Patent Application
20060057237
The present invention relates to the medical field. The invention relates in a first aspect to extracts of the plant Calotropis procera, having a pharmacological activity, in particular an anti-tumor activity and/or anti-poisonous activity and active compounds isolated thereof. In a second aspect, the present invention relates to methods for obtaining said extracts. The invention further relates in a third aspect to a pharmaceutical composition for the treatment of cancer comprising an effective amount of said extracts or an active compound thereof. In a fourth aspect, the present invention concerns the use of said extracts or an active compound thereof as a medicament and the use of said extracts or an active compound thereof for the preparation of a medicament for the treatment of cancer.
Cancer develops in a given tissue when some genomic mutation perturbs cell cycle kinetics by increasing cell proliferation or decreasing cell death, or both. This perturbation leads to unrestrained growth of a genomically transformed cell population. Some cells from this transformed cell population may switch to the angiogenic phenotype, enabling them to recruit endothelial cells from the healthy tissue and leading to the sustained growth of the developing neoplastic tumor tissue. Subsequently, some cells migrate from the neoplastic tumor tissue and colonize new tissues, using blood or lymphatic vessels as major routes of migration. This process is also known as the metastatic process.
In practice, most of the agents used today in hospitals to treat cancer patients are drugs, which more or less directly target the cell kinetics, i.e. cell proliferation, of the cancer to be combated. The working mechanism of such anti-cancer drugs essentially relates to the disruption of the development of malignant cells by acting on cell kinetics. These drugs include alkylating agents, intercalating agents, antimetabolites, etc., most of which target DNA or enzymes regulating the DNA duplication and elongation process. These drugs attack DNA.
A major drawback of these drugs involves that the drugs do not work in a selective manner, i.e. they do not select between normal and neoplastic cells. They are used in accordance with the fact that the DNA of rapidly proliferating cells, i.e. cancer cells, is more sensitive to this type of agents than the DNA of less rapidly proliferating cells, i.e. normal cells. However, rapidly growing tumors are not always tumors exhibiting high levels of cell proliferation. Rapidly growing tumors may also include tumors which exhibit low levels of cell death compared to the normal cell population from which these tumor cells issue. For these types of rapidly growing tumors, the mentioned drugs are not effective.
In addition, the great majority of the drugs used in the standard treatment of cancer using the cell kinetics approach have the drawback of being toxic or even highly toxic, i.e. involving many detrimental side-effects on healthy cells, tissues and organs, and this limits their clinical use to a relatively low number of administrations per patient. In addition, several of these compounds must be combined into a poly-chemotherapeutic regimen in order to have any observable effect against cancer. By way of evidence such anti-cancer drug combinations increase detrimentally the toxicity of the treatment and also limit the number of administrations that can be applied.
Some anti-cancer drugs from natural origins, such as e.g. anti-tubulin compounds, using a therapeutic approach different from the cell kinetics approach, have been proposed. Said drugs aim to prevent the migration of cancer cells which escape from the tumor bulk and first invade neighboring tissue therefore establishing metastases. However, the compounds of this type known so far also show major toxic side-effects, which limits their use over long periods of treatment.
Therefore, there remains an urgent need in the art for finding improved anti-cancer drugs, which overcome at least some of the above-mentioned drawbacks. Consequently, it is a general object of the invention to provide improved anti-cancer drugs. In particular the invention aims to provide an improved anti-cancer drug, showing minimal side effects.
One embodiment of the present invention is an extract of the plant Calotropis procera, characterized in that said extract has a pharmacological activity, in particular an anti-poisonous activity.
Another embodiment of the present invention is an extract as described above obtained using an extraction procedure, comprising the steps of:
Another embodiment of the present invention is an extract as described above obtained using a extraction procedure comprising the steps:
Another embodiment of the present invention is an extract as described above obtained using a extraction procedure comprising the steps:
Another embodiment of the present invention is an extract as described above obtained using a extraction procedure comprising the steps:
Another embodiment of the present invention is a composition comprising:
Another embodiment of the present invention is a product containing
Another embodiment of the present invention is a composition as described above or a product as describe above where one of the said extracts comprises at least two active compounds selected from the group comprising asclepin, calactin, vorusharin, calotropin, calotropagenin, uzarigenin, calotoxin, usharin and usharidin.
Another embodiment of the present invention is a composition as described above, or a product as describe above wherein one of the said extracts comprises at least one of the compounds which are represented in Table 1.
Another embodiment of the present invention is a composition as described above or a product as describe above wherein the weight ratio of extract: therapeutic compound is in the range 0.001:1 to 1000:1.
Another embodiment of the present invention is a composition as described above, or a product as describe above, for use as a medicament.
Another embodiment of the present invention is a composition as described above, or a product as describe above, for use as a medicament for the treatment of cancer.
Another embodiment of the present invention is a composition or product as described above, wherein said cancer is selected from the group comprising breast cancer, lymphoma, sarcoma, pancreatic cancer, melanoma, colorectal cancer, glioma, non small cell lung cancer, small cell lung cancer, skin cancer, bone cancer, ovarian cancer, CNS cancer, renal cancer, bladder cancer, head and neck cancer, prostate cancer, liver cancer, hematological cancers.
Another embodiment of the present invention is a composition as described above or a product as describe above further comprising one or more additional therapeutic compounds.
Another embodiment of the present invention is a composition as described above, or a product as describe above, wherein said therapeutic compound(s) is an anti-cancer agent.
Another embodiment of the present invention is a composition as described above, or a product as describe above, wherein said therapeutic compound is selected from the group comprising adriamycin, alkeran, ara-c, bleomycin, biCNU, busulfan, CCNU, carboplatinum, cisplatinum, cyclophosphamide, cytoxan, daunorubicin, DTIC, 5-FU, fludarabine, gemcitabine (gemzar), herceptin, hexamethylmelamine, hydrea, idarubicin, ifosfamide, irinotecan (camptosar, CPT-11), leustatin, methotrexate, mithramycin, mitomycin, mitoxantrone, muphoran, navelbine, nitrogen mustard, oxaliplatine, rituxan, STI-571, streptozocine, taxol, taxotere, topotecan (hycamtin), velban, vincristine, VP-16, xeloda (capecitabine), or zevelin.
Another embodiment of the present invention is a composition as described above, or a product as describe above, wherein said therapeutic compound is selected from the group comprising adriamycine, vincristine, camptothecin and oxaliplatin.
Another embodiment of the present invention is a composition as described above, or a product as describe above, wherein said therapeutic compound(s) is a cytotoxic antibody or a fragment thereof.
