WO2004056371A1 - Use of aglycon protopanaxatriol in cancer therapy - Google Patents

Abstract

Methods of treating multi-drug resistant tumors in a mammal using of aglycon protopanaxatriol (aPPT) alone or in combination with one or more chemotherapeutic are provided. Also provided are pharmaceutical, as well as non-pharmaceutical, compositions comprising aPPT for use in the treatment of mammalian subjects having a multi-drug resistant cancer and pharmaceutical kits comprising suc h compositions.

Description

USE OF AGLYCON PROTOPANAXATRIOL IN CANCER THERAPY

FIELD OF THE INVENTION

The present invention pertains to the field of cancer therapy and in particular to the use of aglycon protopanaxatriol (aPPT) in the treatment of cancer.

BACKGROUND

The resistance of cancer cells to the killing effects of chemotherapy is one of the central problems in the management of cancer. It is now apparent that at diagnosis many human tumors already contain cancer cells that are resistant to standard chemotherapeutic agents. Spontaneous mutation toward drug resistance is estimated to occur in one of every 10 to 10 cancer cells. This mutation rate appears to be independent of any selective pressure from drug therapy, although radiation therapy and chemotherapy may give rise to additional mutations and contribute to tumor progression within cancer cell populations (Goldie et al, Cancer Treat. Rep., 63:1727, 1979; Goldie et al., Cancer Res., 44:3643, 1984; Nowell, Cancer Res., 46:2203, 1986).

Selective killing of only the tumor cells sensitive to the drugs leads to an overgrowth of tumor cells that are resistant to the chemotherapy. Mechanisms of drug resistance include decreased drug accumulation (particularly in multi-dr g resistance), accelerated metabolism of the drug and other alterations of drug metabolism, and an increase in the ability of the cell to repair drug-induced damage (Curt et al., Cancer Treat. Rep., 68:87, 1984; and Kolate, Science, 231:220, 1986). The cells that overgrow the tumor population not only are resistant to the agents used but also tend to be resistant to other drugs, many of which have dissimilar mechanisms of action. This phenomenon, called multi-drug resistance (MDR), may account for much of the drug resistance that occurs in previously treated cancer subj ects. One of the traditional ways to attempt to circumvent the problem of drug resistance has been combination chemotherapy, which uses the differing mechanisms of action and cytotoxic potentials of multiple drugs. Although combination chemotherapy has been successful in many cases, the need still exists for new anti-cancer drugs, it may still fail in cases involving drug resistant tumors having multiple resistance phenotypes.

Prior work has demonstrated that certain compounds derived from Panax ginseng, Panax quinquefolium L, Panax notoginseng and other species of the ginseng family have shown potential as anti-cancer agents (U.S. Patent Application No. 09/910,887, U.S. Patent No. 5,776,460, Kikuchi Y. et al. (1991) Anti-cancer Drugs. 2: 63-7; Lee KY et al. (1996) Cancer Lett. 110: 193-200; Oh M et al. (1999) h t J Oncol. 18: 869- 75; Ota T et al. (1997) Life Sci. 60: PL39-88; Nakata H et al. (1998) Jpn J Cancer Res. 89: 733-80; Kim HE et al. (1999) Life Sci. 65: PL33-80; Park JA et al. (1997) Cancer Lett. 121 : 73-81, Nakata H et al. (1998) Jpn J Cancer Res. 89: 733-80, U.S. Patent Application No. 60/204,785). The two known sapogenins, protopanaxadiol and protopanaxatriol, which are derived from Panax ginseng have further been demonstrated to be effective in chemosensitizing certain multi-drug resistant (MDR) cancer cells when used with paclitaxel, mitoxantrone or cisplatin in vitro (U.S. Patent Application No. 09/957,082).

This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a use of aglycon protopanaxatriol

(aPPT) in cancer therapy. In accordance with an aspect of the present invention, there is provided a composition comprising a therapeutically effective amount of substantially purified aglycon protopanaxatriol (aPPT) and a physiologically acceptable carrier for the treatment of a multi-drug resistant (MDR) cancer in a mammal.

In accordance with another aspect of the invention, there is provided a composition consisting essentially of a therapeutically effective amount of substantially purified aglycon protopanaxatriol (aPPT) and a physiologically acceptable carrier for the treatment of a multi-drug resistant (MDR) cancer in a mammal.

In one embodiment of the invention, the MDR cancer is a primary or recurrent cancer selected from the group of: pancreatic cancer, lung cancer, stomach cancer, esophagus cancer, colon and rectum cancer, brain cancer, ovary cancer, liver cancer, kidney cancer, larynx cancer, bone cancer, multiple myeloma, melanoma, breast cancer, prostate cancer, bladder cancer, cancer in body of uterus, oral cavity cancer, thyroid cancer, cervix cancer, testis cancer, non-Hodgkin's lymphoma, leukemia, Hodgkin's disease, skin cancer, and soft tissue cancer.

In accordance with another aspect of the invention, there is provided a use of substantially purified aglycon protopanaxatriol (aPPT) in the treatment of a multi- drug resistant (MDR) cancer in a mammal in need thereof.

In accordance with another aspect of the invention, there is provided a use of substantially purified aglycon protopanaxatriol (aPPT) in the manufacture of a medicament for the treatment of a multi-drug resistant (MDR) cancer in a mammal in need thereof.

In accordance with another aspect of the invention, there is provided a use of substantially purified aglycon protopanaxatriol (aPPT) in the manufacture of a non- pharmaceutical composition for the treatment of a multi-drug resistant (MDR) cancer in a mammal in need thereof.

In accordance with another aspect of the invention, there is provided a method of treating a multi-drug resistant cancer in a mammal comprising administering to said mammal an effective amount of substantially purified aglycon protopanaxatriol (aPPT). In accordance with another aspect of the invention, there is provided a kit for the treatment of a multi-drug resistant cancer in a mammal, said kit comprising:

(a) a therapeutically effective amount of substantially purified aglycon protopanaxatriol (aPPT); and optionally (b) instructions for use.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 provides a graphical representation of cell viability of multi-drug resistant cancer cells treated with various concentrations of aPPT;

Figure 2 provides a graphical representation of (A) cell viability of multi-drug resistant cancer cells treated with various concentrations of aPPT and (B) cell viability of multi-drug resistant cancer cells treated with various concentrations of Taxol;

Figure 3 demonstrates the effect of aPPT in the mesothelioma cell line MS-1;

Figure 4 demonstrates the effects of aPPT on the drug resistant pancreatic cancer cell line BXPC in vitro;

Figure 5 demonstrates the effects of aPPT on the drug resistant pancreatic cancer cell line BXPC in vivo;

