DE3701162A1 - NEW THERAPEUTIC USE OF FRUCTOSE-1,6-DIPHOSPHATE FOR PROTECTION AGAINST TOXICITY, CAUSED BY LONG-TERM ADMINISTRATION OF ANTITUMOR MEDICINES OF THE ANTHRACYCLIN SERIES - Google Patents

Description

The present invention relates to a new one therapeutic use of fructose 1,6-diphosphate (FDP) to obtain compositions which have a Have protective effect against the toxic effect which metabolism and cellular integrity (Integrity) through long-term administration of Antitumor drugs of the anthracycline series shows.

The invention also relates to the corresponding pharmaceutical Compositions or preparations.

Among the products with antitumor activity Doxorubicin (DXR) has a high chemotherapeutic effect and is therefore used in the treatment of some solid tumors and malignant lymphadenopathies in humans. However, the use of such a drug is limited because of the cardiotoxicity (heart damage) of the Molecule that occurs as a result of administration, Cardiotoxicity, which is either acute or chronic can be. Acute cardiotoxicity is temporary Phenomenon that occurs immediately after (and in some Cases even during) the administration of DXR. It can be determined electrocardiographically become and seems partially with a temporary  Increase in the plasma concentration of histamine linked to be caused by mastocytes, which by contact with DXR were stimulated, was put into circulation.

Chronic toxicity, on the other hand, is a disease resulting from long-term administration of DXR and is characterized by myocytolysis with histologically clearly identifiable, necrotic areas and through congestive heart failure. The Biochemical mechanisms of these diseases were the subject extensive investigations, which both the change in morphological and metabolic Highlighted cell parameters, but their origin is not yet certain.

Recent studies have concluded that the cardiotoxicity of DXR is mainly caused by the ability of DXR itself to generate radical forms of O ₂ . The DXR semiquinone radical generated "in vivo" actually undergoes mono-electron oxidation using oxygen as the electron acceptor according to the following scheme:

DXR · + O ₂ ---- DXR + O ₂ ·

The reaction therefore generates the DXR semiquinone residue and the O ₂ · superoxide anion, starting a series of chain reactions which lead to the formation of other radical forms of oxygen, such as OH, H ₂ O ₂ and singlet oxygen.

The intracellular increase in these highly reactive radical forms causes the peroxidation of the lipids Cell membrane and a subsequent change in the quaternary structure the same; this causes leakage of the cytoplasm and cell death.  

The depletion of GSH during treatment with DXR and the low activity of the myocardial enzymes, which are intended to remove the O ₂ radicals (catalase, SOD, GSH peroxidase), are the reason for the specific property of the toxic effect of DXR at heart.

Exhaustive investigations and several attempts, which the applicant carried out with fructose 1,6-diphosphate, surprisingly led to the determination of the existence of one previously unknown protective effect of FDP, which is caused by the Experiments, the results of which are shown below, is confirmed.

The clinical trials carried out confirmed that Fructose 1,6-diphosphate (FDP) by interacting with the cell membrane modified the ion permeability and the Activation of phosphofructokinase causes linked with the increase in concentrations of endogenous FDP and cellular concentrations of ATP.

The investigations made showed that FDP the isolated rat mastocytes against release of histamine, which protects through various chemical stimuli, including DXR, and inhibits the generation of super oxides which are caused by Phorbol esters (PMA⁺) in isolated human, polymorphonuclear Leukocytes is produced.

Further experiments on isolated cell systems showed that the presence of FDP in the incubation medium is not the amount of DXR which penetrates into the cell, reduced.

(⁺PMA = phorbol milistate acetate (Merck Index, 10th edition, No. 7217))  

The extremely advantageous is then clearly shown Use of FDP as a protective agent against heart damage (Cardiotoxicity) caused by long-term administration is evoked by DXR in all those cases in which complications are necessary to decrease which is due to long-term administration of antitumor drugs the anthracycline Series of patients suffering from neoplastic (tumor-forming) Illnesses, stop.

The present invention will now be described in detail only by the non-limiting example in relation to the following.

example 1

The experiments were carried out on 100 Swiss mice (50 male and 50 female), which 19 to Weighed 21 g (Charles River), with standard laboratory food (Nossan fodder®) and water ad libitum were and under controlled moisture and Temperature conditions were kept.

