EP1200104A1 - Cadmium containing compositions - Google Patents

Abstract

Methods and compositions are provided for decreasing or regulating ratios of zinc:cadmium and PGE2:PGF2α and regulating the concentration of zinc-containing and PGE2-dependent matrix metalloproteinases in body fluids and tissues of a mammal, comprising administering to the mammal an amount of a pharmaceutically acceptable and bioavailable cadmium salt. Elevated or fluctuating levels of PGE2 and zinc and elevated concentrations of zinc-containing and PGE2-dependent matrix metalloproteinases have been found to be associated with the development of certain diseases.

Description

CADMIUM CONTAINING COMPOSITIONS

This patent application claims priority from provisional patent application serial number 60/142,926, filed July 9, 1999.

FIELD OF THE INVENTION

The present invention relates to methods for decreasing PGE2 : PGF2α and zinc: cadmium ratios and for regulating the concentration of zinc-containing and PGE2-dependent matrix metalloproteinases in the body of mammals. More specifically, the present invention relates to the administration of bioavailable cadmium salt(s) to a mammal to decrease PGE2 : PGF2α and zinc: cadmium ratios and to regulate the concentration of zinc-containing and PGE2-dependent matrix metalloproteinases in the body of the mammal. High or fluctuating levels of zinc and these matrix metalloproteinases have been shown to be associated with the onset of a number of diseases. The invention thus further relates to methods of preventing or treating disease. BACKGROUND OF THE INVENTION

Despite all the advances in medical treatment in recent years, a number of diseases remain difficult to treat. A number of these diseases are ones often associated with advancing age, although certainly they are not exclusively afflictions of the elderly. Cancers, for example, remain a leading cause of death in animals and humans, despite advances in the field of cancer treatment. The leading therapies to date are surgery, radiation and chemotherapy. Many types of chemotherapeutic agents have shown at least some effectiveness against certain cancers and tumor cells, but the efficacy of these agents can vary significantly, and their side effects can be severe. Hormones, in particular estrogen, progesterone and testosterone, and some antibiotics, alkylating agents, and anti-metabolites form the bulk of therapeutic agents available to oncologists. Scientists continue to search for cytotoxic agents that have specificity for cancer and tumor cells, yet do not affect normal cells and have minimal side effects.

Other diseases which remain significant include multiple sclerosis (MS) and diabetes.

Present treatments for diabetes include the administration of insulin either orally or through an injection solution. Also, glucose may be administered either directly as through injection or indirectly, as through ingestion of certain foods or drinks. Other methods of treating diabetes include the use of implantable glucose systems which have been designed to provide continuous measurement of the patient's glucose concentration. The use of implantable insulin pumps also can be used for the treatment of diabetes.

Such above described treatments for diabetes require lifelong treatment of the patient. For many patients insulin injection is an unpleasant process. Also the need for daily injections of insulin is hard on the patient's veins. Insulin treatment is costly and it is only a temporary reliever of diabetic symptoms. Continued treatment is necessary in order to control the disease. Therefore, there is a need for a remedy in treatment for diabetes which is easily administered to or by the patient.

Multiple sclerosis (MS) is a chronic inflammatory disease of the white matter of the human central nervous system and is believed to be of autoimmune etiology. The current treatments for MS involve administration of drugs which act in a non-specific fashion to suppress the immune response in a subject. Examples of such drugs are cyclophosphamide, imuran (azathioprine) and cyclosporin A. Steroid compounds such as prednisone and methyl-prednisolone also are employed in many instances. These drugs have only limited efficacy against MS. The use of such drugs is limited by their toxicity and by the fact that they induce "global" immunosuppression upon prolonged use. Consequently, better and alternative methods and compositions for the treatment of MS have been sought. Research into all of these diseases has indicated that elevated or fluctuating levels of zinc, either per se or in the form of zinc-containing or PGE2-dependent matrix metalloproteinases, are associated with the onset and/or advancement of these diseases. As a result, attempts have been made to inhibit or to regulate the concentration of zinc and these enzymes in the body.

Methods for inhibiting matrix metalloproteinases used in the past include the use of hydroxamic acid derivatives such as alpha-amino sulphonyl hydroxamic acids and carboxy-peptidyl compounds. Natural products such as TIMP-1, TIMP-2 and alpha 2-macroglobulin also are known matrix metalloproteinase inhibitors. Although these inhibitors have been of interest, other easily manufactured compounds and compositions which can regulate the concentrations of matrix metalloproteinases and zinc in the body fluids and tissues of a patient are sought.

