AU713031B2 - Use of griseofulvin for inhibiting the growth of cancers - Google Patents

Description

1 use Of griseofulvin for inhibiting the growth Of cancers TECHNICAL

FIELD

This invention is a pharmaceutical composition that is usefu for the treatment of cancers and twmors particularly in human and warm blooded animals. The composition contains I griseofulvi. It can be used in combination with other chemotherapeutic agents also.

BACKGROUND OF THE INVENTION Cancers including leukemia, are the leading cause of death in animnals and humans. The exact cause of leukemia is not known, but links between certain activities such as smoking or exposure to carcinogens and the incidence of certain types of leukemia and rumors has been shown by a number of researchers.

Many types of chemotherapeutic agents have been shown to be effective against cancers, *etumors and leukmia, but not all tpe of cacr n tumor cells respond to these agents.

Unfortunately. many of ths gnsalso dsrynormal cells. The exact mechanism for. the .action of these chemotherapetic agents are not always known.

Despite advances in the field of cancer and leukemia treatments the leading therapies to date are radiation aria chemotherapy and bone marrow transplants. Chemotherapeutic approaches are said to fight cancers that are particularly aggressive. Such cytocidal or cytostatic agents work best on cancers with large growth factrs. ones whose cells are rapidly divding. To date, hormones, in particular esrogen, progesterne and testosterone, and some antibiotics produced by a variety of microbes, alkylating agents, and anti-rmbolites form the bulk of therapies available 20 to oncologists Idally cytotoxic agents that have specificity for leuktemia, cacer and tumor cells .0 while not affecting normal cells would be extremely desirable. Unfortunately, none have been found and instead agents which target especially rapidly dividing cells (both diseased and normal) have been used I I la Clearly, the development of materials that would target cancer or leukemia cells due to some unique specificity for them would be a breakthrough. Alternatively, materials that were cytotoxic to leukemia or cancer cells while exerting mild effects on normal cells would be desirable. Therefore, it is an aspect of this invention to provide a pharmaceutical composition that is effective in treating leukemia with mild or no effects on normal blood cells.

More specifically, it is an aspect of this invention to provide a compositions comprising a pharmaceutical carrier and a griseofulvin as defined herein along with a method for treating cancer, leukemia and tumors.

0 a.

a* a t The use of griseofulvin in combination with other chemotherapeutc agents which are effective in destroying the tumor is a novel method of treatent. Giseofulvmn can also be used to treat viral infections in the presence of a potentiator.

SUMMARY OF THE ENTION A pharmaceutical composition for treatment of mammals and in panicular warm blooded animals and humans, which are affected by leukemia compising a phanrmaceutical carier I and an effective amount of gnsofuivin. Griseofulvin has the formula: ocH3 o0 OCH3 t0 O0 Cl CH3 These compositions can be used to inhibit the growth of ukmia tumors and can cells in humans or animals by administration of an ective amount eith orally, rectally, topically or parenterally, or tra venously. These compositions do not significantly affect healthy 9* cels.

IS.Potentiatorsan also be used in combination with griscofulvin as can chemotherapeutic agent&.

*2 2a Throughout the description and claims of the specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

DETAILED DESCRIPTION OF THE INVENTION A. DEFINITIONS: As used herein, the term "comprising" means various components can be conjointly employed in the pharmaceutical composition of this invention. Accordingly, the terms "consisting essentially of' and "consisting of' are embodied in the term comprising.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

As used herein, the term "safe and effective amount" refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. The specific "safe and effective amount" will, obviously, vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal being treated, the duration of the treatment, the nature of concurrent 9 C:\WNOIRD\ILONA\SHARONW834SP.DOC WO 97/05870 PCT/US96/12475 -3therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.

As used herein, a "pharmaceutical addition salts" is salt of the anti-leukemia compound with an organic or inorganic acid. These preferred acid addition salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like.

As used herein, a "pharmaceutical carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering the anti-leukemia agent to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind.

As used herein, "cancer" or "leukemia" refers to all types of cancers or neoplasm or malignant disease which attack normal healthy blood cells or bone marrow which produces blood cells which are found in mammals.

