MXPA96002846A - Vanadato compounds for the treatment of proliferative disorders, metastases, and farm-resistant tumors - Google Patents

Abstract

The present invention relates to the use of vanadate compounds or derivatives or analogs of vanadate compounds as antiproliferative and antimetastatic agents, and / or to the treatment of drug-resistant tumors in animals, to compositions containing vanadate compounds adapted for said use to methods for the treatment of proliferative disorders, to methods for reducing the ability of a tumor to metastasize, and to methods for the treatment of drug-resistant tumors. The invention also relates to methods for testing substances that affect cell proliferation.

Description

VANADATO COMPOUNDS FOR THE TREATMENT OF PROLIFERATIVE DISORDERS. MET STASIS, AND TUMORS RESISTANT TO THE F RMACOS FIELD OF THE INVENTION The present invention relates to the use of vanadate compounds, or derivatives or analogues of vanadate compounds, as antiproliferative and antimetastatic agents, and / or for the treatment of drug-resistant tumors in animals; to compositions containing vanadate compounds adapted for said use; to methods for the treatment of proliferative disorders, to methods for reducing the ability of a tumor to metastasize, and to methods for the treatment of drug-resistant tumors. The invention also relates to methods for testing substances that affect cell proliferation.

BACKGROUND OF THE INVENTION Cancer is a global problem that affects an estimate of 5.9 million people worldwide annually. There are many cancers, some of the most common in North America include breast, lung, colon, and lymphatic cancers. Although chemotherapy has had a positive impact on the survival rate of cancer patients over the past 30 years, most human cancers are, or have become resistant to, chemotherapy. Therefore, there is a tremendous need for anticancer drugs that are more effective and can act on drug-resistant tumors. Two important characteristics of cancer cells are their ability to proliferate abnormally leading to the formation and growth of tumors, and to invade other tissues, leading to metastasis. It is thought that genetic damage to specific genes is responsible for the transformation of cells and the development of cancer in humans. The genetic damage found in human cancer cells can be divided into two types. One of these involves the mutation of oncogenes, which results in continuous proto-oncogene activation. The second involves the mutation of the tumor suppressor genes, which results in the loss of their function. Genetic damage to proto-oncogenes or tumor suppressor genes leads to oncogenic activation in the absence of stimuli, and uncontrolled cell proliferation. Damage has been found to some proto-oncogenes and tumor suppressor genes with some consistency in a variety of human malignancies. r It has been shown that two oncogenic transcription factors, fos and jun, are involved and are required for the induction of genes involved in cell proliferation, and in particular, in the cell proliferation of many tumor cell lines. The inhibition of the expression of these two genes leads to the inhibition of cell proliferation. One of the most life threatening aspects of cancer is the development of metastasis. In general, most solid tumors can be removed surgically from the primary site resulting in a local cure. However, if the cancer cells have invaded the vascular channels and metastasized to a different organ, then the possibility of a complete cure is reduced. Accordingly, agents that reduce the metastatic properties of cancer cells for the treatment of cancer would be beneficial. The cellular processes thought to have an important role in metastasis include: increased cell binding, proteolysis of tumor cells from the host tissue, locomotion of tumor cells, and colony formation. These processes are presented in an order in sequence. First, the tumor cells bind to the supporting membrane through their surface receptors of the integrin and non-integrin types with ligands such as collagen, laminin, and fibromectin in the support membrane. After binding, a localized zone of lysis of the supporting membrane at the point of cell attachment occurs. The tumor cells produce and secrete degrading enzymes, such as collagenase, and gelatinase, which degrade the supporting membrane, and allow the infiltration and locomotion of the tumor cells into the host organ. There is a positive association between tumor aggressiveness and the ability of cells to produce a group of enzymes, matrix metalloproteases, involved in the invasive process. Inhibition of certain proteases, such as metalloproteases or serine proteases, have been shown to prevent invasion and metastasis (Alvarez et al., 1990, J. Nati. Cancer Inst. 82: 589-595; Schultz et al 1988, Cancer Res. 48, 5539-5545; and ang & Stearns 1988, Cancer Res. 48, 6262-6271). Ionic vanadium compounds, such as vanadyl or vanadate salts in combination with the thiosulfate or sulfite compounds, have been reported as useful for the treatment of malignant tumors, atherosclerosis, and mental syndromes in the elderly (Patent Application of the United States of America with serial number 5,045,316 of Kaplan). Kaplan describes a daily dose of 0.0043 milligrams / kilogram to 0.14 milligrams / kilogram of vanadyl or vanadate salts. Kaplan does not provide a mechanism for the action of vanadate and thiosulfate in the treatments described. In the background of the Kaplan patent, it is reported that others have reported that vanadium salts have an antineoplastic effect, and dietary vanadyl sulfate has been reported to inhibit chemically-induced mammary carcinogenesis in rats. Saxena et al. (Biochem. Pharmacology 45 (3): 5 539-542, 1993) examined the in vivo effects of vanadate on the antioxidant status of diabetic and alloxan rat livers and control. Diabetic rats were given 0.6 milligrams of sodium orthovanadate / milliliter in drinking water. It should be noted that LT present inventor has discovered that oral administration of orthovanadate to animals at 0.5 milligrams / milliliter results in gastric toxicity (see Example 9 herein). It has been reported that antioxidants such as β-15 carotene, α-tocopherol, vitamin E, vitamin C, and glutathione, have an activity against cancer (G. Shklar et al., Nutrition and Cancer, 1993, page 145). It has also been described that a mixture of antioxidants (β-carotene, dl-a-tocopherol acid succinate (vitamin E), vitamin C, and reduced glutathione), was very effective in preventing carcinogenesis in a cancer model in vivo, and was more effective than the individual components of the mixture as agents for cancer prevention.

