WO2001045740A2

WO2001045740A2 - Compositions and methods for treatment of breast cancer

Info

Publication number: WO2001045740A2
Application number: PCT/US2000/035315
Authority: WO
Inventors: Geoffrey Clark; Joanne Zujewski
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 1999-12-22
Filing date: 2000-12-20
Publication date: 2001-06-28
Anticipated expiration: 2002-06-22
Also published as: AU2292801A; WO2001045740A3

Abstract

The present invention relates to compositions comprising an estrogen receptor modulator and a farnesyl transferase inhibitor and methods for treating and/or preventing cancer. In particular, the present invention provides compositions and methods for the treatment and/or prevention of breast cancer. Specifically, the present invention provides combination drug therapy that acts in a synergistic manner to inhibit the growth of cancer cells (e.g., breast cells). In some embodiments of the present invention, therapeutic agents are administered to subjects suspected of having cancer or being susceptible to cancer, subjects with cancer, subjects experiencing a recurrence of cancer, or subjects who are post-operative for cancer. It is also contemplated that the treatment agents be prophylactically administered to patients at risk for development of cancer.

Description

COMPOSITIONS AND METHODS FOR TREATMENT OF BREAST CANCER

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treating and/or preventing cancer. In particular, the present invention provides compositions and methods for the treatment and/or prevention of breast cancer. Specifically, the present invention provides combination drug therapy that acts in a synergistic manner to inhibit the growth of cancer cells (e.g., breast cells). In some embodiments of the present invention, therapeutic agents are administered to subjects suspected of having cancer or being susceptible to cancer, subjects with cancer, subjects experiencing a recurrence of cancer, or subjects who are post-operative for cancer. It is also contemplated that the treatment agents be prophylactically administered to patients at risk for development of cancer.

BACKGROUND OF THE INVENTION Invasive breast cancer is the most common non-skin cancer observed in

American women (Davidson, "12 Oncology, VII. Breast Cancer," in Dale and Federman (eds.), Scientific American Medicine, Scientific American [1999]). Estimates indicate that the disease will be diagnosed in 176,000 women in 1999, and result in approximately 44,000 deaths (See, Davidson, supra). Although the incidence and mortality of the disease appear to have reached a plateau and may be declining slightly due to use of screening mammography and systemic adjuvant therapy in the U.S. and parts of Europe, the disease continues to cause a high level of morbidity and mortality worldwide.

Due to the increased use of screening mammography and heightened breast cancer awareness in the public, in situ carcinomas presently account for 20% of newly diagnosed cases of breast cancer, with the majority being ductal carcinoma in situ (DCIS). These lesions are associated with a risk of approximately 30% for subsequent invasive breast cancer in the ipsilateral breast. As the risk of metastasis in DCIS cases is relatively small, management decisions are largely centered on the involved breast. The traditional therapy, total mastectomy, has a highly likelihood of cure. However, breast conservation may be appropriate for many women with DCIS. Lumpectomy, in combination with radiotherapy has been shown to reduce the likelihood of recurrence of in situ or invasive breast cancer in these individuals. However, a trial demonstrated a small benefit from the use of a 5 -year regimen of tamoxifen in women who underwent excision and radiotherapy, although ipsilateral breast cancer recurrence and contralateral breast cancer development were reduced by approximately 50% (See, Davidson, supra). In cases of invasive breast cancer, radical mastectomy was the mainstay of breast cancer treatment for many decades. However, it is rarely performed today. Multiple randomized trials have uniformly and unequivocally shown that breast conservation therapy (BCT), utilizing lumpectomy with radiotherapy, and modified radical mastectomy, provide similar survival rates for women with stage I or stage II breast cancer (See, Davidson, supra). Nonetheless, as with DCIS, controversy remains as to the best treatment approaches.