As used herein, "viruses" includes viruses which cause diseases in warm blooded animals including HIV, influenza, rhinoviruses, herpes and the like.

As used herein, "griseofulvin" means 7 -Chloro-2',4,6-trimethoxy-6-methylspiro [benzofuran-2-(3H),'-[2]cyclohexene]-3,4'-dione. It is an antibiotic substance produced by penicillium griseofulvum As used herein "potentiators" are materials such as triprolidine and its cis-isomer which are used in combination with griseofulvin. Potentiators can suppress the immune system or enhance the effectiveness of the drugs.

As used herein "chemotherapeutic agents" includes DNA-interactive Agents, Antimetabolites, Tubulin-Interactive Agents, Hormonal agents and others, such as Asparaginase or hydroxyurea.

B. GRISEOFULVIN Griseofulvin has the following structure: OCH3

O

OCH3

O

Cl CH3 It is prepared according to the method described in U.S. 3,069,328 issued to Hockenhull (1962) and U.S. 3,069,328 issued to Dorey et al. (1962) C. CHEMOTERP~ EUT[C

AGENTS

The chemotherapeutic agents are generally grouped as DNA-interactive Agents, ,ktmctaolites, Tubulin-Inreractive Agents, Hormonal agents and others such as Asparaginase or hydroxyura Each of the groups of chemotherapeutic agents can be further divided by type of activity or compound. The chemotherapeutic agents used in combination with gnseofuj',jn include members of all of these groups. For a detailed discussion of the chemotherapeutic agents and their method of administration, see Dorr, et al. Cancer Chemotherapy Handbook 2d edition, t pages 15-34, Appleton Lange (Connecticut, 1994) herein incorporated by reference.

DNA-Interactive Agents include the alkylating agents, e.g. Cisplatin, Cyclophospamide, Altretaminc; the DNA strand-breakage agents, such as Bleomycin; the intercalating II inhibitors, Dacuinomycin and Doxorubicin); the noninterualating topoisomerase 11 inhibitors such as, Etoposide and Teniposide; and the DNA minor groove binder Picamnydin.

The alkylating agents form covalent chemical adducts with cellular DNA, RNA, and protein molecules and with smaller amino acids, glutathione and similarchmal.Gnrly these alkylating agents react with a nucleopiiic atom in a cellular constituent such as an amino, ca r oxy, phosphate sul yd y group innucleic ad s protei ms m inol acids, r glutatbione. Il mechnis and terole ofthese alywgagents in cancer thrp is niot welunderstood.

Typicail alkylatisi agents include: Nitrogen ,'mustards, such as Chiorambucil, Cyclophosplhamide, Isofamide, Mechiorethamine, Melphalan Uracil mustard; Aziridine such as Thiotepa a. methallestjphointe esters such as Busulfan; a: a nitrmuo ureas, such as Carmustine, Lomustine, Streczocn; a 25 platin complexes, such as Cisplatin, Carboplain bioredutive alkylator, such as Mitomyci, and PrOcarbazine Dacarbaz=n and DNA strand breaking agents include Bleomycin; DNA topoisomerase HI inhibitors include the following: Intercalators, such as Amsocrne, Dactinomycin, Daunorubicin, Doxorubicin, IdarubiciA. and Mitoxantrone; nonintercalators, such as Etoposide and Teniposide.

The DNA minor grov binder is Plcmycia.

The antimetabolites interfere with the production of nucleic acids by one or the Other Of two major mechanism. Some of the drup inhibit production of the deoxyribonucleoside triphosphates that are the immediate precw for DNA synthesis, thus inhibiting

DNA

replication. Some of the compounds arm suffxcdy like purines or pyrimidines to be able to substitute for them in the anabolic nucleotide pathways. These analogs can then be substituted into the DNA and RNA instead of their nlormlfa counterpats The antimem~bolites useful herein include: folate a-ntagoruists such as Methotrexate and trirnetrexate Pyrimiudine antagonists, such as Fluorouracil, Fluorodeoxyuridjne, CB37 17, Azacitidine, Cytarabine, and Floxuridine pwiri antagonists include Mercaptopurine, 6 -Thioguanine, Fludarabine, Pentostatin; sugar modified analogs include cytarabine, Fludarabine; ribonucleotide reductase inhibitors include hydroxyurea.