SUMMARY OF THE INVENTION

In accordance with the present invention, methods and compositions are provided for decreasing or regulating PGE2 : PGF2α and zinc: cadmium ratios and regulating the concentration of zinc-containing and PGE2-dependent matrix metalloproteinases in the body of a mammal. Elevated or fluctuating levels of PGE2 and zinc and elevated concentrations of zinc-containing and PGE2-dependent matrix metalloproteinases have been found to be associated with the development of certain diseases, including prostate cancer, colon cancer, breast cancer, multiple sclerosis and diabetes. By regulating the concentration of zinc and PGE2 in the mammal's body, one can prevent the onset of, or treat the development of, these diseases. In one embodiment of the invention, there is provided a method of decreasing the PGE2 : PGF2α ratio in the body fluids and tissues of a mammal which comprises administering to the mammal one or more cadmium salts in an amount sufficient to lower the concentration of PGE2 in the body of the mammal. In another embodiment of the present invention, there is provided a method for decreasing the zinc: cadmium ratio in the body fluid and tissues of a mammal which comprises decreasing the concentration and activity of zinc in the body fluid of the mammal by administering to the mammal one or more cadmium salts. In a further embodiment, there is provided a method of regulating the concentrations of zinc and zinc-containing and PGE2-dependent matrix metalloproteinases in a mammal by administering one or more cadmium salts.

In another embodiment of the present invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more cadmium salts, said composition being in oral dosage form.

In yet another embodiment of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more cadmium salts in combination with an estrogen, a protease inhibitor or a combination thereof.

In still yet another embodiment of the invention, there is provided a method for treatment or prevention of cancer, particularly prostate, colon or breast cancer, which comprises decreasing the PGE2:PGF2α and zinc: cadmium ratios in the body of a mammal suffering from, or at risk of developing, cancer by administering an amount of one or more cadmium salts effective to inhibit or prevent the growth, division or metastasis of cancer cells.

In still yet another embodiment of the invention, there is provided a method for the prevention or treatment of a disease in a mammal, the onset of which is associated with abnormal fluctuating levels of zinc in the body fluids and tissues of the mammal, which comprises administering one or more cadmium salts at dose levels effective to regulate the concentration of zinc in the body fluids and tissue of a patient suffering from or at risk of developing such a disease.

DETAILED DESCRIPTION OF THE INVENTION

It is known that stallions have low fertility (e.g. unsuccessful equine in vi tro fertilization (1)) and approximately 1/3 the incidence of cancer mortality compared to humans or dogs (8% versus 24% or 23%, respectively (2, 3, 4, 5)). Also, prostate cancer has never been reported in the stallion. Stallions have high levels of unique estrogens (6, 7, 8, 9) paralleling the high levels of unique estrogens of pregnant mares. Stallions, in comparison to man, have a low testosterone: estrogen ratio (stallions 1:13, (10), men 9:1, (10)) which may regulate their postulated low seminal plasma PGE2 : PGF2α concentrations and ratio (11, 12, 13, 14, 15, 16, 17) . It also is known that stallion urine comprises cadmium in the form of cadmium sulfate, as well as protease inhibitors and other chemicals.

It now has been found that bioavailable salts of cadmium can be administered to mammals, particularly humans, as therapeutic agents. Specifically, it now has been found that cadmium salt(s) can be administered to humans as an effective therapy for a number of diseases which are associated with high or fluctuating levels of zinc in body fluids and tissues. Such tissues include multiple sclerosis, diabetes and cancers, such as prostate cancer, colon cancer and breast cancer.

Prostaglandin E2 (PGE2) increases sperm quality (26, 27, 28, 29, 30, 31) and increases cancer cell division (32). The high PGE2 levels in man's seminal plasma also impair the immune systems of individuals who receive this semen (33, 34, 35, 36, 37, 38). In parallel, it is known that zinc increases sperm quality (39) and enhances cancer cell metastasis (40, 41, 42) . Zinc is a key component of proteases that function in cancer cell metastasis (40, 41, 42). Zinc is not retained in tissues (18). In general, zinc is regarded as an essential metal. Zinc has a negative relationship with cadmium. Zinc decreases the amount of cadmium absorbed from the gut (18) and decreases the physiological effects of cadmium (43, 44, 45, 46, 47, 48, 49, 50, 51) .