The doxorubicin used is that which as Raw material is produced by Farmitalia Carlo Erba.

The animals were divided into two treatment groups of 8 mice each.
Group 1: 0.9% NaCl
Group 2: FDP at a dose of 750 mg / kg.
NaCl and FDP were administered daily intraperitoneally 5 days / week.

DXR was given to both groups twice / week, intraperitoneally administered at a dose of 2 mg / kg.  On those days when DXR was administered treatment 20 min before infusion of the anti-tumor Drug administered.

Two animals in each group were 1, 2, 3 and 4 weeks killed after administration of DXR. This treatment pattern was repeated to 10 animals for each week and to have every treatment group. The remaining ones 20 animals which are not subjected to the treatment were used to determine the basic values of the subjects to be examined Parameters used.

The animals were sacrificed by severing the head and blood was collected in heparinized test tubes. The hearts and livers were removed, frozen in liquid nitrogen and then homogenized in 1.15% cold KCl using an Ultra Turrax homogenizer (Janke and Kunkel). After taking a few portions to determine the proteins, X-100 Triton® was added to the samples to a final concentration of 1%. The following parameters were determined:
- decreased glutathione (GSH)
- Lipid peroxidation, determined as malondialdehyde (MDA)
- glucose-6-phosphate dehydrogenase (G-6-PDH)
- 6-phosphogluconate dehydrogenase (6-PGDH)
- Catalase.

The collected blood was centrifuged and the values of certain parameters - with the exception of lipid peroxidation - regarding the red blood cells, the previously washed, packed and with distilled water had been hemolyzed. Furthermore the activity of lactate dehydrogenase (LDH) in the Plasma determined. All measurements were spectrophotometric  performed with a Beckman DU-8 spectrophotometer. All attempts were made on the same day that the Animal was killed. The data were for each individual animal recorded on separate cards. The value of the differences between the treatment groups and the core values and the differences between The two treatment groups were treated with the Wilcoxon One Sample Trial Found.

In the selected test model, the toxic effect long-term administration of DXR through a change the erythrocytic and hepatic G-6-PDH and 6-PGDH, through erythrocyte and cardiac catalase, through plasmatic LDH and shown by lipid peroxidation of the myocardium.

Table 1 shows the erythrocytic values of G-6-PDH, 6-PGDH and catalase from both treatment groups (NaCl and FDP).

Table 2 shows the total plasma LDH values of the two treatment groups.

Table 3 shows the values of the enzymatic activities some enzymes of the hepatic metabolism of the two Treatment groups.

Table 4 shows the values of some parameters of cardiac metabolism.

The administration of DXR affected the Core values clearly indicate the erythrocytic activities of G-6-PDH and 6-PGDH in week four and from Catalase in the second, third and fourth week.

The activity of total plasma LDH increased during the whole four weeks, based on the basic values.  

The activity of the hepatic G-6-PDH was found to be significantly different compared to the baseline in the fourth week.

Total cardiac lipid peroxidation and catalase based on the basic value of the first week of the experiment, clearly.

The activities of the Total plasma LDH in week four, the total Cardiac lipid peroxidation in the second and fourth weeks and the activity of the heart catalase throughout four trial weeks for a significantly different one Result compared to the basic values.

When comparing the two treatment groups, significant Differences in erythrocyte activities of G-6-PDH in the fourth week, of 6-PGDH in the second, third and fourth week and from Catalase in the second, third and fourth week.

The activity of total plasma LDH was demonstrated in the two treatment groups during the entire four weeks of the experiment as clearly different. The Activities the hepatic G-6-PDH and 6-PGDH resulted in the two Treatment groups became clear in the fourth week different result.

The comparison between the values of cardiac lipid peroxidation between the two treatment groups significant differences in all four test weeks clear.

The activity of cardiac catalase resulted in the third and fourth week of experiment at a clearly different Result.  

The data listed above clearly show two different phenomena: the occurrence of the injury, which is caused by the long-term administration of DXR becomes; the protective effect of repeated treatment with FDP.

In the experimental model, the toxicity of DXR was both at the level of blood and tissue (liver, Heart).