Tubulin Interactive agents act by binding to specific sites on tubulin, a protein that polymerizes to form cellular iucrotubules. Mficrotubules are critical cell structure units. When the interactive agents bind on the protein, the cell can not form microtubules Tubulin Interactive agents include Vincristine and Vinbiastine, both alkaloids and Paclitaxel.

Hormonal agents are also usefu in the treatent of cancers and tumors. T'hey are used in hormonally susceptible tumors and are usually derived from natural sources. These include: estrogens, conjugated estrogens and Ethinyl Estradiol -and DiethylsibesteroL, Chlortrianisen and Idenestrol;an v~u *..progestins such as Hydroxyprogesterone carot, Medroyprgsterone, adMgsr ~androgens such as testosterone, testosterone Propionate; fluoxYmesterone, methyltestostelrone;, Adrenal corticosteroids are derived from natural adrenal cortisol or hydrocortisone. They 9* are used because of their anti inflammaory benefits as well as the ability of some to inhibit mitotic divisions and to halt DNA synthesis. These compounds include, Prednisone, Dexamethasone 9 9 Methyipredaisolome and Prednisolone.

9999 25 Latudnizing hormone Melasng hormone agents or gonadotropin-releasing hormone antagonists arm used primarily the treatment of prostate cancer. These include leuprolide acetate and g s ilin acett They prevent the biosynthesis of steroids in the testes Antihormonal antigens include: antiestrogenic agents such as Tamosifen, antiandrogen agents such as Flutamide and antiadrenal agents such as Mitotane and Aminoglutethimide.

Hydroxyurea appears to act pimaril through inhibition of the enzyme ribonucleotide reductase.

Asparaginase is an enzyme which coamu asparagine to nonfunctional aspartic acid and thus blocks protein synthesis in the tumor.

WO 97/05870 PCT/US96/12475 -6- D. POTENTIATORS The "potentiators" can be any material which improves or increase the efficacy of the pharmaceutical composition or acts as an immunosuppressor. One such potentiator is triprolidine and its cis-isomer which are used in combination with the chemotherapeutic agents and the griseofulvin. Triprolidine is described in US 5,114,951 (1992). Another potentiator is procodazole, lH-Benzimidazole.2-propanoic acid; [B-(2-benzimidazole) propionic acid; 2-(2carboxyethyl)benzimidazole; propazol]. Procodazole is a non-specific active immunoprotective agent against viral and bacterial infections and can be used with the compositions claimed herein.

It is effective with griseofulvin alone in treating cancers, tumors, leukemia and viral infections or combined with chemotherapeutic agents.

Propionic acid and its salts and esters can also be used in combination with the pharmaceutical compositions claimed herein.

Antioxidant vitamins such as vitamins A, C and E and beta-carotene can be added to these compositions.

E. DOSAGE Any suitable dosage may be given in the method of the invention. The type of compound and the carrier and the amount will vary widely depending on the species of the warm blooded animal or human, body weight, and the type of cancer or tumor or viral infection being treated.

Generally a dosage of between about 1 milligram (mg) per kilogram (kg) of body weight and about 8000 mg per kg of body weight is suitable for either the griseofulvin or the chemotherapeutic agent. Preferably from 15 mg to about 5000 mg/kg of body weight is used. Generally, the dosage in man is lower than for small warm blooded mammals such as mice. A dosage unit may comprise a single compound or mixtures thereof with other compounds or other cancer inhibiting compounds. The dosage unit can also comprise diluents, extenders, carriers, liposomes and the like. The unit may be in solid or gel form such as pills, tablets, capsules and the like or in liquid form suitable for oral, rectal, topical, intravenous injection or parenteral administration or injection into or around the bone marrow. The range and ratio of griseofulvin to chemotherapeutic agent will depend on the type of cancer or tumor being treated and the particular chemotherapeutic agent.

