MXPA99001505A - Pharmaceutical compositions comprising tyrphostins - Google Patents

Abstract

Compounds useful for countering undesired toxic effects to cells, tissues or organs having formula (I) wherein:Ar is a group of formulae (i) or (ii), n is O or, when Ar has formula (i) above, then n may also be 1, R is CN, -C(S)NH2, -C(O)NHR3 or, when R1 is 4-NO2 and R2 is H or 3-OH, then R may also be a group of formulae (iii), (iv), (v), (vi) where R3 is H, phenyl, phenyl(lower alkyl) or pyridylmethyl;R1 and R2 are each independently H, OH, NO2 or, when R is CN, also CH3, F, or CF3, provided that both R1 and R2 are not simultaneously H.

Description

PHARMACEUTICAL COMPOSITIONS THAT COMPRISE THIRPAROSTOSES DESCRIPTION OF THE INVENTION The present invention relates to compositions which are useful for counteracting damage caused by harmful agents, particularly antineoplastic agents used in the treatment of cancer, for example cytotoxic drugs. The damage in the context of the present invention means adverse effects in either cells, tissue or organs. The present invention also relates to therapeutic methods to counteract such damage. Additionally, the present invention also relates to novel compounds useful in such compositions and methods. Most commonly used antineoplastic treatments, including chemotherapy and radiation, have adverse toxic effects, manifested in dividing cells and in the function of certain organs. Cells that are particularly affected by such treatment are bone marrow cells, epidermal cells and epithelial cells of the gastrointestinal tract. In addition, such treatments often cause damage to specific organs, such as kidney and liver. As a result of such toxic effects, the therapeutic index of such treatment is limited. It is a great desire in medical research to treat and develop treatment modalities and means which will reduce unwanted toxic side effects, without affecting the therapeutic activity of drugs, thus increasing their therapeutic index. Apoptosis or programmed cell death is a fundamental physiological mechanism of cell death regulated during embryonic development and mechanisms of normal homeostasis within the body. Recent data indicate that the antitumor effect of a variety of chemotherapeutic agents is related to their ability to induce apoptosis. The toxicity of these agents may also be related to the induction of apoptosis in normal cells. Preliminary reports have described the use of several pharmacological agents for the prevention of nephrotoxicity resulting from the use of anticancer drugs (Skinner, R., Current Opinion in Oncology, 7: 310-315, 1995). Attempts have been made to counteract the severe chronic proximal tubular toxicity resulting from the use of the cytotoxic drug Cisplatin. In in vivo experiments in rats, para-aminobenzoic acid (PABA) co-administered together with cisplatin results in reduced nephrotoxicity of cisplatin without reducing its antitumor activity. Other agents such as mitimazole, chloropromazine and L-arginine are also administered to animals in various in vivo experiments performed in connection with the toxicity of cisplatin resulting in only preliminary non-conclusive findings. The drug amifostine is also used which is dephosphorylated to produce a thiol portion to reduce antitoxic effects of the antineoplastic drugs (Cancer Res., 55: 4069, 1995). The present invention provides, for a first aspect, a pharmaceutical composition for counteracting, ie reducing or preventing, damage to cells or tissue comprising, as an active agent, an effective amount of a compound of the general formula I: where: - Ar is a group of the formula: R ((«) - n is 0 or, when Ar has the formula (i) above, then n can also be 1, CN, -C (S) NH2, -C (0) NHR3 or, when R is 4-N02 and R2 is H or 3 -OH, then R can also be a group of the formula: * H2'M3n clpQflfe R3 or pyridylmethiFb; - -Rx and R2 are each independently H, OH, N02 or, when R is CN, also CH3, F, or CF3, with the proviso that both Rx and R2 are not simultaneously H, together with a pharmaceutically acceptable carrier The active agent and the pharmaceutical composition can be administered to or brought into contact with cells or tissue in a variety of conditions to reduce or prevent damage to cells, tissues or organs Examples of such conditions are such that they can lead to apoptosis Preventing unwanted apoptosis is a specific embodiment of the invention., tissue or organ to dangerous factors, which can be exogenous or endogenous factors, as well as other physiological conditions, which can lead to damage, for example change in temperature, blood flow damage, exposure to ionizing radiation, etc. The damage to be avoided may also be a result of natural physiological deterioration processes, for example such as occurring in cells, tissues or organs, maintained, grown, or cultivated ex vivo. The present invention also provides a method for treating an individual, to counteract damage to cells, tissue or organ, which comprises administering to the individual an effective amount of a compound having the general formula I.

The term "effective amount" should be understood as meaning an amount of the active compound, which is effective in counteracting the damage manifested in either the destruction of normal (non-diseased) cells or damage to tissue or organ. The harmful factor can be an exogenous agent, for example, a therapeutic drug that has a cytotoxic effect (for example antineoplastic drugs), irradiation, harmful chemicals, etc. In addition, the harmful factor can be endogenous, such as free radicals the level of which increases in the course of various metabolic or other disorders, autoantibodies, cytokines, etc. The pharmaceutical compositions of the invention can be used in the structure of the treatment of various diseases, disorders or conditions such as AIDS, conditions which give rise to heptotoxicity, radiation damage, reduction or inhibition of damage to grafted cells or tissues as a result of graft rejection, for the treatment of intoxications, for example intoxication by paracetamol, to counteract the adverse effects of solvents and harmful carriers of therapeutic drugs, to counteract the damages caused by alcohol, etc. Additionally, the compositions can also be used to counteract undesired immune mediated reactions or an inflammatory response, for example septic shock, to reduce damage caused by autoimmune and other reactions. Finally, the pharmaceutical compositions of the invention may also be used in ex vivo conservation of cells, tissues or organs used for transplantation to reduce or prevent cell or tissue deterioration or death which may otherwise occur during the time they are maintained. ex vivo before the transplant, etc. The pharmaceutical compositions of the invention are especially useful for counteracting the toxic effects of cytotoxic or antimetabolic drugs, particularly as used in cancer therapy. Occasionally, the pharmaceutical composition of the invention will be administered together with the cytotoxic drug. A pharmaceutical composition according to this embodiment can comprise such a drug in combination with a compound of formula I above. The compounds of the formula I belong to a family of compounds known as Tirfostin compounds (Levitxki, A. And Gazit, A. Science, 267: 1782, 1995). In the following text, a compound of Formula I will be referred to as "Tirfostin" and compositions comprising such compounds will sometimes be referred to as "Tirfostin compositions". Of the tyrphostins of the formula I, some are known, although for their uses different from those provided by the invention, and others are novel. The novel tyrphostins, which constitute another, independent aspect of the invention, are those of the formula I, wherein Ar, R R2 and R3 are as defined above, with the conditions that a) R can not be b) When R is CN and n is 0, then (ba) if one of Ri and R2 is H or OH, then the other can not represent N0; (bb) if one of Ri and R2 is H or F, then the other can not represent H or F; and c) When R is 4-N02, R2 is H and n is 0; then R can not represent C (0) NH2 or -C (S) NH2. Preferred compounds of the formula I for use in the compositions are those wherein R is CN, -C (S) NH2, C (0) NHCH2C6H5 or a group of the formula and n is 0, Ri is 4-N02 and R2 is H. Examples of tyrphostins are shown in the compound of Table I. Some of the tyrphostins shown in Table 1 in compound are novel, and these novel tyrphostins are also shown in compound Table II. Compounds Table I The numbers refer to the following list or appended references List of references for several known compounds used according to the invention 1. Mo ry, D.T., J. Am. Chem. Soc. , 67: 1050, 1945 2. Zabichy, J., J. Chem. Soc, 683, 1961. 3. Bronskill, J.S., De A., and Ewing, d.F., J. Chem. Soc. Perkin, Trans. I., 629, 1978. 4. Junek, H., and Olny, B., Monat, Chem., 107: 999, 1976. 5. Novogrodsky, A., Vanichkin, A., Patya, M., Gazit, A., Osherov, N. And Levitzki, A., Science, 264: 1319, 194. 6. Sakamoto, M., et al., J. Chem. Soc., Perkin Trans I., 1759, 1995. 7. (a) Flenner, AL, CA 63:13, 278, 1965. (b) Lythgoe, K. Todd, A. And Tapham, C.J. Chem. Soc. 515, 1944 8. Drabek, J. And Meyer, A., C.A. , 89: 152, 719, 1978. 9. Gazit, A., et al., J. Med. Chem. 34: 1896, 1991. lO.Carson, B.B., and Staughton, R.W. , J. Am. Chem. SOC, 50: 2825, 1928. 11. Gazit, A., et al., J. Med. Chem., 32: 2344, 1989. 12. Weiinberger, M.A. , and Higgin, R.M. , Can. J. Chem., 43: 2585, 1965. 13.Blox Ram, J., Dell, C.P., and Smith, C.W.