Figure 6 demonstrates the tumor inhibitory effect of aPPT on multi-drug resistant B16 melanoma cells;

Figure 7 demonstrates the effects of aPPT on the drug resistant breast cancer cell line MCF7r in vivo;

Figure 8 demonstrates that aPPT enhances BCNU inhibition of growth of U87 human glioma cells;

Figure 9 demonstrates (A) the effect of aPPT on MDR cancer cell MCF7r, (B) the effect of aPPT and paclitaxel on MCF7 non-drug resistant cancer cells and (C) the effect of aPPT on MCF7r multi-drug resistant cancer cells; and Figure 10 demonstrates the effect of aglycon protopanaxatriol on multi-drug resistant cancer cells (A) shows cells pre-aPPT treatment and (B) shows cells 15 min. post- aPPT treatment.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

"Multi-drug resistant (MDR)", as used herein with reference to a cancer, tumor or neoplasm refers to an innate or acquired ability of the cancer, tumor or neoplastic cells to develop resistance to treatment. By convention: primary MDR is untreated cancer that is considered unresponsive to chemotherapy and secondary MDR develops resistance during the course of treatment. One skilled in the art will appreciate that the term "multi-drug resistant cancer" will refer to drug resistance that may be caused by several mechanisms including, but not limited to, decreased drug accumulation (e.g., active excretion of the chemotherapeutic by a protein pump (such as P-glycoprotein)), accelerated metabolism of the drug and other alterations of drug metabolism, and an increase in the ability of the cell to repair drug-induced damage.

A "refractory" cancer or tumor refers to a cancer or tumor that has not responded to treatment. Such cancers or tumors are often also MDR.

"Advanced cancer," as used herein, refers to overt cancer in a subject, wherein such overt cancer is usually not localized, and is not amenable to cure by local modalities of treatment, such as surgery or radiotherapy.

"Primary cancer", as used herein, refers to the original tumor or primary tumor and is usually named for the part of the body in which it begins. "Metastatic cancer", as used herein, refers to a cancer which has spread from an initial site ("primary cancer") to another site(s) ("secondary cancer"). Virtually all cancers can develop metastases. The term, thus, is not limited to any one particular type of cancer.

"Recurrent cancer", as used herein, refers to a reappearance of a cancer that was thought to be cured or inactive (in remission). A cancer may recur after several weeks, several months, a few years, or many years. Recurrent cancer usually starts from cancer cells that were not removed or destroyed by the original therapy, e.g. chemotherapy.

The term "aggressive cancer," as used herein, refers to a rapidly growing cancer. One skilled in the art will appreciate that for some cancers, such as breast cancer or prostate cancer the term "aggressive cancer" will refer to an advanced cancer that has relapsed within approximately the earlier two-thirds of the spectrum of relapse times for a given cancer, whereas for other types of cancer, such as small cell lung carcinoma (SCLC) nearly all cases present rapidly growing cancers which are considered to be aggressive. The term can thus cover a subsection of a certain cancer type or it may encompass all of other cancer types.

"Relapse," as used herein, refers to the relapse of a patient with advanced disease. "Relapse time," as used herein, refers to the time from the initial appearance of a primary cancer to the appearance of advanced disease requiring chemotherapy.

"Adjuvant therapy," as used herein, refers to a treatment that is added to increase the effectiveness of a primary treatment. In cancer, adjuvant therapy usually refers to chemotherapy or radiation therapy after surgery (primary treatment) to increase the likelihood of killing all cancer cells.

The term "neoadjuvant therapy," as used herein, refers to a treatment given before the- primary treatment. Examples of neoadjuvant therapy include chemotherapy, radiation therapy, and hormone therapy. THERAPEUTIC USE OF aPPT

The present invention provides for the use of aglycon protopanaxatriol (aPPT) in the treatment of multi-drug resistant (MDR) cancer in mammalian subjects, including humans. As is known in the art, many chemotherapeutics capable of inhibiting the growth of drug sensitive cells are ineffective in inhibiting the growth of MDR cancer cells. Thus, the ability of a compound to inhibit the growth of drug sensitive cells is not predictive of its ability to attenuate the growth of MDR cancer cells. The present invention relates to the unexpected cytotoxic effect of aPPT as a single agent, as well as in combination with other chemotherapeutics, on a range of MDR cancer cells, which have developed drug resistance through different mechanisms.

In the context of the present invention, aPPT may act on MDR cancer cells by attenuating or decreasing the growth and/or viability of MDR neoplastic cells, or it may act as a chemosensitizing agent. aPPT may, therefore, be used alone or in combination with one or more standard chemofherapeutic agent(s) in the treatment of a range of MDR cancers.

As is known in the art, mechanisms of multi-drug resistance in tumor cells can be generally divided into P-gp dependent and P-gp independent. Different mechanisms determine specific groups of chemotherapy drugs that tumor cells are resistant to and, therefore, different treatments need to be selected in order to try to re-sensitise the cells. aPPD is demonstrated herein to be able to re-sensitise cancer cells that have developed multi-drug resistance through both P-gp dependent and P-gp independent mechanisms.

aPPT

In accordance with the present invention, the aPPT for use in the treatment of MDR cancers is substantially purified. The term "substantially purified" as used herein with reference to an aPPT preparation refers to a preparation in which the aPPT forms the major component, i.e. aPPT constitutes more than about 50% of the preparation.

In one embodiment of the present invention, a substantially purified aPPT preparation refers to a preparation in which the aPPT constitutes more than about 60% of the preparation, i another embodiment, the term refers to a preparation in which the aPPT constitutes more than about 70% of the preparation. In a further embodiment, the term refers to a preparation in which the aPPT constitutes more than about 75% of the preparation. In other embodiments, a substantially purified aPPT preparation refers to a preparation in which the aPPT constitutes more than about 80%, more than about 85%, more than about 90% and more than about 95% of the preparation.

