WO2005077424A2

WO2005077424A2 - Conjugate for destroying cancer cells

Info

Publication number: WO2005077424A2
Application number: PCT/US2005/003464
Authority: WO
Inventors: David Rubin
Original assignee: David Rubin
Priority date: 2004-02-05
Filing date: 2005-02-07
Publication date: 2005-08-25
Also published as: WO2005077424A3

Abstract

A conjugate of methyl glyoxal with methyl glucoside destroys cancer cells while sparing normal cells.

Description

METHOD FOR DESTROYING CANCER CELLS

Field of the Invention The present invention relates to a method for destroying cancer cells by destroying their energy supply.

Background of the Invention Control of cell growth is one of the most important aspects of an animal's physiology. The cells of an adult must divide frequently enough to allow tissues to remain in a steady state, and division must be stimulated at wounds or when special requirements are placed on the tissues. There must be many circulating cell-specific factors that signal individual cell types whether to divide or nor. However, uninhibited cell growth results in malignant tumors. One of the greatest problems associated with treatment of cancers is delivery of a cytotoxic agent directly to the tumor or cancer cells without affecting normal cells in the body. Although it was hoped that monoclonal antibodies could be used as delivery agents for cytotoxic drugs to treat cancers and to inhibit metastasis of existing cancers, monoclonal antibodies have not lived up to their promise. Normal living cells engage in glycolysis, that is, the metabolism of a molecule of glucose to carbon dioxide and water. This is normally first an anaerobic process, namely, converting glucose to pyruvate, and then, after a few steps, it becomes an aerobic process in forming acetyl CoA that enters the Krebs cycle to produce energy in the form of ATP molecules, carbon dioxide, and water. It is crucial in normal cells that energy be conserved. During glycolysis, prior to formation of pyruvate, a molecule of bis-glyceraldehyde 1, 3-diphosphate is formed. This molecule is the result of the catalytic reaction of the enzyme glyceraldehyde 3 phosphate dehydrogenase (GA3PDH) on glyceraldehyde 3-phosphate. In normal cells, GA3PDH is inhibited by ATP; once ATP is formed and accumulates, glycolysis stops. In many cancerous cells, however, the enzyme GA3PDH is mutated, and as a result the cancerous cells continue the process of glycolysis and consume more and more glucose. This phenomenon is currently used in order to detect malignancy, most notably with the fluoro-deoxy-glucose-PET scan imaging technique. Evidently, this glucose derivative molecule is consumed by malignant tumors at a much higher rate than by normal cells. However, this molecule emits a positron that, after reacting with a neighboring electron, forms a gamma ray. This gamma ray can be detected, so that an image of the malignant tumor appears on a film exposed to this radiation. Methyl glyoxal, also known as pyruvic aldehyde, is known to inhibit the enzyme GA3PDH. The inhibition of the aberrant enzyme of the cancerous cells by methyl glyoxal is even a few times higher than inhibition for normal cells. Thus, methyl glyoxal should be an ideal candidate for destroying cancerous cells. However, there are several reasons that methyl glyoxal is contraindicated for treating a patient suffering from cancer. First, methyl glyoxal is also very toxic to normal cells. Secondly, cancerous cells own the enzymatic system methyl glyoxalase. This methyl glyoxalase system converts methyl glyoxal to pyruvic acid, and thus minimizes its.toxicity to cancer cells. Burman et al . , in U.S. Patents 6,596,755 and

6,613,793, disclose using methyl glyoxal and its imino acid conjugates for treating cancer. Burman et al . disclose the broad spectrum anticancer activity of methyl glyoxal in vi tro on cancer of the colon, prostate, larynx, kidney, pancreas, lung, breast, intestine, oral cavity, ovary, as well as glioblastoma, and leukemia. Kiso et al . , in U.S. Patent No. 6,620.424, disclose glycolytic metabolism regulators such as itaconic acid. These glycolytic metabolism regulators are used to prevent obesity as well as to provide an antidiabetic effect and an antilipemic effect. Lampidis et al . , in U.S. Patent No. 6,670,330, disclose a class of glycolytic inhibitors useful for treating solid tumors by attacking anaerobic cells at the center of the tumor. In this protocol, 2-deoxyglucose, oxamate, and analogs thereof have a natural selective toxicity toward anaerobic cells, and are used to increase ' the efficacy of standard cancer chemotherapeutic and radiation treatment regimens as well as new protocols emerging with anti-angiogenic agents.

