WO1993001824A1

WO1993001824A1 - Combination therapy using bioflavonoid compounds with anti-cancer drugs

Info

Publication number: WO1993001824A1
Application number: PCT/US1992/006087
Authority: WO
Inventors: Barry M. Markaverich; Rajender Singh Varma
Original assignee: Baylor College of Medicine
Current assignee: Baylor College of Medicine
Priority date: 1991-07-24
Filing date: 1992-07-17
Publication date: 1993-02-04
Anticipated expiration: 1994-01-24
Also published as: AU2393992A

Abstract

A method of regulating cell growth proliferation in normal and malignant tissues comprising this type of administering therapeutic dose of a combination of compounds. Wherein at least one compound is selected from the group of methyl p-hydroxylphenyllactate, its analogues, chemical derivatives and chemically related compounds. Further, at least one compound is selected from the group of anti-metabolites, antibiotics and alkylating agents. This combination therapy has an enhanced effect on inhibiting the cell growth and proliferation of cancer and nonmalignant cells.

Description

COMBINATION THERAPY USING BIOFLAVONOID

COMPOUNDS WITH ANTI-CANCER DRUGS

Field of the Invention

This invention relates to the use of a combination of anti-cancer drugs with bioflavonoid compounds and related compounds which include methyl p-hydroxyphenyllactate (MeHPLA), its analogs, chemical derivatives and chemically related compounds, phenylmethylene ketones, nitroalkenes, aurones, and chalcones as an enhanced anti-tumor agent and inhibitor of proliferative cell growth.

Background of the Invention

Currently available anti-cancer drugs are typically described as cytotoxic agents which inhibit cancer cell division through a variety of mechanisms. Most of these drugs block tumor cell proliferation by inhibiting DNA synthesis and interfering with the progression of the cell cycle. One popular class of anti-cancer drugs are the anti-metabolites such as arabinocytosylcytosine, 5-fluorouracil (5-FU), 6-mercaptopurine, 6- thioguanine and methotrexate (MTX) which block DNA synthesis through a variety of mechanisms. MTX is an antimetabolite directed against dihydrofolate reductase; 5-FU inhibits tRNA methylation, rRNA maturation, nuclear RNA synthesis, and thymidylate synthetase which results in "thymidineless" cell death and causes DNA damage and strand breaks; and both 5-FU and MTX cause extensive hypermethylation of the nucleic acid. Antibiotics such as bleomycin and adriamycin are DNA intercalating agents which cause DNA strand breaks and arrest of tumor cells in the G2 phase of the cell cycle. Alkylating agents such as the nitrosoureas, cis-platinum and cyclophoshamide (CY), covalently bind to nucleic acids causing DNA-DNA and DNA-protein crosslinking which results in the .arrest of cells during the G2 phase of the cell cycle. Regardless of their mechanism of action, all of the above drugs kill both cancer cells and normal cells simply because they are extremely toxic agents which lack specificity for malignant cells. Consequently, the severe systemic side effects associated with these cytotoxic agents are one of the major drawbacks of these chemotherapeutic drugs.

A recent major finding was the identification of methyl phydroxyphenyllactate (MeHPLA) as a naturally occurring cell growth regulating agent. This compound is present in all mammalian cells and inhibits cell proliferation by binding to nuclear type II sites. The interaction of MeHPLA with type II sites is suspected to be a major factor in the maintenance of a very low level of cell proliferation in all normal tissues. Additional evidence indicates that MeHPLA is metabolized by tumor cells and therefore, the deficiency of this compound in cancer cells is directly correlated to high levels of unoccupied nuclear type II sites and the loss of regulatory control. A number of compounds designed to mimic MeHPLA have been synthesized. These include DHBA and RSV-101 which bind to nuclear type II sites with high affinity and inhibit malignant cell proliferation. These compounds appear to possess little if any non- specific cytotoxicity since they are targeted to the high levels of unoccupied type II sites present in tumor cell nuclei and mimic MeHPLA as a cell growth regulating agents. MeHPLA and related compounds appear to be cytostatic as opposed to cytotoxic agents. MeHPLA and type II sites are present in essentially all non-malignant tissues that have been examined and occupancy of type II sites by MeHPLA in these tissues results in an arrest of cell proliferation. Therefore, drugs which mimic MeHPLA as cell regulatory agents such as RSV-101 and DHBA should not effect non-malignant tissues since type II sites in these cells are already occupied by endogenous MeHPLA. Conversely, mammary cancer preparations (rats, mouse, human) are deficient in MeHPLA and consequently have high levels of unoccupied nuclear type II sites. These nuclear type II sites are available for binding drugs related to MeHPLA like DHBA and RSV-101, resulting in the inhibition of tumor cell proliferation in vitro and mouse mammary tumor growth in vitro. In animal studies, treatments with DHBA and RSV-101 has not resulted in body weight loss, hair loss or other signs of generalized non-specific cytotoxicity. Further, these drugs appear to inhibit mammary cancer cell proliferation without many of the severe side effects normally observed with other standard cytotoxic chemotherapeutic drugs. Since nuclear type II sites appear to be ubiquitous, it is suspected that MeHPLA related compounds will inhibit a broad spectrum of cancers. It has been observed that MeHPLA-related compounds inhibit melanoma, retinoblastoma, lymphoma, cervical cancer and a number of breast cancer cell types in tissue culture.