As is known in the art aPPT can adopt a number of stereoisomeric forms. For example, the natural form of most ginsenosides is 20S, however, during the process of extracting the ginsenosides from plant material, 20R compounds may be formed. The use of various stereoisomers of aPPT is contemplated. One embodiment of the invention, therefore, employs aPPT having the following chemical structure:

Another embodiment of the invention employs aPPT having the following chemical structure:

The use of racemic mixtures of aPPT is also contemplated. Preparation ofaPPT

Methods of preparing aPPT are known in the art. For example, aPPT can be obtained from extracts prepared from plants of the genus Panax using standard techniques. The isolation and purification of aPPT from natural sources has been described in the art, for example, see U.S. Patent No. 4,157,894. Examples of plants from which aPPT may be obtained include, but are not limited to, Panax aureus; Panax bipinnatifidus (also known as: Panax major and Panax pseudoginseng var. bipinnatifidus); Panax ginseng C. A. Meyer (also known as: Panax schinseng); Panax japonicus (also known as: Panax pseudoginseng subsp. japonicus; Panax pseudoginseng var. japonicus and Panax repens); Panax notoginseng (also known as: Aralia quinquefolia var. notoginseng and Panax pseudoginseng var. notoginseng); Panax pseudoginseng (also known as: Aralia pseudoginseng and Panax pseudoginseng var. pseudoginseng); Panax wangianus (also known as: Panax pseudoginseng var. wangianus); Panax quinquefolius (including P. quinquefolius L); Panax stipuleanatus; Panax trifolius; Panax vietnamensis and Panax zingiberensis.

aPPT can be obtained from Panax plant extracts using standard extraction, fractionation and/or purification techniques. Such techniques include, for example, solid-liquid extraction, liquid-liquid extraction, solid-phase extraction (SPE), membrane filtration, ultrafiltration, dialysis, electrophoresis, solvent concentration, centrifugation, ultracentrifugation, liquid or gas phase chromatography (including size exclusion, affinity, and the like) with or without high pressure, thin-layer chromatography, lyophilisation, evaporation, precipitation with various "carriers" (including PVPP, carbon, antibodies, and the like), or various combinations of these techniques. aPPT is also available commercially (Pegasus Pharmaceuticals, Inc., Richmond, British Columbia, Canada) or can be chemically or biologically synthesized using techniques well known to persons of skill in the art (see, for example, Shibata, S. (2001) J. Korean Med. Sci., 16 Suppl.: S28-37, and references therein). aPPT Activity

Preparations of aPPT can be assayed to determine their ability to attenuate the growth of MDR neoplastic cells, or sensitize such cells to a chemotherapeutic, using standard techniques well known to workers skilled in the art. Exemplary testing methods are outlined herein and are not intended to limit the scope of the present invention.

i) In vitro Activity

Initial determinations of the efficacy of the preparations of aPPT can be made using in vitro techniques. Suitable MDR cell lines for testing the ability of the preparations to attenuate MDR cancer cell growth include, for example, LNCaP prostate cancer cells, MS-1 mesothelioma cancer cells, BXPC pancreatic cancer cells, U87 glioma cells and MCF-7r breast cancer cells.

For example, the cytotoxic activities of aPPT can be measured using a standard method for adherent cell lines such as the microculture tetrazolium assay that utilises 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Details of this assay have been published (Alley, M C et al, Cancer Research 48:589-601, 1988). Typically, in this technique, cancer cells are cultured and plated into a 96-well plate. The cells are then exposed to aPPT for 24 h. The medium is removed from the plates and replaced with MTT solution. The plates are analyzed and the number of viable cells in each well containing cells treated with aPPT is calculated from the absorbance at 570 nm compared to untreated controls, or controls treated with a standard chemotherapeutic. The means and standard deviation at each concentration for each agent are calculated using standard statistical methods. For example, using the formula: % viable cells treated/control (% T/C) calculated as OD of treated cells/ OD of control cells x 100 = % T/C. The concentration of aPPT that gives a T/C of 50% growth inhibition is designated as the IC₅₀ value.

An alternative method of evaluating anti-cancer activity of aPPT involves determining the effect of various concentrations on the viability of cancer cells. Exponentially growing cancer cells are treated with various concentrations of aPPT. The cells are further incubated for 24 h and stained with a molecule that allows a distinction between viable and non-viable cells, for example crystal violet. The absorbency of the stained cells at 590nm is then measured. The percent viability of treated cells is then compared to that of control cells. Average absorbency of the control wells (A_c) without any treatment is calculated, average absorbency of each treatment group (A_T;) is determined, and then the average cell viability of each treatment group (V) is derived using the following formula:

A decrease in viability in the treated cells in comparison to the control cells is indicative of anti-cancer activity.

The aPPT preparations can also be tested by determining their ability to inhibit anchorage-independent growth of tumor cells. Anchorage-independent growth is known in the art to be a good indicator of tumorigenicity. In general, anchorage- independent growth is assessed by plating cells from an appropriate cancer cell-line onto soft agar and determining the number of colonies formed after an appropriate incubation period. Growth of cells treated with aPPT can then be compared with that of cells treated with an appropriate control compound and with that of untreated cells.

The ability of the aPPT preparations to sensitise MDR cancer cells to the effects of one or more standard chemotherapeutic can also be assessed using the above- described assays by contacting the cells with both the aPPT preparation and the chemotherapeutic(s). The cells maybe contacted with the aPPT preparation prior to, at the same time as, or subsequent to contact with the chemotherapeutic(s). The effect on the cells is then compared to control cells that were contacted with the chemotherapeutic(s) alone.

If necessary, toxicity of the preparations can also be initially assessed in vitro using standard techniques. For example, human primary fibroblasts can be treated in vitro with the compositions and then tested at different time points following treatment for their viability using a standard viability assay, such as the trypan-blue exclusion assay. Cells can also be assayed for their ability to synthesize DNA, for example, using a thymidine incorporation assay, and for changes in cell cycle dynamics, for example, using a standard cell sorting assay in conjunction with a fluorocytometer cell sorter (FACS).

ii) In vivo Testing

The ability of the compositions to inhibit tumor growth or proliferation in vivo can be determined in an appropriate animal model using standard techniques known in the art (see, for example, Enna, et al, Current Protocols in Pharmacology, J. Wiley & Sons, Inc., New York, NY).

hi general, current animal models for screening anti-tumor compounds are xenograft models, in which a human tumor has been implanted into an animal. Examples of xenograft models of human cancer include, but are not limited to, human solid tumor xenografts in mice, implanted by sub-cutaneous injection and used in tumor growth assays; human solid tumor isografts in mice, implanted by fat pad injection and used in tumor growth assays; experimental models of lymphoma and leukaemia in mice, used in survival assays, and experimental models of lung metastasis in mice.

For example, aPPT can be tested in vivo on solid tumors using mice that are subcutaneously grafted bilaterally with 30 to 60 mg of a tumor fragment on day 0. Alternatively, an appropriate number of cancer cells can be implanted subcutaneously. The animals bearing tumors are mixed before being subjected to the various treatments and controls. In the case of treatment of advanced tumors, tumors are allowed to develop to the desired size, animals having insufficiently developed tumors being eliminated. The selected animals are distributed at random to undergo the treatments and controls. Animals not bearing tumors may also be subjected to the same treatments as the tumor-bearing animals in order to be able to dissociate the toxic effect from the specific effect on the tumor. Chemotherapy generally begins from 3 to 22 days after grafting, depending on the type of tumor, and the animals are observed every day. The aPPT can be administered to the animals, for example, interperitoneally or by bolus infusion. The different animal groups are weighed about 3 or 4 times a week until the maximum weight loss is attained, after which the groups are weighed at least once a week until the end of the trial. The tumors are measured after a pre-determined time period, or they can be monitored continuously by measuring about 2 or 3 times a week until the tumor reaches a predetermined size and / or weight, or until the animal dies if this occurs before the tumor reaches the pre-determined size / weight. The animals are then sacrificed and the tissue histology, size and / or proliferation of the tumor assessed.