Another embodiment of the present invention is a composition as described above, or a product as describe above, wherein said therapeutic compound(s) is a cytotoxic hormone or a fragment thereof.
Another embodiment of the present invention is a composition as described above, or a product as describe above, wherein said therapeutic compound(s) is a cytotoxic peptide or a fragment thereof.
Another embodiment of the present invention is a composition as described above further comprising a pharmaceutically acceptable carrier or a product as describe above wherein the composition and/or therapeutic compound(s) further comprises a pharmaceutically acceptable carrier.
Another embodiment of the present invention is a use of an extract of Calotropis procera as described above for the preparation of a medicament for alleviating the side effects of one or more therapeutic compounds.
Another embodiment of the present invention is a use of an extract of Calotropis procera as described above for the preparation of a medicament for increasing the dose administered to an individual of one or more therapeutic compounds.
Another embodiment of the present invention is a use of an extract as described above wherein said therapeutic compound(s) has anti-cancer activity.
Another embodiment of the present invention is a use of an extract as described above wherein said therapeutic compound(s) is selected from the group comprising adriamycin, alkeran, ara-c, bleomycin, biCNU, busulfan, CCNU, carboplatinum, cisplatinum, cyclophosphamide, cytoxan, daunorubicin, DTIC, 5-FU, fludarabine, gemcitabine (gemzar), herceptin, hexamethylmelamine, hydrea, idarubicin, ifosfamide, irinotecan (camptosar, CPT-11), leustatin, methotrexate, mithramycin, mitomycin, mitoxantrone, muphoran, navelbine, nitrogen mustard, oxaliplatine, rituxan, STI-571, streptozocine, taxol, taxotere, topotecan (hycamtin), velban, vincristine, VP-16, xeloda (capecitabine), or zevelin.
Another embodiment of the present invention is a use of an extract as described above wherein said therapeutic compound(s) is selected from the group comprising adriamycine, vincristine, camptothecin and oxaliplatin.
Another embodiment of the present invention is a use of an extract as described above wherein said therapeutic compound(s) is a cytotoxic antibody or a fragment thereof.
Another embodiment of the present invention is a use of an extract as described above wherein said therapeutic compound(s) is a cytotoxic hormone or a fragment thereof.
Another embodiment of the present invention is a use of an extract as described above wherein said therapeutic compound(s) is a cytotoxic peptide or a fragment thereof.
Another embodiment of the present invention is a use of an extract as described above wherein said therapeutic compound(s) is therapeutic radiation.
Another embodiment of the present invention is a use of an extract as described above wherein said extract comprises at least two active compounds selected from the group comprising asclepin, calactin, vorusharin, calotropin, calotropagenin, uzarigenin, calotoxin, usharin and usharidin.
Another embodiment of the present invention is a use of an extract as described above wherein said extract further contains at least one of the compounds which are represented in Table 1.
Another embodiment of the present invention is a use of an extract as described above wherein said extract is administered prior to said therapeutic compound(s).
Another embodiment of the present invention is a use of an extract as described above wherein said extract is administered after said therapeutic compound(s).
Another embodiment of the present invention is a use of an extract as described above wherein said extract is administered at the same time as said therapeutic compound(s).
Another embodiment of the present invention is a use of an extract as described above wherein the weight ratio of extract:therapeutic compound is in the range 0.001:1 to 1000:1.
Another embodiment of the present invention is an extraction process for obtaining an extract having biologically active components comprising the steps of:
Another embodiment of the present invention is an extraction process for obtaining a several extracts having biologically active components present substantially in the leaves, stems, barks and roots of Calotropis procera, which comprises the following steps:
Another embodiment of the present invention is an extraction process as described above further comprising the steps of:
Another embodiment of the present invention is an extraction process as described above, further comprising the steps of
Another embodiment of the present invention is a process as described above, wherein step b) is performed at a working temperature of between 20 and 80° C.
Another embodiment of the present invention is a process as described above, wherein said step b) is repeated between one and five times.
Another embodiment of the present invention is a process as described above, wherein the duration of step b) is between 4 hours and 48 hours.
Another embodiment of the present invention is a process as described above, wherein the solid phase of step d) is silica gel.
Another embodiment of the present invention is a process as described above, wherein the eluent of step d) is a binary eluent, the ratio between the two components of the eluent being between 100:0 to 0:100.
Another embodiment of the present invention is a process as described above, wherein the components of said binary eluent comprise an alcoholic solvent and a non polar solvent.
Another embodiment of the present invention is a process as described above, wherein step e) is performed at a working temperature of between 20 and 50° C.
Another embodiment of the present invention is a process as described above, wherein the solid phase of step f) is silica gel.
Another embodiment of the present invention is a process as described above, wherein the eluent of step f) is a binary eluent, the ratio between the two components of eluent being between 100:0 to 0:100.
Another embodiment of the present invention is a process as described above, wherein the components of the binary eluent are a non-polar and a more polar solvent.
Another embodiment of the present invention is a process as described above, wherein the ratio between the two components of eluent, non-polar:polar, is between 50:50 and 100:0.
Another embodiment of the present invention is a process as described above, wherein the solvent of step h) is methanol.
Another embodiment of the present invention is a process as described above, wherein step e) is performed at a working temperature of between 20 and 50° C.
Another embodiment of the present invention is an active extract isolated from the process as described above.
Another embodiment of the present invention is a use of an active extract as described above as a medicament.
Another embodiment of the present invention is a use of an active extract as described above for the preparation of a medicament in the treatment of cancer.
Another embodiment of the present invention is a method for treating cancer comprising administering to an individual in need of such treatment a pharmaceutical composition as described above, or a product as described above.
Another embodiment of the present invention is a method as described above, wherein said cancer is selected from the group comprising breast cancer, lymphoma, sarcoma, pancreatic cancer, melanoma, colorectal cancer, glioma, non small cell lung cancer, small cell lung cancer, skin cancer, bone cancer, ovarian cancer, CNS cancer, renal cancer, bladder cancer, head and neck cancer, prostate cancer, liver cancer, hematological cancers.
Another embodiment of the present invention is a composition as described above, or product as described above, wherein the therapeutic compound is radiation.
Another embodiment of the present invention is a kit comprising a container in which an extract of Calotropis procera as described above is present, and a container in which a therapeutic compound is present.
The present invention relates to extracts of the plant Calotropis procera. In a first embodiment the invention relates to extracts of the plant Calotropis procera, which has a pharmacological activity, in particular an anti-tumor activity and/or an anti-poisonous activity. Surprisingly, at least one of the extracts according to the invention combines an anti-tumor effect with an anti-poisonous activity.