F. DOSAGE DELIVERY

FORMS

The chemotherapeutic agents, griseofulvin and, optionally, the potentiators are typically mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid and the type is generally chosen based on the type of administration being used. The active agent can be coadministered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar.

Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid

I

WO 97/05870 PCT/US96/12475 -7forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.

Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms would also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

Specific examples of pharmaceutical acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U. S. Pat. No. 3,903,297 to Robert, issued Sept. 2, 1975. Techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modern Pharmaceutics Chapters 9 and 10 (Banker Rhodes, Editors, 1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976).

G. METHOD OF TREATMENT The method of treatment can be any suitable method which is effective in the treatment of the particular cancer or tumor type being treated. Treatment may be oral, rectal, topical, parenteral or intravenous administration or by injection into the tumor or cancer. The method of applying an effective amount also varies depending on the leukemia, cancer, tumor or virus being treated. It is believed that parenteral treatment by intravenous, subcutaneous, or intramuscular application of the griseofulvin, formulated with an appropriate carrier, additional cancer inhibiting compound or compounds or diluent to facilitate application will be the preferred method of administering the compounds to warm blooded animals.

In addition to the use of chemotherapeutic agents and potentiators, griseofulvin can be combined with fungicides, herbicides or other antiviral agents. Preferred herbicides and fungicides include carbendazim, fluoconazole, benomyl, glyphosate and propicodazole.

Example 1 In an acute HIV in vitro model, griseofulvin inhibited viral replication by 98% at lOg/ml with a therapeutic index of 5.3. AZT, a known HIV drug, also inhibited viral replication by 98% at lug/ml with a therapeutic index of 12,500. The therapeutic index is the ratio of toxic dose of drug to efficacious dose of drug.

WO 97/05870 PCT/US96/12475 -8- Example 2 In an in vivo mouse study for leukemia (P388), griseofulvin showed an increase in the survival time relative to a non treated control of 156% at 4000 mg/kg dose; 188% at 5000 mg/kg dose; and 218% at 6000 mg/kg dose.

Example 3 In an in vivo mouse study for melanoma (B16), griseofulvin showed an increase in the survival time relative to a nontreated control of 165% at 4000 mg/kg dose; 179% at 5000 mg/kg dose; and 201% at 6000 mg/kg dose. Cytoxan at 300 mg/kg showed an increased survival rate of 192%.

Example 4 In an in vitro screening for Rhinovirus, type A-i, cell line WI-38, griseofulvin was effective at 100 pg/ml. The positive control was A-36683 of Abbot Company, (S,S)-1,2-bis(5methoxy-2-benzimidazolyl)-1,2-ethanediol. A-36683 has a therapeutic index of 1000-3200.

Griseofulvin has a therapeutic index of 1-2. (See Schleicher et al, Applied Microbiology, 2, No.

1, 113-116 (1972).

Example Solid tumors removed by patients are minced into 2 to 5 mm fragments and immediately placed in McCoy's Medium 5A plus 10% heat inactivated newborn calf serum plus 1% penicillin/streptomycin. Within 4 hours, these solid tumors are mechanically disassociated with scissors, forced through No. 100 stainless steel mesh, through 25 gauge needles, and then washed with McCoy's medium as described above. Ascitic, pleural, pericardial fluids and bone marrow are obtained by standard techniques. The fluid or marrow is placed in sterile containers containing 10 units of preservative free heparin per ml. of malignant fluid or marrow. After centrifugation at 150 x g for 10 minutes, the cells are harvested and washed with McCoy's medium plus 10% heat inactivated calf serum. The viability of cell suspensions is determined on a hemocytometer with trypan blue.

Cells to be cloned are suspended in 0.3% agar in enriched CMRL1066 supplemented with heat inactivated horse serum, penicillin (100 units/ml), streptomycin (2mg/ml), glutamine (2mM), insulin (3 units/ml), asparagine (0.6 mg/ml), and HEPES buffer (2mM). For the continuous exposure test each compound is added to the above mixture. Cells are placed in mm petri dishes in a top layer of agar over an underlayer of agar to prevent growth of fibroblasts.