The present invention describes a combination therapy using

MeHPLA related drugs and standard chemotherapeutic agents such as the aforementioned antibiotics, alkylating agents and anti-metabolites. This invention has significant benefit as compared to treatment with standard cytotoxic agents alone (which possess significant non-specific cytotoxicity). Combination therapy with both types of compounds will significantly inhibit cancer growth with a substantial reduction in severe non-specific generalized cytotoxicity.

Summary of the Invention

An objective of the present invention is the provision of a combination of compounds for the treatment of cancer.

A further object of the present invention is a method of regulating cell growth and proliferation in normal and malignant cells by using a combination of compounds. An additional object of the present invention is a procedure to inhibit the growth of proliferating cells which include a Type II nuclear estrogen binding site.

A further object of the present invention is a method of inhibiting the growth of estrogen responsive tissues.

An additional object of the present invention is the treatment of human breast cancer, and other malignancies which contain unbound nuclear Type II sites.

Thus, in accomplishing the foregoing objects, there is provided in accordance one aspect of the present invention a method of regulating cell growth and proliferation in normal and malignant cells comprising the step of administering a therapeutic dose of a combination of compounds wherein at least one compound of the combination is selected from group I and at least one compound is selected from group II and wherein the compound from group I is selected from the group consisting of antimetabolites, antibiotics and alkylating agents and the compound from group II is selected from the group consisting of:

Wherein, R₁ and R₄ are selected from the group consisting of H, OH, acylosy and halogens; R₂, R₃ and R₆ are selected from the group consisting of H, OH, OCH₃, amino, alkylamine and alkyl groups containing 1 to 6 carbons, acyloxy, and halogens; R₆ is selected from the group consisting of H, CH₃, OH, OCH₃, acyloxy and halogens; R'₁ and R'₂ are selected from the group consisting of H, OH, CH₃, OCH₃, amino, cyano, alkyl or alkylamine groups of 1 to 6 carbon atoms, acyloxy, halogens, α-azido and aziridine derivatives, 3,4-dihydroxyphenylmethylene, p- hydroxyphenylmethylene and other substituted phenyl groups; R'₃, R'₄, R'₅, R"₁ and R"₂ are selected from the group consisting of H, OH, CH₃,

OCH₃, amino, cyano, alkyl or alkylamine groups of 1 to 6 carbon atoms, acyloxy, halogens, α-azido, aziridine, acyloxy and halogen substituted derivatives.

In further embodiments of the present invention, the antimetabolites are selected from the group consisting of arabinocytosylcytosine, 6-mercaptopurine, 6-thioguanine, 5-fluorouracil, cytoxan, and methotrexate. The antibiotics are selected from bleomycin, adriamycin and vinblastin and the alkylating agents are selected from nitrosoureas, cis-platinum and cyclophosphamide.

In additional embodiments of the present invention, the following compounds can also be used as group II compounds: methyl p- hydroxyphenyllactate, analogues of methyl p-hydroxyphenyllactate, chemical derivatives of methyl p-hydroxyphenyllactate, and chemically related compounds such as the group consisting of the formula:

-6-

wherein,

R₁ is selected from the group consisting of H, alkyl groups containing 1 to 6 carbons and aryl groups;

R₂ and R₃ are selected from the group consisting of H, OH and OCH₃; and

R₄ is selected from the group consisting of H and an alkyl group of 1 to 6 carbons.

In specific embodiments of the present invention Group II compounds have consisted of: 2-(hydroxybenzylidene)-5-methyl- cyclopentanone (MV-1), 2, 6-bis(hydroxybenzylidene-4-methyl- cyclohexanone (MV-17), 2, 6-bis(3,4-dihydroxybenzylidene)-4- methylcylcohexanone (MV-18), 2,6-bis(3-methoxy-4-hydroxybenzylidene) cyclohexanone (RSV-101), 2,6-bis(3,4-dihydroxybenzyhdene) cyclohexanone (MV-3), 4-hydroxy-β -methyl-β -nitrostyrene (MV-N1), 3,4-dihydroxy-β - methyl- β -nitrostyrene (MV-N3), aurone (MV-19), 4'-hydroxyaurone (MV-

