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Title:  Phytochemotherapy for cancer

United States Patent:  6,849,604

Issued:  February 1, 2005

Inventors:  Izevbigie; Ernest B. (Flowood, MS)

Assignee:  Jackson State University (Jackson, MS)

Appl. No.:  756241

Filed:  January 13, 2004

Abstract

The present invention provides for a novel anti-neoplastic pharmaceutical composition. Specifically, the present invention provides for phytochemotherapeutic compositions produced from aqueous extracts (and fractions thereof), derived from Vernonia amygdalina leaves. These pharmaceutical compositions inhibit the growth of neoplastic cells, including human breast cancer cells. Furthermore, the instant invention provides for methods of producing the compositions and methods of using the compositions to inhibit the proliferation of neoplastic cells.

Description of the Invention

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present application relates generally to the treatment of neoplastic disease and the compositions employed therein. The instant invention concerns a novel phytochemotherapeutic composition, methods for producing the composition, and use of the composition in a method for treating cancer and other neoplastic diseases. Specifically, the instant invention provides for an aqueous extract, and fractions thereof obtained by chromatographic separation, isolated from Vernonia amygdalina (V. amydalina) each of which inhibit the growth of cancer cells.

2. Technical Problem Addressed

Breast cancer is the most common form of malignant disease among women in Western countries and, in the United States, is the most common cause of death among women between 40 and 55 years of age (Forrest, 1990). There is a need for improved compositions and protocols for the treatment of human breast cancer and other neoplastic diseases. The instant invention provides phytochemotherapeutic compositions and methods for inhibiting the growth of cancer cells, and specifically for the growth inhibition of human breast cancer cells.

3. Description of Related Art

Vernonia amygdalina, a plant known for its very bitter taste, has been shown to sometimes be ingested by chimpanzees suffering from parasite-related diseases (Koshimizu, 1994).

Previous investigation into the possible antineoplastic properties exhibited by Vernonia amygdalina have focused on chloroform extractable components. For example (Kupchan et al., 1969) suggests that Vernonia amygdalina contains at least three distinct cytotoxic components, namely vernolide, vernodalin, and vernomygdin (see page 3909, Chart I). However, there are no reports of any water-extractable Vernonia amygdalina components which exhibit antineoplastic properties.

SUMMARY OF THE INVENTION

The present invention provides for an anti-neoplastic pharmaceutical composition typically comprising filtrates prepared from aqueous extracts of Vernonia amygdalina leaves, collected from plants in Benin City, Nigeria. Also provided are pharmaceutical compositions prepared from fractions of the Vernonia amygdalina aqueous extracts, prepared by chromatographic separation of the extracts. The instant invention also provides for methods of preparing the Vernonia amygdalina aqueous extracts and chromatographic fractions. Additionally the instant invention provides methods for the treatment of neoplastic disease using these Vernonia amygdalina extracts and chromatographic fractions. These methods are useful for treating neoplastic disease, especially breast cancer, in animals suffering therefrom.

One embodiment of the instant invention provides for a method of preparing a pharmaceutical composition. In one aspect of this embodiment of the invention the pharmaceutical composition is produced by a process comprising the following steps:

a) Providing Vernonia amygdalina leaves.

b) Rinsing the leaves with water.

c) Soaking the leaves in water.

d) Crushing the leaves, preferably in by a gentle means, to produce a mixture.

e) Producing a filtrate by filtering the mixture to remove particulate matter.

f) Optionally, concentrating the filtrate by removing water.

Other embodiments of the instant invention provide for chromatographic separation of the aqueous extract filtrate. This separation may be by any suitable means commonly used, by those of skill in the art, to partition the various components of a complex solution and/or mixture.

Other embodiments of the instant invention provide for anti-neoplastic pharmaceutical compositions prepared from a extract, filtrate, or fraction thereof, isolated by any of the methods described above.

Other embodiments of the invention provide methods of inhibiting the growth and/or proliferation of neoplastic cells, both malignant and non-malignant. According to one aspect of this embodiment of the invention, cancer cell proliferation is inhibited by contacting the neoplastic cells with an effective amount of a pharmaceutical composition produced from an aqueous extraction of Vernonia amygdalina leaves. In another aspect of this embodiment of the invention, neoplastic cell proliferation is inhibited by contacting the cells with an effective amount of a pharmaceutical composition produced from a fraction of an aqueous extraction of Vernonia amygdalina leaves, wherein the fraction was isolated by chromatographic separation of the aqueous extract.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides for pharmaceutical compositions prepared from aqueous extracts of Vernonia amygdalina, or fractions thereof. The instant invention also provides for methods of inhibiting neoplastic cell proliferation, particularly breast-cancer cells, using these pharmaceutical compositions. As used herein, the term "neoplastic cells" refers to both cancerous (malignant) and non-malignant cells.

