance to PDD in the animal treated.

However, these investigations were not pursued because of the low water solubility of all these compounds. Research was thus directed to ligands possessing a third carboxylic group which could enhance solubility of the complex and possibly improve its therapeutic activity without necessarily increasing its toxicity.

These ligands include sodium trans-aconitate and sodium citrate molecules, the latter being present in the living human organism (Krebs cycle) in both the citric and isocitric isomeric forms. Different corresponding complexes have been synthesised in order to compare their properties as will be described below. It was observed that the citrate complex PHIC exhibited a greater solubility than compound (3).

The present invention is concerned with the behaviour and properties of the isocitrate complex, PHIC. In comparison with all the compounds prepared both in previous investigations and in the course of the present study, this isocitrate complex presents the following unexpected advantages:

1. An almost unlimited degree of solubility which is at least several hundred times greater than that of other similar platinum complexes.

2. Extremely low toxicity in mice and baboons.

3. Very marked anti-tumor activity toward L1210 leukemia and sarcoma 180.

The comparative trials described below quantitate these advantages, demonstrate this outstanding combination of properties and consequently show the therapeutic value of this compound in the treatment of tumours.

These comparative studies were undertaken with the following compounds which are identified by abbreviations for greater convenience:

PATA: cis-(t-aconitato) diammineplatinum (II)

PHTA: cis-(t-aconitato) (1,2-diaminocyclohexane) platinum (II)

The compound of the invention, i.e. PHIC, can be prepared from compound (4) as follows:

In the first step, the dihydrate derivative was prepared in accordance with the following reaction:

In the second step, the isocitrate anion was complexed as follows:

The following Example illustates the preparation of PHIC.

In 250 ml of bidistilled water were suspended 5 g of cis-Pt (DAC)Cl.sub.2 and 4.3 g of silver nitrate were added. The reaction medium was protected from light. The reaction itself took place at 50.degree.-55.degree. C. over a period of 4 hours. A white precipitate of silver chloride formed. The reaction medium was then brought to 0.degree.-4.degree. C. for several hours and then filtered on Whatman paper. (The word "Whatman" is a registered Trademark of Whatman Inc., New Jersey, U.S.A.). The precipitate was washed with water (50-75 ml) and the resulting colorless filtrate (300-350 ml) was passed through a Millipore filter (0.45 microns). (The word "Millipore" is a registered Trademark of the Millipore Corporation, Massachusetts, U.S.A.). To the filtrate, 3.85 g of trisodium isocitrate, 2H.sub.2 O were added and the mixture was brought to 60.degree.-65.degree. C. for 20 hours during which time it was shielded from light. The resulting yellow-colored solution (which can be filtered on Whatman paper if necessary) was then concentrated to 25-30 ml using a rotavapor (temperature of the bath =50.degree. C.).

The resulting complex was filtered on a Millipore filter (0.45 microns) and added by means of a pipette to 1000 ml of 1/1 cold ethanol/acetone mixture under continuous stirring. The medium was allowed to stand at 4.degree. C. for 2 hours, then filtered on sintered glass, washed with 100 ml acetone and then with 100 ml ether.

The off-white complex obtained was dried under vacuum and then weighed. In this manner, about 6.5 g of PHIC were collected from 5 g of cis-Pt (DAC)Cl.sub.2, which corresponds to a yield of 85%.

The PHIC so obtained was in the form of a fine off-white powder which began to turn black when heated to 245.degree. and then completely decomposed at 254.degree. C. The following results were obtained in the elementary analysis of PHIC (Pt was determined by atomic absorption):

These analytical results are also in agreement with the formula PHIC.2H.sub.2 O in which the Pt (DAC) moiety does not form two bonds with the isocitrate ion, but is linked with one carboxylate ion, most likely C-1, the other bond being a Pt--OH bond.

This compound has an exceptionally high degree of solubility. In water it is almost unlimited (more than 1500 mg/ml). In a 9% solution of sodium chloride it is about 700 mg/ml, the complex changing in 24 hours to a yellow chlorinated derivative which precipitates. PHIC can be identified by its chromatogram and its spectrum analysis (U.V., I.R. and .sup.13 C) which are illustrated in the accompanying drawings.