20), 3',4-dihydroxyaurone (MV-21), 2'-hydroxychalcone (MV-72), 2',4',4- trihydroxychalcone (RV-40), 4-hydroxychalcone (RV-73), 4,4'- hydroxychalcone (MV-88), α-azido-2'-hydroxychalcone (MV-35), 3,4- dihydroxy-4'-cyanochalcone (MV-46), 4'-cycanochalcone (MV-47), methyl 3-(4-hydroxyphenyl)-2-hydroxypropionate,n-propy3-(4-hydroxyphenyl)-2- hydroxypropionate, n-butyl 3-(4-hydroxyphenyl)-2-hydroxypropionate, 3-(4- hydroxyphenyl)-2-propenoic acid, 1-(4-hydroxyphenyl)-3-butanone, 1-(4- hydroxyphenyl)-3-pentanone, methyl-(4-hydrosyphenoxy) acetate, and methyl 3-(3,4-dihydroxyphenyl)-2-propenoate.

The compounds described herein can be used for inhibiting the growth of proliferating cells which include a Type II nuclear estrogen binding site.

Other and further objects, features and advantages will be apparent and the invention more readily understood from a reading of the following specification and by reference to the accompanying drawing forming a part thereof, wherein the examples of the presently preferred embodiments of the invention are given for the purpose of disclosure. Description of the Drawings

Figure 1 shows the effects of the combination therapy on mouse mammary tumor growth.

Detailed Description of the Invention

It will be readily apparent to one skilled in the art that various substitutions or modifications may be made to the invention disclosed herein within departing from the scope and spirit of the invention.

The term "biologically inhibiting" or "inhibition" of the growth of proliferating cells as used herein means partial or total growth inhibition and also includes decreases in the rate of proliferation or growth of the cells. The biologically inhibitory dose of the compounds of the present invention may be determined by assessing the effects of the test compound on malignant cell growth in tissue culture, uterine growth in the animal or tumor growth in the animal as previously described by Markaverich, et al., Cancer Research 43:3208-3211 (1983), or any other method known to those of ordinary skill in the art. These methods have also been fully described in U.S. Patent Application No. 219,680.

Administration of the compounds useful in the method of the present invention may be by topical, parenteral, oral, intranasal, intravenous, intramuscular, subcutaneous, or any other suitable means.

The dosage administered is dependent upon the age, weight, kind of concurrent treatment, if any, and nature of the malignancy or the pathological condition. The effective compound useful in the method of the present invention may be employed in such forms as capsules, tablets, liquid solutions, suspensions, or elixirs, for oral administration, or sterile liquid forms such as solutions, suspensions or emulsions. Any inert carrier is preferably used, such as saline, or phosphate-buffered saline, or any such carrier in which the compounds used in the method of the present invention have suitable solubility properties.

The compounds of the present invention may be administered in a biologically effective carrier. The biologically effective carriers may include any solvent with which the compounds of the present invention are compatible and which are non-toxic to the individuals treated at the amounts administered.

Most preferably, the compounds of the present invention are administered as an encapsulated composition. Due to the low aqueous solubility of many of the compounds effective in carrying out the present invention they are encapsulated in cyclodextrin, liposomes or as silastic implants. However, the compounds of the present invention may be encapsulated by other methods and with other compounds by methods known to those skilled in the art.

The term "antitumor agent" is meant to include agents which decrease cell growth, or inhibit the proliferation of tumor cells when administered to said tumor cells in a effective dose. As used herein "Methyl 3-(4-hydroxyphenyl)-2-hydroxypropionate" is also known as methyl p-hydroxyphenyllactate or MeHPLA. The term

"MeHPLA" is meant to also include its analogs, chemical derivatives, and chemically related compounds which bind to the nuclear Type II receptors and by so doing inhibit cell proliferation.

The term "chemically related compounds" refers to the derivatives and analogs of p-coumaric acid, p-hydroxyphenylbutanone, (4- hydroxyphenoxy)acetate and the arylpropenaldehydes, alkyl aryletheneyl ketones, aryl arylethenyl ketones, aryl butenaldehydes, alkyl arylpropenyl ketones and arylpropenyl ketones which are structurally related to

MeHPLA and disclosed herein. These chemically related compounds include the cis and trans isomers of said compounds and their esters, ethers, ketones and derivatives containing S or N in place or O atoms. More specifically these structurally related analogs and derivatives include compounds where R₁ represents the methyl, ethyl, n-propyl, n-butyl, isopropyl tert-butyl or aryl group and R₂ and R₃ represent H, OH or OCH₃ groups and R₄ is H or an alkyl group of 1 to 6 carbons. Specific analogs of each class of these structurally related compounds to MeHPLA have been demonstrated to possess biological activity mimicing MeHPLA as an effective inhibitor of cell proliferation and tumor cell growth. Preferably, the analogs and chemically related compounds effective in practicing the present invention include, but are not limited to, phenylmethylene ketones, nitroalkenes, aurones, and chalcones. Most preferably, these analogs are selected from the group consisting of the general formulas:

