GB2185398A - Use of fructose-1,6-diphosphate to protect against the toxic effects of antitumour drugs - Google Patents

Abstract

Fructose-1,6-diphosphate (FDP) is used to achieve a protective action towards the toxicity caused to the metabolism and the cellular integrity by the prolonged administration of anthracyclinic antitumour drugs. The invention relates as well to preparations based on FDP having the above mentioned antitoxic action.

Description

SPECIFICATION Use of fructose-i 6-diphosphate to protect against the toxic effects of antitumour drugs This invention relates to a new therapeutic use of fructose-1,6-diphosphate (FDP) and to compositions containing FDP.

Among products having an antitumour activity, doxorubicin (DXR) has a high chemotherapeutic effect, and is therefore used in the treatment of several solid tumours and of malignant lymphadenopathies in humans. The use of such a drug is, however, limited because of the acute and/or chronic cardiotoxicity of the substance. Acute cardiotoxicity is a transitory phenomenon occurring immediately after (and in some cases even in the course of) administration of DXR and can be detected electrocardiographically. It seems to be partly connected with a transitory increase in plasma concentration of histamine which is released by mastocytes that have been stimulated by DXR. Chronic toxicity results from prolonged administration of DXR, and is characterised by myocytolysis, with necrotic areas well detectable histologically, and congestive cardiac failure.

The biochemical mechanism of these side-effects have been the object of prolonged studies, which have been based on the alteration of both morphologic and metabolic cellular parameters, the origin of which, however, is not yet certain.

More recently, studies have led to the conclusion that DXR cardiotoxicity is mainly caused by the capability of DXR itself to generate free radical 02 species. The "in vivo" generated semiquinonic radical of DXR in fact undergoes monoelectronic oxidation, using oxygen as an electron acceptor according to the following scheme: DXR- +O2DXR+Q- The reaction therefore gives a DXR semiquinonic radical and the 02 superoxide anion, which gives rise to a series of cascade reactions leading to the formation of other 02 radical species such as OH, H2 2 02., singlet oxygen, and the like.The endocellular increase in these highly reactive radicals may cause peroxidation of lipids of the cell membrane, and a consequent change in the quaternary structure thereof; this brings about cytoplasm outflow and cell death.

The depletion of GSH during treatment with DXR and the low activity of myocardium enzymes serving to eliminate 02 radicals (catalase, SOD, GSH-peroxidase) are believed to be the cause of the specificity of the toxic effects of DXR on the heart.

Exhaustive studies are several tests which we have carried out on fructose-l ,6-diphosphate have surprisingly led to the discovery of a previously unknown protective activity of FDP, in particular against the toxic effects of anthracyclinic antitumour drugs.

Thus, in one aspect the invention provides use of fructose-1,6-diphosphate or a physiologically acceptable salt thereof for the preparation of a pharmaceutical composition for protecting against the toxic effects caused by the administration of an anthracyclinic antitumour drug.

In another aspect, the invention provides a pharamaceutical composition for protecting against the injury induced by the administration of an anthracyclinic antitumour drug, comprising lyophilised fructose-l ,6-diphosphate sodium salt and a solvent for intravenous administration.

In a further aspect the invention provides a pharmaceutical composition for protecting against the injury induced by the administration of an anthracyclinic antitumour drug comprising an aqueous solution of fructose-1,6-diphosphate sodium salt.

In a still further aspect, the invention provides a pharmaceutical composition comprising fructose-1,6-diphosphate or a physiologically acceptable salt thereof in association with an anthracyclic antitumour drug.

Clinical tests which we have effected have confirmed that fructose-1,6-diphosphate (FDP) interacts with the cell membrane to modify ionic permeability and cause the activation of phosphofructoquinase. An increase in the concentrations of endogenous FDP and in the cellular concentration of ATP has been observed.

Our research has shown that FDP protects insulated rat mastocytes against the release of histamine induced by different chemical stimulants, including DXR, and inhibits the production of superoxides induced by esters of phorbol (PMA) in insulated human polymorphonucleate leukocytes.

Further experiments carried out on insulated cellular systems have shown that the presence of FDP in the incubation medium does not reduce the amount of DXR penetrating the cells.

It clearly appears then that the employment of FDP as a protective agent against cardiotoxicity induced by prolonged administration of DXR and other anthracyclinic antitumour drugs reduces the risk of complications setting in as a consequence of the prolonged administration of the drugs to patients suffering from neoplastic pathologies.