As many women with primary breast cancer already have distant micrometastases at the time of diagnosis, there has been great interest in the use of systemic therapy for such cases. Indeed, overt metastatic disease develops in many of these women, despite surgery and radiotherapy. Thus, systemic therapy is frequently used to prevent or delay recurrence of disease. Many trials over the past 40 years have evaluated the worldwide experience with use of ovarian ablation, hormonal therapy and chemotherapy. The success rates of each of these approaches has varied, depending upon the age of the patients and other factors. While ovarian ablation has been reported to reduce the annual odds of cancer recurrence by 25% and reduce the odds of death by 24% for women who underwent ovarian ablation in the absence of chemotherapy, the benefits to women who underwent ablation and chemotherapy were more modest (8-10% reduction) (See, Davidson, supra). For example, chemotherapy commonly used for treating cancer or other diseases is difficult to accomplish without incurring significant toxicity. The agents currently in use are generally poorly water soluble, quite toxic, and given at doses that affect normal cells as wells as diseased cells. Although used for a number of years, the risk versus benefit of tamoxifen use remains a consideration in developing treatment options for patients. For example, in large randomized study, the administration of tamoxifen after chemotherapy was associated with a worse outcome than the use of chemotherapy alone in a group of women with node-negative, steroid receptor-negative breast cancer. Indeed, although chemohormonal therapy may be useful for some women, others may find the benefit too small to warrant its toxicity and other risks. Thus, despite the large amount of research into breast cancer treatment and prevention, the need remains for treatment methods that are efficacious and less toxic.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides compositions comprising at least one selective estrogen receptor modulator and at least one famesyl transferase inhibitor. In some preferred embodiments, the composition is therapeutically effective in that the cancer cells are killed. In alternative preferred embodiments, the composition is capable of inhibiting the growth of cancer cells expressing estrogen receptors (i.e., the composition is anti-proliferative). The present invention also provides methods of treatment of cancer. In one embodiment, the methods comprise providing: i) a subject suffering from cancer, ii) at least one selective estrogen receptor modulator and iii) at least one famesyl transferase inhibitor; and b) administering the selective estrogen receptor modulator and famesyl transferase inhibitor to the subject. In one alternative embodiment, the subject is suffering from breast cancer, while in other embodiments the subject is at risk for developing cancer, and in still other embodiments, the subject is experiencing a recurrence of cancer. In a further embodiment, the cancer cells of the subject express estrogen receptors. In another embodiment the selective estrogen receptor modulator is tamoxifen. In still another embodiment, the selective estrogen receptor modulator and famesyl transferase inhibitor are provided in a combination composition such that they are administered together. In yet another embodiment, the selective estrogen receptor modulator and fa esyl transferase inhibitor are provided to the subject in separate compositions, such that they are administered to the subject sequentially. In some embodiments, the selective estrogen receptor modulator and famesyl transferase inhibitor are provided in a single dosage unit (e.g., in a single tablet, pill, or liquid dose), while in other embodiments they are provided in separate units (e.g., each compound is provided in a separate pill, tablet, or liquid foπnulation). In still further embodiments, the selective estrogen receptor modulator and/or the famesyl transferase inhibitor are provided in sub-therapeutic concentrations.

DESCRIPTION OF THE FIGURES

Figure 1 is a graph showing results obtained for MCF7 cells. Figure 2 is a graph showing the results obtained for T47D cells. Figure 3 is a graph showing that tamoxifen and FTI did not act synergistically to inhibit growth of MDA-231 cells.

DEFINITIONS

To facilitate an understanding of the present invention, a number of terms and phrases are defined below: As used herein, the term "selective estrogen receptor modulator" refers to the class of compounds that is capable of binding to the estrogen receptor. It is not intended that the definition be limited to any particular compound. Indeed, it is contemplated that any number of such modulators will find use in the present invention.

As used herein, the term "tamoxifen" refers to the selective estrogen receptor modulator commonly referred to as tamoxifen, as well as by its tradename "Nolvadex." Tamoxifen is a mixed estrogen agonist-antagonist. The major mode of action of the compound is through its interaction with the estrogen receptor. As used herein, the terms "famesyl transferase inhibitor" and "FTI" refer to compounds capable of inhibiting the effects of famesyl transferases (also referred to as "famesyl protein transferases").

As used herein, the term "chemotherapeutic agent" refers to an agent that inhibits the growth or decreases the survival of neoplastic or pathogenic microbial cells or inhibits the propagation (which includes without limitation replication, viral assembly or cellular infection) of a virus. It is intended that the definitions encompass compounds purified from the natural source, as well as compounds prepared synthetically or by semisynthesis. Thus, it is not intended that the present invention be limited to these compounds produced in by a particular method or from any specific source.