Summary of the Invention It is an object of the present invention to overcome the aforesaid deficiencies in the prior art. It is another object of the present invention to provide a treatment for cancer. It is a further object of the present invention to create conjugates of methyl glyoxal that are not toxic to normal cells while still toxic to cancer cells. According to the present invention, a saccharide

molecule is conjugated to methyl glyoxal. Because the

conjugate is a glucose moiety, the conjugate is concentrated and consumed in a much higher rate by cancerous cells than by normal cells. Additionally, it has, surprisingly, been found that, in cancerous cells, the conjugate inhibits the enzyme GA3PDH several times more than it does in normal cells, even more than the parent molecule, methyl glyoxal. This reaction stops the aerobic glycolysis and the main production of ATP. Another advantage of the conjugates of the present invention is that they are not a substrate for the enzymatic system of methyl glyoxalase, and thus are not destroyed by this enzyme system. The present invention also provides a pharmaceutical composition of conjugates of saccharides such as glucose with methyl glyoxal useful for killing or inhibiting multiplication of cancer cells. In a preferred embodiment, a pharmaceutically acceptable carrier, diluent, or solvent is used for the conjugate. The method comprises administering a therapeutically effective dose of the conjugate so as to kill or inhibit the multiplication of cancer or tumor cells. The conjugates of the present invention can be administered alone or in combination with other therapeutic agents such as 5-fluorouracil, methotrexate, etoposide, paclitaxel, taxotere, doxorubuicin, daunarubicin, vincristine, vinblastine, 4-hydroxyanisole, antiangiogenic drugs, and other known therapeutic agents for treating cancer. Detailed Description of the Invention In order to overcome the toxicity of methyl glyoxal to normal cells while still destroying cancer cells, methyl glyoxal was reacted with methyl glucoside. The conjugate produced is called, for purposes of the present invention, pyruviden 1,6-methyl glucoside, or PG. Because PG is a glucose moiety, the PG molecule is concentrated at the site of cancerous cells and is consumed at a much higher rate by cancer cells than by normal cells. Secondly, in cancer cells, the PG molecule surprisingly inhibits the enzyme GA3PDH several times more than it does in normal cells, even more than the parent molecule methyl glyoxal. This reaction stops the aerobic glycolysis and the main production of ATP.

Another major advantage of PG over methyl glyoxal is that PG is not a substrate for the enzyme system of methyl glyoxalase, and thus is not destroyed by this enzyme system.

The safety of PG was established by dosing mice until and LD50 was established. For PG, the LD50 is 4650 mg/kg, indicating an extremely low toxicity for normal cells. Athymic nude mice with human malignant melanoma and bronchiogenic carcinoma were studied. In the control group of ten mice, all of the ten mice in each group died between day 35 and day 45. In the treated melanoma group of ten mice, one mouse died at day 78, one mouse died at day 91, and the remaining eight mice exhibited no sign of melanoma after day 91. In the bronchiogenic carcinoma group of ten mice, after 120 days all ten mice were alive. One mouse appeared to have developed a small tumor, and the other nine mice appeared normal. Pharmaceutical compositions according to the present invention can be administered by any convenient route, including parenteral, subcutaneous, intravenous, intramuscular, intra peritonea, or transdermal . Alternatively or concomitantly, administration may be by the oral route. The dosage administered depends upon the age, heath, and weight of the recipient, nature of concurrent treatment, if any, and the nature of the effect desired. Compositions within the scope of the present invention include all compositions wherein the active ingredient is contained in an amount effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each compound is within the skill of the art. Typical dosages comprise 0.01 to 100 mg/kg body weight. The preferred dosages comprising 0.1 to 100 mg/kg body weight. The most preferred dosages comprise 1 to 50 mg/kg body weight. Pharmaceutical compositions for administering the active ingredients of the present invention preferably contain, in addition to the pharmacologically active compound, suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Preferably, the preparations, particularly those preparations which are administered orally and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.01 to about 99 percent by weight, preferably from about 20 to 75 percent by weight, active compound (s) , together with the excipient. For purposes of the present invention, all percentages are by weight unless otherwise indicated. In addition to the following described pharmaceutical composition, the compounds of the present invention can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes. The pharmaceutically acceptable carriers include vehicles, adjuvants, excipients, or diluents that are well known to those skilled in the art and which are readily available. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active compounds and which has no detrimental side effects or toxicity under the conditions of use. The choice of carrier is determined partly by the particular active ingredient, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical compositions of the present invention. Formulations can be prepared for oral, aerosol, parenteral, subcutaneous, intravenous, intra arterial, intramuscular, intra peritoneal, intra tracheal, rectal, and vaginal administration. Suitable excipients are, in particular, fillers such as saccharides, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hyd oxypropylmethylcellulose, sodium catrboxymethylcelullose, and/or polyvinyl pyrrolidone. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water- soluble form, such as water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides . Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.