Heterocycles, 38: 400, 1994. Particularly preferred tyrphostins according to the invention are the compounds designated AG1714, AG1744, AG1801 and AG1843 in the "Table I of compounds" above. Of these four compounds, the last two are novel. The tyrphostins of the formula I can be administered to the patient in combination with another treatment, for example, in combination with a cytotoxic drug or irradiation. In such combination treatment the tyrphostins can be administered simultaneously with or at a different time to the other treatment, in order to produce a maximum effect. A particular example is the administration of tyrphostins before the other treatment, for example several hours before irradiation or administration of the cytotoxic drug. It has been found according to the invention that when the tyrphostins of the formula I are administered together with another therapeutic agent, they do not reduce the therapeutic activity of the other agent, but if they act by reducing their unwanted side effects on the cells or tissue normal. This means that therapy with the therapeutic agent will still achieve the same desired therapeutic effect, in the same dose. For example, in the case of a chemotherapeutic drug, the administration of a tyrphostin not only may, affect or minimally affect the effect of the drug in reducing the tumor burden or prevent tumor growth or tumor recurrence, in a dose of administration Dadaist. In some chaos, however, the administration of tiffostins even intensifies the therapeutic effect of the treatment. The total effect of tyrphostins, in such a combination therapy, is thus increased in the therapeutic index of another therapy, that is, the ratio between the therapeutic effect of the therapy on its unwanted side toxic effects is increased. The increase in the therapeutic index can sometimes be used to overcome the increase in the dose of the therapeutic agent, for example, the dose of the cytotoxic agent or radiation, without a concurrent increase in unwanted toxic side effects. Tyrphostins according to the invention can be administered either in a single dose or can be given repetitively over a period of time, for example, before, during and after treatment with a cytotoxic agent or radiation. A preferred mode of administration of tyrphostins to humans is intravenously, although by an appropriate formulation, they can also be administered by other modes of administration, for example intramuscularly, intraperitoneally or orally. While tyrphostin compositions will typically contain a single tyrphostin compound, it is sometimes possible to include in the composition and or co-administer two or more tyrphostins which act together in a synergistic or additive manner to counteract damage caused, for example by a therapeutic treatment. BRIEF DESCRIPTION OF THE FIGURES Figure IA is a graphical representation showing the mortality of mice treated with doxorubicin compared to the mortality of mice receiving a single injection of AG1714 two hours before treatment with doxorubicin. Figure IB is a graphical representation showing the mortality of mice treated with cisplatin compared to the mortality of mice receiving a single injection of AG1714 two hours before treatment with cisplatin. Figure 2 is a graphical representation showing the mortality of mice receiving 10 i.p. injections. of doxorubicin (5 mg / kg) for a period of 21 days (cumulative dose 50 mg / kg) compared to the mortality of mice treated as above but receiving an i.p. of AG1714 (5 mg / kg) two hours before each injection of doxorubicin. Figure 3 is a graphical representation showing the mortality of mice treated with cisplatin alone or with cisplatin and AG1801 in a period of ten days after the administration of cisplatin. Figure 4 is a graphical representation showing the mortality of mice treated with doxorubicin alone or with tyrphostins and doxorubicin in a period of ten days after administration of doxorubicin. Figure 5 is a graphical representation showing the effect of a number of Tyrphostins on the toxic effect of cisplatin in kidneys of treated mice. The toxic effect is measured by the level of creatinine detected in the serum of the treated mice, a high level of creatinine that indicates kidney damage (nephrotoxicity). Figure 6 is a graphical representation showing the level of creatinine (Figure 6A) and blood urea nitrogen (ÑUS) (Fig. 6B) (which are indicative of nephrotoxicity) in serum of mice receiving either cisplatin alone, AG1714 alone or a combination of cisplatin and AG1714. The levels of creatinine and US are compared with the levels of the same substances in sera from control mice that receive only the vehicle. Figure 7 is a photograph showing the histopathological analysis of kidneys (Fig. 7A-C) and small intestines (Fig. 7D-F) of mice administered with cisplatin alone or with tyrphostin AG1714 before administration of cisplatin. The final amplification x 400.

Figure 7A kidneys from normal untreated mice; Figure 7B kidneys of mice receiving cisplatin alone showing large proteinaceous plaques in the proximal tubules; Figure 7C shows kidneys of mice receiving AG1714 before administration of cisplatin showing normal kidney structure; Figure 7D thin intestines of untreated mice; Figure 7E Thin intestines of mice receiving cisplatin alone showing severe necrosis and damage; Figure 7F small intestine of mice treated with AG1714 before administration of cisplatin showing normal small bowel structure; Figure 8 is a graphical representation showing the level of two transaminases, aspartic transaminase (AST) and alanine transaminase (ALT) in the serum of mice receiving an injection of anti-FAS antibody alone or in combination with the tyrphostin AG1801. High levels of transaminases indicate that the anti-FAS antibody induces liver damage (hepatoxicity). Figure 9 is a graphical representation showing the level of two transaminases AST and ALT in serum of mice receiving an injection of anti-FAS antibody alone or in combination with tyrphostin AG1714; High levels of transaminases indicate that anti-FAS antibody induces liver damage (hepatoxicity). Figure 10 is a graphical representation showing the serum AST and ALT level of mice treated with Con A alone or in combination with AG1714; Figure 11 is a graphical representation showing the serum AST and ALT level of mice treated with TNF / GalN or "in combination with the tyrphostin AG1714; Figure 12 is a graphical representation showing the number of nucleated cells of bone marrow (Figure 12A) or the number of colony forming units (CFU) (Fig. 12B) in bone marrow of the femur of mice treated with doxorubicin alone, AGI714 alone or with their combination; Figure 13 is a graphical representation showing the number of nucleated cells in bone marrow of mice treated with doxorubicin alone or with AG1714 and doxorubicin. Figure 13A shows the number of nucleated cells in bone marrow of mice receiving different doses of doxorobucin; Figure 13B shows the number of nucleated cells in bone marrow of mice treated at different periods of time after treatment with doxorubicin; Figure 14 is a graphical representation showing the weight of the spleen (Fig. 14A) and thymus (Fig. 14B) of mice treated with either doxorubinca alone or with AG1714 before administration of doxorubicin; Figure 15 is a graphical representation showing the number of nucleated cells of bone marrow in the femurs of mice treated with 5FU alone or with 5FU and AG1714; Figure 16 is a graphical representation showing the number of nucleated cells of bone marrow in the femurs of mice treated with mitomycin-C alone or with mitomycin-C and AG1714; Figure 17 is a graphical representation showing the number of nucleated cells in bone marrow of mice treated with doxorubicin alone or with AG1801 at different doses prior to administration of doxorubicin; Figure 18 is a graphical representation showing the number of nucleated cells in bone marrow from irradiated mice (3OOR) and from mice treated with tyrphostin AG1714 before radiation; Figure 19 is a graphical representation showing the number of cells in bone marrow of irradiated mice (450R) and of mice treated with tirphostin AG1714 before radiation; Figure 20 is a graphical representation showing the mortality of mice after lethal radiation (800R) compared to the mortality of mice treated with tyrphostin AG1714 before irradiation over a period of 23 days after radiation; Figure 21 is a graphical representation showing the weight of the lungs of mice inoculated with MCA-15 fibrosarcoma cells (Fig. 21A), Lewis lung carcinoma cells (Fig. 21B) or B-16 melanoma cells (Fig. 21C) and treated with either cisplatin alone, AG1714 alone or with a combination of cisplatin and AG1714; Figure 22 is a graphical representation showing the weight of nude mice carrying human Sk-28 melanoma tumors and treated with doxorubicin, AG1714 or their combination; Figure 23 is a graphical representation showing the volume of the human ovarian carcinoma tumor (OVCAR-3) in mice treated either with cisplatin alone, with AG1714 alone or with their combination; Figure 24 is a graphical representation showing the volume of OVCAR-3 tumor in mice receiving repetitive administration of the treatments described in Figure 23 above; Figure 25 is a graphical representation showing the weight of lungs with MCA-15 fibrosarcoma tumor carrying mice treated with cisplatin alone, AG1801 alone or with a combination of both (Fig. 25A) or with doxorubicin alone, AG1801 alone or its combination (Fig. 25B); Figure 26 is a graphical representation showing the weight of the lungs of mice treated with cisplatin alone, AG1801 alone or with a combination of both; Figure 27 is a graphical representation showing the number of colony forming units (CFU) in cultures of bone marrow cells grown with or without various concentrations of AG1714; Figure 28 is a graphical representation showing the viability of thymocytes grown with or without AG1714 at various time periods after the addition of AG1714 to the cells; Figure 29 is a graphical representation showing the number of beats per minute in cardiomyocyte cultures grown with or without AG1714 as an indication of the viability of the cells; Figure 30 is a graphical representation showing the percentage of lysis measured per cent of specific 51Cr release of target EL-4 cells by murine peritoneal exudates (LEP) cytotoxic lymphocytes in the presence or absence of AG1714. PREPARATIVE EXAMPLES The preparation of several of the above novel compounds is exemplified in the following preparative examples. In all subsequent examples, the novel compounds are synthesized by Knoevenagel condensation of the aldehyde with malononitrile or the substituted amide.