According to the present invention the term “anti-tumor activity”, refers to the in vitro as well as in vivo anti-tumor effects exerted by at least one of the extracts or isolated compounds thereof. The anti-tumor effects essentially include but are not limited to a dramatic decrease of cell growth and a pro-apoptotic effect. Importantly, the extracts according to the invention exhibits anti-tumor activity on a large number of cancer types, such as breast cancer, melanoma or lymphoma cancers amongst others.
Another feature of the extracts according to the invention encompasses their anti-poisonous activity. The term “anti-poisonous activity” refers to the ability of the extracts according to the invention or isolated compounds thereof to attenuate or reverse the effects of therapeutic compounds or treatments. A “therapeutic compound”, as used herein, refers to a compound that exerts a relevant detrimental, toxic effect(s) on normal, i.e. non-cancer related cells, tissues or organs. The term therapeutic compound may thus also include a compound, drug, physical treatment or medicament, used to treat a particular disease that exerts detrimental toxic side effects. Such side effects are known to the skilled person and include, but are not limited to tiredness, nausea, vomiting, sores, mouth sores, dry skin, sensitive skin, hair loss, reduced red and white blood count. Such therapeutic compounds include, but are not limited to chemotherapy agents, radiation, antibodies and hormones.
Therapeutic compounds useful according to the invention include those used in chemotherapy or are cytotoxic. They include, but are not limited to any of adriamycin, alkeran, ara-c, bleomycin, biCNU, busulfan, CCNU, carboplatinum, cisplatinum, cyclophosphamide, cytoxan, daunorubicin, DTIC, 5-FU, fludarabine, gemcitabine (gemzar), herceptin, hexamethylmelamine, hydrea, idarubicin, ifosfamide, irinotecan (camptosar, CPT-11), leustatin, methotrexate, mithramycin, mitomycin, mitoxantrone, muphoran, navelbine, nitrogen mustard, oxaliplatine, rituxan, STI-571, streptozocine, taxol, taxotere, topotecan (hycamtin), velban, vincristine, VP-16, xeloda (capecitabine), or zevelin.
It is another aspect of the invention that a therapeutic agent comprises one or more anticancer agents which are derived from an extract of the present invention. Possible anti-cancerous compounds according to the invention are any extracted from Calotropis procera such as asclepin, calactin, voruscharin, calotropin, calotropagenin, uzarigenin, calotoxin, uscharin and uscharidin.
In another embodiment of the invention, the anti-cancerous compounds are any extracted from Calotropis procera such as 2″oxo-voruscharin, and derivatives thereof as shown in Table 2.
It is another aspect of the invention that a therapeutic agent is an extract of the invention. Said extract may be identical to an extract that has an anti-poisonous activity. Alternatively, said extract may be identical to an extract that has an anti-poisonous activity, with the exception of the absence of one or more active compounds from either extract. Alternatively, said extract may be identical to an extract that has an anti-poisonous activity, with the exception of the presence of one or more additional active compounds in either extract. Alternatively, said extract may be identical to an extract that has an anti-poisonous activity, with the exception of difference in the concentration of one or more active compounds in either extract.
It is another aspect of the invention that a therapeutic compound is a physical treatment such as radiation. Said radiation may be any used in medicine. For example the radiation may be that used to shrink tumourous growths, to totally irradiate the body as part of a bone marrow transplants program, etc. The radiation may also be that used for diagnosis, for example, in X-rays or radioactive markers.
It is another aspect of the invention that the therapeutic compound is an antibody, or a fragment thereof. Said antibody demonstrates cytotoxic activity. Examples of therapeutic antibodies include, but are not limited to HerceptinR, Prostascint, Rituximab, and Trastuzumab.
It is another aspect of the invention that the therapeutic compound is a hormone, or a fragment thereof. Said hormone demonstrates cytotoxic activity. Examples of therapeutic hormones include, but are not limited to busereline, fluoxymesterone, flutamide, formestane, norethandrolone, norethisterone, prednisolone, prednisone, and tamoxifene
It is another aspect of the invention that the therapeutic compound is a peptide, or a fragment thereof. Said peptide demonstrates cytotoxic activity.
By fragment in reference to a antibody, hormone or peptide means a peptide comprising a portion of the antibody, hormone or peptide that is capable of recognizing the target of the whole antibody, hormone or peptide. The fragment is also capable of recognizing the target of the whole antibody, hormone or peptide. The portion may comprise amino acid substitutions, deletions or insertions that do not substantially change the recognition of the target by portion. The number of substitutions, deletions or insertions may be less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500.
It is another aspect of the invention, that the anti-poisionous activity of the extracts allows the administration of a higher dose of the therapeutic compound. By administering a higher dose of therapeutic compound, the cancer may be more quickly and effectively treated. An individual in receipt of a higher dose, in combination with an extract of the invention would benefit from reduced side-effects of the therapeutic compound.
It is another aspect of the invention that the anti-poisionous activity of the extracts allows the administration of a combination of therapeutic compounds. A combination therapy would target different aspects of the cancer simultaneously, so leading to a more effective and efficient treatment. By administering the combination in accordance with the invention, the patent would suffer less side effects and benefit from the effectiveness of the combination therapy.
In another embodiment, the present invention relates to the active compounds, isolated from the extracts. The term ‘active compounds’ or ‘active components’ of the extracts are used herein as synonyms, and refer to the compounds present in the extracts, which exhibit an activity that is similar to at least one of the above-defined activities of the extracts.
In yet another embodiment, the present invention relates to methods for obtaining the extracts of the plant Calotropis procera.
Due to the fact that at least one of the extracts according to the invention exert an anti-tumor activity the extracts according to the invention are particularly useful for the treatment of diseases such as cancer. Therefore, in another aspect, the present invention relates to pharmaceutical compositions comprising one of the above-described extracts or at least one active compound thereof.
Furthermore, since the extracts according to the invention exert an anti-poisonous activity as well, it is also particularly suitable to be combined with other therapeutic compounds, such as other medicaments, which show toxic side effects. Therefore, in another embodiment, the present invention relates to a pharmaceutical composition comprising one of the above-described extracts or at least one active compound thereof, and a second compound, which exerts a pharmacological activity having toxic side effects.
Furthermore, the present invention relates to the use of one of said extracts and/or at least one active compound thereof as a medicament. The present invention further relates to the use of one of the extracts or at least one active compound thereof in the preparation of a medicament for the treatment of diseases, in particular cancer.