It is known that the highest concentrations of PGE2 in a man's body are in his seminal vesicles (15, 16, 17). Likewise, the highest concentrations of zinc are in his prostate gland (18). A positive feedback occurs between PGE2 and zinc; PGE2 increases the absorption of zinc from the gut (19, 20) and zinc increases the PGE2 levels in tissues (21, 22) . In contrast to zinc, cadmium decreases PGE2 levels (23, 24). Protease inhibitors also decrease PGE2, as secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2 and matrix metalloproteinases (25) . PGF2α has opposing physiologic actions to PGE2. When mammals, particularly humans, are administered compositions of the present invention the levels of zinc and PGE2 in the mammal's body fluids and tissues are regulated. Matrix metalloproteinases are regulated by zinc and PGE2. Therefore, the systemic decrease in zinc and PGE2 levels inhibit these matrix metalloproteinases. By regulating the level of matrix metalloproteinases, the onset of diseases which have been shown to be associated with high or fluctuating levels of zinc and matrix metalloproteinases in the body can be prevented or delayed and the presence of the disease in one who has already contracted it can be treated. As used herein, "treatment" includes halting or slowing the progress of a disease. Also as used herein, "regulating" means to lower abnormally high levels of, or to minimize abnormal fluctuations in the levels of, zinc (and, therefore, zinc-containing and PGE2-dependent matrix metalloproteinases) in body fluids and tissues. Diseases which are influenced by zinc and matrix metalloproteinase levels and, therefore, can be prevented or treated by the present invention include diabetes and multiple sclerosis and cancers, such as prostate, colon and breast cancer.

Although not wishing to be bound by theory, it is theorized that cadmium is relatively high in stallion semen in contrast to man's semen because cadmium is retained at high levels in horse kidneys. In contrast to PGE2 or zinc, cadmium decreases fertility in man (52, 53, 54, 55) and, in an animal study, suppresses tumors in the B6C3F1 mouse liver and lung (56) . Cadmium has a contraceptive action (52) and is in high levels in infertile men (53, 54, 55). In contrast to zinc, which is generally viewed as an essential metal, cadmium is regarded as a toxic metal. Because of cadmium's classification as a toxin, it has been overlooked as a beneficial treatment for humans. In fact, although cadmium is highly toxic and potentially carcinogenic when inhaled, it is relatively innocuous when ingested (57).

The concentration of zinc in human serum normally is within the range of about 0.5 - 1.5 μg/ml and in human semen normally is within the range of about 140 - 202 μg/ml. Researchers have found elevated levels of zinc and, therefore, elevated levels of zinc-containing and PGE2-dependent matrix metalloproteinases, in patients suffering from certain diseases. In accordance with the present invention, it now has been found that one or more bioavailable cadmium salts can be administered to a mammal, preferably a human, to decrease elevated ratios of PGE2 : PGF2α and zinc: cadmium in the mammal's body so as to effect a treatment for, or to prevent or delay the onset of, a disease associated with elevated levels of zinc or zinc- containing or PGE2-dependent matrix metalloproteinases. For example, as noted above, high levels of these proteinases have been shown to be associated with the metastasis of at least certain types of cancer cells. Accordingly, bioavailable cadmium salts can be administered to effect a treatment for, or to prevent or delay the onset of, certain types of cancer, such as prostate cancer, colon cancer or breast cancer. By lowering the concentration of zinc in the mammal's body fluids and tissues, the concentration of zinc- containing and PGE2-dependent matrix metalloproteinases can be decreased and stabilized.

It also has been found that significant fluctuations of normal zinc concentrations are associated with the onset and progression of certain diseases. For example, studies of patients suffering from MS have shown both significant high and low fluctuations in zinc levels in serum and tissues (74, 75, 76) . Diabetes patients have been shown to have approximately double the normal amounts of zinc in their urine and about 15 - 20% lower levels of zinc in their blood (77, 78). The administration of a physiologically and bioavailable cadmium salt can minimize these fluctuations. Although not wishing to be bound by theory, it appears that cadmium competes with zinc in pathways in the body. Thus, in the case of a diabetic patient who has large amounts of zinc in his urine, it is postulated that the administration of cadmium leads to a competition between the processing of cadmium and zinc in the patient's kidneys, resulting in less zinc being excreted in the urine and an accompanying increase in systemic zinc levels to more normal levels. In patients with elevated levels of zinc, the administration of cadmium serves to lower the ratio of zinc to cadmium.