Optionally, the suspension may also contain stabilizers. Other pharmaceutically acceptable carriers for the active ingredients according to the present invention are liposomes, pharmaceutical compositions in which the active ingredient is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipid layers. The active ingredient may be present both in the aqueous layer and in the lipidic layer, inside or outside, or, in any event, in the nonhomogeneous system generally known as a liposomic suspension. The hydrophobic layer, or lipid layer, generally, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surface active substances such as dicetyl phosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature . The compounds may also be formulated for transdermal administration, for example in the form of transdermal patches so as to achieve systemic administration. Formulations suitable for oral administration can consists of liquid solutions such as effective amounts of the compound (s) dissolved in diluents such s water, saline, or orange juice; capsules, tables, sachets, lozenges, and troches, each containing a predetermined amount of the active ingredient as solids or granules; powders, suspensions in an appropriate liquid; and suitable emulsions. Liquid formulations may include diluents such as water and alcohols, e . g. , ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agents, or emulsifying agents. Capsule forms can be of the ordinary hard- or soft- shelled gelatin type containing, for example, surfactants, lubricant, and inert fillers, such s lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscaramellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other preservatives, flavoring agents, and pharmaceutically acceptable disintegrating agents, moistening agents preservatives flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a carrier, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base such as gelatin or glycerin, or sucrose and acacia. Emulsions and the like can contain, in addition to the active ingredient, such carriers as are known in the art . Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compounds can be administered in a physiologically acceptable diluent in a pharmaceutical carriers, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol such as ethanol, isopropanol, or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, glycerol ketals such as 2,2- dimethyl-1, 3-dioxolane-4-methanol, ethers such as poly (ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides, without the addition of a pharmaceutically acceptable surfactants, such as soap or a detergent, suspending agent, such as carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants. Oils which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Fatty acids can be used in parenteral formulations, including oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable salts for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include cationic detergents such as dimethyl dialkyl ammonium halides, and alkyl pyridimium halides; anionic detergents such as dimethyl olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates and sulfosuccinates; polyoxyethylenepolypropylene copolymers; amphoteric detergents such as alkyl-beta-aminopropionates and 2-alkyl-imidazoline quaternarry ammonium salts; and mixtures thereof. Parenteral formulations typically contain from about 0.5 to 25% by weight of the active ingredient in solution. Suitable preservatives and buffers can be used in these formulations. In order to minimize or eliminate irritation at the site of injection, these compositions may contain one or more nonionic surfactants having a hydrophilic-lipophlic balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be present in unit dose or multiple dose sealed containers, such as ampoules and vials, and can be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, e . g. , water, for injections immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Additionally, the active ingredients can be formulated into suppositories by mixing the active ingredient with a variety of bases, including emulsifying bases or water- soluble bases. Formulations suitable for vaginal administration may be in the form of pessaries, tampons, creams, gels, pastes, foam, or spray formulations containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate. The active ingredients can be used as functionalized congeners for coupling to other molecules, such as amines and peptides. The use of such congeners provides for increased potency, prolonged duration of action, and prodrugs. Water solubility is also enhanced, which allows for reduction, if not complete elimination, of undesirable binding to plasma proteins and partition in to lipids. Accordingly, improved pharmacokinetics can be realized. In determining the dosages of the conjugate to be administered, the dosage and frequency of administration is selected in relation to the pharmacological properties of the specific active ingredients. Normally, at least three dosage levels should be used. In toxicity studies in general, the highest dose should reach a toxic level but be sublethal for most animals in the group. If possible, the lowest dose should induce a biologically demonstrable effect. These studies should be performed in parallel for each compound selected. Additionally, the ID₅₀ level of the active ingredient in question can be one of the dosage levels selected, and the other two selected to reach a toxic level . The lowest dose is that dose that does not exhibit a biologically demonstrable effect. The toxicology tests should be repeated using appropriate new doses calculated on the basis of the results obtained. Young, healthy mice or rats belonging to a well- defined strain are the first choice of species, and the first studies generally use the preferred route of administration. Control groups given a placebo or which are untreated are included in the tests. Tests for general toxicity, as outlined above, should normally be repeated in another non- rodent species, e . g. , a rabbit or dog. Studies may also be repeated using alternate routes of administration. Single dose toxicity tests should be conducted in such a way that signs of acute toxicity are revealed and the mode of death determined. The dosage to be administered is calculated on the basis of the results obtained in the above- mentioned toxicity tests. It may be desired not to continue studying all of the initially selected compounds. Data on single dose toxicity, e . g. , ID₅₀, the dosage at which half of the experimental animals die, is to be expressed in units of weight or volume per kg of body weight and should generally be furnished for at least two species with different modes of administration. In addition to the ID₅₀ value in rodents, it is desirable to determine the highest tolerated dose and/or lowest lethal dose for other species, i.e., dog and rabbit. When a suitable and presumably safe dosage level has been established as outlined above, studies on the drug's chronic toxicity, its effect on reproduction, and potential mutagenicity may also be required in order to ensure that the calculated appropriate dosage range will be safe, also with regard to these hazards . Pharmacological animal studies on pharmacokinetics revealing, e. g. , absorption, distribution, biotransformation, and excretion of the active ingredient and metabolites are then performed. Using the results obtained, studies on human pharmacology are then designed. Studies of the pharmacodynamics and pharmacokinetics of the compounds in humans should be performed in healthy subjects using the routes of administration intended for clinical use, and can be repeated in patients. The dose-response relationship when different doses are given, or when several types of conjugates or combinations of conjugates and free compounds are given, should be studied in order to elucidate the dose-response relationship (dose vs . plasma concentration vs . effect) , the therapeutic range, and the optimum dose interval. Also, studies on time-effect relationship, e . g. , studies into the time-course of the effect and studies on different organs in order to elucidate the desired and undesired pharmacological effects of the drug, in particular on other vital organ systems, should be performed. The compounds of the present invention are then ready for clinical trials to compare the efficacy of the compounds to existing therapy. A dose-response relationship to therapeutic effect and for side effects can be more finely established at this point. The amount of compounds of the present invention to be administered to any given patient must be determined empirically, and will differ depending upon the condition of the patients. Relatively small amounts of the active ingredient can be administered at first, with steadily increasing dosages if no adverse effects are noted. Of course, the maximum safe toxicity dosage as determined in routine animal toxicity tests should never be exceeded. The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various application such specific embodiments without undue experimentation and without departing from the generic concept. Therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means and materials for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention. Thus, the expressions "means to..." and "means for..." as may be found in the specification above and/or in the claims below, followed by a functional statement, are intended to define and cover whatever structural, physical, chemical, or electrical element or structures which may now or in the future exist for carrying out the recited function, whether or nor precisely equivalent to the embodiment or embodiments disclosed in the specification above. It is intended that such expressions be given their broadest interpretation.