SUMMARY OF THE INVENTION The present inventor has discovered that the levels of superoxides or H0 in the cell have an important role in the induction of fos and jun expression. The reduction of H20 levels by inhibiting their production with diphenylic iodonium (DPI), or by increasing the levels of intracellular reducing agents such as N-acetyl cysteine and orthovanadate, showed to completely inhibit the expression of fos and jun in response to factors such as IL 1 or arachidonic acid. Under all the conditions examined, the inhibition of fos and jun expression results in the inhibition of collagenase expression. The present inventor also discovered that the orthovanadate and its analogues are extremely toxic to proliferating cell lines, at concentrations that are not toxic to normal nonproliferating cells, indicating that the orthovanadate may be useful as a chemotherapeutic agent. He has also discovered in a significant manner that orthovanadate acts on cell lines resistant to conventional drugs such as colchicine, vinblastine, and doxorubicin, indicating that the drug is useful for the treatment of drug-resistant tumors. The mechanisms that normally expel chemotherapeutic agents from cancer cells that are __ drug resistant do not recognize the vanadate compounds. Orthovanadate and its analogues also showed suppressing tumor growth in a live animal model (model MDAY-D2). Doses of at least 0.2 milligrams / kilogram were required to reach concentrations of orthovanadate or its analogs in the serum of the animals, to be highly toxic to cancer cells. Significant inhibition of < , tumor growth when orthovanadate was administered in combination with an antioxidant, N-acetyl cysteine. The action of orthovanadate and N-acetyl cysteine was more effective in inhibiting tumor growth in vivo than orthovanadate alone. The present inventor also discovered that animals receiving orthovanadate or vanadyl sulfate, did not have detectable levels of metastasis. In accordance with the above, said in a broad manner, the present invention relates to a method for modulating the expression of fos and jun by regulating the concentrations of hydrogen peroxide. In accordance with one embodiment of the invention, the compounds are used to reduce hydrogen peroxide and / or superoxides, thereby effecting a reduction in cell proliferation. Preferably, the compounds are vanadate compounds, or their derivatives or analogues. The invention also contemplates a pharmaceutical composition for the treatment of proliferative disorders, which comprises an amount of a vanadate compound, or a derivative or an analogue thereof, effective to reduce cell proliferation, and one or more of a vehicle, diluent , or pharmaceutically acceptable excipient. In a preferred embodiment of the invention, the pharmaceutical composition is used to reduce tumor growth. The invention further contemplates a method for the treatment of a proliferative disorder, which comprises administering an amount of a vanadate compound, or a derivative or an analogue thereof, effective to reduce cell proliferation. The amount of a vanadate compound or derivative or analogue thereof, effective to reduce cell proliferation, is an amount that results in a "" concentration of the compound in extracellular body fluids such as serum, cerebral spinal fluid, and sinusoidal fluid, of at least 5μM, preferably 5-50μM, more preferably 10-30μM. In general, a dosage of at least 0.2 milligrams / kilogram, preferably 0.2 milligrams / kilogram to 25 milligrams / kilogram, and most preferably 0.2 milligrams / kilogram to 20 milligrams / kilogram will result in the appropriate concentrations in humans and others mammals In a preferred embodiment of the invention, a dosage of at least 1 milligrams / kilogram, preferably between 1.0 milligrams / kilogram to 25 milligrams / kilogram of a vanadate compound or derivative or analog thereof, is used to provide an optimal dosage. The invention also relates to a method for reducing or inhibiting the growth of drug resistant tumors, which comprises administering an amount of a vanadate compound, or a derivative or an analogue thereof, effective to reduce or inhibit the growth of drug-resistant tumors. The invention further contemplates a method for reducing or inhibiting metastasis, which comprises administering an amount of a vanadate compound, or a derivative or an analogue thereof, effective to reduce or inhibit metastasis. The invention also contemplates a composition comprising a vanadate compound, or a derivative or analogue thereof, and at least one antioxidant, preferably N-acetyl cysteine, which enhances the anti-proliferative and anti-metastatic effects of the vanadate compound, and reduces proliferation. cellular and metastasis. Methods for the treatment and prevention of proliferative disorders, the treatment of drug-resistant tumors, and the reduction of metastases using this composition are also provided. The invention also relates to methods for testing a drug for its activity in reducing cell proliferation. These and other aspects of the present invention will become apparent upon a reference to the following detailed description and the accompanying drawings. In addition, various publications are referred to herein, which are incorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS Further details of the invention are further described with the help of the examples illustrated in the accompanying drawings, in which: Figure 1 is a graph showing the FACS analysis of superoxide production in response to IL 1 and inhibition of NADPH oxidase by DPI. Figure 2 is a Northern blot, showing the role of superoxide production on the expression of fos and collagenase. Figure 3 is a Northern blot, showing that hydrogen peroxide stimulates fos expression. Figure 4 is a Northern blot, which shows that the orthovanadate inhibits the expression of fos, jun, and collagenase. Figure 5 is a Northern blot, showing that N-acetyl cysteine inhibits IL 1 induction of fos and collagenase expression. Figure 6 is a graph showing the effect of orthovanadate on proliferating cells. Figure 7 is a graph showing that the orthovanadate is toxic to MDAY-D2 and HTB14 cells. Figure 8 is a graph showing the effect of lí. different forms of orthovanadate on cellular toxicity. Figure 9 is a bar graph showing that H202 potentiates orthovanadate toxicity. Figure 10 is a graph showing that orthovanadate is toxic to cell lines of different drug resistance. Figure 11 is a photograph of tumors of untreated and orthovanadate-treated mice. - * - 'Figure 12 is a graph showing that administration of orthovanadate suppresses tumoral growth 20 in vivo. Figure 13 is a graph showing the effect of orthovanadate administration, vanadyl sulfate, and vanadyl hydroperoxide, on tumor growth in vivo. Figure 14 is a graph showing that the administration of orthovanadate and N-acetyl cysteine completely inhibits tumor growth in vivo. Figure 15 is a photograph showing liver metastasis by MDAY-D2 cells. Figure 16 are photographs showing the effect of vanadium orthovanadate and vanadium sulfate on metastases. Figure 17 is a graph showing a comparison of a prior art treatment and the "orthovanadate / N-acetyl cysteine treatment of the present invention. Figure 18 is a Northern blot showing the inhibition by orthovanadate of the expression of c-fos and c-jun induced by IL 1, PMA, and AA. Figure 19 are Northern blots showing the effect of H202 on the mRNA levels of c-fos (A), and showing that the NAC and Ase antioxidants inhibit the mRNA levels of c-fos induced by TNF-a and bFGF (B). Figure 20 are graphs showing that DPI 20 inhibits ROS production induced by TNF-a and bFGF in chondrocytes. Figure 21 are immuno-broad showing that DPI inhibits the induction of c-fos expression by TNF-a and bFGF. Figure 22 is a graph showing the effect ^ of DPI on cell proliferation. Figure 23 is a graph showing the effect of BMOV on tumor growth.