Example 1 - AG1801: 0.45 g of 3 mM p-nitrobenzaldehyde, 0.61 g of 3.5 mM N- (cyanoacetyl) benzylamide (reference 9) and 50 mg of β-alanine in 20 ml of ethanol for 5 hours are refluxed. Cooling and filtering gives 0.67 g, 73% yield, of a light yellow solid, m.p. 164 °. NMR (CDC13) d 8.44 (1H, S, vinyl), 8.35, 8.07 (4H, AB, JAB = 8.8 Hz), 7.35 (5H, m), 6. 6 (1H, br.t, NH), 4.63 (2H, d, J = 5.8 Hz). MS-m / e 307 (m +, 50%), 290 (M-HCN, 63), 260 (MH-N0291), 201 (M-NHCH 2 C 6 H 5, 15), 173 (M-CONHCH 2 C 6 H 5, 13), 155 (22 ), 127 (21), 105 (100). Example 2 - AG1798: 0.4 g of 2.65 mM p-nitrobenzaldehyde, 0.57 g of 2.8 mM N- (cyanoacetyl) 3-propylphenylamide (reference 9) and 40 mg of β-alanine in 30 ml of ethanol per 4 are refluxed. hours. The solution is concentrated by evaporation, cooled and the filtrate gives 0.38 g, 43% yield, of a light yellow solid, m.p. 98 °. NMR (CDC13) d 8.38 (ÍH, S, vinyl), 8.35, 8.06 (4H, ABq, JAB = 8.6 Hz), 7.35 (5H, m), 3.50 (2H, J = 8.0 Hz), 2.74 (2H, t , J = 8.0 Hz), 2.0 (2H, quintet, J = 8.0 Hz). Example 3 - AG1719: 146 mg of 0.87 mM 3-hydroxy-4-nitrobenzaldehyde, 48 mg of 0.89 mM malononitrile dimer and 20 mg of β-alanine in 10 ml of ethanol for 1 hour are refluxed. Evaporation, trituration with CH2C12 hexane and filtration gives 190 mg, 78% yield, of a yellow solid, m.p. 105 °. NMR (acetone d6) d 8.32 (HH, d, J = 8.6 Hz), 8.20 (HH, S, vinyl), 7.77 (1H, d, J = 2.2 Hz), 7.65 (1H, dd, J = 8.6, 2.2 Hz). Example 4 - AG1762: 170 mg of 1 mM 3-fluoro-4-nitrobenzaldehyde, 80 mg of 1.2 mM malononitrile and 15 mg of β-alanine for 1 hour are refluxed. Evaporation, trituration with hexane and filtration gives 202 mg, 93% yield, of a pink solid, m.p. 120 °. NMR (CDC13) d 8.22 (HH, m), 7.83 (3H, m). MS-m / e 217 (m +, 100), 187 (M-NO, 78), 171 (m-N02, 19), 159 (M-NO-HCN-H, 80), 151 (M-N02-HF, 33), 144 (M-N02-HCN, 71), 132 (75), 124 (M-N02-HCN -HF, 81) Example 5 - AG1799 0.4 g of 2.65 mM 4-nitrobenzaldehyde, 0.51 g of 2.8 mM phenylsulfonylacetonitrile and 40 mg of β-alanine in 30 ml of ethanol for 4 hours are refluxed. Cooling, filtering and washing with ethanol gives 0.68 g, 82% yield, of a light yellow solid, m.p. 158 °. NMR (CDC13) d 8.36 (2 H, d, J = 8.6 Hz), 8.31 (H, S, vinyl), 8.07 (4 H, m), 7.70 (3 H, m). Examples of biological and therapeutic activities The biological and therapeutic effect of some of the compounds which can be used in the composition of the invention in the following non-limiting examples will be exemplified with occasional reference to the appended figures.

I. Reduction of mouse mortality caused by doxorubicin or cisplatin by tyrphostins Example 6 Female CDL mice of 6 weeks of age are divided into the following groups: i. Mice injected intraperitoneally (i.p.) with doxorubicin (20 mg / kg); ii. Injected mice i.p. with cisplatin (14 mg / kg); iii. Mice receiving a single injection i.p. of AG1714 (20 mg / kg) two hours before to doxorubicin; and iv. Mice receiving a single injection i.p. of AG1714 (20 mg / kg) two hours before receiving cisplatin. As seen in Figure 1, treatment of mice with AG1714 significantly reduces the mortality of the mice compared to the mortality of mice receiving doxorubicin alone (p = 0.001) (Fig. 1A) or of mice receiving cisplatin alone (Fig. p <0.005) (Fig. IB). EXAMPLE 7 Female CDI mice of six weeks of age are divided into the following two groups: i. Mice receiving 10 injections i.p. of doxorubicin in a concentration of 5 mg / kg for each injection for 21 days (cumulative dose of 50 mg / kg); and ii. Mice receiving 10 injections i.p. of doxorubicin at 5 mg / kg for 21 days with the addition of a single injection i.p. of AG1714 (5 mg / kg) two hours before each injection of doxorubicin. Each group consists of 10 mice and the% mortality of each group is tested as explained above. As seen in Figure 2, the mortality of mice receiving injections of AG1714 before each administration of doxorubicin is significantly reduced compared to the high mortality of mice receiving treatment with doxorubicin alone. Example 8 Two groups of CDl mice are injected with 14 mg / kg of cisplatin as described above. One group receives a single injection i.p. of AG1801 at a low dose of 1 mg / k two hours before receiving the cisplatin injection. As seen in Figure 2, all mice in the group receiving cisplatin only die within ten days of injection. Against this, in the group of mice receiving a single injection of AG1801 in a low dose before the cisplatin injection, the% mortality is only 20%. Example 9 The CDl mice are divided into the following five groups: i. Mice receiving a single injection i.p. of 20 mg / kg doxorubicin; ii. Mice receiving a single injection of 20 mg / kg AG1714 and two hours later an i.p. of 20 mg / kg doxorubicin; iii. Mice receiving a single injection i.p. of 20 mg / kg of AG1782 and two hours later a single injection i.p. of 20 mg / kg doxorubicin; iv. Mice receiving a single injection i.p. of 20 mg / kg of AG126 and two hours later an i.p. of 20 mg / kg doxorubicin. The% mortality in each of the previous groups is determined by classifying the number of dead mice each day up to ten days after the injection. As seen in Figure 10, in the group of mice receiving doxorubicin alone (i), 90% of the mice die within one week after injection. In the other groups of mice receiving the tyrphostins before the administration of doxorubicin, the% mortality is reduced to between 50% (iii) to only 10% (ii). II. Reduction of the toxic effect of the various dangerous agents in organs (kidneys, liver, intestines, heart) of mice treated with tyrphostins Example 10 i.p. groups of CDl mice, each comprising three mice, with a dose of 14 mg / kg cisplatin. The mice are injected in each of the groups except one with a single i.p. of tyrphostin in a dose of 10 mg / kg two hours before the injection of cisplatin. In order to evaluate the toxic effect of cisplatin on the kidneys of the treated mice (nephrotoxicity), the creatinine level in the serum of each mouse is determined four days after the administration of cisplatin using a commercial Sigma kit. As seen in Figure 5, the detected creatinine level of approximately 2 mg / dL in the serum of mice receiving cisplatin alone indicates that there is approximately a 50% reduction in kidney function of these mice.