One embodiment of the instant invention provides for an anti-neoplastic pharmaceutical composition. In one aspect of this embodiment of the invention the pharmaceutical composition is produced by a process comprising the following steps:

a) Providing Vernonia amygdalina leaves.

b) Rinsing the leaves with water.

c) Soaking the leaves in water.

d) Crushing the leaves, preferably in a gentle manner, to produce a mixture.

e) Producing a filtrate by filtering the mixture to remove particulate matter.

f) Optionally, concentrating the filtrate by removing water.

In this embodiment of the invention it is contemplated that the leaves may be rinsed and soaked in any water of suitable purity. For example, washing may be carried out in distilled or deionized water. Preferably, the water is sterilized (e.g. by either heat or filter sterilization) prior to use.

According to one aspect of the present embodiment the Vernonia amygdalina leaves may be crushed by any suitable means. Exemplary methods for crushing the leaves to produce the mixture, which are contemplated for use according to this aspect of the invention, include by are not limited to smashing the leaves using a mortar and pestle and pressing the leaves through a sieve.

In order to separate the particulate matter from the mixture the mixture may be filtered using any means which is compatible with the instant invention. Such means may include but is not limited to filtering through a small mesh sieve, filtering through gauze or a paper filter or filtration through any other commercially available filter.

Following filtration the filtrate may, optionally, be concentrated by any suitable means, including, but not limited to evaporative concentration and lyophilization.

In another aspect of the current embodiment, the process for preparing the pharmaceutical composition may further comprise separating the filtrate (concentrated or not) into multiple fractions using one or more chromatographic methods. Any chromatographic method suitable for the present invention may be used. Exemplary potential chromatographic method(s) include, but are not limited to, partition chromatography (such as hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC), reverse phase chromatography (RPC), including preparative reverse phase chromatography, and affinity chromatography) and size exclusion chromatography.

In another aspect of this embodiment, the filtrate may be further separated by two or more sequential chromatographic modes. Similarly, in yet another aspect of this embodiment of the invention, the filtrate may be separated into fractions, sub-fractions, and sub-sub-fractions by sequential separation using preparative reverse phase high-performance liquid chromatography (referred to hereinafter as PRPC), IEC, and RPC or any other suitable separation technique. These modes of separation may be performed in any order consistent with the instant invention.

In one particularly preferred aspect of this embodiment of the invention the filtrate is first separated by PRPC to produce PRPC-fractions. The PRPC fractions are then analyzed to determine which fraction(s) has/have the greatest potency against neoplastic cells (as used herein, "potency against neoplastic cells" means having the ability to prevent or reduce the ability of the neoplastic cells to grow, divide (including the ability to replicate its genomic DNA), and/or metastasize). Following the analysis the fraction(s) exhibiting the greatest potency against the neoplastic cells are sub-fractionated by IEC to produce IEC sub-fractions. Next the IEC sub-fractions are analyzed to determine which sub-fraction(s) has/have the greatest potency against neoplastic cells. Once identified, the sub-fraction(s) having the greatest potency against neoplastic cells are further separated by RPC to produce RPC sub-fractions. Finally, as with the IEC sub-fractions, the RPC sub-fractions are analyzed to determine which sub-fractions have the greatest potency against neoplastic cells and these fractions are used to produce an anti-neoplastic pharmaceutical composition. In an even more particular aspect of the invention the composition comprises one or more peptides having the sequence of SEQ ID NO:1 and/or SEQ ID NO:2

According to the instant invention, fractionation of the Vernonia amydalina aqueous extract filtrate is typically carried out in order to concentrate and/or further purify the efficacious components of the identified fractions. It is believed that the efficacious components of the current fractions may comprise any water extractable molecule, including but not limited to proteins and/or peptides.

The potency against neoplastic cells, exhibited by the isolated fractions, may be determined by measuring the inhibition of cell growth and/or division of cells treated with the fractions. This analysis may be done using any suitable neoplastic cell line, such cell lines are available from cell culture repositories such as American Type Culture Collection (Manassas, Va.). In one particular aspect of the present embodiment the potency against cancer cells is determined by measuring the pharmaceutical composition's ability to inhibit the growth and/or division of MCF-7 cells (MCF-7 is a human cell line derived from an adenocarcinoma of the breast).