DETAILED DESCRIPTION OF THE INVENTION As previously mentioned herein, the present invention relates to a method for modulating phos and jun expression by regulating hydrogen peroxide concentrations. The increase in hydrogen peroxide concentrations should result in an increased expression of fos and jun, and in accordance with the same, an increase in cell proliferation. An increase in cell proliferation would be useful in the treatment of conditions involving damaged cells, and in particular may be useful in the treatment of conditions in which tissue degeneration occurs such as arthropathy, bone resorption, disease * 'Inflammatory, degenerative disorders of the central nervous system, and to promote the healing of wounds. In accordance with one embodiment of the invention, the compounds are used to reduce hydrogen peroxide and / or superoxides, thereby effecting a reduction in cell proliferation. Preferably, the compounds are vanadate compounds, or derivatives or analogs thereof. The vanadate compounds suitable for use in the present invention are the oxidative forms of vanadate, preferably orthovanadate. Also derivatives of vanadate compounds, preferably pharmaceutically acceptable salts, esters, and complexes of vanadate compounds, including potassium and sodium salts, and amino acid complexes, carbohydrate, and fatty acid, for example, can be used in the present invention. , vanadate complexed with cysteine, dihydroxamate, and glucuronate. In a preferred embodiment, BMOV, an organo-vanadium compound is used in the present invention. Suitable analogs can be selected based on their functional similarity to the vanadate compounds, including the ability to interact with hydrogen peroxide to produce hydroxyl radicals, or to generally reduce hydrogen peroxide. Examples of these compounds include metal ions, such as iron, titanium, cobalt, nickel and chromium complexes, tin, glutathione, and diphenyl iodonium. The analogs of the vanadate compounds can also be selected based on their three-dimensional structural similarity to the vanadate compounds. For example, the vanadyl vanadyl forms in the present invention, preferably vanadyl sulfate, can be used. Compounds that affect the synthesis of hydrogen peroxide and / or superoxides, such as inhibitors of flavenoid-containing enzymes, can also be used in the present invention to modulate cell proliferation. For example, DPI can be used in the present invention. More preferably, orthovanadate and vanadyl sulfate are used in the pharmaceutical compositions, in the therapeutic treatments, and in the methods of the present invention. Selected derivatives and analogs of vanadate compounds can be tested for their ability to reduce hydrogen peroxide, their ability to affect the growth of proliferating cell lines, non-proliferating cell lines, and drug-resistant cell lines. , and its ability to inhibit growth tumor or metastasis in animal models following the methods described herein. The composition of the invention may contain one or "" more antioxidants in combination with a vanadate compound or analog or derivative thereof. The antioxidants are are selected based on their ability to increase the efficacy of vanadate compounds and reduce toxicity on normal cells using the methods described herein. Suitable antioxidants for use in the enhancement composition of the invention include acetylc-N-25 cysteine, glutathione, vitamin E (α-tocopherol), vitamin C (ascorbic acid), β-carotene, ergothioneine, zinc, selenium, copper, manganese, flavonoids and estrogens, or derivatives thereof, preferably N-acetyl cysteine. The administration of the vanadate compounds or 5 analogues or derivatives thereof, and optionally one or more antioxidants, in the forms and modes described herein, reduces hydrogen peroxide to effect a reduction in cell proliferation, and also reduces the metastasis of tumors. Accordingly, the compositions can be used for the treatment of proliferative disorders, including different forms of cancer, such as leukemias, lympholas (of Hodgkins and non-Hodgkins), sarcomas, melanomas, adenomas, solid tissue carcinomas, hypoxic tumors, Squamous cell carcinomas of the mouth, throat, larynx, and lung, genitourinary cancers such as cervical and bladder cancer, hematopoietic cancers, cancers of the head and neck, and cancers of the nervous system, benign lesions such as papilloma, atherosclerosis, angiogenesis, and viral infections, in particular HIV HE has shown that the compositions of the invention are specifically effective in inhibiting the growth of hematopoietic tumors, human glioma, and primary astrocyte tumors. Vanadate compounds or analogues or derivatives thereof, and optionally one or more antioxidants, in the compositions described herein, can also be used for the treatment of drug resistant tumors. Examples of drug-resistant tumors are tumors that express high levels of p-glycoprotein, which is known to confer resistance to multiple cancer drugs, such as colchicine, vinblastine, and doxorubicin, or tumors that express protein. resistant to multiple drugs, as described in R. Deeley et al., Science, 258: 1650-lvs, 1654, 1992. The compositions of the invention contain vanadate compounds or derivatives or analogs thereof, and optionally one or more antioxidants. , either alone or together with other substances. These pharmaceutical compositions can be for topical, parenteral (intravenous, subcutaneous, intramuscular, or intramedullary), or local use. Preferably, a mode of administration that results in a slow and continuous release of the active substances is employed. This can be achieved through intravenous administration, subcutaneous administration, or using control release mechanisms, such as implants or pumps. The control release methods generally use control release polymers, and the "release of the active ingredient is based on the solubility properties, and in the pore size of the polymers and the active ingredients. In the case of parenteral administration, solutions, suspensions, emulsions, or powders of the vanadate compound and / or derivative and / or analogue thereof, and optionally antioxidants, may be employed, using one or more suitable pharmaceutically acceptable excipients or diluents. for the aforementioned uses, and with an osmolarity that is compatible with physiological fluids. For local use, preparations should be considered in the form of creams or ointments for local use or in the form of sprays. The preparations of the invention can be pretended to be administered to humans and to other different mammals, such as sheep, cattle, equines, pigs, canines, and felines. The amount of a vanadate compound or derivative or analogue thereof, effective to reduce proliferation Cellular, and / or to reduce metastases, or to treat drug-resistant tumors, is the minimum dose Suitable for achieving a reduction in cell proliferation, a reduction or inhibition of metastases, and / or the growth of drug-resistant tumors. A dose that results in a concentration of the compound in extracellular body fluids such as Serum, senovial fluids, or cerebral spinal fluid, of at least 5μM, preferably 5-50μM, and more preferably 10-30μM, to reduce cell proliferation, and in accordance with the same, provide effective treatment of disorders proliferative In general, a dose of when less than 0.2 milligrams / kilogram, preferably from 0.2 milligrams / kilogram to 25 milligrams / kilogram, and most preferably from 0.2 milligrams / kilogram to 20 milligrams / kilogram, will provide an appropriate concentration in humans and others mammals In a modality _. _. of the invention, a dose of at least 1.0 milligrams / kilogram, and preferably between 1.0 milligrams / kilogram and 25 milligrams / kilogram, will provide an optimal dosage in humans and other mammals. The above mentioned doses can be used to reduce metastasis and to treat drug-resistant tumors. The doses selected will also depend on the individual needs and the mode of administration. It will be appreciated that standard procedures can be employed to quantitate the concentration of the vanadate compound or the derivative or analogue thereof in the extracellular body fluids. When the vanadate compound or the analog or derivative thereof is used in combination with one or more antioxidants, the doses of the vanadate compound or analogue or derivative thereof and the antioxidants are selected in such a manner that the compound of Vanadate and antioxidants alone do not show a full effect. In general, the effective doses of the vanadate compound and antioxidants are the minimum doses suitable for improving antiproliferative or antimetastatic effects. The vanadate compound and antioxidants can be administered concurrently, separately, or in sequence. The vanadate compound and the antioxidant can be prepared and administered as a complex. For example, i-a vanadate can be complexed with glutathione or N-acetyl cysteine. In one embodiment of the invention, a dose of orthovanadate compound that provides a concentration of the compound in body fluids is administered. extracellular such as serum, sinusoidal fluid, or cerebral spinal fluid, of at least 5μM, preferably 5-50μM, and more preferably Iμ-30μM. The N- "acetyl cysteine is administered before (preferably 20 minutes before), and during administration of the orthovanadate, in a dose that provides a concentration of the compound between 0.5 mM and 15.0 mM, preferably 5 mM to 12.5 mM. In general, a dose of between 40.0 milligrams / kilogram and 1000 milligrams / kilogram of N-acetyl cysteine will provide an appropriate concentration in humans and other mammals.