Three plates are prepared for each data point. The plates are placed in a 37°C incubator, and are removed on day 14 for counting of the number of colonies in each plate. The number of colonies (defined as 50 cells) formed in the 3 compound treated plates is compared to the number of colonies formed in the 3 control plates, and the percent colonies surviving at the concentration of compound can be estimated. Three positive control plates are used to determine survival rate.

WO 97/05870 PCT/US96/12475 -9- Orthosodium vanadate at 200 pg/ml is used as the positive control. If there is <30% colonies in the positive control when compared to the untreated control, the test is evaluated.

At concentration of 0.5 and 5.0 pg/ml in a single dose experiment griseofulvin was not effective against tumors in this test. At concentration of 50.0 gg/ml in a continuous exposure experiment griseofulvin was effective against colon, lung, non-small cell, and ovarian cancers.

Over all 5 of 6 had <50% survival.

Claims (17)

1. A pharmaceutical composition for treating cancer and tumors in a mammal comprising a pharmaceutically acceptable carrier and a safe and effective amount of griseofulvin, a chemotherapeutic agent and a potentiator.

2. A pharmaceutical composition according to claim 1 wherein said chemotherapeutic agent is selected from the group consisting of DNA-interactive Agents, Antimetabolites, Tubulin-lnteractive Agents, Hormonal Agents, So Asparaginase or hydroxyurea. o

3. A pharmaceutical composition according to claim 1 or 2 wherein said chemotherapeutic agent is selected from the group consisting of Asparaginase, hydroxyurea, Cisplatin, Cyclophosphamide, Altretamine, Bleomycin, Dactinomycin, Doxorubicin, Etoposide, Teniposide and Plicamycin.

4. A pharmaceutical composition according to claim 1 or 2 wherein said chemotherapeutic agent is selected from the group consisting of Taxol, Methotrexate, Fluorouracil, Fluorodeoxyuridine, CB3717, Azacitidine, Cytarabine, 20 Floxuridine, Mercaptopurine, 6-Thioguanine, Fludarabine, and Pentostatin.

A method of treating cancer or tumors in mammals comprising administering a composition according to any one of claims 1 to 4.

6. A method of treating cancer or tumors in a mammal comprising administering a composition according to claim 1, comprising an amount of griseofulvin to deliver from 15 mg to 5000 mg of griseofulvin per kg body weight of said warm blooded mammal.

7. A method of treating viral infections in a mammal comprising administering a composition comprising a pharmaceutical carrier and a safe and effective -amount of griseofulvin. VAT A:66834SP.DOC 11

8. A method of treating viral infections in a mammal comprising administering a composition comprising an amount of griseofulvin to deliver from 1 mg to 8000 mg of griseofulvin per kg body weight of said warm blooded mammal.

9. A method of treating viral infections in a mammal comprising administering a composition comprising an amount of griseofulvin to deliver from 15 mg to 5000 mg of griseofulvin per kg body weight of said warm blooded mammal.

10. Use of a composition according to any one of claims 1 to 4, for the S manufacture of a medicament for treating cancer and tumors.

11. Use of a pharmaceutical composition according to claim 1, wherein griseofulvin is at the amount of 15 mg to 5000 mg per kg body weight of said mammal, for the manufacture of a medicament for treating cancer and tumors.

12. Use of a composition comprising a pharmaceutical carrier and a safe and S effective amount of griseofulvin, for the manufacture of a medicament for treating viral infections in a mammal.

S13. An use according to claim 12, wherein said griseofulvin is from 1 mg to 8000 mg per kg body weight of said mammal.

14. An use according to claim 12, wherein said griseofulvin is from 15 mg to 5000 mg per kg body weight of said mammal.

A pharmaceutical composition according to claim 1 substantially as hereinbefore described with reference to any one of the examples.

16. A method according to claim 5 substantially as hereinbefore described with reference to any one of the examples. VAT A:6834SP.DOC 12

17. An use according to claim 10 substantially as hereinbefore described with reference to any one of the examples. DATED: 15 septeiiber, 1999 PHILLIPS ORMONDE FITZPATRICK Attorneys for: THE PROCTER GAMBLE COMPANY 0 0 VAT MM84SP DOC