For the study of the effect of the aPPT preparations on leukaemias, the animals are grafted with a particular number of leukaemic cells, and the anti-tumor activity is determined by the increase in the survival time of the treated mice relative to the controls.

To study the effect of the aPPT preparations on tumor metastasis, tumor cells are typically treated with aPPT ex vivo and then injected into a suitable test animal. The spread of the tumor cells from the site of injection is then monitored over a suitable period of time.

The ability of the preparations to sensitise MDR tumors to the effects of one or more standard chemotherapeutic can also be assessed in the above models. The aPPT preparation can be administered prior to, in combination with, or subsequent to the chemotherapeutic(s) and the effects compared to control animals that received the chemotherapeutic(s) alone.

In vivo toxic effects of the aPPT preparations can be evaluated by measuring their effect on animal body weight during treatment and by performing haematological profiles and liver enzyme analysis after the animal has been sacrificed.

Use ofaPPT in Treatment of MDR Cancers

The present invention provides for methods of treating a MDR cancer in a mammal comprising administering to the mammal an effective amount of aPPT, either alone or in combination with one or more chemotherapeutic. Forms of cancer with a high malignancy potential, or present with metastasis at diagnosis, typify the MDR category. A variety of tumors with a frequency of primary MDR have been reported (Yin L., et al; 19(6) :420-2, 1997), for example: lung (55%), stomach (33%), esophagus (37%), colorectal (31%), and thyroid (40%). Examples of other forms of cancer that develop secondary MDR after treatment include: breast, prostate, Hodgkins & Non-Hodgkins lymphoma, bladder, leukemia, endometrial, oropharyngeal, cervical, testis, skin, and soft tissue cancer; this does not exclude the occurrence of a primary MDR in these groups.

In addition, a disparity has been shown in the frequency of MDR in the specific types of cancer. For example, 20-35% pancreatic cancer patients (Permert J., et al., Acta Oncol; 40(2-3):361-70, 2001) versus 60-80% of breast cancer patients (Hortobagyi G.N., et al, Semin Oncol;23(lSuppl 1):53-7,1996), respond to chemotherapy. Pancreatic cancer is one example of a tumor impervious to conventional chemotherapy, i.e. a MDR cancer, h contrast, breast cancer often achieves high remission rates in response to chemotherapy, but relapses are exclusively secondary MDR (Liu X., et al; Zhonghua YiXue Za Zhi 77(7):488-90, 1997).

In one embodiment of the present invention, aPPT is used to treat primary cancers that are usually resistant to conventional chemotherapeutics, including without limitation: pancreatic cancer, lung cancer, stomach cancer, esophageal cancer, colon and rectal cancer, brain cancer (including gliomas), ovary cancer, liver cancer, kidney cancer, larynx cancer, bone cancer, multiple myeloma, mesothelioma and melanoma.

In another embodiment of the present invention, aPPT is used to treat MDR cancers developed in subjects who have undergone prior chemotherapy, including without limitation, the above cancers as well as: breast cancer, prostate cancer, bladder cancer, cancer in body of uterus, oral cavity cancer, thyroid cancer, cervix cancer, testis cancer, non-Hodgkin's lymphoma, leukemia, Hodgkin's disease, skin cancer, and soft tissue cancer.

In a further embodiment of the present invention, aPPT is used to treat advanced and metastatic cancers, which are usually drug resistant, including the above cancers when in an advanced and/or metastatic stage. The aPPT may also be used to treat aggressive, refractory and recurrent cancers. One skilled in the art will appreciate that many of these categories may overlap, for example, aggressive cancers are typically also metastatic. Additional cancers encompassed by the present invention include, for example, primary and metastatic multi-drug resistant leukaemias, carcinomas, adenocarcinomas, melanomas and sarcomas. Carcinomas, adenocarcinomas and sarcomas are also frequently referred to as "solid tumors," examples of commonly occurring solid tumors include, but are not limited to, cancer of the brain, breast, cervix, colon, head and neck, kidney, liver, lung, ovary, pancreas, prostate, stomach and uterus, non-small cell lung cancer and colorectal cancer.

The term "leukaemia" refers broadly to progressive, malignant diseases of the blood- forming organs. Leukaemia is typically characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow but can also refer to malignant diseases of other blood cells such as erythroleukaemia, which affects immature red blood cells. Leukaemia is generally clinically classified on the basis of (1) the duration and character of the disease - acute or chronic; (2) the type of cell involved - myeloid (myelogenous), lymphoid (lymphogenous) or monocytic, and (3) the increase or non-increase in the number of abnormal cells in the blood - leukaemic or aleukaemic (subleukaemic). Leukaemia includes, for example, acute nonlymphocytic leukaemia, chronic lymphocytic leukaemia, acute granulocytic leukaemia, chronic granulocytic leukaemia, acute promyelocytic leukaemia, adult T- cell leukaemia, aleukaemic leukaemia, aleukocythemic leukaemia, basophylic leukaemia, blast cell leukaemia, bovine leukaemia, chronic myelocytic leukaemia, leukaemia cutis, embryonal leukaemia, eosinophilic leukaemia, Gross' leukaemia, hairy-cell leukaemia, hemoblastic leukaemia, hemocytoblastic leukaemia, histiocytic leukaemia, stem cell leukaemia, acute monocytic leukaemia, leukopenic leukaemia, lymphatic leukaemia, lymphoblastic leukaemia, lymphocytic leukaemia, lymphogenous leukaemia, lymphoid leukaemia, lymphosarcoma cell leukaemia, mast cell leukaemia, megakaryocytic leukaemia, micromyeloblastic leukaemia, monocytic leukaemia, myeloblastic leukaemia, myelocytic leukaemia, myeloid granulocytic leukaemia, myelomonocytic leukaemia, Naegeli leukaemia, plasma cell leukaemia, plasmacytic leukaemia, promyelocytic leukaemia, Rieder cell leukaemia, Schilling's leukaemia, stem cell leukaemia, subleukaemic leukaemia, and undifferentiated cell leukaemia. The term "sarcoma" generally refers to a tumor which originates in connective tissue, such as muscle, bone, cartilage or fat, and is made up of a substance like embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas include soft tissue sarcomas, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented haemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

The term "melanoma" refers to a tumor arising from the melanocytic system of the skin and other organs. Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.