The compositions can be prepared by methods known per se for the preparation of pharmaceutically acceptable compositions that can be administered to patients, and in such a way that an effective amount of the active substance is combined in a mixture with a pharmaceutically acceptable carrier. Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the pharmaceutical compositions include, but are not limited to, solutions of the vanadate compounds, derivatives or analogs thereof, in association with one or more pharmaceutically acceptable carriers or diluents, and are contained in regulated solutions with a suitable pH, and iso-osmotic with physiological fluids. The compositions and treatments are indicated as therapeutic agents or treatments either alone or in conjunction with other therapeutic agents or other forms of treatment. In particular, the compositions and treatments described herein may be employed to reduce the toxicity of other therapeutic agents. For example, the compositions of the invention may be used in combination with radiotherapy and chemotherapy, such as multi-drug chemotherapy for Hodgkins disease, or combination radiotherapy, and chemotherapy for the treatment of breast cancer.

As mentioned hereinabove, the invention also relates to methods for testing substances that affect cell proliferation. The method involves determining the effect of the substance on the growth of the non-proliferating cells, and comparing the effect with that observed for the substance with the proliferating cells. In one embodiment, a substance suspected of affecting cell proliferation is tested by preparing a non-proliferating primary cell culture, coating chondrocytes or non-proliferating fat cells preferably human or bovine, in a high cell density, preferably from 2 x 106 to 4 x 10 6 cells / well on a six-well plate, and prepare a proliferating cell culture by coating the proliferating cells, preferably chondrocytes at a low density, preferably from 5 x 105 to 1 x 106 cells / well on a six-well plate; incubating each of the cell cultures in a medium containing the substance suspected of affecting cell proliferation, preferably for 1 to 48 hours at about 37 ° C, harvesting the cells and quantifying the number of viable cells, and compare the number of viable cells in proliferating and non-proliferating cell cultures. The following non-limiting examples are illustrative of the present invention: EXAMPLES Example 1 Identification of signaling mechanisms that regulate the expression of fos, jun, and collagenase. The sequence of events or second messengers responsible for stimulating the expression of fos and jun was investigated.

A. IL 1 induces a transient increase in the fos and jun mRNA. The cytokine interleukin 1 (IL 1) has been used to identify the second intermediary messengers that regulate the expression of fos and jun. The reason for using IL 1 is that it has been shown to stimulate the expression of fos and jun, and produces all the signals required to induce the expression of matrix metalloproteases. It was discovered that IL 1 induces a transient growth in the mRNA levels of fos and jun which have their peaks at 30 minutes to 1 hour, whereas the appearance of mRNA of collagenase is detected at 9 hours, and continues to increase. until 12 o'clock These data are consistent with studies demonstrating that fos and jun expression is required for the production of collagenase.

B. IL 1 stimulates the production of reactive oxygen intermediates. Chondrocytes (ie bovine chondrocytes coated as described in Kandel RA and 5 co-workers, Biochim, Biophys. Acta. 1053, 130-134, 1990) were incubated with dihydroxirrodamine for 5 minutes (DHR) or for 4 hours in the absence (- IL 1) or in the presence of IL 1 (+ IL 1), or in the presence of both IL 1 and the inhibitor NADPH, iodonium diphenyl (+ IL 1, + DPI). Figure 1 shows that IL 1 does it? stimulates the production of reactive oxygen intermediates by FACS analysis. The oxidase inhibitor NADPH, the DPI (diphenyl iodonium), completely inhibits the constitutive reactive oxygen intermediates and induced by IL 1 in chondrocytes. These data indicate that IL ~ 1 stimulates production of intracellular superoxides and of intermediates that react with oxygen.

C. Effect of DPI on the levels of fos and iun mRNA induced by IL 1 20 Although IL 1 stimulated the production of superoxide, it was not known if the expression of fos and jun induced by IL 1 depended on the production of superoxides. In order to elucidate this possibility, the effect of the DPI on the mRNA levels of fos and jun induced by IL 1 was investigated. analyzed RNA from chondrocytes treated with IL 1 in the presence and in the absence of DPI by Northern blot analysis, using either fos or collagenase cDNA probes. The results showed that the induction with IL 1 of fos and collagenase is suppressed by the DPI, indicating that the production of superoxide has a role in the induction of these genes (Figure 2). Similar data have been obtained for the expression of jun induced by IL 1. In addition, the inhibition of fos and jun expression by DPI was sufficient to suppress the expression of collagenase induced by IL 1 and le. constitutive These data indicate that the inhibition of the production of superoxides or H202 prevents the induction of the expression of fos, jun, and collagenase.