As used herein, the temi "pharmaceutical composition" refers to any composition or compound suitable for administration to a subject for treatment and/or prevention of disease. It is contemplated that such compositions encompass drugs that are effective in reducing or eliminating signs and symptoms of disease. In particularly preferred embodiments, the pharmaceutical compositions of the present invention are provided as compositions comprising at least one SERM and at least one FTI that are capable of acting synergistically to suppress the growth of cancer cells. In other particularly preferred embodiments, the compositions comprising at least one SERM and at least one FTI are capable of acting synergistically to kill cancer cells. It is not intended that the present invention be limited to any particular dosage or administration strategy. It is also intended that the term encompass various regimens, including, but not limited to regimens in which at least one SERM and at least one FTI are provided in separate vehicles (e.g., separate tablets, pills, liquids, etc.).

As used herein, the term "therapeutically effective amount" refers an amount of a therapeutic agent that inhibits the growth or decreases the survival of neoplastic cells.

As used herein, the terms "subtherapeutic amount" and "subtherapeutic concentration" refer to an amount of a therapeutic that is administered at a concentration that would not be expected to exhibit anti-tumor effects. In particularly preferred embodiments, subtherapeutic amounts of at least one selective estrogen receptor modulator and at least one famesyl transferase inhibitor are provided in conjunction with each other. The combination of these subtherapeutic concentrations of at least one selective estrogen receptor modulator and at least one famesyl transferase inhibitor act synergistically such that an anti-tumor effect is produced.

As used herein, the term "anti-proliferative" refers to a therapeutic agent that suppresses or inhibits the growth of abnormal (e.g., tumor, malignant, or transformed) cells, but does not necessarily kill the cells.

As used herein, the term "cytotoxic" drug or agent refers to a therapeutic agent useful in treating cancer that results in the death of cancer cells. In some preferred embodiments, cytotoxic drugs are particularly effective against rapidly dividing cells. As used herein, the term "IC₅₀" refers to the concentration at which 50% cytotoxicity is obtained. Cytotoxicity can be measured by the method of Alley et al, Cancer Res. 48: 589-601, 1988 or Scudiero et al, Cancer Res., 48:4827, 1988, or by any other suitable method known in the art. In one embodiment, cytotoxicity can be measured based on the drug concentration at which a 50% reduction in the activity of mitochondrial enzymes is observed.

As used herein, the term "subject" refers to a human or other animal that is capable of being treated with a pharmaceutical composition.

As used herein, the term "cancer" refers to a neoplasm characterized by the uncontrolled growth of anaplastic cells that tend to invade surrounding tissue and/or to metastasize to distant body sites. Although it is not intended that the present invention be so limited, in particularly preferred embodiments, the present invention provides methods and compositions to treat and/or prevent breast cancer.

As used herein, the term "suspected of having cancer" refers to a patient or subject who has been diagnosed as having cancer or tested positive in one or more tests diagnostic for cancer.

As used herein, the term "susceptible to cancer" refers to a subject or patient having one or more πsk factors for cancer, is currently being treated for cancer, or is in remission from a previously diagnosed cancer

As used herein, the term "post-operative for removal of a cancer" means the peπod of time after a tumor has been removed from a subject

As used herein, the term "recurrence of cancer" refers to a subject who is experiencing the recurrence of a cancer The term encompasses recurrences of the same type of cancer (e g , the same cell types are affected), as well as a second or subsequent episode of cancer in a subject in which different cell type(s) are involved As used herein, the term "biological agent" refers to an agent that is useful for diagnosing or imaging or that can act on a cell, organ or organism, including but not limited to drugs (e g , pharmaceuticals) to create a change in the functioning of the cell, organ or organism Such agents can include, but are not limited to nucleic acids, polynucleotides, antibacterial agents, antiviral agents, antifungal agents, anti-parasitic agents, tumoπcidal or anti-cancer agents, proteins, toxins, enzymes, hormones, neurotransmitters, glycoproteins, immunoglobulins, immunomodulators, dyes, radiolabels, radio-opaque compounds, fluorescent compounds, polysacchandes, cell receptor binding molecules, anti-mflammatoπes, anti-glaucomic agents, mydπatic compounds and local anesthetics As used herein, the term "sample" is used in its broadest sense In one sense it can refer to a composition compπsing a therapeutic agent In another sense, it is meant to include a specimen or culture obtained from any source Biological samples may be obtained from animals (including humans) and encompass tissues (e g , biopsy samples), fluids, solids, tissues, and gases Biological samples also include blood products, such as plasma, serum and the like. These examples are not to be construed as limiting the sample types applicable to the present invention.