In FIG. 1 are shown the high pressure liquid chromatograms of the following compounds:

A: PDD

B, C, D and E: PHIC

F and H: trisodium isocitrate

G: PAC

obtained under the following experimental conditions:

In FIG. 2 are shown the U.V. spectra of the indicated compounds namely:

trisodium citrate

PAC

PHC

trisodium isocitrate

PAIC

PHIC

obtained under the following experimental conditions:

The U.V. spectra were recorded on a CARY 14 spectrophotometer. All of the complexes as well as the unreacted ligands absorbed between 203 and 204 nm. The concentrations and molecular absorption coefficients .epsilon. in 1. mol.sup.-1.cm.sup.-1) were the following:

trisodium citrate: 10 mg/100 ml H.sub.2 O; .epsilon.=590

PAC: 2.5 mg/100 ml H.sub.2 O; .epsilon.=7500

PHC: 2.5 mg/100 ml H.sub.2 O; .epsilon.=10100

trisodium isocitrate: 6 mg/100 ml H.sub.2 O; .epsilon.=620

PAIC: 3 mg/100 ml H.sub.2 O; .epsilon.=6200

PHIC: 3 mg/100 ml H.sub.2 O; .epsilon.=9400

In FIG. 3 is shown the I.R. spectrum of PHIC recorded on a Perkin Elmer 577 spectrophotometer. The solid compound was diluted to 1% with potassium bromide and compressed into pellets. The two standard peaks correspond to .nu..sub.1 =2850.7 cm.sup.-1 and .nu..sub.2 =906.7 cm.sup.-1.

Finally, in FIG. 4 are shown the .sup.13 C spectra of trisodium isocitrate, PHS and PHIC obtained under the following experimental conditions:

The .sup.13 C resonance spectra with proton decoupling, were obtained using a pulsed Fourrier Transform apparatus, Bruker WH 90, working at 22.62 MHz. The number of scans was 400 for isocitrate, 3000 for PHS and 17000 for PHIC, with a sweepwidth of 6 KHz and a 8K memory. In all cases a 90.degree. pulse of 12 .mu.s was used with a repetition rate of 3s.

The chemical shifts .delta. Ci are expressed in ppm and are compared to TMS. The compounds were dissolved in D.sub.2 O at the concentration of 1 g/ml for isocitrate, 0.44 g/ml for PHS 1.1 g/ml for PHIC and the solutions were filtered on a Millipore filter (0.45 microns) before the spectrum was recorded. The C.sub.6 D.sub.6 was used as external standard. The chemical shifts are given in the table below. The PHS was included in the study in order to obtain the .sup.13 C spectrum of the 1,2-diaminocyclohexane ligand complexed with Pt (II). CHEMICAL SHIFTS .delta. .sup.13 C (ppm/TMS) OF ISOCITRATE, PHS AND PHIC

The anti-tumour properties of PHIC were evaluated, in comparative studies, using classical tests on two kinds of experimental tumours in mice, leukemia L 1210 and sarcoma S 180. The experimental conditions used and the results obtained are described below:

DAB mice/2 females (CNRS, Orleans) of 20 to 22 g

10 mice per dose

i.p.=intraperitoneal route; i.v.=intravenous route;

s.c.=subcutaneous route

(a) i.p. graft--i.p. treatment

10.sup.5 cells were grafted intraperitoneally on D.sub.0 and intraperitoneal treatment was carried out 24 hours later, D.sub.1, in one single injection.

(b) i.p. graft--i.v. treatment

10.sup.5 cells were grafted intraperitoneally on D.sub.0 and intravenous treatment in the tail vein was carried out 24 hours later, D.sub.1, in one single injection.

(c) s.c. graft--i.p. treatment

10.sup.6 cells were grafted under the skin behind the head on D.sub.0 and intraperitoneal treatment was carried out in one single injection either on D.sub.1 or on D.sub.4.