Wherein, R₁ is selected from the group consisting of H, OH, acyloxy, alkyl groups containing 1 to 6 carbons, and aryl groups; R₂ and R₃ are selected from the group consisting of H, OH and OCH₃ and R₄ is selected from the group consisting of H, or alkyl group containing 1 to 6 carbons. Preferred compounds which may be used to practice the present invention may be selected from the group consisting of methyl 3-(4- hydroxyphenyl)-2-hydroxypropionate, n-propyl 3-(4-hydroxyphenyl)-2- hydroxypropionate, n-butyl 3-(4-hydrorjφhenyl)-2-hydroxypropionate, 3-(4- hydroxyphenyl)-2-propanoic acid, 4-(4-hydroxyphenyl)-2-butanone, 1-(4- hydroxyphenyl)-3-pentanone,methyl(4-hydroxyphenoxy)acetate,methyl3- (3,4-dihydroxyphenyl)-2-propenoate.

Another aspect of the invention is a method of regulating cell growth proliferation in normal and malignant cells comprising the step of administering a therapeutic dose of a combination of compounds, wherein at least one compound is selected from group I and one compound is selected from group II, and wherein the compound from group I is selected from the group consisting of antimetabolites, antibiotics and alkylating agents and the compound from group II is selected from the group consisting of:

Wherein, R₁ and R₄ are selected from the group consisting of H and OH, acyloxy and halogens; R₂, R₃ and R₆ are selected from the group consisting of H, OH, OCH₃, amino, alkylamino and alkyl groups containing 1 to 6 carbons, acyloxy, and halogens; R₅ is selected from the group consisting of H, CH₃, OH, OCH₃, acyloxy and halogens; R'₁ and R'₂ are selected from the group consisting of H, CH₃, OH, OCH₃, amino, cyano, alkyl or alkylamino groups of 1 to 6 carbon atoms, acyloxy, halogen, α- azido and aziridine derivatives, 3,4-dihydroxyphenylmethylene, p- hydroxyphenylmethylene and other substituted phenyl groups; R'₃, R'₄, R'₅, R"₁ and R"₂ are selected from the group consisting of H, OH, CH₃,

OCH₃, amino, cyano, alkylamino groups of 1 to 6 carbon atoms, acyloxy, halogens and α-azido and aziridine derivatives. Preferred compounds which may be used to practice the present invention maybe selected from phenylmethylene ketones, nitroalkenes, aurones and chalcones.

Examples of the type of group I compounds which can be used for antimetabolites include arabinocytosylcytosine, 6-mercaptopurine, 6- thioguanine, 5-fluorouracil, cytoxan, and methotrexate. For Antibiotics the compounds which can be used include bleomycin, adriamycin and vinblastin. Some alkylating agents which can be used are nitrosoureas, cis-platinum and cyclophoshamide.

Among group II compounds, the preferable compounds include the phenyl methylene ketones selected from the group consisting of:

Among the nitroalkenes, the most preferable are:

Among the aurones most preferable for practicing the present invention are:

Among the chalcones most preferable for practicing the present invention are:

2'-hydroxychalcone 2',4',4-trihydroxychalcone

4-hydroxychalcone 4-4'-dihydroxychalcone

Other preferred compounds, which may be used to practice the present invention may be selected from the group consisting of methyl 3-

(4-hydroxyphenyl)-2-hydroxypropionate, n-propyl 3-(4-hydroxyphenyl)-2- hydroxypropionate, n-butyl 3-(4-hydroxyphenyl)-2-hydroxypropionate, 3-(4- hydroxyphenyl)-2-propanoic acid, 4-(4-hydroxyphenyl)-2-butanone, 1-(4- hydroxyphenyl)-3-pentanone, methyl (4-hydroxyphenoxy) acetate, methyl

3-(3,4-dihydroxyphenyl)-2-propenoate, p-coumaric acid, 3-(4- hydroxyphenyl)-2-propanoic acid, and its esters, caffeic acid and 3-(3,4- dihydroxyphenyl)-2-propanoic acid.

Preferred compounds which may be used to practice the present invention may be selected from the group consisting of methyl 3-(4- hydroxyphenyl)-2-hydroxypropionate, n-propyl 3-(4-hydroxyphenyl)-2- hydroxypropionate, n-butyl 3-(4-hydroxyphenyl)-2-hydroxypropionate, 3-(4- hydroxyphenyl)-2-propanoic acid, 4-(4-hydroxyphenyl)-2-butanone, 1-(4- hydroxyphenyl)-3-pentanone, methyl (4-hydroxyphenoxy) acetate, and methyl 3-(3,4-dihydroxyphenyl)-2-propenoate.