Claims

WHAT IS CLAIMED IS: 1. A conjugate of methyl glyoxal and methyl glucoside.

2. A composition for destroying cancer cells comprising the conjugate according to claim 1 and a pharmaceutically acceptable carrier.

3. The composition according to claim 2 further including at least one anti-cancer drug.

4. The composition according to claim 3 wherein the at least one anti-cancer drug is selected from the group consisting of anti-angiogenic agents.

5. The composition according to claim 3 wherein the at least one anti-cancer drug is selected from the group consisting of 5-fluorouracil, methotrexate, etoposide, paclitaxel. Taxotere, doxorubicin, daunarubicin, vincristine, vinblastine, 4-hydroxyanisole, and mixtures thereof.

6. A method for destroying cancer cells comprising administering to a patient in need thereof an effective amount

of the conjugate according to claim 1.

7. The method according to claim 6 wherein the conjugate is administered in combination with at least on other anti-cancer drug.

8. The method according to claim 7 wherein the at least one anti-cancer drug is selected from the group consisting of anti-angiogenic agents.

9. The method according to claim 7 wherein the at least one anti-cancer drug is selected from the group consisting of 5-fluorouracil, methotrexate, etoposide, paclitaxel. Taxotere, doxorubicin, daunarubicin, vincristine, vinblastine, 4-hydroxyanisole, and mixtures thereof.