DESCRIPTION OF THE INVENTION

A. Selective Estrogen Receptor Modulators Selective estrogen receptor modulators (SERMs) are a relatively newly defined category of therapeutic agents that bind to estrogen receptors. In particularly preferred embodiments, these modulators bind with high affinity to estrogen receptors and mimic the effect of estrogens in some tissues but act as estrogen antagonists in others. Tamoxifen was the first clinically available selective estrogen receptor modulator, although additional SERMs have been developed and have been used in the treatment of estrogen receptor positive cancers. Examples of newer SERMs include, but are not limited to idoxifene, raloxifene, trans-2,3-dihydroraloxifene, levormeloxifene, droloxifene, and MDL 103,323. Indeed, much research has been conducted to identify compounds that are tissue-selective modulators of estrogen receptors useful with post-menopausal women where the agonistic action of estrogen is needed for bone maintenance and brain function, but is not desirable in the breast and uterus. In cancer treatment, the goal is to develop SERMs that efficiently target malignant cells with an estrogen antagonist that would spare other estrogen-responsive tissues. The term SERM encompasses numerous pharmaceuticals, including phytoserms (i.e., phytoestrogens), and environmental pollutants (xenoestrogens or "xenoserms").

Tamoxifen, the most prescribed antineoplastic drug worldwide (Goldstein, Am. J. Obstet. Gyn., 179:1479-1484 [1998]) has anti-estrogenic properties and is widely used in the treatment of breast cancer. Although an understanding of the mechanism of action is not necessary in order to use the present invention, tamoxifen acts a competitive inhibitor of estradiol binding to the estrogen receptor. When bound to the estrogen receptor, tamoxifen induces a change in the receptor's three-dimensional shape, which inhibits its binding to the estrogen-responsive element (ERE) on DNA.

Under normal physiologic conditions, estrogen stimulation of cells results in increased tumor cell production of transforming growth factor β (TGF-β), an autocrine tumor growth inhibitor (Goodman and Gilman, Goodman & Gilman's The Pharmacological Basis of Therapeutics. 9th ed., McGraw Hill, New York, New York [1996], pages 1424-1426, 1275-1276). By blocking these pathways, tamoxifen treatment decreases the autocrine stimulation of breast cancer growth by capturing the cells in G,. In addition to these effects, tamoxifen decreases the local production of insulin-like growth factor 1 (IGF-1); IGF-1 is a paracrine growth factor for breast cancer cells (Goodman and Gilman, supra). However, the effects of tamoxifen on breast cancer cells appear to be more complex than a simple competitive blockade of estrogen receptor binding, as some estrogen receptor-negative cells also respond to tamoxifen.

After initial studies documented its efficacy in treatment of advanced breast cancer, a number of trials were conducted in order to investigate its usefulness in treating women with early-stage breast cancer. For those women with tumors that had low or no expression of estrogen receptors, administration of tamoxifen had little effect on outcome. However, in patients with tumors that responded to tamoxifen, the benefit of tamoxifen increased with the duration of treatment, with proportional reductions in 10-year recurrence in mortality reported at 47% and 26% respectively for 5-year tamoxifen treatment therapy (See, Davidson, supra). Thus, the benefit of tamoxifen therapy was shown to extend beyond the treatment period. Benefits in node-positive women have been observed regardless of age, menopausal status, tamoxifen dose, and chemotherapy use. A 5-year treatment regimen with tamoxifen has been shown to be superior to regimens of shorter duration; regimens lasting longer than 5 years have failed to show an advantage over the 5-year regimens. An increase in uterine cancer and a decrease in contralateral breast cancer have also been associated with tamoxifen use, although tamoxifen had no effect on the incidence of other cancers or on the incidence of death from other cancers.

Nonetheless, tamoxifen remains the endocrine treatment of choice for women with hormone receptor-positive breast cancer and those with a high risk of disease recurrence. In this setting, the presence of estrogen receptors is the best predictor for a response to endocrine therapy. Approximately half of the patients with estrogen receptor positive tumor cells will respond to tamoxifen therapy, while less than 15% of those with estrogen receptor-negative cells will respond (Goodman and Gilman, supra). Although tamoxifen is also used in premenopausal women with estrogen receptor- positive cells, other alternatives may also be used that help eliminate ovarian estrogen production.