D. Hydrogen peroxide mimics the effect of IL 1 on the induction of fos expression Since superoxides are rapidly converted to hydrogen peroxide in cells by superoxide dismutase, it was investigated whether hydrogen peroxide could mimic the effect of IL 1 on the induction of expression of fos. RNA was extracted from chondrocytes (Kandel et al., Supra) treated with H202 for 30, 60, and 90 minutes, and examined by Northern blot analysis using a fos cDNA probe. As demonstrated in Figure 3, the addition of H202 to the chondrocytes also stimulates the expression of fos, suggesting that this molecule may be a second key messenger in the induction of transcription factors, fos and jun.

E. Effect of the orthovanadate and N-acetyl cysteine on the expression of fos, iun, and collagenase The effect of orthovanadate and N-acetyl cysteine on the expression of fos was examined., jun, and collagenase. Bovine atrial chondrocytes were isolated and coated as described previously (Kandel R.A. et al., Biochi, Biophys. Acta. 1053, 130-134, 1990). In order to determine the effect of orthovanadate on the responses induced by IL 1 and PMA (phorbol ester), chondrocytes were incubated with orthovanadate (100 μM) for 2 hours before stimulation with IL 1 (10 nanograms / milliliter) or PMA (100 milligrams / milliliters). Collagenase production was determined by incubating the chondrocytes for 24 hours with IL 1 or PMA, and the conditioned cell medium was "" assayed for the collagenase activity using an ELISA procedure as described previously (Kandel et al., supra). The activity of PLA2 was measured by the incorporation of 3H-arachidonic acid (3H-AA) into the cells, and then incubating the cells with medium containing 1 milligram / milliliter of BSA, either alone or in the presence of IL 1 or PMA, for 10 minutes, as described previously (Conquer, JA 1192, Biochim, Biophys. 1134, 1-6). The amount of 3H-AA released to the supernatant was determined. To measure the production of PGE2, chondrocytes were incubated for 6 hours in Ham's F12 medium, either alone or with IL 1 or PMA. The supernatant was analyzed by RIA using an antibody specific for PGE2 (Dr. S.A. Jones, Mount Sinal Hospital, Toronto, Canada). In order to examine the expression of c-fos and c-jun, the chondrocytes were incubated for 1 hour in the presence of IL-1, PMA, or AA (3 μM). The chondrocytes were washed in PBS, and the l, total RNA was extracted as described previously (Cruz et al, 1991, Biochem J. 277, 327-330). The RNA samples were tested on formaldehyde-agarose gels, and transferred to a nylon membrane for Northern analysis using cDNA probes for c-fos and c-jun. 15 IL 1 and PMA induced the release of 3 H-AA, as well as the production of PGE 2 and collagenase by the chondrocytes in the single layer culture. Although the "*" orthovanadate (100 μM) completely inhibited collagenase, did not inhibit IL-1 or PMA-induced release of 3H-AA, or production of PGE2. These data would suggest that the effect of the orthovanadate is occurring downstream from the release of 3H-AA, or that the mechanisms that regulate the activity of PLA2 and the production of PGE2 are separate from those that regulate the production of collagenase. The expression of 5 c-fos and c-jun was stimulated by IL 1, PMA, as well as AA, in bovine chondrocytes. Orthovanadate completely inhibited the expression of c-fos and c-jun induced by IL 1, PMA, and AA. which may be responsible for the inhibition of collagenase production. These data (see Figures 4 and 18) suggest that the inhibition by orthovanadate of collagenase production may be presenting downstream from the release of 3H-AA induced by IL 1, by inhibiting the expression of c-fos and c-Jun in chondrocytes.- The data that demonstrate that orthovanadate is a potent inhibitor of K_r expression of fos, jun, and collagenase, indicate that agents that reduce H202 levels in cells can serve as potent inhibitors of fos and jun expression. The cells were also incubated as described above, with 20 mM N-acetyl cysteine for 20 minutes. minutes, and then incubated with IL 1 for 1 or 12 additional hours. The RNA was extracted and examined by Northern blot analysis using cDNA probes for c-fos and collagenase. It was also discovered that N-acetyl cysteine that converts to GSH intracellularly reduces levels of the expression of fos and collagenase in response to IL 1 (Figure 5). Presumably, the higher intracellular levels of GSH reduced H02 and superoxide levels, and suppressed the expression of fos and collagenase expression. In summary, the results show that both N-acetyl cysteine and orthovanadate, indirectly reduce the levels of superoxides and, H202 in the cells.

Example 2 Vanadate compounds as potent chemotherapeutic agents in vitro. The effect of a class of vanadyl derivatives on cell proliferation in vitro is described below.

A. In vitro effects of vanadyl derivatives on normal and proliferating non-proliferating cells. As described in Example 1, the orthovanadate inhibited the expression of fos, jun, and collagenase. If the expression of fos and jun is required for cell proliferation, then the orthovanadate should inhibit the proliferation of chondrocytes. In order to compare the effect of "-" "orthovanadate on non-proliferating and proliferating chondrocytes, chondrocytes were coated at both high cell density (2 x 106 to 4 x 106 cells / well on a six well plate) (non-proliferating), and at a density lower cell (5 x 105 to 5 x 10 6 cells / well on a six-well plate) (proliferating), and then maintained for 48 hours, then the cells were incubated in a medium (HAMS F12) containing 0- 50 μM of orthovanadate for an additional 48 hours The cells were harvested and the number of viable cells was determined Figure 6 shows that the orthovanadate did not affect the chondrocytes that were coated in a high cell density, but was toxic to the coated cells in low cell density These data suggest that proliferating cells are sensitive to orthovanadate, while non-proliferating cells are resistant to orthovanadate toxicity. on proliferating tumor cell lines. The activity of fos and jun is also required for cell proliferation in many cell lines tumor. In accordance with the foregoing, the effect of orthovanadate on adherent cells and cell suspensions was examined. MDAY-D2 cells were incubated (a ^ mouse lymphoid cell line grown in suspension) and HTB14 (a human or adherent primary astrocytoma cell line) in a medium containing 0-50 μM of orthovanadate for 48 hours. The cells were harvested and the number of viable cells was determined. Figure 7 demonstrates the effect of orthovanadate on HTB14 and MDAY-D2 cells. Treatment with orthovanadate resulted in a concentration dependent on the increase in cell death. Although there are slight differences in the sensitivity to orthovanadate between cell types, all the cell lines examined were annihilated by the orthovanadate at concentrations 5 to 10 times lower than those used in 5 studies with normal nonproliferating cells (previous) Orthovanadate-induced cell death was observed at 24 hours, and was completed (over 98 percent) within 3 days of continuous treatment.In conclusion, the treatment of cancer cell lines with orthovanadate leads to cell death at concentrations that did not have significant toxic effects on normal nonproliferating cells.