Against this, the creatinine level measured in the serum of many of the mice receiving an injection of a tyrphostin prior to the cisplatin injection is lower than the creatinine levels in the serum of mice receiving cisplatin alone. This indicates that the injection of tyrphostins before the injection of cisplatin reduces the toxic effect of cisplatin on kidney function. The reducing effect of nephrotoxicity of cisplatin varies between different tyrphostins. For example, AG1824, AG1744, AG1751, AG1714, AG1823, AG1782, AG1801, markedly reduce the nephrotoxicity of cisplatin by bringing creatinine levels in the serum to those of control mice. EXAMPLE 11 CDI female mice are injected with either cisplatin alone or with a single injection comprising either the tyrphostin AG1843 or AG1714 two hours before the cisplatin injection and the level of creatinine in their serum is tested as explained above . As seen in Table 1 below and in accordance with the results of Example 10 above, cisplatin alone has a nephrotoxic effect in mice as observed by its high levels of serum creatinine (1.53). This toxic effect of cisplatin is significantly reduced (to between 0.45-0.70) by injecting tyrphostins two hours before d, and the injection of cisplatin. In this example, the effect of tyrphostin AG1843 administered at a low dose (5 mg / kg) is more effective than the administration of a high dose (20 mg / kg) of the same tyrphostin. Table 1 Example 12 CDl (i.p.) mice are injected with cisplatin (14 mg / kg) or first with AG1714 (10 mg / kg) and two hours later with cisplatin as explained above. Mice in the control group receive vehicle injections only (a stock solution of ethanol: burner (50:50) diluted with saline). Four days after the administration of cisplatin, several parameters indicating nephrotoxicity and liver damage are measured in the sera of each of the mice 4 days after the administration of cisplatin. The parameters that show nephrotoxicity of cisplatin are creatinine and blood urea nitrogen (ÑUS) and the parameters that indicate liver damage are hepatic transaminase alanine transaminase (ALT) and aspartic transaminase (AST) measured by standard methods. Each group consists of three mice and the results are shown as average levels of the parameter measured ± SD. As seen in Figure 6, administration of cisplatin alone to the mice results in elevated levels of creatinine (Fig. 6A) and ÑUS (Fig. 6B). Table 2 below shows that ALT and AST levels are also elevated in these mice. These results indicate both nephrotoxicity as well as damage to the liver of the mice as a result of the administration of cisplatin. As seen in Figure 6 and Table 2, administration of AG1714 two hours before the cisplatin injection results in a reduced level of all four parameters at their normal level as measured in the control mice. Table 2 Prevention of kidney and liver damage induced by cisplatin by AG1714 Example 13 CDl mice are injected with 14 mg / kg of cisplatin or first with a single injection of the tyrphostin AG1714 and two hours later an injection of 14 mg / kg of cisplatin. Histopathological analysis of the kidneys and small intestines of the untreated and treated mice is carried out using slides from fixed tissues with formalin stained with hematoxylin eosin. As seen in Figure 7, the kidneys of mice receiving cisplatin alone show large proteinaceous plaques in the proximal tubules (7B) as well as the appearance of granular material in the columnar epithelium. Against this, the kidneys of the mice receiving an injection of AG1714 before the administration of cisplatin (7C) show no damage and their structure is similar to that of the control mice (7A). As can also be seen in Figure 7, the thin intesttin of mice receiving cisplatin alone (7E) show severe necrosis and disintegration of the columnar intestinal epithelial cells compared with the small intestines of mice receiving AG1714 before the administration of cisplatin (7F) which shows a normal structure similar to that observed in the small intestine of untreated mice (7D). Example 14 In order to test the protective effect of AG1714 against doxorubicin-induced cariotoxicity, cultures of 7-day primary rat cardiomycites are prepared according to the standard procedure (described in: In Vitro Toxicology, Model System and Methods, Eds. C. McQueen, Telford Press New Jersey, 1990, page 163, Primary cultures of neonatal rat myocardial cells, and Kessler-Icekson, G., J. Mol. Cell Cardiol, 20: 649-755, 1988). The cardiomyocyte cultures are divided into the following four groups that receive: i. Vehicle only (control); ii. AG1714 as a final concentration of 20 μM; iii. Cells receiving doxorubicin in a concentration of 10 μM; and iv. Cells receiving AG1714 at a final concentration of 20 μM followed by an hour later by the addition of doxorubicin (10 μM). The beating rate of cardiomyocyte clots ("mini-hearts") is determined 24 hours after the start of incubation. As seen in Table 3, in cell cultures incubated with doxorubicin alone, the number of beats per minute is reduced significantly to about 20% of that of the control cultures incubated with the vehicle alone. Against this, in cultures incubated with doxorubicin together with AG1713, the number of beats per minute is similar to the number of beats per minute in the control cultures. Similar results are observed (not shown) in the cultures of previous cardiomyocytes 16 hours and 40 hours after incubation. In this way, incubation of cultures with AG1714 one hour before the addition of doxorubicin avoids the cardiotoxic effect of doxorubicin in cell cultures. Table 3 Protective effect of AG1714 against doxorubicin-induced cardiotoxicity, in vitro Example 15 The FAS antigen is a cell surface protein that belongs to the two factors of the nerve growth factor receptor family and has been shown to mediate the apoptosis (Itoh, N. et al., Cell., 66: 233-243 (1991)). It is shown that intraperitoneal administration of an anti-FAS antibody in mice causes severe damage of the liver by apoptosis. In order to test the effect of the tyrphostin AG1801 and AG1714 on the hepatoxicity induced by the anti-FAS antibody, the Balb / C i.p. mice are injected. either with AG1801 (5 mg / kg) or with AG1714 (5 mg / kg) and two hours later with an i.p. of an anti-FAS antibody in a dose of 5 μg / mouse. Another group of mice receives only one injection of anti-FAS antibody without prior treatment with the tyrphostins. Five hours after the injection of the anti-FAS antibody, the animals are sacrificed and the level of the two transaminases AST and ALT in the serum of the sacrificed mice is determined: a high level of these transaminases indicating hepatoxicity (Ogasaware, J., et al., Nature, 364: 806-809, 1993). As seen in Figures 8 and 9, the level of AST and ALT in the serum of mice treated with the anti-FAS antibody is only very high indicating hepatoxicity induced by the FAS antibody. Against this, the level of the two transaminases AST and ALT in serum of mice is significantly lower, which are treated with AG1801 (Fig. 8) or with AG1714 (Fig. 9) before the anti-FAS injection. In this way, the administration of tyrphostins to mice results in their protection against the hepatoxicity induced by the anti-FAS antibody. Example 16 T-cell mediated hepatoxicity is a complication of several disorders and diseases such as infection by Hepatitis B virus. Such hepatoxicity can be induced by injection of Conavalin A (Con A). The effect of AG1714 on hepatoxicity induced by Con A in mice is determined in the following manner (see Tiegas, G. et al., J. Clin. Invest., 90: 196-203, 1992). The CDl mice are divided into the following groups: i. Mice receiving vehicle injection only (control): ii. Mice receiving an injection i.p. of AG1714 (10 mg / kg); iii. Mice receiving an i.v. injection of With A (35 mg / mouse); and iv. Mice receiving an injection i.p. of AG1714 (10 mg / kg) and two hours later an i.v. of Con A (35 mg / mouse). 6 hours after injection of Con A into the mice, the serum levels of the AST and ALT enzymes of the liver of treated mice are determined as explained above. The increase in serum levels of these enzymes reflects the damage to the liver. As seen in Figure 10While administration of AG1714 alone has no effect on mice, injection of Con A causes significant hepatoxicity in mice. The administration of AG1714 prior to the administration of Con A significantly reduces the hepatoxicity induced by Con A in these mice. Example 17 It is now known that damage to the liver can be induced by various immune mechanisms such as cytokines as well as by a variety of other agents known to exert an apoptotic effect (for example alcohol or paracetamol). A model for such damage to the liver induced by apoptosis is developed in which the in vivo liver damage is induced by the injection of galactosamine and TNF-α (see Leist, M., et al., J. Immunol., 153: 1778 -1788, 1994). The effect of AG1714 on liver damage induced by TNF in mice is determined as follows: The mice are divided into the following four groups: i. Mice receiving an injection of the vehicle only; ii. Mice receiving an injection i.p. of AG1714 (10 mg / kg); iii. Mice receiving an i.v. injection of TNF-α (0.25 μμμμg / mouse) in combination with galactosamine (GalN) (18 ml / mouse, i.p.); and iv. Mice receiving the treatment of group iii above together with an injection of AG1714 (10 mg / kg) injected i.p. two hours before the injection of TNF / GalN, the serum levels of the liver enzymes AST and ALTR are determined in the serum of the various mice treated as described above. As can be seen in Figure 11, the injection of AG1714 before the administration of TNF / GalN to the mice significantly reduces the hepatoxicity induced by the administration of TNF / GalN. Tyrphostins can therefore be useful in reducing liver damage induced by various immune mechanisms. III. Reduction of the toxic effect of several harmful agents in nucleated bone marrow cells (myelotoxicity) and lymphocytes (lymphotoxicity) by tyrphostins. Example 18 The CDl mice are divided into the following groups: i. Mice receiving an injection i.p. of doxorubicin (10 mg / kg) alone; ii. Mice receiving an injection i.p. of AG1714 (20 mg / kg) and two hours later an i.p. of doxorubicin (10 mg / kg) and iii. Mice receiving an injection of the vehicle and two hours later an injection i.p. of doxorubicin (10 mg / kg). 3 days later, the number of bone marrow nucleated cells and colony forming units (CFU) in the femurs of the treated mice is determined (Nikerich, DA, et al., J. Immunopharmacol., 8: 299-313, 1986). Each group contains 3 mice and the results are expressed as a mean + DE. As seen in Figure 12, the injection of doxorubicin to the mice results in 3 days later in myelotoxicity as manifested by the 47% reduction in the number of nucleated cells (Fig. 12A) and 55% reduction in the UFC (Fig. 12B). Mice that are pretreated with AG1714 two hours before injection with doxorubicin are fully protected against their effects of myelotoxicity.