The present invention will now be illustrated by way of non-limiting example, with reference to the following RESUL TS Tests were carried out on 100 Swiss mice (50 male and 50 female) weighing 19-21 g (Charles River), fed on a standard laboratory diet (Nossan fodder) and water ad libitum. The mice were kept under controlled humidity and temperature conditions.

Doxorubicin employed in the tests is manufactured by Farmitalia Carlo Erba.

The animals were divided into two sets of treatment groups, each formed by 8 mice: Group 1: 0.9% Na Cl Group 2: FDP at a dose rate of 750 mg/kg NaCI and FDP were administered daily by the intraperitoneal route for five days a week.

Both groups were given DXR twice a week, intraperitoneally at a dose of 2 mg/kg.

On those days when DXR was administered, treatment with saline or FDP was effected 20 minutes before the infusion of the antitumour drug.

Two animals from each group were killed 1,2,3,4 weeks after administration of DXR commenced, respectively. This pattern of treatment was repeated so as to have 10 animals for each week for each treatment group. The remaining 20 animals not treated were used as controls for the parameters to be studied.

The animals were decapitated and their blood was collected in heparinised test-tubes. The hearts and livers were removed, frozen in liquid nitrogen and then homogenized in 1.15% cold KC1 using an Ultra Turrax (Janke and Kunkel) homogenizer. After removing a quantity for determining proteins, X-100 Triton at a final concentration of 1% was added to the samples.

The following parameters were then assessed: -reduced glutathione (GSH) -lipidic peroxidation determined as malondialdehyde (MDA) -glucose-6-phosphate dehydrogenase (G-6-PDH) --6-phosphogluconate dehydrogenase (6-PGHD) -catalase.

The collected blood was centrifuged, and the amounts of the desired parameters-except lipidic peroxidation-were assessed with respect to the red blood cells that had been previously washed, packed and haemolyzed with distilled water. In addition, the lactic dehydrogenase (LDH) activity was determined in the plasma. All measurements were made spectrophotometrically using a Beckman DU-8 spectrophotometer. All the tests were carried out on the same day in which the animals were killed. The data were recorded on individual cards for each single animal.

The differences between the treatment groups and the controls, and the differences between the two treatment groups, were assessed by the Wilcoxon one-sample test.

In the chosen experimental model the toxic effect of prolonged administration of DXR is indicated by the alteration in the erythrocitary and hepatic G-6-PDH and 6-PGDH, by the erythrocitary and heart catalase, by the plasmatic LDH and by the lipidic peroxidation of the myocardium.

Table 1 (below) shows the erythrocitary values of G-6-PDH, 6-PGDH and catalase for both treatment groups (NaC1 and FDP).

Table 2 (below) shows the amounts of total plasmatic LDH for the two treatment groups.

Table 3 (below) shows enzymatic activities for some enzymes of the hepatic metabolism for the two treatment groups.

Table 4 (below) shows parameters of heart metabolism.

It will be seen that the administration of DXR is significantly affected compared to the control values, the erythrocitary activities of G-6-PDH and 6-PGDH at the fourth week, and of catalase at the second, third and fourth week.

The total plasmatic LDH activity increased in all four weeks compared to the controls.

The hepatic G-6-PDH activity proved to be significantly different compared to the control value at the fourth week.

The total cardiac lipidic peroxidation and catalase increased significantly compared to the control value from the first week of experimentation.

In the FDP group only the activities of the total plasmatic LDH at the fourth week, of the total heart lipidic peroxidation at the second and fourth week and the activity of the heart catalase over the whole four weeks of experimentation were significantly different compared to the control values.

By comparing the two treatment groups, significant differences are shown in the erythrocitary activities of G-6-PDH at the fourth week, of the 6-PGDH at the second, third and fourth week, and of catalase at the second, third, and fourth week.

The total plasmatic LDH activity proved to be significantly different in the two treatment groups for the whole four weeks of experimentation. The hepatic G-6-PHD and 6-PGDH activities were significantly different in the two treatment groups at the fourth week. Comparison of the values of the heart lipidic peroxidation between the two treatment groups shows significant differences in all four weeks of experimentation.

The heart catalase activity was significantly different at the third and fourth week of experimentation.

The data clearly show two distinct phenomena: the setting in of injury induced by the prolonged adminstration of DXR; and the protective action of the repeated treatment with FDP.

In the experimental model, the toxicity of DXR was exhibited at both hematic and at tissue (liver, heart) level.