The term "carcinoma" refers to a malignant growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colorectal carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, haematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma meduUare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, non-small cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small- cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

The term "carcinoma" also encompasses adenocarcinomas. Adenocarcinomas are carcinomas that originate in cells that make organs which have glandular (secretory) properties or that originate in cells that line hollow viscera, such as the gastrointestinal tract or bronchial epithelia. Examples include, but are not limited to, adenocarcinomas of the breast, lung, pancreas and prostate.

Further examples of cancers encompassed by the present invention include, for example, Hodgkin's Disease, Non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, gliomas, testicular cancer, thyroid cancer, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, mesothelioma and medulloblastoma.

In one embodiment of the present invention, aPPD is used in the treatment of primary, advanced, recurrent or metastatic MDR cancers selected from the group of: solid tumors, adenocarcinomas, melanomas, cancers of the mesothelium and brain cancers.

In another embodiment of the present invention, aPPD is used in the treatment of primary, advanced, recurrent or metastatic MDR cancers selected from the group of: ovarian cancer, renal cancer, colorectal cancer, uterine cancer, liver cancer, adenocarcinoma of the lung, lung cancer, breast cancer, pancreatic cancer, melanoma, mesothelioma and glioma.

In a further embodiment, aPPD is used in the treatment of primary, advanced, recurrent or metastatic MDR cancers selected from the group of: breast cancer, pancreatic cancer, melanoma, mesothelioma and glioma.

The present invention contemplates the use of aPPT in adjuvant therapy after primary therapy as well as in neoadjuvant therapy, i.e. before the primary treatment. aPPT can be used to treat subjects who have undergone prior chemotherapy or it may be used to treat chemotherapy naive subjects. Thus, in one embodiment of the invention, aPPT is used as part of an adjuvant therapy. In a further embodiment, aPPT is used to treat subjects who have already undergone one or more courses of prior chemotherapy.

The present invention further contemplates that aPPT may be used as part of a combination chemotherapy regimen to treat a subject having a multi-drug resistant cancer. Thus, aPPT according to the present invention can be used either alone or in combination with other pharmacologically active chemotherapeutic agents or other anti-cancer therapeutics to treat multi-drug resistant cancers. In combination therapy, aPPT may be active in its own right or it may at as a "sensitizing agent," which selectively inhibits the growth of cancer cells. In the latter case, aPPT alone may not have a cytotoxic effect on the cancer cell, but provides a means of weakening the cancer cells, and thereby facilitates the benefit from conventional anti-cancer therapeutics. An "anti-cancer therapeutic" is a compound, composition or treatment that prevents or delays the growth and or metastasis of cancer cells. Such anti-cancer therapeutics include, but are not limited to, chemotherapeutic drug treatment, radiation, gene therapy, hormonal manipulation, immunotherapy and antisense oligonucleotide therapy.

A wide range of cancer chemotherapeutic agents known in the art are suitable for use in the methods of the present invention. Chemotherapeutic agents can be specific for the treatment of a particular type of cancer or they may be applicable to a range of cancers, for example doxorubicin, mitoxantrone, and irinotecan (CPT-11) are generally applicable chemotherapeutics.

Examples of chemotherapeutic agents suitable for the treatment of breast cancer include, but are not limited to, cyclophosphamide, ifosfamide, cisplatin, carboplatin, 5-fluorouracil (5-FU), taxanes such as paclitaxel and docetaxel and various anthracyclines, such as doxorubicin and epi-doxorubicin (also known as epirubicin). Combination therapies using standard cancer chemotherapeutics may also be used in conjunction with aPPT and are also well known in the art, for example, the combination of epirubicin with paclitaxel or docetaxel, or the combination of doxorubicin or epirubicin with cyclophosphamide, which are used for breast cancer treatments. Polychemotherapeutic regimens are also useful and may consist, for example, of doxorubicin/cyclophosphamide/5-fluorouracil or cyclophosphamide/epirubicin/5-fluorouracil.

Cyclophosphamide, mitoxantrone and estramustine are known to be suitable for the treatment of prostate cancer. Cyclophosphamide, vincristine, doxorubicin and etoposide are used in the treatment of small cell lung cancer, as are combinations of etoposide with either cisplatin or carboplatin. In the treatment of stomach or oesophageal cancer, combinations of doxorubicin or epirubicin with cisplatin and 5- fluorouracil are useful. For colorectal cancer, CPT-11 alone or in combination with 5- fluorouracil-based drugs, or oxaliplatin in combination with 5-fluorouracil-based drugs can be used. Other examples include the combination of cyclophosphamide, doxorubicin, vincristine and prednisone in the treatment of non-Hodgkin's lymphoma; the combination of doxorubicin, bleomycin, vinblastine and DTIC in the treatment of Hodgkin's disease and the combination of cisplatin or carboplatin with any one or a combination of gemcitabine, paclitaxel, docetaxel, vinorelbine or etoposide in the treatment of non-small cell lung cancer.

Carmustine (N,N'-bis(2-hydroxyethyl)-N-nitτosourea or BCNU) is useful in the treatment of brain tumors (such as gliomas), multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma and other malignant neoplasms.

Other suitable chemotherapeutic agents include, but are not limited to, mitomycin C, IL-2-II and IL-2-I, novantrone, DTIC, hydroxyurea, busulphan, chlorambucil, melphalan, Ifosphamide, danorubicin, Navelbine® (vinorelbine), teniposide, cytosine, arabinoside, neocarcinostatin, suramin and the like.

In one embodiment of the present invention, aPPT is used in a chemotherapy regimen that involves the use of a taxane chemotherapeutic (such as paclitaxel, docetaxel or taxol), an alkylating agent (such as BCNU, cyclophosphamide, melphalan, mitomycin C or chlomambucil) or an antimetabolite (such as gemcitabine, methotrexate, 5-FU or cytarabine).

In another embodiment, aPPT is used in a chemotherapy regimen that involves the use of paclitaxel, BCNU or gemcitabine. The chemotherapy regimen can be polycheniotherapeutic and involve the use of other chemotherapeutic drugs in addition to those listed above.