Example 19 The effect of AG1714 on the doxorubicin-induced myelotoxicity at different doses is investigated. The mice are treated as described in Example 18 above. The number of nucleated bone marrow cells of the femur was determined in the mice treated three days (Fig. 13A) and several times (Fig. 13B) after administration of doxorubicin. As seen in Figure 13A, administration of doxorubicin to mice at a concentration of 10 mg / kg and 15 mg / kg induces myelotoxicity in the treated mice. The pretreatment with AG1714 reduces the myelotoxicity induced with doxorubicin significantly. As seen in Figure 13B, pretreatment of mice administered with doxorubicin with AGI714 also results in rapid recovery of bone marrow cells after administration with doxorubicin. 5 days after treatment with doxorubicin (15 mg / kg) sol, the number of bone marrow cells in the femurs of treated mice is still significantly reduced. The pretreatment of the mice with AG1714 before the administration of doxorubicin, completely reconstitutes their bone marrow at that time. Example 20 CDl mice receive either a single i.p. of doxorubicin (10 mg / kg) or first a single injection i.p. of AG1714 (20 mg / kg) and two hours later an i.p. of doxorubicin (10 mg / kg). 72 hours after the administration of the doxorubicin the mice are sacrificed and the weight of the spleen and thymus of the mice are determined as lymphotoxicity parameters. Each experimental group consists of three mice and the results are no. cell medium +. FROM. As a control, mice receive an injection of the vehicle only in place of doxorubicin. As seen in Figure 14 below, the administration of doxorubicin alone causes a significant reduction in the weight of the spleens (Fig. 14A) and the scams (Fig. 14B) of the mice. This myelotoxic and lympho-toxic effect of doxorubicin is significantly reduced by the administration of AG1714 to mice two hours before administration of doxorubicin. Example 21 CDl mice are injected with a single i.p. injection. of cyclophosphamide in a dose of 50 mg / kg and is divided into the following groups: i. Mice receiving a single injection i.p. of the vehicle only two hours before the administration of cyclophosphamide; ii. Mice receiving a single injection i.p. of AG1714 (20 mg / kg) two hours before to cyclophosphamide; iii. Mice receiving a single injection i.p. of AG1801 (2.5 mg / kg) two hours before the administration of cyclophosphamide. 72 hours after the administration of the cyclophosphamide the mice are sacrificed and the nucleated cells are counted in the bone marrow, spleen and thymus of the mice. Each experimental group consists of three mice and the results shown are the average number of cells in a group ± DE. As observed in Table 4a and B, the administration of cyclophosphamide causes a significant reduction in the number of nucleated cells in the bone marrow, spleen and thymus indicating myelotoxic and lymphatic effect of cyclophosphamide. The administration of tyrphostins AG1714 or AG1801 before administration of cyclophosphamide results in a reduction in the myelotoxic and lymphatic effect of cyclophosphamide in the three anterior organs. The number of nucleated cells in these organs is higher than in organs of mice receiving cyclophosphamide alone although the level of nucleated cells in the bone marrow, spleen and thymus of the mice treated with the tyrphostins do not reach the level of control.

Table 4a and b AG1714 and AG1801 prevent myelotoxicity and linfolysis induced by cyclophosphamide or EXAMPLE 22 Female CDl mice are divided into the following groups: i. A control group that receives a single injection i.p. of the vehicle alone (cosolvent: propylene carbonate cremophor saline solution); ii. Mice receiving a single injection i.p. of 5-fluorouracil (5 FU) in a dose of 80 mg / kg; iii. Mice receiving a single injection i.p. of AG1714 in a dose of 20 mg / kg; iv. Mice receiving a single injection i.p. of AG1801 in a dose of 2.5 mg / kg; v. Mice receiving a single injection i.p. of AGÍ843 in a dose of 5 mg / kg; saw. Mice receiving a single injection i.p. of AG1714 (20 mg / kg) and two hours later a single injection i.p. of 5 FU (80 mg / kg); vii. Mice receiving a single injection i.p. of AG1801 (2.5 mg / kg) and two hours later a single injection i.p. of 5 FU (80 mg / kg); and viii. Mice receiving a single injection i.p. of AG1843 (mmg / kg) and two hours later a single injection i.p. of 5 FU (80 mg / kg). Five days after administration of 5 FU to the mice, the mice are sacrificed and the number of nucleated cells in the bone marrow of a femur is counted. Each group of mice consists of two or three mice and the results shown are the mean number of cells ± SD, as shown in Figure 15, the administration of FU to mice results in a decrease in the number of nucleated cells in the bone marrow of these mice. Administration of AG1714 before administration of 5 FU to the mice results in a significantly lower reduction in the number of cells in the bone marrow. In this way AG1714 reduces the myelotoxic effect of 5 FU in these mice. EXAMPLE 23 Female CDl mice are divided into the following groups: i. A control group that receives a single injection i.p. of the vehicle alone (cosolvent: propylene carbonate cremophor saline solution); ii. Mice receiving a single injection i.p. of mitomycin-C in a dose of 2 mg / kg; iii. Mice receiving a single injection i.p. of AG1714 (20 mg / kg) and two hours later a single injection i.p. of mithomycin-C (2 mg / kg); As described in Example 22 above, five days after the administration of mitomycin-C to the mice, mice are sacrificed and the number of nucleated cells in the bone marrow of a femur is counted. Each group of mice consists of two or three mice and the results shown are the mean number of + cells. FROM. As seen in Figure 16, mice receiving mitomycin C alone show a very low count of nucleated cells in their bone marrow. While administration of AG1714 alone has no effect on mice, its administration prior to the administration of mitomycin C to mice, results in a reduction in the myelotoxic effect of mitomycin C in these mice and provides almost total protection from the harmful effects of mitomycin C leading to the count of nucleated cells in the bone marrow of these mice at the level of control mice receiving only the cosolvent. Example 24 The effect of various doses of AG1801 against the myoxotoxicity induced by doxorubicin in mice is determined. The CDl mice are divided into five groups: i. Mice that receive the vehicle sun; ii. Mice receiving doxorubicin (10 mg / kg) alone; iii. Mice receiving an injection i.p. of AG1801 (0.5 mg / kg) two hours before the administration of doxorubicin; iv. Mice receiving an injection i.p. of AG1801 (1 mg / kg) two hours before administration of doxorubicin; and V. Mice receiving an injection i.p. of AG1801 (2 mg / kg) two hours before administration of doxorubicin. Three days after the administration of doxorubicin, the mice are sacrificed and the number of nucleated cells in the bone marrow is counted as described above. As seen in Figure 17, administration of AG1801 to mice has a protective effect against doxorubicin-induced myelotoxicity which is dose-dependent. IV Reduction of tyrphostin-induced toxicity Example 25 In order to test the protective activity of tyrphostin AG1714 in irradiated mice, CDI mice irradiated with a radiation dose of 3 OOR were divided into two groups using a cobalt source : i. Mice that do not receive additional treatment; and ii. Mice receiving a single injection i.p. of the tyrphostin AG1714 in a dose of 20 mg / kg two hours before irradiation. A third group of mice receives only one injection of AG1714 (20 mg / kg) and a fourth group of mice serves as control mice which are not treated. At different periods of time after radiation, the mice are sacrificed and the number of cells in the bone marrow of a femur is counted. As seen in Figure 18, irradiation of mice with 3 OOR causes a decrease in the number of cells in the bone marrow of these mice compared to untreated mice or mice treated only with AG1714. The reconstitution of cells in the bone marrow of the irradiated mice begins approximately five days after their radiation. The treatment of mice irradiated with AG1714 prevents the decrease in the number of cells in the bone marrow of the irradiated mice and causes a very significant improvement in the number of cells in their bone marrow. In this way, the tyrphostin AG1714 has a protective effect against the myelotoxicity caused by the irradiation. Example 26 The protective effect of tyrphostin AG1714 against myelotoxicity caused by a high dose of radiation (450R) is tested as described in Example 25 above except that the radiation dose is 450R instead of 300R. As seen in Figure 19, the number of cells in the bone marrow of mice irradiated with 450R was significantly reduced five days after radiation indicating a severe myelotoxic effect of radiation in these mice. Five days after radiation the reconstitution of the number of cells in the bone marrow begins but 18 days after radiation, the number of cells in the bone marrow of the irradiated mice still remains significantly lower than the number of cells in the bone marrow of untreated mice or mice receiving AG1714 alone. In the bone marrow of mice receiving AG1714 before their radiation, the myelotoxic effect is also observed up to five days after radiation. However, after five days, a significant reconstitution is observed in the number of cells in the bone marrow of these mice and 18 days after radiation, the number of cells in the bone marrow of the irradiated mice receiving AG1714 is more high that the number of cells in the control mice. In this way the administration of AG1714 increases the reconstitution of the number of cells in the bone marrow of mice irradiated by a high dose of radiation. Example 27 CDl mice are irradiated with a lethal dose 800R using a cobalt source, and divided into two groups: i. Mice that receive only radiation; and ii. Mice receiving a single injection i.p. of AG1714 in a dose of 20 mg / kg one hour before radiation. Each group of mice consists of 10 mice and the% mortality in each group is tested by classifying the number of mice killed each day after radiation. As seen in Figure 20, in the group of mice receiving 800R radiation, the mortality of the mice begins 10 days after radiation and the% mortality in this group reaches the 80% proportion 23 days after its radiation. Against this, the% mortality in the group of mice receiving AG1714 before their radiation is significantly reduced to 20% mortality 20 days after radiation. Mortality in both groups does not change further until 50 days after radiation. V. Tfphostin Ephesus in the Antitumor Activity of Chemotherapeutic Agents Example 28 The effect of AG1714 on the antitumor effect of chemotherapy is determined using a variety of experimental tumor models in mice. 2xl05 / mouse MCA-105 fibrosarcoma cells or Lewis lung carcinoma cells and 5xl04 / mouse B-16 melanoma cells are injected into the tail vein of C57BL mice.