The estrogenic effect of tamoxifen lowers the total serum cholesterol, LDL cholesterol, and lipoproteins, and raises apolipoprotein Al levels, potentially decreasing the risk of myocardial infarction in patients taking the drug. In addition, tamoxifen may help slow the development of osteoporosis in postmenopausal women. While the side effects are minimal in comparison with many other cancer therapies, high doses of tamoxifen have been associated with retinal degeneration. The most frequent adverse reactions include hot flashes, nausea, and vomiting. Up to 25% of patients experience these effects, while menstrual iπegularities, vaginal bleeding and discharge, pruritus vulvae, hypercalcemia, edema, anorexia, dermatitis, depression, light-headedness, dizziness, and headaches are less frequently observed. More significantly, there is an increasing concern regarding the potential for tamoxifen to cause endometrial cancer, as the incidence of this cancer appears to be at least two-fold greater in women who receive 20 mg tamoxifen per day for at least 2 years, as compared to controls (See, Goodman and Gilman, supra). In addition, there are other potential side effects associated with tamoxifen therapy, including an increased risk of thromboembolic events, the occurrence of retinal deposits, decreased visual acuity, and cataracts Also, like estrogen, tamoxifen acts as a hepatic carcinogen in animals, although increases m pnmary hepatocellular carcinoma have not been reported in patients (Goodman and Gilman, supra) Thus, it is contemplated that other SERMs without these potentially seπous side effects will find use m the present invention.

B. Farnesyl Transferase Inhibitors (FTI)

Although an understanding of the mechanism of action is not necessary in order to use the present invention, farnesyl transferases are known to be important regulators in cellular signalling mechanisms For example, famesyl transferases are involved in the function of Ras proteins Ras proteins are plasma membrane-associated GTPases that function as relay switches m the transduction of biological information from extracellular signals to the cell nucleus In normal cells, these proteins cycle between the inactive (GDP) and active (GTP)-bound forms in the regulation of cellular proliferation and differentiation Vaπous tπggers are involved in this process In a large number of human cancers, Ras is locked in its GTP -bound form due to ammo acid mutations As a result of these mutations, the Ras pathway no longer requires an upstream growth signal and the enzymes in the pathway (e g , Raf, MEK, and MAPK) are constitutively activated However, in addition to its inability to hydrolyze GTP, oncogenic Ras must associate with the plasma membrane to cause malignant transformation. This post-translational modification is vital for the function of Ras proteins, as without it the Ras protein cannot stably associate with the plasma membrane and loses the ability to transform cells Ras membrane association is mediated through a seπes of post-translational modifications The first step in this seπes is catalyzed by a cytosohc heterodimer farnesyl transferase (FTase), which attaches farnesyl to the thiol group of cysteine of the carboxyl-terminal tetrapeptide CAAX, where "A" is isoleucine or vahne, and "X" is seπne or methionme Because farnesylation is required and sufficient for Ras transformation, FTAse has been considered a target for the development of a new class of anti-cancer agents Although CAAX peptides are potent competitive inhibitors of FTase, their rapid degradation and low cellular uptake have limited their use as therapeutic agents (See, Lemer et al, J. Biol. Chem., 270:26802-26806 [1995]). However, recent developments have produced CAAX peptidomimetics that potently inhibit FTase in vitro and Ras processing in vivo, but retain several peptidic features. Additional work has resulted in the design of non-peptide CAAX mimectics that have several desirable features. While these non-peptide mimectics and CAAX peptidomimectics potently inhibit FT A, their ability to disrupt Ras processing in whole cells occurs at micromolar concentrations that would not be easily achievable in vivo. Thus, improved FTase inhibitors (FTIs) have been developed, including FTI-277 (See, Lemer et al, supra).

FTIs are a class of designed drugs that were specifically developed to inhibit the Ras oncogene. A striking characteristic and significant advantage of this class of compounds is the lack of toxicity associated with the compounds (i.e., as observed in most model systems). Interestingly, it now appears that the anti-tumor activity exhibited by FTIs is largely Ras-independent. This hypothesis is based on two observations. First, the kinetics of inhibition of transformation do not follow the kinetics of Ras de-famesylation. Second, the ability of FTIs to inhibit the soft agar growth of a particular tumor cell line appears to have no correlation with its Ras mutation status. Thus, the true mechanism of FTI function remains to be elucidated. Nonetheless, an understanding of the mechanism of action of FTIs is not necessary in order to make and use the present invention. Moreover, a variety of different FTI compounds have been proven effective in model studies and are now in clinical trials.