Cadmium salts are administered to a patient so as to regulate the PGE2 : PGF2 and zinc: cadmium ratios in the patient's blood and other body fluid, thereby regulating the patient's systemic levels of zinc and PGE2. The cadmium salts are administered in a series of daily doses and can be administered at a dose of about 1 μg to about 100 μg per day. Preferably, the cadmium salts are administered at a daily dose of about 10 to about 100 μg per day, and most preferably within the range of about 10 to about 50 μg per day.

Suitable salts are bioavailable cadmium salts. Such salts include the sulfate, nitrate, chloride and acetate salts. A single salt can be administered or a combination of salts can be used.

Optionally, the one or more cadmium salts can be administered in combination with at least one further therapeutic agent. In one embodiment of the invention, the cadium salts are administered in combination with at least one estrogenic compound. This combined administration of cadmium salt and estrogen can be beneficial in the treatment or prevention of prostate cancer. The estrogenic compound (s) act synergistically with the cadmium salt(s) to directly prohibit PGE2 production in semen, blood and other bodily fluids. A single estrogen can be administered or a mixture of estrogens can be used. Suitable conjugated estrogenic compounds include those found in commercially available estrogen preparations, such as Premarin®, sold by Wyeth Ayerst . The principal estrogen in Premarin® is sodium estrone sulfate. The estrogenic compound (s) can be administered at a daily dose of about 0.1 mg to about 0.5 mg, preferably about 0.3 mg per day. The actual amount of estrogen (s) used will depend upon the particular estrogen selected and its relative potency. Another embodiment of the present invention comprises the administration of cadmium salt(s) in combination with one or more protease inhibitors to synergistically decrease levels of PGE2 concentration in blood, and other body fluids. Proteases play pivotal roles in sperm function (58, 59, 60, 61, 62) and cancer cell metastasis (64, 65, 66, 67, 68, 69, 70, 71, 72, 73) . The administration of protease inhibitors can block these proteases and thereby decrease sperm and cancer cell function. A number of protease inhibitors are known and have been proposed or used as therapeutic agents. For example, several studies have described the negative effects of protease inhibitors on fertility. Protease inhibitors can decrease the penetration rate of human sperm into oocytes (58) and, in seminal plasma, can act as decapacitation factors (61, 62).

In addition, a number of patents, such as U.S. Patent 5,830,888, U.S. Patent 5,708,004, U.S. Patent 5,602,175, and U.S. Patent 5,527,829, describe the administration of protease inhibitors as anti-HIV therapeutics. A number of protease inhibitors (e.g., invirase, ritonavir, indinavir sulfate, and nelfinavir mesylate) have been found to prevent reverse transcription of HIV (63).

U.S. Patent 4,906,457 reports the use of protease inhibitors for cancer protection. A serine protease inhibitor, FOY-305, inhibits the invasion of tumor cells through interference with the u-PA activity of tumor cells (64). Selective protease inhibitors at 10 nM concentration in the culture medium inhibit the migration of tumor cells in a Matrigel assay (65). Dietary phytochemicals have protease inhibitory activity, and also inhibit cancer (66). The recognized relationship between protease inhibitors and cancer is demonstrated by a second conference on ^Proteases and Protease Inhibitors in Cancer' that was held in 1998 (68) . Useful protease inhibitors in accordance with the present invention include those found in stallion urine. U.S. Patent 3,912,704, incorporated herein by reference, describes protease inhibitors from stallion urine. The molecular weight of the protease inhibitors from stallion urine are 26,000-28,000 by gel filtration, 17,000 by polyacrylamide gel electrophoresis, and 20,400 by ultracentrifuge . Other suitable protease inhibitors include those described in the patents cited above, including indinavir sulfate, commercially available as Crixivan® (Merck) ; ritonavir, commercially available as Norvir® (Abbott) ; invirase, commercially available under the name Saquinavir® (Immunet) ; and nelfinavir mesylate, commercially available as Viracept® (Agouron) . When one or more protease inhibitors is administered in combination with the cadmium salt(s), the dose of the protease inhibitor varies depending upon the particular inhibitor or inhibitors chosen, but typically is in the range of from about 600 mg to about 2400 mg per day, depending upon the strength of the particular compound (s) of interest. For example, a preferred daily dose of indinavir sulfate typically is about 800 mg orally every eight hours, a preferred daily dose of ritonavir is within the range of about 300 mg to about 600 mg twice a day, a preferred daily dose of invirase is about 200 mg three times a day and a preferred daily dose of nelfinavir mesylate is about 750 mg three times per day. If desired, the protease inhibitor can be administered in combination with an estrogenic compound in addition to a cadmium salt.