Cisplatin (4 mg / kg) i.p. 4 days after the tumor inoculation. AG1714 (20 mg / kg) i.p. 2 hours before to cisplatin. Twenty-four days after the tumor inoculation, the mice are sacrificed, the lungs are weighed and the number of metastases is classified. Each group contains 5 mice and the results are expressed as the average weights of the lungs ± SD. As seen in Figure 21A, cisplatin markedly inhibits MCA-105 fibrosarcoma growth. AG1714 by itself has a small antitumor effect. The combined treatment with AG1714 and cisplatin is as effective as cisplatin alone in suppressing the growth of established lung metastasis of MCA-105. Cisplatin and AG1714 by themselves have a partial antitumor effect against Lewis lung carcinoma. The combined treatment with AG1714 and cisplatin is more effective than cisplatin alone in suppressing the growth of established lung metastases from this tumor (Fig. 21B). Cisplatin (4 mg / kg) or AG1714 (20 mg / kg) alone has no effect against the established lung metastasis of melanoma B-16 (Fig. 21C). The combined treatment with these agents is more effective than the treatment with each agent alone. Example 29 The effect of doxorubicin and AG1714 on xenografts of SK-28 human melanoma tumors in nude mice is determined as follows: CDI (un / un) i.v. nude mice are inoculated. with 4xl05 / human melanoma mouse SK-28. Doxorubicin (4 mg / kg) i.p. 4 days after the tumor inoculation. AG1714 (20 mg / kg) or vehicle is injected 2 hours before to doxorubicin. The lung weights of treated mice and the number of metastases in the lungs are determined 24 days after the tumor inoculation. Each group contains five mice and the results are expressed as mean ± SD. As seen in Figure 22, doxorubicin markedly suppresses SK-28 tumor growth while AG1714 itself has a small antitumor effect. Pretreatment with AG1714 does not damage the effect of doxorubicin alone in suppressing the growth of this tumor. EXAMPLE 30 The nude mice are inoculated subcutaneously (s.c.) with human ovarian carcinoma cells (OVCAR-3) (3xlOG cells / site). Cisplatin (4 mg / kg) is injected 4 days after tumor inoculation. AG1714 (20 mg / kg) or i.p. vehicle is injected. two hours before the cisplatin. Twenty days post tumor inoculation, the short (c) and long (L) diameters of the tumor are measured and the tumor volume (V) is calculated according to the formula: V =. { [S2xL] / 2].

Each group contains 5 mice and the results are expressed as the mean ± SD. As seen in Figure 23, cisplatin has a marked antitumor effect in the mouse bearing the OVCAR-3 tumors while AG1714 itself is less effective. The administration of AG1714 does not damage the chemotherapeutic effect of cisplatin. As seen in Figure 24, the difference in diameter of the tumor volume of tumors in mice receiving repeated injections of cisplatin and AG1714 compared to the volume of tumors in mice receiving repeated injections of cisplatin alone or repeated injections of AG1714 alone. Example 31 The effect of AG1714 on the high dose efficacy of doxorubicin is measured by determining the mortality of the mice carrying the B-16 melanoma and the number of lung metastases or lung weight. The i.v. mice are injected. with 2xl05 B-16 melanoma cells and 4 days after tumor inoculation each mouse receives a single injection i.p. of doxorubicin (4 mg / kg 8 mg / kg). Two hours before the injection of doxorubicin, each i.p. mouse is injected. with AG1714 at a dose of 20 mg / kg. As seen in Table 5 a and B showing two experiments performed as explained above, administration of doxorubicin at low dose results in low mortality of the mice and a significant therapeutic effect. The high dose administration of doxorubicin results in a high mortality rate of the treated mice. AG1714 administered alone shows some therapeutic effect by itself. The administration of doxorubicin in a high dose together with AG1714 results in the high therapeutic effect of high doxorubicin, however, the mortality of the mice is significantly reduced. Table 5 Effect of AG1714 on the efficacy of doxorubicin in high dose and mortality in mice carrying melanoma B-16 A.

B.