Pharmaceutical Compositions

The present invention further provides for compositions comprising aPPT and an appropriate physiologically acceptable carrier, diluent, excipient or vehicle. The pharmaceutical compositions may also be formulated to contain aPPT and one or more other chemotherapeutic agents for simultaneous administration to a subject. The pharmaceutical compositions of the present invention may be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

The pharmaceutical compositions may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to methods known to the art for the manufacture of pharmaceutical compositions and may contain one or more agents selected from the group of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with suitable non- toxic pharmaceutically acceptable excipients including, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch, or alginic acid; binding agents, such as starch, gelatine or acacia, and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets can be uncoated, or they may be coated by known techniques in order to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

Pharmaceutical compositions for oral use may also be presented as hard gelatine capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active compound in admixture with suitable excipients including, for example, suspending agents, such as sodium carboxymethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum fragacanth and gum acacia; dispersing or wetting agents such as a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethyene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, hepta-decaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol for example, polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example, polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl -hydroxy- benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and/or flavouring agents may be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti- oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.

Pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example, liquid paraffin, or it may be a mixtures of these oils. Suitable emulsifying agents may be naturally-occurring gums, for example, gum acacia or gum fragacanth; naturally-occurring phosphatides, for example, soy bean, lecithin; or esters or partial esters derived from fatty acids and hexitol, anhydrides, for example, sorbitan monoleate, and condensation products of the said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monoleate. The emulsions may also contain sweetening and flavouring agents. i Syrups and elixirs may be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and/or flavouring and colouring agents.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known art using suitable dispersing or wetting agents and suspending agents such as those mentioned above. The sterile injectable preparation may also be sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer's solution, lactated Ringer's solution and isotonic sodium chloride solution. Other examples are, sterile, fixed oils which are conventionally employed as a solvent or suspending medium, and a variety of bland fixed oils including, for example, synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy," Gennaro, A., Lippincott, Williams & Wilkins, Philidelphia, PA (2000) (formerly "Remingtons Pharmaceutical Sciences").

Non-Pharmaceutical Compositions

The invention also provides for aPPT to be administered as a non-pharmaceutical composition in an appropriate physiologically acceptable medium such as a buffer, a solvent, a diluent, an inert carrier, an oil, a creme, or an edible material. The non- pharmaceutical composition may be in the form of, for example, a nutraceutical composition, a food, a health food, a natural health product, a functional food, a nutritional supplement, a dietary supplement, an herbal supplement, an herb, an alternative medicine, and a naturopathic product.

In one embodiment of the invention, the non-pharmaceutical composition comprises a therapeutically effective amount of aPPT in a physiologically acceptable medium. The non-pharmaceutical compositions disclosed herein can be provided in various forms, for example, as a tablet, a capsule, or an ointment.

Administration and Dosage Protocols

In accordance with the present invention, a therapeutically effective amount of aPPT is administered to a subject in order to treat a multi-drug resistant cancer. aPPT or a pharmaceutical composition comprising aPPT may be administered in a manner consistent with conventional chemotherapeutic practice.

The dosage of aPPT to be administered will be dependent upon the type of cancer to be treated and the size of the subject and can be readily determined by a skilled practitioner. It is to be understood, however, that the dosage and frequency of administration may be adapted to the circumstances in accordance with known practices in the art, for the treatment of different cancers.

Daily dosages of the compounds of the present invention will typically fall within the range of about 0.01 g to about 50g per 70 kg bodyweight per day. However, it will be understood that the actual amount of the compound(s) to be administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. The above dosage range is given by way of example only and is not intended to limit the scope of the invention in any way. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing harmful side effects, for example, by first dividing the larger dose into several smaller doses for administration throughout the day. In one embodiment of the present invention, daily dosages of aPPT are within the range of about 0.02g to about 40g per 70 kg bodyweight per day. In another embodiment, daily dosages of aPPT are within the range of about 0.03g to about 30g per 70 kg bodyweight per day. In a further embodiment, daily dosages of aPPT are within the range of about 0.04g to about 20g per 70 kg bodyweight per day. In other embodiments, daily dosages of aPPT are within the range of about 0.05g to about 15g, about 0.05g to about lOg, about 0.05g to about 0.9g and about 0.05g to about 0.8g per 70 kg bodyweight per day.

The present invention further contemplates the administration to a subject of a therapeutically effective amount of aPPT in combination with one or more anti-cancer therapeutics for the treatment of a MDR cancer. aPPT can be administered before, during or after treatment with the anti-cancer therapeutic.

Kits

The present invention additionally provides for therapeutic kits containing aPPT for use in the treatment of multi-drug resistant cancer. Individual components of the kit would be packaged in separate containers and, associated with such containers, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

When the components of the kit are provided in one or more liquid solutions, the liquid solution can be an aqueous solution, for example a sterile aqueous solution. In this case the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the composition may be administered to a subject.

The components of the kit may also be provided in dried or lyophilised form and the kit can additionally contain a suitable solvent for reconstitution of the lyophilised components. Irrespective of the number or type of containers, the kits of the invention also may comprise an instrument for assisting with the administration of the composition to a subject. Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.

The kit may further comprise one or more other chemotherapeutic agents for administration to a subject in conjunction with aPPT.

To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in any way.

EXAMPLES

EXAMPLE 1: Effect of Aglycon Protopanaxatriol on Multi-Drug Resistant Cancer Cells

Cytotoxicity was measured using a standard microculture tetrazolium assay (MTT) (Alley, M C et al, Cancer Research 48:589-601, 1988). Exponentially growing cultures of tumor cells, including multi-drug resistant cell lines, were used to make microtiter plate cultures. Cells were seeded at 1.2 x 10³ cells per well in 96-well plates, and grown overnight at 37°C. aPPT was then added. Cells were treated for 24 hours. To determine the number of viable cells in each well, MTT dye (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in saline) was added in accordance with the standard practice known in the art.

Multi-drug resistant human breast cancer (MCF7r) and mesothelioma (MS-1) cells were treated with aPPT at various concentrations. The cell viabilities were examined with MTT at 24 hours.

Figure 1 shows the comparison of the effect of the various concentrations of aPPT on a human mesothelioma cell line (MS-1) and a multi-drug resistant human breast cancer cell line (MCF7R). Each cell line was treated with 6, 12, 24, and 48 μg/ml of aPPT for 24 hours prior to determining cell viability. It is clear that aPPT caused cancer cell death in a dose-response fashion and reached near 100% cell killing at high concentrations. EXAMPLE 2: Comparative Effect of Aglycon Protopanaxatriol to Taxol

In this study, the effect of aPPT on a multi-drug resistant cell line was compared to the effect of the known chemotherapeutic Taxol to demonstrate the efficacy of aPPT on multi-drug resistant cancer cells.

Figure 2a illustrates the effect of aPPT on a multi-drug resistant human breast cancer cell line (MCF7r). Human breast cancer cells both multi-drug resistant (MCF7r) and drug sensitive (MCF7) were treated with aPPT at various concentrations. The cells were treated with 6, 12, 24, and 48 jUg/ml of aPPT for 24 hours prior to determining cell viability using the MTT method as described in Example 1. Unlike Taxol, aPPT showed equal potency on inhibiting both drug sensitive and drug resistant cancer cells.