Specific examples of compounds are 1-(4-hydroxyphenyl)-3~ pentanone and 1-(4-hydroxyphenyl)-3-butanone. These compounds have been shown to bind to Type II sites and to have antitumor and anti- proliferative activity in the uterotropic assay.

Another group of compounds which show anti-proliferative activity in the rat uterus are methyl (4-hydroxyphenoxy) acetate and 2-(4- hydroxyphenoxy) ethyl methyl ether.

Preferred compounds which may be used to practice the present invention are:

2'-hydroxychalcone 2',4\4-trihydroxychalcone

4-hydroxychalcone

4-4'-dihydroxychalcone

MV-35 MV-46

Most preferred compounds of this group for practicing the present invention are 3-(4-hydroxyphenyl)-1-phenyl-2-propen-1-one and 4-(4- hydroxyphenyl)-3-buten-2-one, analogs, chemical derivatives and chemically related compounds and pharmaceutically acceptable salts thereof. In addition to being used as a treatment for cancer, these antitumor agents are also useful as inhibitors of cell growth and proliferation in those cells which include a Type II nuclear estrogen binding site.

These compounds bind to Type II nuclear estrogen binding sites and regulate cell growth. Specific proliferating cells which are sensitive to the binding of these compounds include estrogen responsive tissues such as uterus, mammary gland, uterine tumors and mammary tumors. The above-described compounds inhibit the proliferative capacity of human breast cancer cells and thus provide an effective therapy for this disease. Benign prostatic hyperplasia is another example of a proliferative tissue disease in which the above-described compounds can successfully be used in the treatment.

A further embodiment includes a method of regulating cell growth and proliferation in normal and malignant cells, comprising the step of administering a therapeutic dose of a combination of compounds, wherein at least one compound is selected from group I and at least one compound is selected from group II and wherein the compound from group I is selected from the group consisting of antimetabolites, antibiotics and alkylating agents and the compound from group II is selected from the group consisting of methyl p-hydroxyphenyllactate, its analogues, chemical derivatives and chemically related compounds. Compounds of group II which are used include:

Wherein R₁ is from the group consisting of H, alkyl groups containing 1 to 6 carbons, and substituted or unsubstituted aryl groups; and R₂ and R₃ are selected from the group consisting of H, OH and OCH₃.

Preferred compounds of this group which may be used to practice the present invention are:

2'-hydroxychalcone 2',4',4-trihydroxychalcone

4-hydroxychalcone

4-4'-dihydroxychaicone

The following example is offered by way of illustration and not intended to limit the invention in any manner.

Example 1

COMBINATION THERAPY

In this example, mice bearing estrogen-independent mammary tumors (T-511R) were segregated into 6 experimental groups (5 mice per group) at the time of tumor transplantation. Eleven days following transplantation, the animals were treated with the MV-88, RSV- 101 or 5-FU administered in the drinking water. MV-88 and RSV-101 were dissolved in the drinking water as a complex in a 2-hydroxypropyl β-cyclodextrin-vehicle by adding the drug in excess to an aqueous solution of 2-hydroxypropyl-β-cyclodextrin (Aldrich). The suspension was stirred at room temperature and the excess non-solubilized drug removed by centrifugation and ultrafiltration. The aqueous cyclodextrin-drug complex was lyophillized, weighed and dissolved in a known concentration of methanol and the drug concentration (mg drug/ mg dry cyclodextrin-drug complex) determined by high performance liquid chromatography (HPLC) utilizing a C₁₈ reversed phase column by methods known to those reasonably skilled in the art. Sample peak areas were compared to those of known concentrations of authentic standards. Typical values were 1 mg

MV-88 or RSV-101 per 11-15 mg dry cyclodextrin. These values were used for determining the dosage of the drug-cyclodextrin complex to be dissolved in water for oral delivery. An equivalent amount of 2- hydroxypropyl β-cyclodextrin was also added to the drinking water of the controls and 5-FU treated animals. The concentration of the drug delivered to the animals was determined by measuring the volume of the drinking water consumed (miUiHters/day/Kg body weight/mouse) throughout the study. These date demonstrated that MV-88, RSV-101 and 5-Fu were delivered at a daily rate of approximately 5mg/Kg/day/mouse in these studies. Tumor size was monitored by measuring the tumors (length x width) with vernier calipers at the indicated times following treatment.

Since 100% (5/5) of the MV-88 + 5-FU treated animals and 80% (4/5) of the RSV-101 + 5-FU treated animals had no signs of tumor on days 32 and 46 respectively, the combination treatments were discontinued at this time for these two experimental groups. This was done to determine if tumors would regrow after the discontinuation of treatment and to definitively establish that the drugs were suppressing the growth of these tumors. By day 87 all of the animals in the MV-88 + 5-

FU and RSV-101 + 5-FU treatment groups had palpable tumors demonstrating that treatment had indeed blocked the growth of this tumor as opposed to inhibiting its establishment.