In this way, the combined administration of AG1714 together with doxorubicin results in the intensification of the chemotherapeutic effect of doxorubicin. The significant therapeutic effect of doxorubicin in high dose administered with AG1714 can be achieved while neutralizing the high mortality rate of doxorubicin in high dose by combined administration. Such an intensification effect can also be observed in mice carrying MCA-105 fibrosarcoma tumors and treated with either doxorubicin alone (Table 6A) or cisplatin alone (Table 6B) or with a combination of each of the above with AG1714 . Table 6 Effect of AG1714 on the efficacy of doxorubicin in high dose and mortality in mice carrying fibrosarcoma MCA-105 B. Example 32 The effect of AG1801 on the anti-metastatic activity of cisplatin in established lung metastasis of mice bearing fibrosarcoma MCA-105 is determined as follows: C57BL mice are divided into the following groups: i. Injected mice i.v. at the age of six weeks with 2xl05 MCA105 cells of firbosarcoma and 4 days 1 later, they receive an injection comprising the vehicle only; ii. Injected mice i.v. at the age of 6 weeks with 2 xlO5 cells MCA105 of fibrosarcoma and 4 days1 later, injected with 10 mg / kg of cisplatin; iii. Injected mice i.v. at the age of 6 weeks with 2xl05 MCA105 cells of fibrosarcoma and 4 days1 later, injected with 10 mg / kg of doxorubicin; iv. Injected mice i.v. at the age of 6 weeks with 2xl05 MCA105 cells of fibrosarcoma and 4 days1 later, injected with 5 mg / kg of AG1801; v. Injected mice i.v. at the age of 6 weeks with 2xl05 MCA105 cells of fibrosarcoma and 4 days1 later, injected with 5 mg / kg of AG1801 and 2 hours1 later, with 10 mg / kg of cisplatin; saw. Injected mice i.v. at the age of 6 weeks with 2xl05 MCA105 cells of fibrosarcoma and 4 days1 later, injected with 5 mg / kg of AG1801 and 2 hours1 later, with 10 mg / kg of doxorubicin; and vii. C57BL mice not treated. As seen in Figure 25A and 25B, the weight of the lungs of the mice carrying the tumor is significantly greater than that of the control mice. While the injection of AG1801 alone has no effect, the injection of cisplatin (Fig. 25A) or doxorubicin (Fig. 25B) reduces the weight of the lungs of the mice to a weight similar to that of mice that do not carry control tumor. . The combined injection of cisplatin and AG1801 (Fig. 25A) and the combined injection of doxorubicin and AG1801 (Fig. 25B) results in the reduction of tumor burden in the lungs of mice similar to the reduction in tumor load of mice who receive cisplatin alone. The results of the four previous examples clearly show that the administration of tyrphostins to mice carrying the tumor together with cisplatin does not affect the antitumor effect of cisplatin and in some cases even increases it. Example 33 The effect of cisplatin and AG1801 on SK-28 human melanoma tumor xenografts in nude mice is determined as explained in Example 29 above. As seen in Figure 26, the administration of cisplatin alone reduces the weight of the lungs of the mice bearing the tumor to a certain degree while the administration of AG1801 has no therapeutic effect by itself. The combined administration of AG1801 with cisplatin significantly reduces the proportion of the lungs of the treated mice and in this way the administration of AG1801 before the administration of cisplatin increases its therapeutic effect. The above examples clearly show that the administration of tyrphostins to mice carrying the tumor together with cisplatin or doxorubicin does not damage the antitumor effect of the therapeutic agents and in some cases even increases it. Therefore tyrphostins may be useful in potentiating and intensifying the chemotherapeutic effect of such agents. SAW. The effect of AG1714 on the viability of several cells in vitro is determined as follows: EXAMPLE 34 Nucleated murine bone marrow cells are prepared by gradient centrifugation in histoplaque. The cells are then divided into two groups: i. Cells incubated for one hour in growth medium alone (control); ii. Cells incubated for one hour in growth medium with the addition of several concentrations (5 μM, 10 μM or 20 μM) of the tirphostin AGÍ714. The cells are plated on semi-solid agar and the number of colony forming units (CFU) is determined after a period of 14 days of incubation (see: Nikerich et al., Supra, 1986). As seen in Figure 27, the efficiency of plaque formation (determined by the number of CFUs per 105 nucleated cells) of the cultures to which it is added AG1714 is significantly increased compared to that of cells grown in growth medium alone. In this way, tyrphostin AG1714 allows greater conservation of bone marrow cell culture. EXAMPLE 35 Thymocytes are prepared from thymuses of young CDl mice at a concentration of 3 x 10 cells / ml in RPN11640 growth medium containing 5% FCS. The cells are divided into two groups: i. Cells grown in the presence of the growth medium only; and ii. Cells grown in growth medium with the addition of AG1714. The cell viability of the thymocytes in each of the above cultures is determined using the trypsin blue exclusion test at various time periods after the addition of AG1714 to the cells. As seen in Figure 28, the addition of AG1714 to the thymocytes in the culture significantly increases its viability. Example 36 Cultures of primary rat myocytes are prepared from rats as described in Kessler-Iceksan, G., J. Mol. Cell. Cardiol., 20: 649-755, 1988. The cardiomyocytes are grown in culture for seven days and then the cultures are divided into two groups. i. Cultures grown in culture medium alone; ii. Cultures grown in growth culture with the addition of 20 μM of the tyrphostin AGÍ144. The number of beats per minute of the myocyte clots in each culture is determined 16 hours and 40 hours after the addition of the tyrphostins to the cells. As shown in Figure 29, the number of beats per minute in the cultures of cardiomyocytes to which the tyrphostin AG1714 is added is significantly higher than the number of beats per minute in the cultures grown in the growth medium alone. AG1714 increases the viability of cardiomyocytes in culture. VII. Tyrphostin toxicity study Example 37 In order to study the toxic effects of tyrphostins AG1714 and AG1801, tyrphostins are injected into ICR mice 3 times a week for a period of five weeks. Each injection contains 20 ml of the tyrphostins which is prepared in the propylene carbonate vehicle: cremophor (40/60) diluted 1:20 with saline / bicarbonate. In order to test the toxic effect of the cumulative administration of tyrphostins, several parameters of the injected mice are determined including their weight, weight of their internal organs (thymus, spleen, kidney), total blood chemistry, number of marrow cells bone, etc. As seen in Table 7 below, the injection of the tyrphostins AG1714 and AG1801 does not cause significant changes in the parameters tested in the injected mice compared to the same parameters in control mice injected with saline or the vehicle alone. Therefore, toxic effects of tyrphostins are not apparent after cumulative administration in mice. In addition, typhostin injection does not cause mortality in mice. Table 7 Subchronic intraperitoneal administration of AG1714 and AG1801 (female ICR mice, injection 3 times in a week, 5 weeks) Clots (in 8170 ± 697 15000 + 2140 13410 + - 1740 femur) Vehicle - propylene carbonate: Cremophor (40:60) Diluted 1:20 with saline / bicarbonate VIII. Inhibition of specific immune activities Example 38 Cytotoxic lymphocytes of murine peritoneal exudates (LEP) capable of causing specific lysis of EL-4 cells are prepared and tested as described in Lavy, R. et al., J. Immunol. 154: 5039-5048, 1995. The EL-4 cells are divided into cells that are incubated with LEP alone, cells that are incubated with AG1714 (20 μM) and then with LEP and cells that are incubated with AG1714 (50 μM) and then with LEP. The lysis of target EL-4 cells is determined using the specific 51C release assay. As seen in Figure 30, the addition of AG1714 reduces the specific cytotoxic activity of the LEPs against the target EL-4 cells. The inhibitory effect of AG1714 is dose dependent. The above results indicate that tyrphostins may be useful for reducing the immune mediated rejection of transplants, in cases of autoimmunity and in conditions involving specific immune activity against cells (for example anti-CD4 cells such as in the case of AIDS).

IX. Anti-inflammatory effect of tyrphostins One of the main complications of an inflammatory reaction in an individual is the development of septic shock which results from the effects of the immune reaction which is mediated mainly by cytokines such as TNF and IL-1. The implication of tyrphostins in TNF and IL-1 related to immune responses is determined. Example 39 A. Incubation of mouse fibroblast cells with RNF-a results in apoptosis of these cells. In order to test the effect of tyrphostins on apoptosis induced by TNF-α cells, the mouse A9 fibroblast cells are incubated for two hours with cycloheximide (50 μg / ml) after which the various tyrphostins are added. to the final concentration of 5 μM. After one hour of incubation with tyrphostins, TNF-α is added to the cells in a concentration of 0.2 ng / ml and the cells are incubated for an additional ten hours. At the end of ten hours, the cell cultures are analyzed by FACS using propidium iodide and the degree of apoptosis in each of the cell cultures is determined (see as DNA fragmentation) (see Novogrodsky A., et al., Science, 264: 1319-1322, 1994). As seen in Table 8 below, the addition of the various tirfostins to mouse fibroblast cells incubated with TNF, reduces the apoptotic effect of TNF significantly. The degree of reduction of the apoptotic effect of TNF by the various tyrphostins is slightly different. Table 8 Prevention of TNF-α induced apoptosis of mouse fibroblast cells (A-9) by typhostins * All tyrphostins are added to a final concentration of 20 μM, except AG1801, which is added to a final concentration of 5 μM. B. As seen in Table 9, cell cycle analysis of the above cell cultures shows that treatment of the cells with TNF-α induces G arrest. There is a direct correlation between the reduction of tyrphostins from the apoptotic effect caused by TNF and their ability to prevent the arrest of Gi in cell cultures. Table 9 Prevention by AG1714 of Go / Gl arrest induced by TNF-α and apoptosis in mouse fibroblast cells (A-9) Example Q The effect of tyrphostins on TNF-induced cytotoxicity in vitro is determined using A-9 cells as described in Example 39 above. In addition, the effect of tyrphostin on TNF-mediated cytotoxicity in vivo in mice injected with LPS (which induces TNF toxicity in vivo) is determined. It injects i.p. LPS of E. coli at the concentration of 1.3 mg / mouse to CDl mice. While the control mice are also injected with the vehicle alone, the remaining mice are injected with the various tyrphostins (20 mg / kg) i.p. two hours before injection with LPS. As seen in Table 10 a, in accordance with the results shown in Example 39 above, the addition of tyrphostins to cells incubated with TNF significantly reduces the cytotoxic effect of TNF. As seen in Table 10B, most tyrphostins show a significant protective effect against the toxicity induced by LPS in vivo which correlates with their in vitro activity. Table 10 Effect of tyrphostins on TNF-induced cytotoxicity (in vitro) and lethal toxicity induced by LPS (in vivo) A B Example 41 IL-1 is one of the major mediators of septic shock in vivo. In order to determine the inhibitory activity of AG1714 in the production of IL-1 from cells incubated with LPS, the human peripheral blood monocytes (PBM) are divided into a concentration of 2x10 cells / ml in the following groups: i. Cells grown in growth medium alone; ii. Cells grown in growth medium to which AG1714 (20 μM) is added; iii. Cells grown in growth medium to which LPS (10 mg / ml) is added; and iv. Cells grown in growth medium with the addition of LPS (10 mg / ml) and AG1714 (20 μM). The cells are incubated for 24 hours and the amount of IL-1β is determined using the Genzyme ELISA kit. As seen in Table 11, which shows two experiments performed as described above, the addition of AG1714 to cells incubated with LPS significantly reduces the amount of IL-lβ produced by the cells. Table 11 Inhibition of AG1714 of IL-lβ production induced by LPS in human peripheral mononuclear cells IL-lβ pg / ml AG1714 (20 μM) μ Exp. 1 - + None 2522 1174 LPS (10 μg / ml) 10187 1230 Exp. 2 - + None 583 648 LPS (10 μg / ml) 9309 970 The above results indicate that tyrphostins may be useful in reducing the effect mediated by unwanted IL-1 involved in septic shock. The results of the three previous examples indicate that tyrphostins may be useful in reducing or preventing the development of septic shock during an inflammatory response. Tyrphostins can also be useful as anti-inflammatory agents and in counteracting the pathogenic effects mediated by various cytokines (for example TNF and IL-1) which occurs for example in autoimmunity.