Swiss female mice (Evic-Ceba, Bordeaux) of 20 to 22 g

10 mice per dose

10.sup.6 cells intraperitoneally grafted on D.sub.0

intraperitoneal treatment 24 hours later, D.sub.1, in one single injection.

The compounds investigated, namely PHIC, PDD, PAC, PAIC, PHC and PHTA, were dissolved in a 9% solution of sodium chloride. Two other compounds, namely the cis-sulfatomonoaquo(1,2 diaminocyclohexane) platinum (II), abbreviated PHS, and the cis-malonato (1,2 diaminocyclohexane) platinum (II), abbreviated PHM, have already been reported and will be included in this study for comparison purposes. PHD and PHM were suspended in a 4% Klucel (hydroxy propyl cellulose) solution (the word "Klucel" is a registered trademark of Hercules Incorporated, Delaware, U.S.A.).

PHS was dissolved in water because its dissolution in 9% aqueous sodium chloride gave rise almost instantaneously to the formation of a yellow insoluble chlorinated compound, most likely monochlorinated then dichlorinated PHD. The solutions of these compounds were prepared extemporaneously. The increase in life span, ILS (%), were calculated in comparison with the controls by the following formula: ##EQU1## Deaths were recorded until day 30 for L 1210 tumour and until day 60 for S180 tumour. Survivals were included in the ILS calculation by assigning a 30 day life span in the L 1210 experiments and a 60 day life span in the S 180 experiments It should be added that most of the mice reaching 30 days of survival treatment with PHM and PHIC developed peritonitis as PHM and PHIC developed peritonitis as judged by adhesion and hardening of the abdominal viscera. This effect was also observed in healthy mice when the compound was injected by intraperitoneal route. In contrast, this delayed toxic effect was never encountered when the compound was administered by intravenous route. This type of toxicity seems to be correlated with the presence of 1,2-diaminocyclohexane (PHD, PHS, PHM, PHC and PHIC) since it was never observed with the diammino derivatives (PPD, PAC, PAIC).

The compounds PAC, PAIC and PHTA gave 17% (100 mg/kg), 74% (75 mg/kg) and 13% (500 mg/kg) of ILS in leukemia L 1210 (10.sup.5 cells, intraperitoneal graft on D.sub.0, treatment in one single injection on D.sub.1). It is important to note the following:

(a) PHIC exerts the anti-tumour activity in leukemia L 1210 and sarcoma S 180, intraperitoneal graft, but causes peritonitis intraperitoneal treatment (Tables I and II).

(b) PHIC is active against leukemia L 1210 when the graft is intraperitoneal and the treatment is intravenous (Table III).

(c) The therapeutic index of PHIC, defined by the ratio T.I.=LD.sub.50 /MED (MED=Minimum effective dose, i.e., the dose corresponding to an ILS of 25% for both tumours) in 5.5 times greater than that of PDD in the case of leukemia L 1210 and 4.0 times greater in the case of sarcoma S 180.

Comparative studies were also performed in order to evaluate the in vitro and in vivo toxicities of the compound PHIC of the invention. The results of the trials are summarized:

A. L 1210 CELLS IN CULTURE

In vitro toxicity was determined using L 1210 cells in culture by measuring the inhibitory dose 50, ID.sub.50, which represents the concentration of the platinum compound which reduces by 50% the rate of growth of the cells after prolonged contact, usually 24 to 48 hours, with the compound to be tested. At the ID.sub.50, all the cells were viable as judged by exclusion with trypan blue. The experimental part of this study is described in a recently published article (P. LECOINTE, J. P. MACQUET and J. L. BUTOUR (1979) Biochem. Biophys. Res. Commun., 90, 209-213). The results are shown in Table IV.

B. TOXICITY IN THE MOUSE AND IN THE RAT.

1. Toxicity i.p., i.v. and p.o.

Acute toxicity, one single injection of the compound, was measured using Swiss female mice (Evic-Ceba, Bordeaux) of 21 to 23 g. and Sprague-Dawley rats. Ten animals were treated for each dose studied. The toxicity of the different compounds is given in Table V.