In accordance with this invention, for purposes of the prevention or treatment of prostate cancer, breast cancer or colon cancer, the cadmium salts are administered in a series of doses in an amount sufficient to prevent or inhibit the growth, division, or metastasis of cancer cells. For purposes of the prevention or treatment of MS or diabetes, the cadmium salt(s) are administered in a series of doses in an amount sufficient to regulate fluctuations in the levels of zinc and the zinc-containing and PGE2- dependent matrix metalloproteinases in the patient's body which have been found to be associated with these diseases .

Cadmium salt(s) and optional estrogenic compound (s) and/or protease inhibitor (s) can be administered orally, parenterally or by inhalation. Oral administration, such as capsules, tablets, suspensions or solutions, is preferred. Cadmium salt(s) conveniently are administered in the form of tablets. The cadmium salt(s) can be mixed with one or more lubricants, such as stearic acid or magnesium stearate, flavor ameliorating agents, disintegrating elements, including potato starch and alginic acid, binders, such as gelatin and corn starch, and/or tablet bases, such as lactose, corn starch and sucrose, and then pressed into tablets. Alternatively, the cadmium salt(s) can be given in the form of capsules, prepared by mixing the salt(s) with a pharmaceutically acceptable excipient and then filling gelatin capsules with the mixture in accordance with conventional procedures .

As an alternative to oral administration cadmium salt(s) can be administered parenterally, provided in injectable doses of a solution or suspension in a physiologically acceptable diluent with a pharmaceutical carrier. The carrier can comprise water or an oil and also optionally can comprise a surfactant or other pharmaceutically acceptable adjuvant. As a further alternative, the cadmium salt(s) can be administered by inhalation. The salt(s) are provided in an aerosol spray and administered via an inhaler.

The examples set forth below are intended to further illustrate the present invention and are not intended to be limiting.

Examples As shown in Example 1, 1) PGE2, PGF2 , and zinc occur at higher concentrations in man's than in stallion' s semen and cadmium occurs at a lower concentration in man's than in stallion's semen, and 2) the ratio of PGE2 : PGF2 is higher in man's semen than in stallion's semen and the ratio of zinc: cadmium is higher in man's semen than in stallion's semen. As shown in Example 2, observed sperm motility in

5 stallions was higher when the zinc: cadmium ratio was higher. Example 1 Evaluation of Semen Samples

SEMEN COLLECTION: Sperm-rich fractions of semen were collected from 8, healthy Morgan stallions aged 2-15 years old. The collected semen was immediately diluted 1:1 with 20 mg/ml aspirin in ultra-pure water and transported on ice to the laboratory. The stallion semen samples were frozen until analysis. Just prior to assay, samples were thawed and purified by centrifugation and then assayed for PGE2, PGF2, zinc and cadmium.

Nine semen samples were collected from a healthy, 46-year-old man over a 45-day period which began on November 13. The first five of the samples were collected over the first 27 days and were collected as controls. Then, over an 18 day period, the man was administered 350cc of stallion urine twice daily. The urine previously had been harvested from three Arabian stallions and frozen. The remaining four samples of the man's semen were collected during this 18 day period. Each semen sample was purified by centrifugation and the top 1 ml was aspirated and frozen. The remaining semen also was frozen, then, just prior to assay, the samples were thawed and purified by centrifugation and then assayed for PGE2, PGF2, zinc and cadmium.

Centrifugation of both stallion and man' s semen samples consisted of a primary centrifugation to remove cells and debris (at 500 x g for 10 minutes at 4°C) and then a secondary centrifugation to remove large proteins (at 80,000 x g for 90 minutes at 4°C). The stallion semen and all human semen samples were assayed for PGE2 and PGF2 by radioimmunoassay or enzyme immunoassay .