Claims (27)

1. CLAIMS 1. Pharmaceutical compositions for counteracting damage to cells or tissue characterized in that it comprises an effective amount of a compound of the general formula: where: Ar is a group of the formula: 0) (ü) - n is 0 or, when Ar has the formula (i) above, then n can also be 1, CN, -C (S) NH2, -C (0) NHR3 or, when Ri is 4- N0 and R2 is H or 3 -OH, then R can also be a group of the formula: wherein R3 is H, phenyl, phenyl (lower alkyl) or pyridylmethyl; -Ri and R2 are each independently H, OH, N02 or, when R is CN, also CH3, F, or CF3, with the proviso that both Rx and R2 are not simultaneously H, together with a pharmaceutically acceptable carrier.
2. 2. Pharmaceutical compositions according to claim 1, characterized in that they comprise a compound of the formula I wherein R is CN, -C (S) NH2, -C (O) NHCH2C6HS or a group of the formula and n is 0, Rx is 4-N02 and R2 is H.
3. 3. Pharmaceutical compositions according to claim 1 or 2, characterized in that they counteract the damage caused by a cytotoxic drug.
4. 4. Pharmaceutical compositions according to claim 3, characterized in that they counteract the damage caused by an antineoplastic drug.
5. 5. Pharmaceutical compositions according to claim 3 or 4, characterized in that they comprise an effective amount of compounds of the formula I in claim 1, in combination with a cytotoxic drug.
6. 6. Pharmaceutical compositions according to any of claims 3-5, characterized in that the drug is selected from the group consisting of cisplatin, doxorubicin, cyclophosphamide, mitomycin C and 5-fluoroacyl.
7. 7. A pharmaceutical composition according to any of claims 1-6, characterized in that it counteracts myelotoxicity and lymphotoxicity.
8. 8. Pharmaceutical compositions according to any of the preceding claims characterized in that they are in a form suitable for intravenous administration.
9. 9. Pharmaceutical compositions according to any of the preceding claims characterized in that they are in a form suitable for oral administration.
10. 10. Pharmaceutical compositions according to claim 1 or 2, characterized in that they are used in ex vivo conservation of cells, tissues or organs.
11. 11. Pharmaceutical compositions according to claim 1 or 2, characterized in that they counteract the damage caused by an immune-mediated inflammatory response.
12. 12. A pharmaceutical composition according to any of claims 1-10, characterized in that it counteracts unwanted apoptosis.
13. 13. A method for treating an individual, to counteract damage to cells, tissues or organs, the method characterized in that it comprises administering to the individual an effective amount of a compound of the general formula. where: Ar is a group of formulas 0) (ü) is 0 or, when Ar has the formula (i) above, then n can also be 1, CN, -C (S) NH2, -C (0) NHR3 or, when Ri is 4-N02 and R2 is H or 3 -OH, then R can also be a group of the formula: wherein R3 is H, phenyl, phenyl (lower alkyl) or pyridylmethyl; -Ri and R2 are each independently H, OH, N02 or, when R is CN, also CH3, F, or CF3, with the proviso that both Ri and R2 are not simultaneously H, together with a pharmaceutically acceptable carrier.
14. 14. A method according to claim 13, characterized in that the compound of the formula I wherein R is CN, -C (S) NH2, -C (0) NHCH2C6H5 or a group of the formula and n is 0, Rx is 4-N02 and R2 is H.
15. 15. A method according to claim 13 or 14, characterized in that the dangerous agent is a cytotoxic drug.
16. 16. A method in accordance with the claim 15, characterized in that the anti-toxic drug is an antineoplastic drug.
17. 17. A method in accordance with the claim 16, characterized in that the cytotoxic drug is selected from the group consisting of cisplatin, doxorubicin, cyclophosphamide, mitomycin C and 5-fluoroacyl.
18. 18. A method according to any of claims 13-17, characterized in that it counteracts myelotoxicity and lymphotoxicity.
19. 19. A method according to claim 13 2, characterized in that it counteracts undesired hazardous effects of the radiation treatment.
20. 20. A method according to any of claims 13 or 19, characterized in that the compound of the formula I is administered in combination with the cytotoxic drug or radiation treatment.
21. 21. A method according to any of claims 1 to 20, characterized in that the compound of the formula I is administered before the administration of the cytotoxic drug or radiation treatment.
22. 22. A method according to any of claims 13 to 21, characterized in that the compound of the formula I is administered intravenously.
23. 23. A method according to any of claims 13 to 21, characterized in that the compound of the formula I is administered orally.
24. 24. A method according to claim 13 or 14, characterized in that it counteracts the damage caused by an inflammatory or immune mediated response.
25. 25. A method for the ex vivo preservation of cells, tissues or organs, characterized in that it comprises contacting the organ, tissue or cells ex vivo with a compound of the general formula. where: - Ar is a group of the formulas: 0) (ii) - n is 0 or, when Ar has the formula (i) above, then n can also be 1, CN, -C (S) NH2, - C (0) NHR3 or, when Rx is 4-N02 and R2 is H or 3 -OH, then R can also be a group of the formula: wherein R3 is H, phenyl, phenyl (lower alkyl) or pyridylmethyl; -Ri and R2 are each independently H, OH, N02 or, when R is CN, also CH3, F, or CF3, with the proviso that both Ri and R2 are not simultaneously H, together with a pharmaceutically acceptable carrier.
26. 26. A compound of the general formula. where: Ar is a group of formulas: R (i) (ü) n is 0 or, when Ar has the formula (i) above, R is different from CN, then n can also be 1, - R is CN, -C (S) NH2, -C (0 ) NHR3 or, when Rx is 4-N02 and R2 is H, then R can also be a group of the formulas: -Ri and R2 are each independently H, OH, N02 or, when R is CN, also CH3, F, or CF3, with the proviso that both Rx and R2 are not simultaneously H, or OH; and R3 is H, phenyl, phenyl (lower alkyl) or pyridylmethyl; With the conditions that a) When R is CN and n is 0, then (aa) if one of Ri and R2 is H or OH, then the other can not represent N02; (ab) if one of Rx and R2 is H or F, then the other can not represent H or F; and b) When Rx is 4-N02, R2 is H and n is 0; then R can not represent C (0) NH2 or -C (S) NH2.
27. 27. A compound according to claim 26, characterized in that R is CN, -C (S) NH2, C (0) NHCH2C6H5 or a group of the formula and n is 0, R is 4-N02 and R2 is H.