Example 3 15 Efficacy of different forms of orthovanadate. Three different forms of vanadyl compounds were examined for their effect on the viability of the lines of "Cancer cells. MDAY-D2 cells were incubated in a medium containing 0-50 μM of orthovanadate, sodium sulfate vanadyl, or vanadyl hydroperoxide, for 48 hours. The cells were harvested, and the number of viable cells was determined. Figure 8 demonstrates the effect of the orthovanadate, vanadyl sulfate, and vanadyl hydroperoxide on MDAY-D2 cells. The results show that all these 5 agents were equally effective in killing these cells. Although there are slight differences in sensitivity, overall cell death was similar.

Example 4 In view of the investigations described in the Examples 1 to 3, it was thought that the orthovanadate reacts with H202 to form hydroxyl radicals, which are extremely toxic. If the formation of hydroxyl radicals induced by orthovanadate is responsible for cellular toxicity, then the addition of exogenous H202 should improve the effects of the orthovanadate. In accordance with the foregoing, cells were incubated in a medium alone, or containing 1 mM H202, or 10 μM orthovanadate, or both, for 24 hours. Cells were harvested and cell viability was determined. Figure 9 demonstrates the combined effects of low concentrations of orthovanadate and H202 on cellular toxicity. The addition of H202 only had a small effect. Without - "" - However, the addition of H202 in combination with orthovanadate increased cellular toxicity in a significant way compared to orthovanadate alone. The enhancement of cellular toxicity by H202 suggests that hydroxyl free radicals generated by orthovanadate treatment may be responsible for cell death.

Example 5 Orthovanadate is toxic to drug resistant cell lines. In many different cancers, tumor cells can not be removed by conventional chemotherapeutic agents, and these tumors are designated as drug resistant. Although the mechanisms involved in this process are not very well understood, it is thought that these cancer cells express a protein that removes the lt "drug from inside the cell, and reduces its intracellular toxicity. Patients who have a tumor resistant to drugs have a very poor prognosis. Therefore, agents that would be toxic to drug-resistant tumors would be a valuable therapeutic agent for the treatment of these patients. The effect of orthovanadate was compared on three ovarian cancer cell lines, KB8, KB8-5, and KB85-11, which have an increasing resistance to drugs, respectively, in relation to the cell line of origin, KB3-1. These drug-resistant cell lines are not annihilated by different classes of chemotherapeutic agents, such as colchicine, vinblastine, and doxorubicin. In the study, cell lines of increasing drug resistance were incubated (KB8, KB8-5, and KB-85-11), and the cell line of origin, KB3-1, in a medium (DMEM) containing 0-50 μM of orthovanadate, for 48 hours. The cells were harvested, and the number of viable cells was determined. As demonstrated in Figure 10, the orthovanadate was equally effective in killing all drug-resistant cell lines. Minor differences in the sensitivity to orthovanadate between the cell lines were observed, but they did not depend on the property of resistance to the drug, and after three days of administration of orthovanadate, these differences could no longer be seen, since most of the cells had dead. In conclusion, the data indicate that orthovanadate is lethal to drug-resistant cell lines, and may be particularly useful for the treatment of drug-resistant tumors.

EXAMPLE 6 LIVE EFFECTS OF TREATMENT WITH VANADILO COMPOUNDS In order to examine the ability of the vanadyl compounds to reduce tumor formation, growth, and metastasis, an animal model was selected that allows the investigation of the three processes in the same animal. This model involves the injection of a metastatic hematopoietic cell line MDAY-D2 into mice subcutaneously. These cells form a tumor at the site of injection, and their size can be easily determined. In, > In addition, these cells are etastasized to the liver, and metastases can be detected histologically after the day. 17 to 19. This model provides a very sensitive and reproducible approach for investigating the effect of vanadyl compounds on tumor growth and metastasis.

A. Effect of orthovanadate treatment on tumor growth in vivo. Using the animal model described above, the effect of subcutaneous orthovanadate administration on tumor growth was investigated. A total of 15 mice were injected subcutaneously with 1 x 10 5 MDAY-D2 cells on day 1. On day 5, small tumors could be observed at the site of injection. Five mice were injected daily with 50 microliters of water alone, and 10 mice were injected daily with water containing 10 milligrams / milliliter of orthovanadate. On day 14, the mice were sacrificed. Tumors were removed from all animals, they were photographed, and weighed. Figure 11 compares tumor sizes of 2 untreated mice and 2 mice treated with orthovanadate. The tumors of mice treated with orthovanadate were either undetectable or considerably smaller. Figure 12 shows the size of the tumors for each mouse. In animals treated with water alone, 4 mice had tumors that weighed between 1.18 and 1.68 grams. In mice treated with orthovanadate, 2 mice had no detectable tumors, and 5 mice had tumor sizes that were less than 0.16 grams.

B. Efficacy of the orthovanadate administration. vanadyl sulfate. and vanadyl hydroperoxide. on the reduction of tumor growth in vivo. In a separate experiment using the same animal model, the effect of the administration of orthovanadate, vanadyl sulfate, and vanadyl hydroperoxide on tumor growth in vivo was examined. On day 1, 20 mice were injected with 2 x 10 5 MDAY-D2 cells subcutaneously. The mice were divided into four groups of 5 mice. On day 5, the animals were injected subcutaneously with 50 microliters of water alone or containing 10 milligrams / milliliter of orthovanadate, 10 milligrams / milliliter of vanadyl sulfate, or 10 milligrams / milliliter of vanadyl hydroperoxide. This treatment was continued daily for 16 days. On day 21, the mice were sacrificed, and the tumors were dissected and weighed. One animal died in each of the groups treated with orthovanadate and vanadyl sulfate, and all 5 died in the group treated with vanadyl hydroperoxide. As shown in Figure 13, untreated mice developed tumors that had weights of 2.32 to 4. 79 grams. Although the effects of the vanadyl sulphate treatment were very variable, the treatment reduced the size of the tumors in all the animals. The tumors had a size of 0.14 grams to 2.18 grams. In the group treated with orthovanadate, one mouse had no detectable tumors, and the remaining 3 mice had tumors that varied in size from 0.15 to 0.38 grams. These data indicate that orthovanadate was most effective in reducing tumor growth, vanadyl sulfate was less effective, and vanadyl hydroperoxide was too toxic to evaluate its efficacy.