PGE2, PGF2α, ZINC, AND CADMIUM ASSAYS: Concentrations of PGE2 in human semen were quantified using acetylcholinesterase competitive enzyme immunoassay kits (Cayman Chemical Company, Ann Arbor, MI) . The assay uses 96-well microtiter plates, with goat anti-mouse polyclonal antibody previously bound to each well, and pure acetylcholinesterase from the electric eel {Electrophorus electricus) that is covalently coupled to PGE2 as the enzymatic tracer. The human semen PGE2 assays were performed according to manufacturer's directions. Briefly, semen samples were thawed and further purified by chromatography (using Waters Sep-Pak cartridges (Millipore Corp. Milford, MA) ) . Purified samples then were reconstituted with 450 μl of EIA buffer, and diluted to final concentrations of 1/10,000 to 1/100,000. Fifty microliters of each sample (from each dilution) were added to each well, followed by 50 μl of PGE2 acetylcholinesterase tracer and 50 μl of PGE2 monoclonal, anti-PGE2 antibody per well. Each dilution from each of the 9 semen samples was assayed in duplicate. Plates were incubated for 18 hours at 4°C, were then rinsed thoroughly with wash buffer, and 200 μl of Ellman' s reagent was added to each well. Development occurred after 60-90 minutes in darkness. Plates were read at 405-420 nm with a spectrophotometer . Simultaneously incubated were wells with 50 μl of PGE2 standard with 50 μl of tracer and 50 μl of anti-PGE2 antibody. Nonspecific binding was determined by incubating 100 μl of EIA buffer and 50 μl of tracer, and maximum binding (B₀) was determined by incubating 50 μl EIA buffer, 50 μl of tracer, and 50 μl of anti-PGE2 antibody. Concentrations of PGF2 in stallion semen samples and in human semen samples were quantified by radioimmunoassay, with [5, 6, 8, 9, 11, 12, 14, 15 (n)-³H]-PGF2α (sa 200 Ci/mmol; New England Nuclear, Boston, MA), rabbit anti-PGF2α antibody (1:20,000 final dilution; supplied by D.H. Dubois & F.W. Bazer) and Lutalyse (Upjohn Co., Kalamazoo, MI) for standards (94). Briefly, just prior to assaying, diluted, previously frozen stallion semen samples were thawed and purified by centrifugation (as described above) . Human semen samples (which had been centrifuged prior to freezing) were thawed and diluted 1:1 with 20 mg/ l aspirin in ultra-pure water. Assay tubes received 25 μl of each sample plus 100 μl of [3H]-PGF2 , 200 μl of assay buffer (50 mM Tris HC1 and 0.01% NaN₃ at pH 7.5, 4°C), and 100 μl of anti-PGF2α. Tubes were incubated at room temperature for 30 minutes and then 18-24 hr at 4°C. Free PGF2α was then separated from antibody-bound PGF2α by addition of 0.5 ml of 0.25% Norit A charcoal and 0.025% dextran (in assay buffer) for 4 min at 4°C, followed by centrifugation for 10 min at 1,800 x g and at 4°C. The amount of antibody-bound [3H]-PGF2 in 0.5 ml of supernatant was determined by liquid scintillation spectrometry . A log-logit regression program was used to calculate concentrations of PGF2 (95). Assay sensitivity is 2.5 pg/tube (p<0.01).

Cross-reactivity of the antisera is 63.0% for PGFl , 0.3% for PGE2, and <0.1% each for 6-keto-PGFl , PGFM and arachidonic acid (94).

After dilution, freezing, thawing, and purification of the stallion semen samples, concentrations of PGE2 were quantified similarly using the same radioimmunoassay techniques as for PGF2α, except with [5, 6, 8, 11, 12, 14, 15 (n) -³H] -PGE2 (sa 200 Ci/mmol; New England Nuclear, Boston, MA) , rabbit anti- PGE2 (1:20,000 final dilution; supplied by D.H. Dubois & F.W. Bazer) and PGE2 (Sigma Chemical Co., St. Louis, MO) for standards.

STATISTICS: The mean concentrations of PGE2, PGF2α, zinc, and cadmium, and the mean ratios of PGE2:PGF2 and zinc: cadmium were contrasted by the Student T test between stallion semen and human semen (control samples) and among human samples.

RESULTS

Tables 1 and 2 show the results of experiment 1.