In another embodiment, the present invention further relates to a method for treating cancer.
Other objects and advantages of the present invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings.
FIGS. 2 to 11 show the in vitro effects of an extract according to the invention on different types of human cancer cell lines, being breast cancer cell lines in
FIGS. 12 to 16 represent the in vitro effects of an extract according to the invention on the cell cycle kinetics of cells corresponding to five different human cell lines, HCT-15 (
FIGS. 17 to 20 represent the in vitro apoptotic-inducing and necrosis-inducing effects of an extract according to the invention on cells corresponding to four different human cell lines, HCT-15 (
FIGS. 28 to 35 illustrate the anti-poisonous effects of extracts according to the invention when combined with anti-tumors compounds adriamycine or vincristine or camptothecin or oxaliplatin.
The Calotropis procera plant, belonging to the family of the Asclepiadaceae, is a plant growing in Africa and Asia. This plant is used in traditional folk medicine and has also been studied with respect to a considerable number of uses such as an anti-pyretic, anti-malarial, anti-diarroeal, analgesic, anti-inflammtory gastric, mucosal protector and as an insecticidal, anti-tussive, antibacterial, wound healing, muscle relaxant. The stems, flowers and leaves of Calotropis procera plant are known to contain certain compounds known as cardenolides. Recently, cardiotoxic activity has been attributed to these cardenolides, and they are exploited in human therapies for treating cardiac insufficiencies.
Unexpectedly, the present invention relates to at least one extract according to the invention of the Calotropis procera plant showing another type of activity, in particular an “anti-tumor activity”. Moreover, it was demonstrated that at least one extract according to the invention of shows in vitro as well as in vivo anti-tumor effects. The said extract according to the invention induces a dramatic reduction in cell growth and shows a pro-apoptotic activity. These properties are further illustrated in the examples 3 and 4. In addition, it has been in-vivo shown by the inventors that at least one of the said extracts according to the invention is highly active against several types of cancers. As is shown in the examples described below, the said extract according to the invention, exerts significant anti-tumor effects on several tumor models tested, which represent a broad panel of histological tumor types, including breast cancer, lymphomas, melanomas. These models are clinically relevant because they mimic specific clinical stages of human cancers.
Surprisingly, the said extract according to the invention is highly active at relatively low doses. The said extract can exert an anti-tumor activity at low doses in the range of 0.4 to 2.2 mg/kg and preferably in the range of 0.5 to 2 mg/kg. The high anti-tumor activity at low doses indicates that the said extract is highly active. Surprisingly, as will also become clear when reading the examples given below, the said extract according to the invention shows significantly higher anti-tumor effects when assayed at chronically low doses, i.e. 0.6 mg/kg to 1.25 mg/kg, than at high doses, i.e. at doses of 5 mg/kg to 10 mg/kg. The Maximum Tolerated Dose (MTD) index of the said extract according to the invention, referring to the maximum amount of the said extract, which can be administered acutely to healthy animals, is 20 mg/kg extract. This value indicates that the said extract according to the invention has a broad therapeutic window.
The terms “toxicity” or “toxic effects” relate to the detrimental effect(s) a compound may have on healthy cells, tissues or organs. Another important property of an extract according to the invention includes its low toxicity level. It was demonstrated that the said extract does not induce in vivo hematological changes, does not induce weight loss and shows minor side effects on different types of organs and tissues. In fact, the toxicity level of the said extract according to the invention is surprisingly low. The said extract according to the invention combines the essential features of a good anti-tumor activity, a low level of toxicity and a minimal induction of detrimental side effects.
Furthermore, the extracts of the plant Calotropis procera according to the invention also show an “anti-poisonous activity”. As mentioned above, the term “anti-poisonous activity”, as used herein, refers to the ability of the extracts according to the invention to attenuate or reverse the detrimental side effects of compounds. Surprisingly, the inventors have demonstrated that combination of an extract according to the invention with other therapeutic compounds, having detrimental side effects enables undesirable side effects of the therapeutic compound to be reduced. For instance, example 8 illustrates that the combination of an extract according to the invention with well known anti-cancer compounds, such as vincristine or adriamycine, camptothecin or oxaliplatin, enables the reduction of some side effects caused by the toxicity to normal cells induced by these anti-cancer compounds.
The anti-poisonous activity is evident when at least one of the extracts according to the invention is administered prior to the therapeutic compound i.e. they are administered sequentially. It is an aspect of the invention to administer an extract prior any time before the therapeutic compound is administered. It is a further aspect of the invention to administer an extract no more than 336, 312, 288, 264, 240, 216, 192, 168, 144, 120, 96, 72, 48, 24, 20, 16, 12, 8, 4, 2, 1, or 0.5 hours before the therapeutic compound is administered.
The anti-poisonous activity is also evident when at least one of the extracts according to the invention is administered at the same time as the therapeutic compound i.e. as a composition, by simultaneous or separate administration.
The anti-poisonous activity is also evident when at least one of the extracts according to the invention is administered after the therapeutic compound i.e. they are administered sequentially. It is an aspect of the invention to administer an extract any time after the therapeutic compound has been administered. It is a further aspect of the invention to administer the extract less than 0.5, 1, 2, 4, 8, 12, 16, 20, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, or 366 hours after the therapeutic compound has been administered.
In sequential administration, the said extracts may be administered once, or any number of times and in various doses before and/or after administration of the therapeutic compound. Sequential administration may be combined with simultaneous or sequential administration.
Composition of the Extract
The extracts according to the invention comprise one or several active compounds, i.e. compounds present in an extract according to the invention, which exhibit an activity similar to at least one of the above-defined activities of an extract according to the invention. The main activities of at least one of the extracts are an anti-tumor activity and an anti-poisonous activity. As it will be understood by the one skilled in the art, an active compound present in the said extracts may exerts only one of these properties, or even both of these properties.
In another embodiment, at least one of the extracts according to the invention comprise at least two of the active compounds selected from the group comprising asclepin, calactin, voruscharin, calotropin, calotropagenin, uzarigenin, calotoxin, uscharin and uscharidin, 2″oxo voruscharin.
As it will be understood, the extracts according to the invention may further contain one or more active compounds, which do not belong to the group of the cardenolides, and other compounds. In another embodiment, the extracts contain at least one of the compounds represented in Table 1.
In another embodiment, the present invention relates to an active compound isolated from the extracts according to the invention.