C. Combination Treatment With SERM and FTI

Although it has been shown that an FTI can cooperate with taxol (paclitaxel) to inhibit tumor growth in some cell lines (Sepp-Lorenzino et al, supra), until the development of the present invention, the synergistic effect of FTIs and SERMs was not known. As FTIs typically exhibit an unusually low toxicity in animal studies, these compounds were used in the development of the present invention in combination with other anti-cancer compounds, including those that inhibit estrogen receptor positive cells (e.g., tamoxifen).

For instance, as indicated in Example I, the combination of tamoxifen and FTI was found to act synergistically in inhibiting the growth of cells positive for estrogen receptors (e.g., MCF7 cells). In addition, the surprising observation was made that the combination of tamoxifen and FTI synergistically inhibited the growth of cells resistant to FTI inhibition of transformation (e g., T47D cells). As would be expected, the combination of tamoxifen and FTI did not exhibit synergistic inhibition of cells negative for estrogen receptors (e g , MDA-231 cells) It is contemplated that the combination therapy of the present invention will provide significant advantages m the treatment of breast cancer, as compared to the administration of SERMs or FTIs alone. For example, it is contemplated that the present invention will find use m treatment regimens that involve the use of lower doses of SERMs or a shorter duration of SERM (e.g., tamoxifen) treatment. It is contemplated that the efficacy of the compositions of the present invention be demonstrated in an animal model. For example, a muπne mammary carcinoma model is implemented to demonstrate the effectiveness of combination therapy with famesyl transferase inhibitors and selective estrogen receptor modulators It is further contemplated that pharmacokinetics studies be performed in healthy animals (e.g., mice)

In addition, it is intended that the present invention encompass administration of at least one SERM and at least one FTI either as a single composition (i.e., both compounds are present in the same composition) or in the alternative, in separate compositions. It is also not intended that the present invention be limited to the administration of at least one SERM and at least one FTI at the same time, as it is contemplated that these compounds may be administered to subjects in a sequential manner (e g , one composition is taken as the morning dose and the other composition is taken as the evening dose). In addition, it is contemplated that sequential administration encompasses other treatment regimens. For example, it is contemplated that one compound (e.g., at least one SERM or at least one FTI) will be administered for a certain time period (e.g., days, weeks, or months), followed the administration of the other compound (i.e., at least one SERM or at least one FTI) for another time period. It is further contemplated that each of these compounds will be given in conjunction with other compounds as appropriate for each individual subject.

Thus, it is also contemplated that various other agents may be incorporated into the combination composition of the present invention. For example, various other therapeutic agents (e.g., growth factors, vitamins, nutrients, nutritional supplements, receptor agonists and/or antagonists, etc.), stabilizers, detectable molecules, and the like, will find use in certain embodiments of the present invention.

D. Administration Of Pharmaceutical Compositions

It is contemplated that administration of pharmaceutical compositions including the SERM and FTI combinations of the present invention be accomplished orally or parenterally. Methods of parenteral delivery include topical, intra-arterial (e.g., directly to a tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and other compounds that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences" (Maack Publishing Co, Easton PA).

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally gπnding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores Suitable excipients such as carbohydrate or protein fillers include, but are not limited to sugars, including lactose, sucrose, manmtol, or sorbitol, starch from com, wheat, πce, potato, or other plants, cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose, and gums including arable and tragacanth, as well as proteins such as gelatin and collagen If desired, disintegrating or solubilizmg agents may be added, such as the cross-linked polyvmyl pyrrohdone, agar, algimc acid, or a salt thereof, such as sodium algmate

Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arable, talc, polyvmylpyrrohdone, carbopol gel, polyethylene glycol, and or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to charactenze the quantity of active compound (i e , dosage)

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubπcants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' s solution, Ringer's solution, or physiologically buffered saline Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. The compositions of the present invention may be manufactured in a manner that known in the art (e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes). The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5, that is combined with buffer prior to use. After pharmaceutical compositions comprising a compound of the invention formulated in an acceptable carrier have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of compositions of the present invention, such labeling would include amount, frequency and method of administration. Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in any appropriate animal model. The animal model is also used to achieve a desirable concentration range and route of administration Such information can then be used to determine useful doses and routes for administration in humans. The sub-therapeutically effective dose optimally suited for vaπous embodiments of the present invention is also determined In particular, it is contemplated that the subtherapeutic doses of SERM(s) and/or FTI(s) for optimal synergistic effect in vivo and in vitro are also determined