In experiment 1, PGE2 is 4,556 (368,570.0/80.9) times greater (p<0.01) in concentration (ng/ml) in man's than in stallion semen. PGF2α is 116 (231.6/2.0) times greater (p<0.01) in concentration (ng/ml) in man's than in stallion semen. The PGE2:PGF2α ratio is 1597:1

(368,570.0/231.6) in man' s semen, and 39:1 (80.9/2.0) in stallion semen. Therefore, the PGE2 : PGF2α ratio is

41 (1597/39) times greater (p<0.01) in man's than in stallion semen.

Also, from experiment 1, zinc is 76

(68,640.0/897.3) times greater (p<0.05) in concentration (ng/ml) in man's than in stallion semen.

In contrast, cadmium was below detectable levels (<0.2 ng/ml) in all of the man's control semen samples, but was detected (1,6 to 11.4 ng/ml) in 5 of the 8 stallion semen samples. Therefore, cadmium is at least 13 (2.6/0.2 ng/ml) times greater (p<0.01) in concentration (ng/ml) in stallion than in man's semen. The zinc: cadmium ratio is 301 (343,200/1,139) times greater (p<0.05) in man's than in stallion semen.

Table 1. Prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2α) , zinc, and cadmium concentrations (mg/ml) in stallion semen.

CONCLUSIONS

The concentration of PGE2 is 4,556 times greater and the concentration of PGF2 is 116 times greater in man's than in stallion's semen. Also, the PGE2 : PGF2 ratio is 41 times greater in man's than in stallion's semen. Therefore, the stallion has regulatory mechanisms that maintain a 4,000 fold lower concentration of PGE2 and a 100 fold lower concentration of PGF2α than in the semen of man. Because the PGF2 concentration is only 100 fold lower in stallions than in man, the ratio of PGE2 : PGF2 is 41 times lower in stallion's semen than in man's semen.

The concentration of zinc is 76 times greater and the concentration of cadmium is at least 13 times lower in man's than in stallion's semen. Also, the zinc: cadmium ratio is at least 301 times greater in man's than in stallion's semen. Therefore, the stallion has regulatory mechanisms that maintain a 76 fold lower concentration of zinc and a 13 fold higher concentration of cadmium than in the semen of man.

Example 2 Materials and Methods:

Two ejaculates were collected from each of five Quarter Horse stallions. Each ejaculate was evaluated for gel-free volume, sperm concentration, and sperm motility. In addition, each sample was chemically analyzed for the concentration of cadmium. Table 2. Concentration of cadmium and sperm motility in paired ejaculates from five Quarter Horse stallions

Results

In all five stallions, when the concentration of cadmium increased, the sperm motility decreased. Motility of sperm correlates to sperm viability and fertility and these data show that the sperm motility is higher in semen from paired semen samples when the naturally occurring concentration of cadmium is lower in the semen. Sperm viability is an indicator of the proliferation environment of the stallion' s prostate gland. Thus, the higher cadmium values from semen decreases the proliferation (metastasis) of prostate cells and replication of viruses within the prostate environment. This test supports the thesis that elevating the cadmium concentration in man' s prostate gland to mimic that of the stallion' s relatively high cadmium concentration in his prostate gland decreases man' s incidence of prostate cancer, decreases his fertility, and . protects against viral (AIDS) infections and age-onset diseases.

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Claims

IT IS CLAIMED:

1. A method of decreasing the PGE2 : PGF2 ratio and zinc: cadmium ratio in the body fluids of a mammal which comprises administering to the mammal an amount of a pharmaceutically acceptable and bioavailable cadmium i salt sufficient to lower the concentration of PGE2 and concentration of zinc in the mammal's body fluids.

2. A method in accordance with claim 1, wherein said mammal is a human.

3. A method in accordance with claim 2, wherein said cadmium salt is administered in a series of daily doses at dose levels of about 1 μg to about 100 μg per day.

4. A method in accordance with claim 2, wherein said cadmium salt is administered in a series of daily doses at dose levels of about 1 μg to about 50 μg per day.

5. A method in accordance with claim 2, wherein said cadmium salt is administered in a series of daily doses at dose levels of about 10 μg to about 50 μg per day.

6. A method in accordance with claim 1, wherein said cadmium salt comprises the sulfate, nitrate, chloride or acetate cadmium salt.

7. A method in accordance with claim 1, wherein said cadmium salt is administered in combination with at least one estrogenic compound.