Inhibition of cell growth and/or division may be determined by any suitable means. Exemplary methods for determining inhibition of cell growth and/or division include evaluation of cell membrane using a trypan-blue exclusion assay; determination of DNA replication by measuring the uptake of tritium-labeled thymidine, measurement of cell viability using the methyl thiazolyl tetrazolium (MTT) cell proliferation assay.

Another embodiment of the invention provides for a pharmaceutical composition comprising peptide(s) having the sequence of SEQ ID NO:1 and/or SEQ ID NO:2.

Another embodiment of the instant invention provides for a method of preparing an anti-neoplastic pharmaceutical composition. According to various aspects of this embodiment of the invention, anti-neoplastic pharmaceutical compositions may be prepared by any of the methods, described above. Specifically, the methods may comprise producing a filtrate from an aqueous extract from Vernonia amygdalina leaves. According to other aspects of this embodiment of the invention, the method may further comprise subsequent separation and/or concentration of the efficacious components of the filtrate (concentrated or not), accomplished by any suitable chromatographic mode. Chromatographic modes, contemplated as being suitable for this aspect of the invention, include, but are not limited to: partition chromatography (such as hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC), reverse phase chromatography (RPC), including PRPC, and affinity chromatography) and size exclusion chromatography.

Another aspect of this embodiment of the invention provides for a pharmaceutical composition comprising a purified peptide of SEQ ID NO:1 or SEQ ID NO:2, wherein the peptides are either purified from V. amygdalina, or another natural source, or are chemically synthesized.

Another embodiment of the invention provides for a method of inhibiting the proliferation of neoplastic cells and/or the treatment of an animal suffering from neoplastic disease. As used herein the term "neoplastic disease" refers cancer as well as other diseases caused by malignant or benign tumors.

According to this embodiment of the invention, neoplastic cell proliferation is inhibited and/or neoplastic disease is treated by contacting the cells with a pharmaceutical compositions of the present invention, in an amount effective to inhibit and/or reduce cell proliferation. The neoplastic cell inhibition may occur either in vitro or in vivo. Furthermore, the treatment of any susceptible neoplastic cell type is envisioned. One aspect of this embodiment of the invention contemplates treating an animal afflicted with a neoplastic disease, with the pharmaceutical compositions of the instant invention. Particularly, mammals and mammalian cells may be treated using the instantly described pharmaceutical compositions. More particularly, the instant compositions may be used to treat humans. Even more particularly, according to one specific aspect of this embodiment of the invention, human breast cancer cell proliferation may be inhibited by contacting the breast cancer cells with an effective amount of a pharmaceutical composition produced according to the teachings of the instant application. In all embodiments of the instant invention the pharmaceutical compositions is prepared by a process comprising generating a filtrate from an aqueous extraction of Vernonia amygdalina leaves.

As used herein an "effective amount" is defined as a dose of the pharmaceutical composition sufficient to measurably reduce cell proliferation. Preferably, an amount sufficient to reduce cell proliferation by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

As used herein, the phrase "contacting the cells" includes any suitable means for delivering the pharmaceutical composition to the target cell(s). Contemplated delivery methods include, but are not limited to: direct delivery to the cell growth medium, delivery to an animal via intravenous, or subcutaneous injection, or any other suitable parenteral or non-parenteral delivery method well known to those skilled in the art.

Various additional aspects of this embodiment of the invention are also contemplated. These aspects comprise inhibiting the proliferation of neoplastic cells by contacting the neoplastic cells with a pharmaceutical composition produced by a process which comprises further fractionation of the filtrate by any of the modes described, supra. That is, wherein the filtrate is subjected to further fractionation by one or more chromatographic modes, such as PRPC, HIC, IEC, and/or RPC. In one particular aspect of this embodiment of the invention human breast cancer cells are inhibited by contacting the cells with pharmaceutical composition prepared from constituents isolated by sequential fractionation of the Vernonia amygdalina aqueous extract filtrate by PRPC, IEC, and RPC. In one particular aspect of this embodiment of the invention neoplastic cell proliferation is inhibited and/or neoplastic disease is treated by contacting the cells with a pharmaceutical composition comprising a purified protein having the sequence of SEQ ID NO:1 and/or SEQ ID NO:2. The peptides of SEQ ID NO:1 and/or SEQ ID NO:2 may be either purified from Vernonia amygdalina or another natural source or may be chemically synthesized.

Claim 1 of 17 Claims

What is claimed is:

1. A pharmaceutical composition comprising one or more purified polypeptide(s), the polypeptide(s) comprising the sequence(s) of SEQ ID NO:1 and/or SEQ ID NO:2.


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