For red blood cells the administration of DXR caused an increase in activities of the key enzymes of the pentose phosphates (G-6PDH and 6-PGDH) pathway because of an increase in oxidative stress.

The increase in total plasmatic LDH dependent on the total administered dose of DXR unmistakably indicates injury produced by the toxicity of the antitumour drug on cellular integrity.

Electrophoretic analysis emphasised an increase in hepatic and heart isoenzymes, confirming organ tropism of anthracycline.

In liver, DXR causes an increase in the activities of G-6-PDH and of 6-PGDH as a result of the induction of the hepatic microsomal system.

In the course of our experiments it was shown that the toxicity of DXR causes greatest injury to the integrity and the functionality of the myocardium cells. The 3.5 times increase in catalase activity compared to the control values shows the large quantity of H202 deriving from the spontaneous or catalyzed (by the cardiac SOD) production of superoxides as a result of the accumulation of high concentrations of DXR in the myocardium tissue.

The modification of all the measured parameters by DXR, both erythrocytary and tissue ones, was positively affected by treatment with FDP.

The non-activation, in red blood cells, of the pentose phosphates (G-6-PDH and 6-PGDH) pathway is a consequence of the activation of glycolysis controlled by administered FDP, which positively regulates phosphofructoquinase, thus preventing glucose from being oxidized via a pathway different from the glycolytic one.

The decrease in DXR-induced tissue- injury after treatment with FDP is clearly shown by the results for plasmatic LDH, which are generally indicative of cellular necrosis. In the FDP-treated group, no significant variations were recorded in respect of the control value until the third week of DXR administration.

The protective action of FDP appears to prevent any increase in enzymatic activities of the enzymes for the pentose phosphates pathway in liver. The mechanism by which FDP probably acts upon these parameters is connected with its ability to stimulate cellular glucose metabolism.

The protective effect of FDP showed most strikingly at the cardiac level, where the injury deriving from prolonged administration of DXR is most apparent. The catalase activity increased compared to the control value in the FDP group, but was about 2 times less than the value found in the NaCI group. An analogous situation occured for lipidic peroxidation, which is certainly the basic biochemical index of the structural and therefore functional integrity of the cell membrane. In fact, the MDA value measured in the FDP group in the fourth week of experimentation is less than half the MDA value for the NaCI group.

TABLE 1 Effect of 750 mg/kg i.p. FDP and of 0.9% i.p. NaCI on the injury caused by prolonged administration of 2 mg/kg i.p. DXR in relation to the activities of some enzymes of erythrocyte metabolism in the mouse.

Average values and (d.s.)

Week of 0-6-POR 6-PGDH catalase Treatment treatment (UI/g Hb) (UI/g Hb) (UIxlO /g treatment (UI/g Hb) (UI/g Hb) (UIxlO /g Hb) 0 (n=20) 43.85 8.59 81.57 ( 9.31) (3.47) (17.78) 1 (n=10) 38.38 8.51 80.02 (15.21) (3.21) (31.01) FDP 2 (n=10) 40.70 6.61 * 81.98 (14.54) (2.43) (15.76) 3 (n=10) 41.26 7.22 * 82.52 * ( 7.91) (2.35) (30.94) 4 (n=lO) 42.30* 7.27* 80.16 * (7.34) (2.40) (14.01) 0(n=20) 43.85 8.59 81.57 (9.31) (3.47) (17.78) 1 (n=10) 43.73 8.69 107.63 (22.21) (5.17) (35.95) NaCl 2 (n=10) 45.39 9.45 106.13 + (14.1S) (2.90) (20.98) 3 (n=10) 43.52 10.18 109.54 + ( 7.48) ( 3.93) (25.67) 4 (n=10) 53.94 + 11.94 + 107.38 + ( 7.48) ( 2.82) (20.01) * p < 0.05 FDP vs NaCl + p 0.01 FDP and NaCI vs control value TABLE 2 Effect of 750 mg/kg i.p. FDP and of 0.9% i.p. NaCI on the injury induced by prolonged adminstration of 2 mg/kg i.p. DXR in relation to the total plasmatic LDH activity in the mouse.