8. A method in accordance with claim 7, wherein the estrogenic compound comprises a conjugated estrogen or a mixture of conjugated estrogens.

9. A method in accordance with claim 7, wherein the estrogenic compound is administered at a level of about 0.1 mg to about 0.5 mg per day.

10. A method in accordance with claim 1, wherein said cadmium salt is administered in combination with at least one protease inhibitor.

11. A method in accordance with claim 10, wherein the protease inhibitor comprises indinavir sulfate, ritonavir, invirase or nelfinavir mesylate.

12. A method in accordance with claim 10, wherein the protease inhibitor is administered at a level of about 600 mg to about 2400 mg per day.

13. A method of inhibiting the concentration of zinc-containing and PGE2-dependent matrix metalloproteinases in the body of a mammal which comprises administering to said mammal one or more > pharmaceutically acceptable and bioavailable cadmium salts in an amount sufficient to lower the concentration of PGE2 and the concentration of zinc in the mammal's body.

14. A method in accordance with claim 13, wherein the mammal is a human.

15. A method in accordance with claim 13, wherein said cadmium salt is administered in a series of daily doses at dose levels of about 1 μg to about 100 μg per day.

16. A method in accordance with claim 13, wherein said cadmium salt is administered in a series of daily doses at dose levels of about 1 μg to about 50 μg per day.

17. A method in accordance with claim 13, wherein said cadmium salt is administered in a series of daily doses at dose levels of about 10 μg to about 50 μg per day.

18. A method of regulating the concentration of zinc in body fluids and tissues of a mammal which comprises administering to a mammal suffering from fluctuations in the level of zinc in his body fluids and tissues a bioavailable and physiologically acceptable cadmium salt in a doasage regimen sufficient to minimize said fluctuations.

19. A method in accordance with claim 18, wherein said cadmium salt is administered in a series of daily doses at dose levels of about 1 μg to about 100 μg per day.

20. A method in accordance with claim 19, wherein said cadmium salt is administered in a series of daily doses at dose levels of about 1 μg to about 50 μg per day.

21. A method in accordance with claim 18, wherein said cadmium salt comprises the sulfate, nitrate, chloride or acetate cadmium salt.

22. A method for preventing or delaying the onset of prostate, colon or breast cancer in a human at risk of developing said cancer which comprises administering to the human a pharmaceutically acceptable and

> bioavailable cadmium salt in an amount sufficient to prevent or delay the growth, division or metastasis of cancer cells.

23. A method for preventing or delaying the onset of multiple sclerosis in a human at risk of developing multiple sclerosis which comprises administering to the human a pharmaceutically acceptable and bioavailable cadmium salt in an amount sufficient to regulate the concentration of zinc in the body fluids and tissues of said human.

24. A method for preventing or delaying the onset of diabetes in a human at risk of developing diabetes which comprises administering to the human a pharmaceutically acceptable and bioavailable cadmium salt in an amount sufficient to regulate the concentration of zinc in the body fluids and tissues of said human.

25. A method for inhibiting cancerous tumor growth in a human which comprises administering to a human with a cancerous tumor a pharmaceutically acceptable and bioavailable cadmium salt at dose levels effective to sufficiently decrease the ratio of PGE2 : PGF2 and ratio of zinc: cadmium in the patient's body fluids such that the growth, division or metastasis of cancer cells is inhibited or prevented.

26. A method for the treatment of multiple sclerosis in a human suffering therefrom which comprises administering a pharmaceutically acceptable and bioavailable cadmium salt at dose levels effective to minimize fluctuations in levels of zinc in body fluids and tissues of said human that are associated with multiple sclerosis.

27. A method for the treatment of diabetes in a human suffering therefrom which comprises administering a pharmaceutically acceptable and bioavailable cadmium salt at dose levels effective to minimize fluctuations in zinc levels in body fluids and tissues of said human that are associated with diabetes .

28. A method in accordance with claim 1, 13 or 18 wherein said cadmium salt is administered orally, parenterally, or by inhalation.

29. A pharmaceutical composition comprising a combination of one or more cadmium salts and a pharmaceutically acceptable carrier, said composition in oral dosage form.

30. A pharmaceutical composition comprising a combination of a) one or more cadmium salts; b) an estrogenic compound, a protease inhibitor, or a combination thereof; and c) a pharmaceutically acceptable carrier.