A therapeutically effective dose refers to that amount of protein or its antibodies, antagonists, or inhibitors which ameliorate the symptoms or condition Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or expeπmental animals (e g , ED₅₀, the dose therapeutically effective in 50% of the population; and LD₅₀, the dose lethal to 50% of the population) The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀ Pharmaceutical compositions which exhibit large therapeutic indices are preferred The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration The present invention also encompasses compositions that are administered at subtherapeutic concentrations For example, it is contemplated that a sub-therapeutic concentrations of at least one SERM and at least one FTI will be administered to a patient under condition such that the SERM and FTI act synergistically against the subject's tumor cells Indeed, the exact dosage is chosen by the individual physician in view of the patient to be treated Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect Additional factors which may be taken into account include the seventy of the disease state (e g , tumor size and location, age, weight and gender of the patient, diet, time and frequency of administration, drug combιnatιon(s), reaction sensιtι\ ιtιes, and tolerance/response to therapy). Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation. Guidance as to particular dosages and methods of delivery is provided in the literature (See, US Patent Nos. 4,657,760; 5,206,344; and 5,225,212, each of which is herein incorporated by reference).

EXPERIMENTAL

The following example is provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. In the experimental disclosure which follows, the following abbreviations apply: N (normal); M (molar); mM (millimolar); μM (micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg (micrograms); ng (nanograms); 1 or L (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); MW (molecular weight); °C (degrees Centigrade); FTI (famesyl transferase inhibitor); ATCC (American Type Culture Collection, Manassas, VA); and Sigma (Sigma Chemical Co., St. Louis, MO).

EXAMPLE 1 Inhibition of Cell Growth by Tamoxifen and FTI In these experiments, the ability of tamoxifen and famesyl transferase inhibitors to inhibit cell growth was determined. Three cell lines were used, including two estrogen receptor positive lines and one estrogen receptor negative line. The cell lines, their estrogen receptor status, and sensitivity to tamoxifen and FTI are shown in Table 1, below, obtained from the ATCC (HTB-26 [MDA-MB-231)], HTB-22 [MCF7], and HTB-133 [T-47D]). TABLE 1. Cell Lines Tested

As T47D and MCF7 are estrogen receptor positive, they are both sensitive to inhibition by tamoxifen, which works at least primarily, by blocking the estrogen receptor. T47D cells have previously been shown to be resistant to the effects of FTI compounds (Sepp-Lorenzino et al, Cancer Res., 55:5302-5309 [1995]). MDA-231 cells have lost expression of the estrogen receptor. Thus, they are resistant to the effects of tamoxifen. These cells are also reported to be resistant to FTI (Sepp- Lorenzino et al, supra).

The cell lines were all grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, as well as penicillin/streptomycin (10 units/μl penicillin and 10 μg/ml streptomycin) and insulin (10 μg/ml). Cells were grown as monolayers in 6 well plates at 2 x 10⁵ cells per well and allowed to grow for 48 hours before commencing drug treatment. Tamoxifen was dissolved in water to a final concentration of 5 μg/ml. FTI-277 (Lemer et al, J. Biol. Chem., 270:26802- 26806 [1995]) was dissolved in a solution of dimethylsulfoxide (DMSO) and 10% dithiothreitol (DTT). Tamoxifen was added to the cells at a final concentration of 5 μg/ml, while FTI was added to the cells at a final concentration of 5 μM. The DMSO/DTT carrier was added to non-FTI treated wells as the control.

After 5 days incubation at 37°C in an atmosphere containing 10% C0₂, the cells were rinsed and suspended by trypsmization as known in the art. The cells were then stained with trypan blue and counted using an inverted microscope and counted using a hemocytometer. Trypan blue-stained cells were counted as dead. Typically, dead cells comprised less than 5% of the total cell count. Dose response curves were performed (data not shown) to determine the dose at which tamoxifen alone was just sub-inhibitory. This dose was determined to be 5 μg/ml. Thus, this dose was used in the experiments illustrated in Figures 1-3.