Average values and (d.s.) Week of LDH Treatment treatment (UI/1 plasma) 0 (n=20) 211.12 (49.95) 1 (n=10) 226.19 * (41.59) FDP 2 (n=10) 267.84 * (71.24) 3 (n=10) 280.42 * (75.50) 4 (n=10) 302.13 * + (42.38) (42. 38) 0 (n=20) 211.12 (49.95) 1 (n=10) 361.22 + (54.60) NaCl 2 (n=10) 379.41 + (76.36) 3 (n=10) 402.73 + (86.23) 4 (n=10) 410.98 + (61.66) * p < 0.01 FDP vs NaCl + p < p < 0.001 FDP or NaCl vs control value

TABLE 3 Effect of 750 mg/kg i.p. FDP and of 0.9% i.p. NaCI on the injury induced by prolonged administration of 2 mg/kg i.p. DXR in relation to the activities of some enzymes of hepatic metabolism in the mouse,

Average values and (d.s.) Week of G-6-PDH 6-PGDH Treatment Treatment treatment (UI/g prqteins) (UI/g proteins) 0 (n=20) 3.86 3.67 (1.42) (1.28) - 1 (n=l0) 3.40 3.25 (1.44) (0.89) FDP 2 (n=10) 3.60 3.26 (1.64) (1.20) 3 (n=10) 3.35 3.15 (1.14) (1.14) 4 (n=10) 3.48 * 3.11 + (1.30) (1.33) 0 (n=20) 3.89 3.67 (1.42) (1.28) 1 (n=10) 4.37 3.92 (1.59) (1.33) NaC1 2 (n=10) 4.60 4.09 (2.25) (1.98) 3 (n=10) 4.15 3.27 (1.79) (1.38) 4 (n=10) 5.84 + 4.50 (2.15) (0.84) * p c 0.05 FDP vs NaC1 + p c 0.01 FDP and NaCl vs control value TABLE 4 Effect of 750 mg/kg i.p.FDP and of 0.9% i.p. NaCI on the injury induced by prolonged administration of 2 mg/kg i.p. DXR in relation to some parameters of cardiac metabolism in the mouse.

Average values and (d.s.) treatment (UIx10 /g prot.) treatment treatment (No.of moles MDA/g prot.) (UIxlO / g prot.) 0 (n=20) 27.86 14.75 (7.56) (8.31) 1 (n=l0) 33.53 * 23.14 + (9.05) (10.90) FDP 2 (n=lO) 36.44 * + 25.81 + (4.72) (10.29) 3 (n=10) 37.32 * 20.55 * + (15.69) (4.67) 4 (n=10) 43.82 * + 23.92 * + (12.12) (6.32) 0 (n=20) 27.86 14.75 (7.56) (8.31) 1 (n=10) 47.83 + 28.36 + (5.37) (10.87) NaC1 2 (n=10) 54.29 + 26.66 + (5.01) (13.11) 3 (n=10) 83.90 + 37.51 + (23.79) (6.74) 4 (n=10) 88.64 + 40.13 + (9.52) (5.00) * p c 0.002 FDP vs NaCl + p < 0.05 FDP and HaCl vs control value

The use of anthracyclinic antitumour drugs is widespread, and therefore the possibility offered by the present invention to prevent the setting in of harmful side-effects by treatment with fructose-1,6-diphosphate may be advantageous in many therapeutic situations.

Claims (11)

1. Use of fructose-1,6-diphosphate or a physiologically acceptable salt thereof for the preparation of a pharmaceutical composition for protecting against the toxic effects caused by the administration of an anthracyclinic antitumour drug.

2. Use according to claim 1 wherein the antitumour drug is doxorubicin.

3. Use according to either of claims 1 and 2 wherein the fructose-1,6-diphosphate is administered in an amount of not less than 250 mg/kg body weight.

4. A pharmaceutical composition for protecting against the injury induced by the administration of an anthracyclinic antitumour drug, comprising lyophilised fructose-l ,6-diphosphate sodium salt and a solvent for intravenous administration.

5. A composition according to claim 4 wherein the lyophilised salt is dissolved in the solvent.

6. A pharmaceutical composition for protecting against the injury induced by the administration of an anthracyclinic antitumour drug comprising an aqueous solution of fructose-i 6-diphos- phate sodium salt.

7. A pharmaceutical composition according to any one of claims 4 to -6 in a form for administration of not less than 250 mg/kg body weight of fructose-1,6-diphosphate sodium salt.

8. A pharmaceutical composition comprising fructose-1,6-diphosphate or a physiologically acceptable salt thereof in association with an anthracyclic antitumour drug.

9. A composition according to claim 8 wherein the anti-tumour drug is doxorubicin.

10. Use according to claim 1 substantially as herein described.

11. A composition according to any one of claims 4 to 9 substantially as herein described.