In another embodiment of the invention, an active compound of the extract is 2″oxo-voruscharin. An aspect of the invention is the anti-cancer and/or anti-poisonous activity of said compound (compound A) and the derivatives (compounds B to H) thereof as shown in Table 2.
According to another embodiment, an extract according to the invention can be obtained by an alcoholic extraction, in particular by a methanol extraction.
In another embodiment, the present invention relates to a method for obtaining an extract according to the invention comprising the steps of:
In a preferred embodiment, the starting material of said Calotropis procera plant is selected from subterranean parts, in particular from roots.
In another embodiment, the aliphatic alcohol used to extract the starting material according to the invention is methanol. In yet another embodiment, the residue obtained in the extraction process is taken up in a solvent, in particular in a pharmaceutically acceptable solvent.
In another embodiment, the present invention relates to a method for obtaining extracts according to the invention, comprising the steps of:
Extracts according to the present invention include those obtained from step c), step g), and step i).
It is an aspect of the invention that the extractions are performed in a manner compatible with preserving the integrity of the biologically active compounds contained in said extract. Such precautions are known to the skilled person.
Applicability of the Extracts
Due to the interesting properties of at least one of the extracts disclosed herein, in particular its anti-tumor activity, its ant-poisonous activity, its low level of toxicity and/or its high activity at low doses, the extract(s) according to the invention or active compounds thereof are particularly suitable for use as a medicament for the treatment of diseases, and in particular for treating cancer.
In another embodiment, the invention relates to the use of an extract according to the invention, or an active compound thereof, as a medicament.
In yet another embodiment, the invention also relates to the use of at least one extract according to the invention, or an active compound thereof, for the preparation of a medicament in the treatment of cancer. In particular the said extract according to the invention, or an active compound thereof is used for the preparation of a medicament in the treatment of a cancers. Examples of cancers that may be treated according to the invention include, but are not limited to breast cancer, lymphoma, sarcoma, pancreatic cancer, melanoma, colorectal cancer, glioma, non small cell lung cancer, small cell lung cancer, skin cancer, bone cancer, ovarian cancer, CNS cancer, renal cancer, bladder cancer, head and neck cancer, prostate cancer, liver cancer and hematological cancer.
As mentioned above, the extracts according to the invention also comprise an anti-poisonous activity. The inventors have demonstrated that a combination of an extract according to the invention or active compounds thereof with a second compound having relevant detrimental side effects enables a reduction of the toxic activity of this second compound, without reducing its therapeutic activity. Therefore, the Calotropis procera extracts according to the invention may also be combined in a medicament with other compounds, in particular with other anti-cancer compounds.
Pharmaceutical Compositions Comprising the Extract
In another embodiment, the present invention relates to a pharmaceutical composition for the treatment of cancer comprising a therapeutic effective amount of an extract of Calotropis procera according to the invention or an active compound thereof, and a pharmaceutical acceptable carrier.
In another embodiment, the present invention relates to a pharmaceutical composition for the treatment of cancer comprising
The term “therapeutically effective amount” as used herein means that amount of at least one extract or active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease being treated.
The pharmaceutical composition can be prepared in a manner known to one of skill in the art. For this purpose, an extract according to the invention and/or any active compound thereof, one or more solid or liquid pharmaceutical carriers and, if desired, in combination with other pharmaceutical active compounds, are brought into a suitable administration form or dosage form which can then be used as a pharmaceutical in human medicine or veterinary medicine.
Particular forms of the pharmaceutical composition may be, for example, solutions, suspensions, emulsions, creams, tablets, capsules, nasal sprays, lipsomes or micro-reservoirs, especially compositions in orally ingestible or sterile injectable form, for example, as sterile injectable aqueous or oleaginous suspensions or suppositories. The preferred form of composition contemplated is the dry solid form, which includes capsules, granules, tablets, pills, boluses and powders. The solid carrier may comprise one or more carriers, e.g. lactose, fillers, disintegrating agents, binders, e.g. cellulose, carboxymethylcellulose or starch or anti-stick agents, e.g. magnesium stearate, to prevent tablets from adhering to tabletting equipment Tablets, pills and boluses may be formed so as to disintegrate rapidly or to provide slow release of the active ingredient.
Furthermore, due to their anti-poisonous activity, the extracts according to the invention or active compounds thereof, are also particularly suitable to be combined with other therapeutic compounds which exert a pharmacological activity having toxic side effects, such as other medicaments, which show toxic side effects.
The “therapeutic compound, which exerts a pharmacological activity having toxic side effects” may include any compound that is used for the treatment of any disease but which induces unwanted toxic effects. Preferably, such compounds are compounds that are used in the treatment of cancer. For instance, an extract according to the invention or active compounds thereof can be combined with one or more than one other compound that has an anti-tumor effect. Since two or more compounds having an anti-tumor effect are combined, an improved anti-tumor activity can be obtained. In addition, such combinations also enable to reduce the toxic side effects, which are induced by one or several of the anti-tumor compounds.
A combination of an extract according to the invention or active compounds thereof with another therapeutic compound may involve a separate use of the said extract or active compound thereof and the other therapeutic compound. In particular, the combination may involve the use of an extract according to the invention or active compounds thereof prior (pre-treatment) to, at the same time, or after (post-treatment) the use of the other therapeutic compound.
Alternatively, a combination of an extract or active compounds thereof with another therapeutic compound may also involve a mixture of both elements. Therefore, in a preferred embodiment, the present invention relates to a pharmaceutical composition comprising a first component, said first component being the above-described extract or at least one active compound thereof, and a second component, said second component comprising a therapeutic compound, which exerts a pharmacological activity having toxic side effects.
Product
One aspect of the invention is a product containing an extract of Calotropis procera and a therapeutic compound for simultaneous, separate or sequential administration to a subject. It is an aspect of the invention that the product may be used according to the invention.
By simultaneous administration means the two components are administered to a subject at the same time. For example, as a mixture of the two components. Examples include, but are not limited to a solution administered intraveneously, a tablet, liquid, topical cream, etc., wherein each preparation comprises both components.
By separate administration means the two components are administered to a subject at the same time or substantially the same time, but the components are present in the product as separate, unmixed preparations. For example, the two components may be present in the product as individual tablets. The tablets may be administered to the subject by swallowing both tablets at the same time, or one tablet directly following the other.
By sequential administration means the two components are administered to a subject sequentially and the components are present in the product as separate, unmixed preparations. There is a time interval between doses. For example, one component might be administered up to 336, 312, 288, 264, 240, 216, 192, 168, 144, 120, 96, 72, 48, 24, 20, 16, 12, 8, 4, 2, 1, or 0.5 hours after the other component.