Example 7 Combination therapy of orthovanadate and N-acetyl cysteine completely inhibited growth and tumor formation. The studies described in the previous examples indicated that the orthovanadate was 80 to 100 percent effective in preventing tumor growth in mice. Since N-acetyl cysteine is converted into glutathione in cells, higher levels of glutathione can not only reduce the toxicity induced by the orthovanadate, but can also reduce tumor formation. Accordingly, it was examined whether the administration of N-acetyl cysteine in combination with orthovanadate was more effective in reducing animal toxicity and tumor growth in vivo. 20 mice were injected subcutaneously with 2 x 10 5 cells per day. On day 4, the mice were divided into four groups of 5 mice. Group one (control) received subcutaneous injections of 50 microliters of water. Group two received daily intraperitoneal injections of 50 microliters of 250 mM N-acetyl cysteine. Group three received daily subcutaneous injections of 50 microliters of 10 milligrams / milliliter of orthovanadate. Group four received daily intraperitoneal injections of 50 microliters of 250 mM N-acetyl cysteine, and 20 minutes later received a subcutaneous injection of 50 microliters of orthovanadate. On day 10, the treatment was stopped. The animals were sacrificed on day 13, and analyzed for tumor growth. An animal treated with orthovanadate died during the experiment. J Tumors were dissected from control mice and mice treated with orthovanadate (V04) or N-acetyl cysteine (NAC) or both (NAC-V04). The data shown in Figure 14 represent the weight of each tumor. As shown in Figure 14, untreated mice had tumors that weighed between 0.87 and 1.69 grams. In comparison, mice treated with N-acetyl cysteine had tumors that weighed between 0.23 and 1.18 grams, indicating that this agent was only able to reduce tumor growth to some degree. Of the 4 mice treated with orthovanadate, 2 had no detectable tumors, and the other 2 had tumors that weighed from 0.13 to 0.35 grams. On the other hand, the 5 animals that received the administration of orthovanadate and N-acetyl cysteine had no detectable tumors. These experiments clearly indicated that the combination therapy of orthovanadate and N-acetyl cysteine was the most effective therapy for inhibiting tumor growth in vivo. In addition, N-acetylic cysteine appeared to reduce the slight toxic effects observed in animals treated with orthovanadate alone.

EXAMPLE 8 VANADILO COMPOUNDS AS ANTIMETASTASIC AGENTS It was discovered that vanadate compounds inhibit the metastatic potential of cancer cells by reducing their ability to invade other organs. More particularly, it was discovered that the metastases of the MDAY-D2 cells are presented in the animal model described in Example 6. Figure 15 shows a control liver and a liver with metastasis. The metastatic liver was obtained from an animal 24 days following the administration of MDAY-D2 cells. The nodules were very numerous and large. In the animals slaughtered between 19 and 23 days, the number and size of the nodules were very variable from animal to animal, indicating that in order to examine the antimetastatic potential of the orthovanadate, the animals had to be kept for a minimum of 23 days in followed by the injection of MDAY-D2 cells. The preliminary results of the histological examination of the livers obtained immediately after one of the experiments described above in Example 6, suggested that the orthovanadate and the vanadyl sulfate were both effective in preventing metastasis. The livers of the treated animals were removed as previously discovered, and prepared for histological examination. Figure 16 compares liver sections from untreated animals (C), treated with orthovanadate (VO) (500 micrograms / day), and treated with vanadyl sulfate (VS) (500 micrograms / day). Nodes are identified by an arrow. Infiltration of MDAY-D2 cells and colony formation in untreated animals was observed. Animals that received orthovanadate and vanadyl sulfate did not have acceptable levels of metastasis.

Example 9 It was found that oral administration of orthovanadate at 0.5 milligrams / milliliter results in gastric toxicity in laboratory mice. In addition, it was also discovered that the intraperitoneal administration of high doses of orthovanadate is toxic to animals. However, subcutaneous injections of up to 500 micrograms of orthovanadate are tolerated by animals. Slow administration of the orthovanadate would decrease the toxicity, and the animals could tolerate higher doses.

Example 10 Comparison with U.S. Patent Application Serial No. 5,045,316 to Kaplan Ir. It was found that the concentration of vanadate used by Kaplan is too low to be effective in inhibiting tumor growth or metastasis. In order to determine if the optimal conditions of Kaplan were effective, the effect of the concentrations was investigated. highs of orthovanadate alone, or thiosulfate only, or orthovanadate and thiosulfate administered together, on tumor growth in mice. Kaplan reported daily doses of 0.0043 milligrams / kilogram to 0.14 milligrams / kilogram of vanadyl or vanadate salts that are required for 0 treatment. Assuming an equal distribution in body fluids, and a water content of 56 percent, the maximum concentration of orthovanadate in the serum with these doses at the time of administration is from .04 μM to 1.3 μM. As demonstrated in Figure 17, no decrease in tumor growth was observed with any of the agents described by Kaplan alone or in combination, at the doses disclosed by Kaplan. Under the optimal treatment conditions of the present invention, the tumor growth was not apparent, or was less than 80 percent of the control.

Example 11 Figure 19 shows the effects of H202 and antioxidants on the expression of c-fos. In particular, Figure 19A shows the effect of H202 on the mRNA levels of c-fos. Chondrocyte cultures were stimulated with H202 (100 μM) at different points of time, as indicated. Figure 19B shows that NAC and Ase antioxidants inhibit c-fos mRNA levels induced by TNFa and bFGF. The chondrocyte cultures were previously incubated with NAC (30 mM) or Ase (100 μM) for 2 hours before the addition of bFGF (10 nanograms / milliliter) or TNFa (30 nanograms / milliliter) for 30 minutes. Recombinant human TNFa and bFGF were dissolved in phosphate-regulated serum, with 0.1 percent bovine serum albumin. First NAC and Ase were dissolved in HAM F12 medium containing 5 percent (volume / volume) of fetal bovine serum, and then neutralized with sodium hydroxide. The total RNA from the bovine atrial chondrocytes was isolated, and mRNA levels of c-fos were determined by Northern blot analysis, as described herein. The spots were subsequently separated from the DNA, and re-probed with rat β-tubulin cDNA labeled with 32p.