Figure 1 shows the effects of tamoxifen and FTI, alone and in combination on the growth of MCF7 cells. The data shown in Figure 1 represent the average of three experiments. The standard error was within 20%. In Figure 1, column 1 provides the results for the carrier alone (DMSO DTT), while column 2 provides the results for tamoxifen and carrier, column 3 provides the results for FTI alone, and column 4 provides the results for tamoxifen and FTI. The growth of MCF7 cells was also inhibited by the combination of tamoxifen and FTI in a synergistic manner.

Figure 2 shows the effects of tamoxifen and FTI, alone and in combination on the growth of T47-D cells. As with Figure 1, the data shown in Figure 2 represent the average of three experiments. The standard error was within 20%. In Figure 2, column 1 provides the results for the carrier alone (DMSO/DTT), while column 2 provides the results for tamoxifen and carrier, column 3 provides the results for FTI alone, and column 4 provides the results for tamoxifen and FTI. As indicated in Figure 2, there was a decrease in cell growth for the experiments in which tamoxifen and FTI were provided together. As indicated, the tamoxifen/FTI combination acted synergistically in T47-D cells. This cell line was included in these experiments as it had been reported to be resistant to FTI action (Sepp-Lorenzino et al, supra).

Figure 3 shows that the combination of tamoxifen and FTI did not act synergistically in decreasing the growth of MDA-231 cells. The data shown in Figure 3 represent the average of two experiments and the standard error was within 20%. The MDA-231 cells were assayed as a control cell line, as these cells are highly transformed and very metastatic. They are no longer dependent upon stimulation of the estrogen receptor for their growth and do not express the receptor protein. Consequently, they are immune to tamoxifen inhibition.

As can be seen, the combination compositions of the present invention provide an improved system for the synergistic action of therapeutic agents such as famesyl transferase inhibitors and selective estrogen receptor modulators for treatment of cancer cells that express estrogen receptors.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in oncology, pharmacy, organic chemistry, and related fields are intended to be within the scope of the following claims.

Claims

CLAIMSWe claim:

1. A composition comprising at least one selective estrogen receptor modulator and at least one famesyl transferase inhibitor.

2. The composition of Claim 1, wherein said composition is therapeutically effective.

3. The composition of Claim 1, wherein said at least one estrogen receptor modulator is present in a sub-therapeutically effective amount.

4. The composition of Claim 1, wherein said at least one famesyl transferase inhibitor is present in a sub-therapeutically effective amount.

5. The composition of Claim 1, wherein said composition is capable of inhibiting the growth of cancer cells expressing estrogen receptors.

6. The composition of Claim 1, wherein said at least one selective estrogen receptor modulator comprises tamoxifen.

7. The composition of Claim 1, wherein said at least one famesyl transferase inhibitor comprises FTI-277.

8. A method for treatment of cancer, comprising: a) providing: i) a subject suffering from cancer, ii) at least one selective estrogen receptor modulator, and iii) at least one farnesyl transferase inhibitor; and b) administering said selective estrogen receptor modulator and said famesyl transferase inhibitor to said subject.

9. The method of Claim 8, wherein said subject is suffering from breast cancer.

10. The method of Claim 8, wherein the cancer cells of said subject express estrogen receptors.

1 1. The method of Claim 8, wherein said selective estrogen receptor is tamoxifen.

12. The composition of Claim 8, wherein said at least one famesyl transferase inhibitor comprises FTI-277.

13. The method of Claim 8, wherein said selective estrogen receptor modulator and said famesyl transferase inhibitor are provided to said subject in a combination composition.

14. The method of Claim 8, wherein said selective estrogen receptor modulator and said famesyl transferase inhibitor are provided to said subject in separate compositions.

15. The method of Claim 8, wherein said subject is post-operative for cancer.

16. The method of Claim 8, wherein said subject is pre-operative for cancer.

17. The method of Claim 8, wherein said subject is at risk for developing cancer.

18. The method of Claim 8, wherein said subject is experiencing a recurrence of cancer.

19. The method of Claim 8, wherein said at least one selective estrogen receptor modulator is provided in a sub-therapeutic concentration.

20. The method of Claim 6, wherein said at least one farnesyl transferase inhibitor is provided in a sub-therapeutic concentration.

21. The method of Claim 8, wherein said at least one selective estrogen receptor modulator and said at least one famesyl transferase inhibitor are provided in a sub-therapeutic concentration.