In sequential administration, the extract may be administered once, or any number of times and in various doses before and/or after administration of the therapeutic compound. Sequential administration may be combined with simultaneous or sequential administration.
Kit
Another aspect of the present invention is a kit comprising a container in which an extract of Calotropis procera is present, and a container in which a therapeutic compound is present. An extract according to the invention and therapeutic compound according to the invention are described above.
By mentioning a kit comprising a container in which an extract of Calotropis procera is present, and a container in which a therapeutic compound, it is to be understood that the kit may contain a single dose, or a plurality of doses, reflected in two single containers, two single containers each capable of holding a plurality of doses or a plurality of single containers each capable of holding a single dose. It is to be understood that the kit may comprise more than one therapeutic compound, each therapeutic compound in a separate container, or a mixture of therapeutic compounds in a single container.
The containers may be any known in the art of medicines. They may be vials, blister packs, syringes, resealable bottles, bottles with dispensing means, aspirators, dropping bottles, patches, applicators, suppositories.
The separate containers enable to extract and therapeutic compound to be administered simultaneously, separately, or sequentially.
Method of Treatment
Due to the favorable anti-tumor properties of at least one of the extracts according to the present invention, said extracts are particularly useful in the treatment of individuals suffering from cancer. In another embodiment, the present invention also relates to a method of treatment of cancer comprising administering to an individual in need of such treatment a pharmaceutical composition according to the invention. Due to its low level of toxicity and its minimal side effects, use of one said extract in a pharmaceutical composition for the treatment of cancer will involve minimal side effects. As a consequence, a said the extract according to the invention may be used during a longer period of time during the treatment of cancer.
In particular, in a preferred embodiment, the invention relates to a method for treating cancer, wherein the cancer is selected from the group comprising, but not limited to breast cancer, lymphoma, sarcoma, pancreatic cancer, melanoma, colorectal cancer, glioma, non small cell lung cancer, small cell lung cancer, skin cancer, bone cancer, ovarian cancer, CNS cancer, renal cancer, bladder cancer, head and neck cancer, prostate cancer, liver cancer and hematological cancer.
For these purposes, the pharmaceutical composition of the present invention may be administered orally, parenterally, i.e. including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques, by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
In accordance with the method of the present invention, said pharmaceutical composition can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The present invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term “administering” is to be interpreted accordingly.
Essentially, the primary modes of treatment of solid tumor cancers comprise surgery, radiation therapy and chemotherapy, separately and in combination. The extracts according to the invention are suitable for use in combination with these medicinal techniques. The extracts according to the invention may be useful in increasing the sensitivity of tumor cells to radiation in radiotherapy and also in potentiating or enhancing damage to tumors by chemotherapeutic agents. The extracts according to the invention may also be useful for sensitizing multidrug-resistant tumor cells. The extracts according to the invention are useful therapeutic compounds for administration in conjunction with other DNA-damaging therapeutic compounds including radiation used in radiotherapy to potentiate their effect.
It will be understood, that the pharmaceutical composition according to the invention can be administered to humans in specific dose levels and at specific frequency of dosage which may be varied for any particular patient and which will depend upon a variety of factors including the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition of the patient undergoing therapy.
The following examples are meant to illustrate the present invention. These examples are not to be considered as limiting the scope of the invention. A first example illustrates the extraction of extracts according to the invention form the plant Calotropis procera. Examples 2 to 4 relate to the in vitro anti-tumor characterization of an extract according to the invention. Examples 5 to 7 describe the in vivo anti-tumor characterization of an extract according to the invention. Example 8 illustrates the anti-poisonous effects of extracts according to the invention.
An extract (Extract A) according to the invention was isolated form the roots of Calotropis procera plant (Asclepiadiaceae family) by methanol. The said extract is further referred as Extract A (to adapt). About 10 grams of plant was put in an erlenmeyer with 150 ml of methanol. The suspension was shaken magnetically for 12 hours and then filtered on a glass frit. The remaining solid was extracted a second time by methanol for 2 hours. Both filtrates were combined and evaporated under vacuum by using rotavapor. The residue constituted the methanolic extract is referred hereafter as Extract A.
Analysis of Extract A revealed the presence of several types of compounds. A particular group of compounds identified in said Extract A comprises cardenolides such as asclepin, calactin, vorusharin, calotropin, calotropagenin, uzarigenin, usharin and usharidin. In addition, some other compounds, present in Extract A comprise, but are not limited to, 3-O-ACETYL-CALOTROPIN, ALPHA-AMYRIN, ALPHA-AMYRIN-BENZOATE, ALPHA-CALOTROPEOL, ALPHA-LACTUCEROL, ALPHA-LACTUCERYL-ACETATE, ALPHA-LACTUCERYL-ISOVALERATE, ARABINOSE, BENZOYLISOLINEOLONE, BENZOYLLINEOLONE, BETA-AMYRIN, BETA-AMYRIN-BENZOATE, BETA-CALOTROPEOL, BETA-SITOSTEROL, CAOUTCHOUC, COROGLAUCOGENIN; COROTOXIGENIN; D-GLUCOSAMINE, FRUGOSIDE, GIGANTEOL, GIGANTIN, GLUCOSE, HISTAMINE, ISOGIGANTEOL, ISOLACTUCEROL, ISOLINEOLONE, LAURANE LINEOLONE, LINOLEIGACID, LINOLENIC-ACID, MELISSYL-ALCOHOL, MUDARINE, OLEIC-ACID, PALMITIC-ACID, PROCEROSIDE, PSEUDOCALOTROPAGENIN, RHAMNOSE STIGMASTEROL, SYRIOGENIN TARAXASTEROL, TARAXASTEROL-BENZOATE and TRYPSIN.
Other extracts according to the invention, where isolated by a method, comprising the steps of:
The extract isolated at step c) is referred hereafter as Extract B. The extract isolated at step g) is referred hereafter as Extract C. The extract isolated at step i) is referred hereafter as extract D.
This example illustrates the anti-tumor activities of Extract A according to the invention on different types of cancer.
In order to characterize the in vitro activities of the Extract A, MTT tests were carried out. The MTT test, which is a well known test in the art, is an indirect technique that rapidly measures, i.e. within 5 days, the effect of a given product on the overall growth of a cell line. This test measures the number of metabolically active living cells that are able to transform the MTT product (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide), having a yellowish color, to the blue product formazan by mitochondrial reduction only performed by living cells. The amount of formazan obtained at the end of the experiment is measured with a spectrophotometer and is directly proportional to the number of living cells.