In figure 1, the results obtained with combination therapy with a chalcone (MV-88) and MeHPLA related enone (RSV-101) in a transplantable mouse mammary tumor model system are displayed. Doses (~5 mg/Kg/day) of MV-88, RSV-101 and 5-FU were chosen as levels which would only slightly inhibit tumor growth following singular drug administration. These doses would then be combined for assessment of antitumor activity. The results demonstrated that singular administration of MV-88 or RSV-101 at these doses failed to inhibit the growth of this tumor relative to control (cyclodextrin-water vehicle), and only slight inhibition was observed with a low dose of 5-FU. However, combination therapy with MV-88 + 5-FU or RSV-101 + 5-FU resulted in complete inhibition of these tumors in 90% of these animals (100% for MV-88 + 5- FU and 80% for RSV-101 + 5-FU). These tumors were non-palpable from days 11-58 of the study in the combination therapy treatment groups (except for one animal treated with RSV-101 + 5-FU).

These results further suggest that the combined therapy will result in a decreased cytotoxicity. The reduction in cytotoxicity results from the fact that much lower doses of 5-FU are employed to inhibit or regress tumors because an additive or synergistic effect was observed when 5-FU was combined with MeHPLA related compounds. The results in Figure 1 demonstrated that singular administration of sub-inhibitory doses of MV- 88 (DHBA), RSV-101 (MeHPLA related compounds) or 5-FU alone failed to substantially inhibit mammary tumor growth, but combination therapy with MV-88 plus 5-FU or RSV-101 plus 5-FU blocked mammary tumor growth. Therefore, MV-88 and RSV-101 enhanced the activity of 5-FU such that substantial therapeutic response was obtained with lower doses of 5-FU than those normally required to inhibit tumor cell proliferation.

Similar responses are obtained following combination therapy with other MeHPLA related compounds and other standard cytotoxic anti-cancer drugs.

The advantage of combination therapy with MeHPLA related compounds and 5-FU is that relatively non-toxic agents (MV-88, RSV-101) can be combined with a cytotoxic drug (5-FU) to inhibit mammary cancer growth. Because of the enhancement of 5-FU activity by MeHPLA related compounds, the dose level of 5-FU required to inhibit mammary cancer growth could be reduced to decrease generalized systematic cytoxicity. Although combination therapy with a variety of cytotoxic anti-cancer drugs is in widespread use, these procedures are limited by a high degree of non-specific cytotoxicity and severe side effects. The present study suggests that combination therapy with MeHPLA related drugs and a variety of anti-cancer drugs such as arabinocytosylcytosine, 5-FU, 6- mercaptopurine, 6-thioguanine, MTX, cis-platinum, adriamycin and cyclophoshamide and other cytotoxic drugs used for cancer therapy and known to inhibit cell cycle are of significant benefit. Such combination therapy may result in significant clinical response, increased survival and an improvement in the quality of life for cancer patients.

All patents and publications mentioned in this specification are indicated with levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The compounds, methods, procedures and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary, and are not intended as limitations on the scope of the present invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the appended claims.

What is claimed is:

Claims

1. A method of regulating cell growth and proliferation in normal and malignant cells, comprising the step of administering a therapeutic dose of a combination of compounds, wherein at least one compound of the combination is selected from group I and at least one compound is selected from group II and wherein the compound from group I is selected from the group consisting of antimetabolities, antibiotics and alkylating agents and the compound from group II is selected from the group consisting of:

Wherein, R₁ and R₄ are selected from the group consisting of H, OH, acyloxy and halogens; R₂, R₃ and R₆ are selected from the group consisting of H, OH, OCH₃, amino, alkylamine and alkyl groups containing 1 to 6 carbons, acyloxy, and halogens; R₅ is selected from the group consisting of H, CH₃, OH, OCH₃, acyloxy and halogens; R'₁ and R'₂ are selected from the group consisting of H, OH, CH₃, OCH₃, amino, cyano, alkyl or alkylamine groups of 1 to 6 carbon atoms, acyloxy, halogens, α-azido and aziridine derivatives, 3,4-dihydroxyphenylmethylene, p- hydroxyphenylmethylene and other substituted phenyl groups preferably with acyloxy or halogen substituents; R'₃, R'₄, R'₅, R"₁ and R"₂ are selected from the group consisting of H, OH, CH₃, OCH₃, amino, cyano, alkyl or alkylamine groups of 1 to 6 carbon atoms, acyloxy, halogens, α- azido, aziridine, acyloxy and halogen substituted derivatives.