The toxicity by intraperitoneal (i.p., 10 animals per dose), intravenous (i.v., 10 animals per dose) and oral (p.o., 10 animals per dose) routes of PHIC is reported in Table VI. The loss of weight on the 4th day of the animals i.p. treated was 3.5 g of the LD.sub.10 dose, 4.3 g for the LD.sub.50 dose.

It should be added that between 50 and 80% of the surviving animals at the i.p. treatment developed peritonitis as judged by adhesion and hardening of the abdominal viscera within the month following the injection of PHIC. As discussed above this problem seems to be linked to the presence of the 1,2-diaminocyclohexane ligand. When PHIC is injected intravenously this long-term toxic effect is not observed.

2. Renal and hepatic toxicity

The nephrotoxicity of PDD is well known. It was thus of interest to compare the effects of the novel compound of the invention with those of the already known compounds listed above on the kidney and the liver, i.e. the organs in which the greatest amount of platinum accumulates.

Swiss female mice (Evic-Ceba, Bordeaux) of 20 to 22 g were treated by intraperitoneal route D.sub.0 and then sacrificed on D.sub.1, D.sub.4 and D.sub.10. Nine mice were used per compound and the doses administered are given in Table VII.

It should be noted that these doses corresponded to the maximum non-toxic doses in the healthy mouse (.about.LD.sub.0). The renal and hepatic parenchyma were observed in organs fixed with a Duboscq-Brasil mixture and embedded in paraffin. Slices 5 microns thick were coloured with hemalun eosine stain. In most cases, the alterations were located in the cortex and in the proximal and distal tubules. They were:

(a) Nuclear alterations:

marked anisocaryosis

alteration of the chromatin either washed out or lumpy with hyperchromatism and/or pyknosis

the nuclear membrane was irregular and sometimes incised

(b) Alterations of the cytoplasm:

Anisocytosis with alteration of the brush border of the proximal tubules and intracytoplasmic eosinophilic inclusions.

Some scattered patches suggested isolated cellular necrosis in the proximal and distal tubules. In one case an abscess was observed on D.sub.10 after treatment with PHM. Tubular dilation with hyaline casts in the nephron as a whole were found with, at times, a pseudovesicular aspect. These alterations, when they were observed, were at their maximum on D.sub.4 and stabilized on D.sub.10. Variations in the same series suggested variations in sensitivity.

These alterations were observed with:

(a) PDD (8 mg/kg), PAC (100 mg/kg) and PHM (50 mg/kg).

(b) PHD (4 mg/kg): vascular alterations without any hyaline casts.

(c) PHS (5 mg/kg): vascular dilation and proximal distorted tubules.

PHC (120/mg/kg) and PHIC (150 mg/kg) did not provoke any significant alterations on this type of preparation. The splenic and even suprarenal parenchyma which were sometimes dissected, showed no abnormalities.

From the foregoing it can be concluded that the novel compound of the invention, PHIC, does not cause any renal lesion even at a dost 20 times greater to that of PDD.

Comparative studies of the behavious of the compound of the invention, PHIC and of compound PDD have also been made with respect to cellular penetration in vivo in the mouse. The results are given below. DBA/2 mice received 10.sup.6 L 1210 cells on D.sub.0 and were treated three days later with an amount of platinum compound equal to the LD.sub.0 in the healthy mouse. Two hours after treatment on D.sub.3, the animals were sacrificed. About 20.10.sup.6 cells were extracted from the peritoneum and washed several times so as to eliminate the extra-cellular platinum and also any intracellular platinum which was not bound in a covalent manner with the cellular organelles. Platinum was monitored by atomic absorption for whole cells and also on the DNA which was purified by phenol extraction. In the case of PDD (9 mg/kg) only 0.4% of the platinum injected into the animal entered the cells. This corresponds to 180.10.sup.-16 g of platinum per leukemic cell. Under these conditions, one fixed platinum atom per 3000 nucleotides was found, i.e. a total of 1% of the amount which penetrated the cells was complexed to DNA. For animals treated with 150 mg/kg PHIC, 0.15% penetrated the cells, which corresponds to 700. 10.sup.-16 of platinum per cell. For this compound one fixed platinum atom was found for 3500 nucleotides. As in the case of PDD, 1% of the amount of PHIC which entered the cells was complexed to DNA. The results are given in Table VIII.