Forty-eight human cancer cell lines, described in Table 2, were tested in the presence of Extract A. These cell lines covered ten histological types, being pancreatic cancer, sarcoma, breast cancer, melanoma, colon cancer, glioma, lung cancer, bladder cancer, prostate cancer and head and neck cancer. The cells were allowed to grow in flat bottomed 96-well micro-wells with 100 μl of cell suspension per well and between 3,000 and 5,000 cells/well depending on cell type. Each cell line was seeded in its own cell culture medium as indicated in Table 2.
Where AA means “Amino Acids”
After a 24-hour incubation period at 37° C. the culture medium was replaced by 100 μl of fresh medium in which Extract A was dissolved at the different concentrations of 0.01 μg/ml, 0.05 μg/ml, 0.1 μg/ml, 0.5 μg/ml, 1 μg/ml, 5 μg/ml, 10 μg/ml, 50 μg/ml and 100 μg/ml. Each experimental condition was carried out in sextuplicate.
After 72 hours of incubation at 37° C. with the drug, i.e. experimental conditions or without the drug, i.e. control, the medium was replaced by 100 μl MTT at the concentration of 1 mg/ml dissolved in RPMI. The micro-wells were then incubated for 3 hours at 37° C. and centrifuged at 400 g for 10 minutes. The MTT was removed and the formed formazan crystals were dissolved in 100 μl DMSO. The micro-wells were shaken for 5 minutes and read on a spectrophotometer at 2 wavelengths at 570 nm corresponding to the maximum formazan absorbance wavelength, and at 630 nm, which is the background noise wavelength.
For each experimental condition, the mean OD associated with the standard error of the mean (SEM) for each condition, i.e. 6 wells, was calculated. The percentage of remaining living cells was calculated in comparison with control. Results of these experiments are represented in FIGS. 1 to 11.
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The overall growth of 1 prostate cancer cell line (the PC3 cell line) was evaluated by MTT assay when the cells had been treated with Extract A. Extract A induced an inhibition of overall growth of about 90% at between 100 μg/ml and 0.5 μg/ml. The IC50 value was around 0.1 μg/ml (
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Summarized, Extracts A according to the invention exerts a dramatic anti-tumor effect on forty-six of the forty-eight human cancer cell lines assayed in the experiments described above. These anti-tumor effects correspond to marked decreases in the overall growth of these human cancer models representing a very broad panel of histological types.
This example illustrates the cytostatic effect of Extract A according to the invention. According to the experiments performed by means of the MTT calorimetric assay described in example 2, it is clear that Extract A dramatically decreases the overall growth of most of the fourty-eight human cancer cell lines submitted to the MTT assay. In the following examples it was investigated whether this extract induced decrease in overall growth corresponds either to modifications occurring at the levels of the cell cycle kinetics (example 3), or the induction of apoptosis (see example 4), or of both.
The cell cycle is in general divided in several phases comprising a G0/G1, S and G2/M phase. Modifications taking place around the proteins controlling cell proliferation and/or cell death may constitute one target of the active compounds, present in Extract A. Modifications to cell cycle kinetics can be investigated by means of flow cytometry using different fluorophores f.e. propidium iodide, orange acridine and ethidium bromide etc. Flow cytometry enables each cancer cell (running to several thousands) to be located into the cell cycle.
Changes in the DNA histogram pattern are thus used to characterize the mechanism of action of various therapeutic compounds. The data resulting from the flow cytometry analysis is processed graphically or mathematically in order to derive meaningful estimates of the G1, the S and the G2/M compartments. Most mathematical processing is based on certain models and assumptions.
Cell lines were seeded in flasks (25 cm2 area) containing 7 ml of culture medium. After 48 hours incubation at 37° C. the cell culture medium was replaced by fresh medium in which Extract A had been dissolved at concentrations which kill 50%, (IC50), 30% (=IC30) and 10% (IC10) of the cell population. After 24 or 72 hours of treatment the cells were harvested in suspension, washed in Phosphate Buffer Saline (PBS) at 4° C. and permeabilized with 70% ethanol (at 4° C.) overnight at −20° C. The cells were then washed with PBS and incubated with propidium iodide solution (80 μg/ml) for 30 minutes at 37° C. and afterwards kept at 4° C. overnight. Ribonuclease A (3% V/V) was added to induce a double-stranded DNA break. The portrait of the cell cycle was established for each sample. A specific software program incorporated into the flow cytometer was used to define precisely the percentage of cells in the different cell cycle phases. Each cell cycle phase was reported in terms of peak surface and calculated as a percentage. The surface of the entire cell cycle was 100%. Each experiment was carried out 3 times. The mean percentage of each different phase and the standard error of the related mean was calculated. Each cell cycle phase of a given condition was compared with the same cell cycle phase of control cells, i.e non-treated cells.
Extract A is highly effective against human tumor cell lines. The concentrations used in our flow cytometry experiments were chosen in accordance with the MTT results (example 2). The five human cancer cell lines Hs683 (glioma), J82 (bladder) A172 (glioma), RPMI (head-and-neck) and HCT-15 (colon) were tested and doses that killed 10, 30 and 50 percent of the cells were determined (Table 3) in order to investigate whether Extract A promoted an accumulation in one of the cell cycle phases when the cultures were treated for 24 or 72 hours with increasing concentrations of Extract A.
The HCT-15 cell cycle analyses (
The analyses of the RPMI cell cycle (
The A172 cell line was treated with Extract A at 10 and 25 μg/ml, which corresponds to the IC10 and IC50. The results (
The J82 cell line is hardly sensitive to the extract (cfr. MTT assay). The concentrations chosen for flow cytometry corresponded to about IC10 and IC30 (
After 24 hours of treatment with Extract A at 0.05 μg/ml the Hs683 cells had accumulated in the G0/G1 phase and attained 70% as compared to control (58%) (
In conclusion, Extract A principally induces an accumulation in the G0/G1 phase and under such circumstances this indicates that Extract A has a cytostatic effect.
This example illustrates the pro-apoptotic effect of Extract A according to the invention.
Cell death may occur in two different ways, either accidentally or as genetically programmed. Accidental cell death, also referred to as necrosis essentially occurs as the results of e.g. physical or biological aggression. Apoptosis refers to the form of cell death which is genetically programmed cell death and which occurs under normal physiological conditions. After the induction of apoptosis, a cascade of events is induced in the cell, which comprises the activation of cell death receptors, the activation of a serie of cytosolic proteases, the formation of apoptotic bodies, the fragmentation of the DNA.