Example 12 Figure 20 shows that DPI inhibits ROS production induced by TNFa and bFGF in chondrocytes. Over time, the DHR by itself caused a change in fluorescence to the right, as shown in panels a to c. A dotted line was drawn through the average fluorescence intensity of the control (panel c) with DHR only for 4 hours. After incubation with TNFa or bFGF for 4 hours in the presence of DHR, there was an additional change in logarithmic fluorescence intensity as indicated in panel d or f. In panels e and g, DPI abolished the fluorescent change stimulated by TNFa and bFGF, respectively.

Example 13 Figure 21 shows that diphenylene iodonium also inhibits the induction of c-fos expression by TNFa and bFGF. The chondrocyte cultures were previously treated with DPI (2 μM) for 30 minutes before the addition of TNFa (30 nanograms / milliliter) or bFGF (10 nanograms / milliliter) for 30 minutes. Measurements of the mRNA levels of c-fos and tubulin were as described in Example 11.

Example 14 Figure 22 shows the effect of iodonium of diphenyl (DPI) on cell proliferation. Non-proliferating cells (chondrocytes), and proliferating adherent cells (HTB14) or in suspension (MDAY-D2), were incubated in the presence and in the absence of 2 mM diphenyl iodonium for 24 hours. The medium was removed from the chondrocytes and from the HTB14 cell cultures, tipped and centrifuged. The MDAY-D2 cells were centrifuged. Cells were resuspended in PBS containing trypan blue, and cell viability was determined by light microscope. The results represent a typical experiment in quadruplicate.

Example 15 Figure 23 shows the effect of the organovanadium compound, bis (maltolate) oxovanadium (IV) (BMOV), on the tumor growth of MDAY-D2. Mice were injected subcutaneously with 5 x 10 5 MDAY-D2 cells. After 5 days, the animals were treated twice daily with 250 micrograms of BMOV. On day 16, the animals were sacrificed, the tumors were removed and weighed. The results represent the tumor weights for each animal. From the foregoing, it will be appreciated that, while the specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. In accordance with the above, the invention is not limited except as attached.

Claims (22)

1. The use of a vanadate compound, or a derivative or an analogue thereof, as an active substance, for the manufacture of a medicament for the therapeutic treatment of a proliferative disorder in a mammal, wherein the amount of the vanadate compound, or an analogue or derivative thereof, effective to reduce cell proliferation, is about 1 to 25 milligrams / kilogram of body weight of the mammal.

2. The use as claimed in claim 1, wherein the proliferative disorder is cancer.

3. The use as claimed in claim 1, wherein the proliferative disorder is arthropathy.

4. The use as claimed in claim 1, wherein the compound of vanadate is orthovanadate or vanadyl sulfate.

5. The use of a vanadate compound, or a derivative or an analogue thereof, as an active substance, for the manufacture of a medicament for the therapeutic treatment of metastases in a mammal, wherein the amount of the vanadate compound, or an analogue or derivative thereof, effective to reduce metastasis, is about 1 to 25 milligrams / kilogram of body weight of the mammal.

6. The use as claimed in claim 5, wherein the vanadate compound is orthovanadate or vanadyl sulfate.

7. The use as claimed in claim 5, wherein the medicament provides an extracellular concentration of orthovanadate in the mammal between 5 and 50 μM.

8. The use of a vanadate compound, or a derivative or an analogue thereof, as an active substance, for the manufacture of a medicament for the therapeutic treatment of a drug-resistant tumor in a mammal, wherein the amount of the composed of vanadate, or an analogue or derivative thereof, is about 1 to 25 milligrams / kilogram of body weight of the mammal.

9. The use as claimed in claim 8, wherein the medicament provides an extracellular concentration of orthovanadate in the mammal between 5 and 50 μM.

10. The use of a vanadate compound, or a derivative or an analogue thereof, in combination with an antioxidant as active substances, for the manufacture of a medicament for the therapeutic treatment of metastases in a mammal, wherein the amount of the compound of vanadate, or an analogue or derivative thereof, effective to reduce metastasis, is from about 1 to 25 milligrams / kilogram of body weight of the mammal. The use as claimed in claim 10, wherein the amount of the antioxidant is from about 40.0 milligrams / kilogram to 1000 milligrams / kilogram of body weight of the mammal. 12. The use as claimed in claim 10, wherein the antioxidant is N-acetyl cysteine, glutathione, vitamin E (a-tocopherol), vitamin C (ascorbic acid), "β-carotene, ergothioneine, zinc, selenium, copper , manganese, a flavonoid, or an estrogen 13. The use as claimed in claim 10, wherein the vanadate compound is orthovanadate, and the antioxidant is N-acetyl cysteine 14. Use as claimed in the claim 13, wherein the medicament provides an extracellular concentration of orthovanadate in the mammal between 5 and 50 μM, and of N-acetyl cysteine between 0.5 μM and 15 μM 15. The use of a vanadate compound for the manufacture of a medicament for the therapeutic treatment of a proliferative disorder in a mammal, wherein the medicament consists of an amount of a vanadate compound effective to reduce cell proliferation, and a pharmaceutically acceptable carrier 16. Use as claimed in claim 15, wherein the proliferative disorder is cancer. 17. The use as claimed in claim 15, wherein the vanadate compound is orthovanadate or vanadyl sulfate. 18. The use as claimed in claim 15, wherein the amount of the vanadate compound, or an analog or derivative thereof, is from about 1 to 25 milligrams / kilogram of body weight of the mammal. 19. Use as claimed in claim 15, where the medicine also consists of an antioxidant. The use as claimed in claim 19, wherein the antioxidant is N-acetyl cysteine, glutathione, vitamin E (α-tocopherol), vitamin C (ascorbic acid), β-carotene, ergothioneine, zinc, selenium, copper , manganese, a flavonoid, or an estrogen. S 21. Use as claimed in claim 19, wherein the antioxidant is N-acetyl cysteine. 22. The use as claimed in claim 19, wherein the amount of antioxidant is from about 40.0 milligrams / kilogram to 1000 milligrams / kilogram of body weight of the mammal.