Finally, a preliminary toxicological study was conducted with PHIC in the baboon so as to determine the folowing parameters:

(a) Nephrotoxicity

(b) Hematotoxicity

(c) Gastro-intestinal toxicity

(d) Hepatic toxicity

(e) Ototoxicity

as well as the maximum non-lethal dose.

In order to find out the maximum tolerable dose, two baboons, identified by no 1 and no 2, received 100 mg/kg and 150 mg/kg respectively without prehydration. Hydration was performed on baboon no 3 which received a dose of 200 mg/kg.

The PHIC was dissolved in a 9% solution of sodium chloride at the concentration of 100 mg/ml and then filtered on a 0.22 microns millipore filter. The compound was injected into the femoral vein during 5 to 10 minutes. Baboon No. 3 (200 mg/kg) received 700 ml of physiological serum by intravenous route. Baboons Nos. 1 and 2 received 1000 ml by sub-cutaneous route.

Gastro-intestinal toxicity was the first type of toxicity to appear and generally manifested itself 1 to 2 hours after injection of the compound. In all instances, vomiting was not profuse and lasted between 4 and 5 hours. Blood samples were taken at regular intervals and the results are listed in Tables IX, X and XI. D.sub.0 was the day of treatment and a sample was taken before the compound was injected.

The results on D.sub.0 were therefore control values.

D.sub.1 =first day after treatment,

D.sub.2 =second day, etc.

Baboon No. 2 treated with 150 mg/kg PHIC died on D.sub.11, most certainly from the nephrotoxicity of the compounds as indicated by a creatine level of 1552. This baboon had neither eaten nor drunk for 11 days. Prolonged hydration of the animal (2 to 3 days) by subcutaneous route was also tested but appeared not to be the best solution. At 100 mg/kg, under these conditions, no nephrotoxic effect was observed, in the absence of hydration. At a dose of 200 mg/kg with hydation, nephtotoxicity is detected but creatinine levels return to normal values on day 18.

An ototoxicity test was carried out on baboon No. 3 (200 mg/kg) on D.sub.31. This test comprised the following: (a) Registering the auditory potentials when the ear was submitted to 1500 stimuli at 90, 80, 70, 60, 50, 40, 30, 20 and 10 decibels. The results gave the latency times for conduction from the ear to the brain. (b) Performing the same kind of test by echocochleography. This analysis was much more accurate than the previous one because it was only carried out in the Corti organ (cochlea). Frequencies of 8000, 4000, 2000 and 1000 Hertz at 90, 70, 50, 30, 20 and 10 decibels were used. Measurements were made using a Racia Medelec Empled MK.sub.4 1026. No ototoxic effect was detected at the dose of 200 mg/kg of PHIC. The properties of PHIC compared to those of PDD can be summarized as follows:

The compound of the invention is superior to PDD by the criteria of solubility, toxicity and anti-tumor activity. (Cf. Tables IX, X and XI pp. 22, 23, 24, 25 and 26).

It will be recognized by one skilled in the art that the administrable amount depends on the seriousness of the condition to be treated and therefore is generally administered in that amount which is effective to control the tumours, which amount is in the range of 1 to 400 mg per kg of body weight per day.

For information relating to cancer and current forms of cancer treatment, the following publications are incorporated by reference:

Thorn, George W. et al., Harrisons Principals of Internal Medicine, Eighth Edition, McGraw-Hill, Inc. (New York, 1977) pp. 1731-1788).

Goodman, Louis S. et al. The Pharmacological Basis of Therapeutics, Fifth Edition, McMillan Publishing Co., Inc. (New York, 1975) pp. 1248-1253.

With regard to other platinum compounds having activity against leukemia L 1210, reference is made to NTIS document No. PB-299 427, (N-phosphonacetyl-L-aspartate) (1,2-diamino-cyclohexane) platinum (II) or Alkali Metal Salt, U.S. Ser. No. 6-058,287, filed July 7, 1979, by Meischen, Gale and Noff.