2. The method of claim 1, wherein the antimetabolite is selected from the group consisting of arabmocytosylcytosine, 6-mercaptopurine, 6- thioguanine, 5-fluorouracil, cytoxan, and methotrexate.

3. The method of claim 1, wherein the antibiotic is selected from the group consisting of bleomycin, adriamycin and vinblastin.

4. The method of claim 1, wherein the alkylating agent is selected from the group consisting of nitrosoureas, cis-platinum and cyclophoshamide.

5. A method for inhibiting the growth of proliferating cells which include a Type II nuclear estrogen binding site comprising the step of administering a biologically inhibiting dose of a combination of compounds, wherein at least one compound is selected from group I and at least one compound is selected from group II and wherein the compound from group I is selected from the group consisting of antimetabolites, antibiotics and alkylating agents and the compound from group II is selected from the group consisting of:

Wherein, R₁ and R₄ are selected from the group consisting of H, OH, acyloxy and halogens; R₂, R₃ and R₆ are selected from the group consisting of H, OH, OCH₃, amino, alkylamine and alkyl groups containing 1 to 6 carbons, acyloxy, and halogens; R₆ is selected from the group consisting of H, CH₃, OH, OCH₃, acyloxy and halogens; R'₁ and R'₂ are selected from the group consisting of H, OH, CH₃, OCH₃, amino, cyano, alkyl or alkylamine groups of 1 to 6 carbon atoms, acyloxy, halogens, α-azido and aziridine derivatives, 3,4-dihydroxyphenylmethylene, p- hydroxyphenylmethylene and other substituted phenyl groups preferably with acyloxy or halogen substituents; R'₃, R'₄, R'₆, R"₁ and R"₂ are selected from the group consisting of H, OH, CH₃, OCH₃, amino, cyano, alkyl or alkylamine groups of 1 to 6 carbon atoms, acyloxy, halogens, α- azido, aziridine, acyloxy and halogen substituted derivatives.

6. The method of Claim 5, wherein said proliferating cells are estrogen responsive tissue selected from the group consisting of uterus, mammary gland, uterine tumors and mammary tumors.

7. The method of Claim 5 wherein said estrogen responsive tissue is human breast cancer cells.

8. The method of claim 5, wherein the antimetabolite is selected from the group consisting of arabinocytosylcytosine, 6-mercaptopurine, 6- thioguanine, 5-fluorouracil, cytoxan, and methotrexate.

9. The method of claim 5, wherein the antibiotic is selected from the group consisting of bleomycin, adriamycin and vinblastin.

10. The method of claim 5, wherein the alkylating agent is selected from the group consisting of nitrosoureas, cis-platinum and cyclophoshamide.

11. A method of treating benign prostatic hyperplasia, comprising the step of administering a therapeutic dose of a combination of compounds, wherein at least one compound of the combination is selected from group I and at least one compound is selected from group II, and wherein the compound from group I is selected from the group consisting of antimetabolities, antibiotics and alkylating agents and the compound from group II is selected from the group consisting of:

Wherein, R₁ and R₄ are selected from the group consisting of H, OH, acyloxy and halogens; R₂, R₃ and R₆ are selected from the group consisting of H, OH, OCH₃, amino, alkylamine and alkyl groups containing 1 to 6 carbons, acyloxy, and halogens; R₅ is selected from the group consisting of H, CH₃, OH, OCH₃, acyloxy and halogens; R'₁ and R'₂ are selected from the group consisting of H, OH, CH₃, OCH₃, amino, cyano, alkyl or alkylamine groups of 1 to 6 carbon atoms, acyloxy, halogens, α-azido and aziridine derivatives, 3,4-dihydroxyphenylmethylene, p- hydroxyphenylmethylene and other substituted phenyl groups preferably with acyloxy or halogen substituents; R'₃, R'₄, R'₆, R"₁ and R"₂ are selected from the group consisting of H, OH, CH₃, OCH₃, amino, cyano, alkyl or alkylamine groups of 1 to 6 carbon atoms, acyloxy, halogens, α- azido, aziridine, acyloxy and halogen substituted derivatives.

12. The method of claim 11, wherein the antimetabolite is selected from the group consisting of arabinocytosylcytosine, 6- mercaptopurine, 6-thioguanine, 5-fluorouracil, cytoxan, and methotrexate.

13. The method of claim 11, wherein the antibiotic is selected from the group consisting of bleomycin, adriamycin and vinblastin.

14. The method of claim 11, wherein the alkylating agent is selected from the group consisting of nitrosoureas, cis-platinum and cyclophoshamide.