In red blood cells, the administration of DXR an increase in key enzyme activity the pentose phosphate pathway (G-6-PDH and 6-PGDH) the increase in oxidative stress.

The increase in total plasma LDH depending on the total administered dose of DXR shows unmistakable damage to cell integrity caused by the toxicity of the antitumor drug is generated. The electrophoretic analysis highlights the increase in hepatic and cardiac isoenzymes, which one Confirm organ tropism of anthracyclines.

DXR increased activity in the liver of G-6-PDH and of 6-PGDH as a result of triggering of the hepatic microsomal system.

During the course of this experiment it was also proven that the toxicity of DXR is the most damaging to the Integrity (integrity) and functionality of the myocardial cells.

The increase in the catalase activity, 3.5 times as much as the basic values, proves the large amount of H ₂ O ₂ which is derived from the spontaneous or cardiac SOD-catalyzed dismutation (disproportionation) of the superoxides, which is caused by the accumulation of high Concentrations of DXR are generated in the myocardial tissue.

In contrast, everyone's behavior through DXR modified parameters, both the erythrocytic as also that of the tissues, by treatment with FDP positive influenced.

The non-activation of red blood cells along the way Pentosephosphate (G-6-PDH and 6-PGDH) is a result of Activation of glycolysis by the administration is controlled by FDP, which is the phosphofructokinase positively regulated, thus the oxidation the glucose through a cycle path that is different from the glycolytic is prevented.

The decrease in tissue damage caused by DXR through treatment with FDP becomes clear through the values of plasma LDH shown are indicative of Are cell necrosis. In the FDP-treated group no significant changes related to the base value, recorded until the third week of DXR administration.

The protective effect of FDP prevented any increase the enzymatic activities of the enzymes along the way Pentosephosphate in the liver. The mechanism by which FDP is likely to always affect the parameters with its ability to control the cell metabolism of glucose stimulate, connected. The protective effect of FDP was shown particularly clearly at the level of the heart where the damage that results from long-term administration  derives from DXR, was most obvious. Catalase activity increased compared to baseline, in the FDP group, but resulted in one twice lower result than the value in the NaCl group was found. An analogous situation occurred for the Lipid peroxidation, which is certainly the basic one biochemical indicator (index) of structural and therefore is functional integrity of the cell membrane. Indeed is the fourth in the FDP group Trial week value of MDA less than half of the MDA in the NaCl group.  

Table 1

Effect of 750 mg / kg ip FDP and 0.9% ip NaCl against the damage caused by long-term administration of 2 mg / kg ip DXR to the activities of some enzymes of the erythrocytic metabolism in the mouse

Table 2

Effect of 750 mg / kg ip FDP and 0.9% ip NaCl against the damage to the overall activity of plasma LDH in the mouse caused by long-term administration of 2 mg / kg ip DXR

Table 3

Effect of 750 mg / kg ip FDP and 0.9% ip NaCl against the damage caused by long-term administration of 2 mg / kg ip DXR to the activities of some enzymes of hepatic metabolism in the mouse

Table 4

Effect of 750 mg / kg ip FDP and 0.9% ip NaCl against the damage caused by long-term administration of 2 mg / kg ip DXR to some parameters of cardiac metabolism in the mouse

The importance of such results is broad Use of antitumor drugs of anthracycline Row connected and thus with the possibility of which is offered by the present invention that Adverse effects caused by the treatment with fructose 1,6-diphosphate in many therapeutic Prevent situations.

Claims (4)

1. Therapeutic use of fructose 1,6-diphosphate to protect against the toxicity caused by Administration of antitumor drugs of anthracycline Row is caused. 2. Pharmaceutical composition with protective effect against damage caused by the administration of Anthracylin antitumor drugs consisting of fructose-1,6-diphosphate sodium salt in the form of a lyophilic powder and from one solvents suitable for intravenous administration. 3. Pharmaceutical composition with protective effect against damage caused by the administration of Anthracylin antitumor drugs are, consisting of an aqueous solution of Fructose 1,6-diphosphate sodium salt.   4. Pharmaceutical composition according to claims 2 and 3 to not under in daily doses 250 mg / kg body weight, based on the content of Fructose 1,6-diphosphate sodium salt administered to will.