Figure 2b illustrates the effect of Taxol on the same human breast cancer cell lines as was used in Figure 2a. Human breast cancer cells both multi-drug resistant (MCF7r) and drug sensitive (MCF7) were treated with the chemotherapy drug Taxol at various concentrations. The cells were treated with 0.1, 1, 10, 100, and 1000 nM of Taxol for 24 hours prior to determining cell viability using the MTT method. Taxol showed significant cytotoxicity on drug sensitive cells but was much less effective for the drug resistant cells.

As indicated in Figures 2a and 2b, the cytotoxic efficacy of aPPT on the multi-drug resistant cancer cells was comparable to its effect on the drug-sensitive control, hi contrast, however, the efficacy of Taxol was markedly inhibited in the drug resistant cancer cells.

EXAMPLE 3: Cytotoxic Activity of aPPT in Mesothelioma Cancer Cells

The cytotoxic activity of aPPT was examined in the MS-1 mesothelioma cancer cell line using the MTT assay outlined in Example 1. MS-1 is highly drug resistant, but does not express P-gp or P-gp like membrane pumps.

aPPT was shown to be cytotoxic to the MS-1 cell line in vitro (Figure 3). EXAMPLE 4: aPPT Sensitizes Pancreatic Cancer Cells to Gemcitabine.

The effect of aPPT on gemcitabine (Gemzar) efficacy was examined in vitro using the drug resistant pancreatic cancer cell BXPC and the MTT assay as described in Example 1.

As shown in Figure 4, aPPT sensitizes BXPC cells to gemcitabine (Gemzar). BXPC cells were not sensitive to gemcitabine alone, but were sensitive when treated with a combination of aPPT and gemcitabine.

Resistance to gemcitabine does not correlate to the level of P-gp expressed in cancer cells and, therefore, likely develops by a P-gp independent mechanism.

EXAMPLE 5: Anti-tumor Efficacy of Gemcitabine (Gemzar) and aPPT In Vivo.

The anti-tumor efficacy of Gemcitabine and aPPT was examined in vivo in the BXPC pancreatic cancer model. Briefly, SCID mice were implanted with BXPC pancreatic cancer cells in the flank region. Once the tumor had grown to 1 mm in size, animals received an i.p. injection of either i) gemcitabine (7.5mg/kg) once a week, ii) aPPT (7.5mg/kg) every second day, or iii) gemcitabine (7.5mg/kg) and aPPT (7.5mg/kg) in combination. Tumor weights were measured at day 20 post-treatment.

As shown in Figure 5, treatment with aPPT and gemcitabine in combination was significantly more effective than either compound alone, indicating that aPPT significantly enhanced the efficacy of gemcitabine.

EXAMPLE 6: Effect of aPPT on Multi-Drug Resistant B16 Melanoma Cancer Cells

The tumor inhibitory effect of aPPT was determined in the highly drug resistant B16 melanoma cell line. Briefly, cells were treated with various concentrations of aPPT and the viability was measured 24 hours post treatment as described previously.

As shown in Figure 6, aPPT has a potent tumor inhibitory effect on the B16 melanoma cell line. EXAMPLE 7: Effect Of aPPT Alone and In Combination with Paclitaxel on MDR MCF7r Cancer In Vivo.

The anti-tumor efficacy of aPPT and paclitaxel was examined in vivo using a human breast cancer SCID mouse model. Briefly, human breast cancer cells MCF7r cells were implanted subcutaneously in SCID mice and tumor size was measured every second day for 30 days post-implantation. Animals received either i) saline, ii) paclitaxel i.p. (5 mg/kg) once a week, iii) aPPT (lOmg/kg) orally every second day or iv) both paclitaxel and aPPT.

As shown in Figure 7, paclitaxel and aPPT combination treatment resulted in significant inhibition in tumor growth compared to the other three groups (p<0.05 at all the data points day 23 forward). The results indicate that aPPT exhibited anti- tumor efficacy in the drug resistant MCF7r SCID model. Further, aPPT enhanced the anti-cancer effect of paclitaxel in this model.

EXAMPLE 8: Effect Of aPPT Alone and In Combination with BCNU on Drug Resistant U87 Glioma Cells In Vivo.

The anti-tumor efficacy of aPPT alone or in combination with BCNU was examined in a human glioma cell SCID mouse model. Briefly, human malignant U87 glioma cells were implanted subcutaneously in SCID mice and tumor size were measured every second day for 33 days post-implantation. Animals received either i) saline, ii) N,N'-bis(2-hydroxyethyl)-N-nitrosourea (BCNU) 5 mg/kg i.p. once, iii) aPPT lOmg/kg orally every second day, or iv) both BCNU and aPPT.

As shown in Figure 8, BCNU and aPPT combination treatment resulted in significant inhibition in tumor growth compared to the other three groups (p<0.001 at all the data points day 17 forward). The results indicate that aPPT exhibited anti-tumor efficacy in the glioma SCID model. Further, aPPT enhanced the anti-cancer effect of BCNU in this model.

EXAMPLE 9: The Cytotoxic and Chemosensitising Activity of aPPT. The cytotoxic activity of aPPT alone was examined in the multi-drug resistant breast cancer cell line MCR7r cells in vitro using the MTT assay described in Example 1.

aPPT alone was shown to be cytotoxic to MDR MCF7r cells in vitro (Figure 9A)

In addition, the ability of aPPT to enhance the efficacy of paclitaxel in the drug sensitive breast cancer cell line MCF7 and the multi-drug resistant breast cancer cell line MCR7r was examined in vitro. Briefly, MCF7 or MCF7r cells were treated with various concentrations of paclitaxel alone or in the presence of 6 μg/ml aPPT.

As can be seem from Figures 9B and 9C aPPT enhanced the chemotherapy efficacy of paclitaxel in cancer cells that are drug resistant due to by P-gp independent mechanisms. This enhanced effect was most prominent in drug resistant tumor cells (MCF7r).

Table 1 illustrates enhancement effect of aPPT on the IC₅₀ of paclitaxel on drug sensitive (MCF7) and drag resistant (MCF7r) cell lines. The enhancement effect of aPPT was much more dramatic in drug resistant cell lines than in the drug sensitive cells, especially aPPT.