15. A method of regulating cell growth and proliferation in normal and malignant cells, comprising the step of administering a therapeutic dose of a combination of compounds, wherein at least one compound of the combination is selected from group I and at least one compound is selected from group II and wherein the compound from group I is selected from the group consisting of antimetabolities, antibiotics and alkylating agents and the compound from group II is selected from the group consisting of: 2-(hydroxybenzylidene)-5-methyl- cyclopentanone (MV-1), 2,6-bis(hydroxybenzylidene-4-methyl- cyclohexanone (MV-17), 2,6-bis(3,4-dihydroxybenzylidene)-4- methylcylcohexanone (MV-18), 2,6-bis(3-methoxy-4-hydroxybenzylidene) cyclohexanone (RSV-101), 2,6-bis(3,4-dihydroxybenzylidene) cyclohexanone

(MV-3), 4-hydroxy-β -methyl-β -nitrostyrene (MV-N1), 3,4-dihydroxy-β - methyl- β -nitrostyrene (MV-N3), aurone (MV-19), 4'-hydroxyaurone (MV-

20), 3',4-dihydroxyaurone (MV-21), 2'-hydroxychalcone (MV-72), 2',4',4- trihydroxychalcone (RV-40), 4-hydroxychalcone (RV-73), 4,4'- hydroxychalcone (MV-88), α-azido-2'-hydroxychalcone (MV-35), 3,4- dihydroxy-4'-cyanochalcone (MV-46) and 4'-cycanochalcone (MV-47).

16. The method of claim 15, wherein the antimetabolite is selected from the group consisting of arabinocytosylcytosine, 6- mercaptopurine, 6-thioguanine, 5-fiuorouracil, cytoxan, andmethotrexate.

17. The method of claim 15, wherein the antibiotic is selected from the group consisting of bleomycin, adriamycin and vinblastin.

18. The method of claim 15, wherein the alkylating agent is selected from the group consisting of nitrosoureas, cis-platinum and cyclophoshamide.

19. The method of Claim 15 for inhibiting the growth of proliferating cells wherein said cells include a Type II nuclear estrogen binding site.

20. The method of Claim 15 wherein the cells are benign prostatic hyperplasia cells.

21. A method of regulating cell growth and proliferation in normal and malignant cells, comprising the step of administering a therapeutic dose of a combination of compounds, wherein at least one compound is selected from group I and at least one compound is selected from group II and wherein the compound from group I is selected from the group consisting of antimetabolities, antibiotics and alkylating agents and the compound from group II is selected from the group consisting of methyl p-hydroxyphenyllactate, analogues of methyl p- hydroxyphenyllactate, chemical derivatives of methyl p- hydroxyphenyllactate, and chemically related compounds.

22. The method of Claim 21, wherein at least one compound from group II is selected from the group consisting of the formulae:

wherein,

R₂ is selected from the group consisting of H, alkyl groups containing 1 to 6 carbons and aryl groups; R₂ and R₃ are selected from the group consisting of H, OH and OCH₃; and

23. The method of claim 22, wherein the antimetabolite is selected from the group consisting of arabinocytosylcytosine, 6- mercaptopurine, 6-thioguanine, 5-fluorouracil, cytoxan, andmethotrexate.

24. The method of claim 22, wherein the antibiotic is selected from the group consisting of bleomycin, adriamycin and vinblastin.

25. The method of claim 22, wherein the alkylating agent is selected from the group consisting of nitrosoureas, cis-platinum and cyclophoshamide.

26. The method of Claim 22, wherein at least one compound from group II is selected from the group consisting of methyl 3-(4- hydroxyphenyl)-2-hydroxypropionate, n-propy 3-(4-hydroxyphenyl)-2- hydroxypropionate, n-butyl 3-(4-hydroxyphenyl)-2-hydroxypropionate, 3-(4- hydroxyphenyl)-2-propenoic acid, 1-(4-hydroxyphenyl)-3-butanone, 1-(4- hydroxyphenyl)-3-pentanone, methyl-(4-hydroxyphenoxy) acetate, and methyl 3-(3,4-dihydroxyphenyl)-2-propenoate.

27. The method of Claim 22 for inhibiting the growth of proliferating cells, wherein said cells, include a Type II nuclear estrogen binding site.

28. The method of Claim 27, wherein said proliferating cells are estrogen responsive tissue selected from the group consisting of uterus, mammary gland, uterine tumors or mammary tumors.

29. The method of Claim 27, wherein said estrogen responsive tissue is human breast cancer cells.

30. The method of Claim 22, wherein the cells are benign prostatic hyperplasia cells.

31. The method of Claim 22, wherein the compound from group I is 5-fluorouracil and the compound from group II is MV-88 or RSV-101.

32. The method of Claim 22, wherein at least one compound from group II is selected from the group consisting of 3-(4- Hydroxyphenyl)-1-phenyl-2-propen-1-one and 4-(4-Hydroxyphenyl)-3- buten-2-one, analogues, chemical derivatives and chemically related compounds and pharmaceutically acceptable salts thereof and at least one compound from group I is 5-fluorouracil.