Table 1

EXAMPLE 10: Aglycon Protopanaxatriol Directly Inhibits P-glycoprotein (P- gp)

The effect of aPPT on P-glycoprotein (P-gp) was examined in vitro. Human breast cancer cells MCF7r that over express P-gp were cultured and incubated with calcein- AM, a P-gp substrate, that shows fluorescence when accumulated inside the cell but has no fluorescence outside of cells. 45μM of aPPT was added into the culture medium and fluorescent microscopic microphotograph was taken at 15 min. post treatment.

As shown in the Figure 10 A, in the control, most cells did not contain any calcein- AM due to high activity of P-gp that prevented the accumulation of the compound inside of cells. In aPPT treated cells (Figure 10B), every cell became fluorescent in 15 min, indicating aPPT can directly block P-gp to allow drug accumulation inside of tumor cells.

The results from Examples 9 and 10 indicate that aPPT is capable of enhancing the effect of chemotherapeutics on MDR cancer cell lines irrespective of the mechanism by which drug resistance evolved.

The disclosure of all patents, publications, including published patent applications, and database entries referenced in this specification are specifically incorporated by reference in their entirety to the same extent as if each such individual patent, publication, and database entry were specifically and individually indicated to be incorporated by reference.

The embodiments of the invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A composition comprising a therapeutically effective amount of substantially purified aglycon protopanaxatriol (aPPT) and a physiologically acceptable carrier for the treatment of a multi-drug resistant (MDR) cancer in a mammal.

2. A composition consisting essentially of a therapeutically effective amount of substantially purified aglycon protopanaxatriol (aPPT) and a physiologically acceptable carrier for the treatment of a multi-drug resistant (MDR) cancer in a mammal.

3. The composition according to claim 1 or 2, wherein said MDR cancer is selected from the group of: solid tumors, adenocarcinomas, melanoma, cancer of the mesothelium and brain cancer.

4. The composition according to claim 1 or 2, wherein said MDR cancer is selected from the group of: ovarian cancer, renal cancer, colorectal cancer, uterine cancer, liver cancer, adenocarcinoma of the lung, lung cancer, breast cancer, pancreatic cancer, melanoma, mesothelioma and glioma.

5. The composition according to claim 1 or 2, wherein said MDR cancer is breast cancer, pancreatic cancer, melanoma, mesothelioma or glioma.

6. The composition according to any one of claims 1 to 5, wherein said treatment is combination chemotherapy with one or more chemotherapeutic drugs.

7. The composition according to claim 5, wherein said one or more chemotherapeutic drug is selected from the group of: taxanes, alkylating agents and antimetabolites.

8. The composition according to claim 5, wherein said one or more chemotherapeutic drug is paclitaxel, docetaxel, taxol, BCNU, cyclophosphamide, melphalan, mitomycin C, chlomambucil, gemcitabine, methotrexate, 5-FU or cytarabine.

. The composition according to claim 5, wherein said one or more chemotherapeutic drug is paclitaxel, taxol, BCNU or gemcitabine.

10. The composition according to any one of claims 1 to 9, wherein said mammal is a human.

11. Use of substantially purified aglycon protopanaxatriol (aPPT) in the treatment of a multi-drug resistant (MDR) cancer in a mammal in need thereof.

12. Use of substantially purified aglycon protopanaxatriol (aPPT) in the manufacture of a medicament for the treatment of a multi-drug resistant (MDR) cancer in a mammal in need thereof.

13. Use of substantially purified aglycon protopanaxatriol (aPPT) in the manufacture of a non-pharmaceutical composition for the treatment of a multi- drug resistant (MDR) cancer in a mammal in need thereof.

14. The use according to any one of claims 11 to 13, wherein said MDR cancer is a primary or recurrent cancer selected from the group of: solid tumors, adenocarcinomas, melanoma, cancer of the mesothelium and brain cancer.

15. The use according to any one of claims 11 to 13, wherein said MDR cancer is selected from the group of: ovarian cancer, renal cancer, colorectal cancer, uterine cancer, liver cancer, adenocarcinoma of the lung, lung cancer, breast cancer, pancreatic cancer, melanoma, mesothelioma and glioma.

16. The use according to any one of claims 11 to 13, wherein said MDR cancer is breast cancer, pancreatic cancer, melanoma, mesothelioma or glioma.

17. The use according to any one of claims 11 to 16, wherein said treatment is combination chemotherapy with one or more chemotherapeutic drugs.

18. The use according to claim 17, wherein said one or more chemotherapeutic drug is selected from the group of: taxanes, alkylating agents and antimetabolites.

19. The use according to claim 17, wherein said one or more chemotherapeutic drug is paclitaxel, docetaxel, taxol, BCNU, cyclophosphamide, melphalan, mitomycin C, chlomambucil, gemcitabine, methotrexate, 5-FU or cytarabine.

20. The use according to claim 17, wherein said one or more chemotherapeutic drug is paclitaxel, taxol, BCNU or gemcitabine.

21. The use according to any one of claims 11 to 20, wherein said mammal is a human.

22. A method of treating a multi-drug resistant cancer in a mammal comprising administering to said mammal an effective amount of substantially purified aglycon protopanaxatriol (aPPT).

23. The method according to claim 22, wherein said MDR cancer is a primary or recurrent cancer selected from the group of: solid tumors, adenocarcinomas, melanoma, cancer of the mesothelium and brain cancer.

24. The method according to claim 22, wherein said MDR cancer is selected from the group of: ovarian cancer, renal cancer, colorectal cancer, uterine cancer, liver cancer, adenocarcinoma of the lung, lung cancer, breast cancer, pancreatic cancer, melanoma, mesothelioma and glioma.

25. The method according to claim 22, wherein said MDR cancer is breast cancer, pancreatic cancer, melanoma, mesothelioma or glioma.

26. The method according to claim 22, wherein said aPPT is administered at a dosage of about O.Olg to about 50g per 70kg bodyweight per day.

27. The method according to any one of claims 22 to 26, wherein said treatment is combination chemotherapy with one or more chemotherapeutic drugs.

28. The method according to claim 27, wherein said one or more chemotherapeutic drug is selected from the group of: taxanes, alkylating agents and antimetabolites.

29. The method according to claim 27, wherein said one or more chemotherapeutic drug is paclitaxel, docetaxel, taxol, BCNU, cyclophosphamide, melphalan, mitomycin C, chlomambucil, gemcitabine, methotrexate, 5-FU or cytarabine.

30. The method according to claim 27, wherein said one or more chemotherapeutic drug is paclitaxel, taxol, BCNU or gemcitabine.

31. The method according to any one of claims 22 to 30, wherein said mammal is a human.

32. A kit for the treatment of a multi-drug resistant cancer in a mammal, said kit comprising:

(c) a therapeutically effective amount of substantially purified aglycon protopanaxatriol (aPPT); and optionally

(d) instructions for use.