The effect of Extract A on the apoptosis pathway was investigated on the four human cancer cell lines: Hs683 (glioma), J82 (bladder), A172 (glioma) and HCT-15 (colon).
Cells were seeded in flasks (25 cm2 area) containing 7 ml of culture medium. After a 48-hours incubation at 37° C. the cell culture medium was replaced by fresh medium in which Extract A according to the invention was dissolved at different concentrations that killed 50% and 30% of cell population. After 24 or 72 hours of treatment cells were harvested in suspension and counted. 250.000 cells were centrifuged for 10 minutes at 1700 rpm at 4° C. The pellet was washed with PBS at 4° C. and incubated with annexin V-FITC and propidium iodide solution for 15 minutes at 4° C. in the dark. For each sample, data from approximately 10,000 cells were recorded on logarithmic scale. Software incorporated into the flow cytometer was used to define precisely the percentage of cells in apoptotic and/or necrotic pathway, and the normal cells. Each experiment was realized two times. The mean of percentage of both the apoptotic and the necrotic way and the standard error of the related mean was calculated. Each condition was compared to the control, being non-treated cells.
Four-fold increase of apoptotic HCT-15 cells was observed when cells were treated by Extract A at 0.25 μg/ml for 24 hours as compared to the control cells (
In conclusion, Extract A principally induces an increase in the percentage of apoptotic cells. An effect on the necrosis pathway was obtained under certain conditions.
In the present example, the MTD index was determined for Extract A according to the invention. The Maximum Tolerated Dose (MTD) of a given drug is defined as the maximum amount of a drug which can be administered acutely, i.e. in one intraperitoneal, intravenous, subcutaneous or per os single dose, to healthy animals, i.e. animals not grafted with tumors.
The experimental conditions to determine the MTD index of Extract A were the following. The survival times of mice, which are not grafted with a tumor, were recorded up to 14 days post-Injection. Six different doses of Extract A were used for the determination of the MTD index. The highest dose administered to tumor-bearing mice was 160 mg/kg. Other doses comprised 5 mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg and 80 mg/kg. Each experimental group was composed of two mice for the determination of the MTD index. Table 4 below shows the data obtained for the MTD index using Extract A.
where x means one dead mouse and — means all the animals remained alive
According to the definition given above, the MTD index for Extract A is 20 mg/kg for single administration in mice. Thus, the Maximum Tolerated Dose (MTD) index of Extract A, referring to the maximum amount of Extract A, which can be administered acutely to healthy animals, is 20 mg/kg extract. This value indicates that Extract A has a broad therapeutic window.
This example illustrates the in vivo effects of the Extract A according to the invention on three different cancer models.
The in vivo effects of Extract A were studied on mice grafted with different types of tumors, including a lymphoma cancer, a melanoma cancer and a breast cancer. The in vivo effects were evaluated with three types of parameters:
Extract A was evaluated on the aggressive P388 lymphoma cancer model. In a first experiment three doses, 10 mg/kg, 5 mg/kg and 2.5 mg/kg were compared to control. The mice were inoculated subcutaneously with 106 P388 cells at day D0 and treated nine times during the three following weeks at days D5, D7, D9, D12, D14, D16, D19, D21 and D23 post-graft. Each experimental group contained nine mice.
In a second set of experiments, the number of administrations was increased from nine to sixteen, accompanied by a concomitant decrease in the dose per single administration. The subcutaneously-administered doses were 2.5 mg/kg, 1.25 mg/kg and 0.63 mg/kg. Extract A was administered daily, five days a week, for five consecutive weeks (5×5=25).
In conclusion, Extract A according to the invention exerts significant anti-tumor effects on the aggressive P388 lymphoma cancer model without any loss of body weight in the animals concerned. Also, unexpectedly, the Extract A according to the invention exerts higher anti-tumor activity when assayed chronically at low doses, i.e. around 1.25 to 0.63 mg/kg, than at high doses, i.e. around 10 mg/kg to 5 mg/kg.
In Vivo Effects of Extract A on a Melanoma Cancer Model
Extract A was evaluated on the aggressive B16 melanoma cancer model. Extract A was subcutaneously administered to mice at doses of 2.5 mg/kg, 1.25 mg/kg and 0.63 mg/kg. Extract A was administered daily, five days a week, for five consecutive weeks.
In a second set of experiments, Extract A was administered orally (per os).
In conclusion the two sets of experiments provide evidence that Extract A according to the invention exerts significant anti-tumor effects on the B16 melanoma model independent of the mode of administration.
In Vivo Effects of Extract A on a Breast Cancer Model
Extract A was evaluated on the MXT-HI breast cancer model, i.e. a hormone insensitive variant of breast cancer. The MXT-HI model corresponds to an undifferentiated carcinoma with dramatic metastatic processes towards the liver. It therefore corresponds to the late clinical stages of human breast cancer. The MXT-HI represents a very aggressive biological tumor model.
The MXT-HI breast cancer is induced in mice by subcutaneously injecting MXT tumor fragments into the flanks of B6D2F1 mice. Without treatment, the inoculated mice die between the fourth and seventh week after the inoculation. Extract A was assayed at 10 mg/kg, 5 mg/kg and 2,5 mg/kg with nine administrations, i.e. three times a week for three consecutive weeks.
In
In conclusion the experiments described above provide evidence that Extract A exerts significant anti-tumor effects on the MXT-HI breast cancer tumor.
Conclusions on Example 6
The Extract A according to the invention has a significant anti-tumor effect on the tested cancer models. These models represent a broad panel of histological tumor types, including carcinomas, lymphomas and melanomas. These models are clinically relevant because they mimic specific clinical stages of human cancers. Apart from this, they are also biologically aggressive and invasive because two of them metastasize dramatically to the liver.
While having a very significant anti-tumor effect, Extract A according to the invention exhibits negligible toxic effects since the tumor-bearing mice did not lose body weight during the treatments. The semi-purified extract can therefore include an “antidote” in addition to the anti-tumor compound responsible for all the anti-tumor activities reported here.
Surprisingly, Extract A induces significantly higher anti-tumor activities when assayed chronically at low doses i.e. around 1.25 to 0.63 mg/kg, than at high doses, i.e. around 10 mg/kg to 5 mg/kg.
This example illustrates the toxicological effects of Extract A according to the invention, in particular the in vivo hematotoxicity and general toxicity.
Hematotoxicity
Hematotoxicity is evaluated by establishing hematological profiles for each animal species. Particular attention was paid to the numbers of blood platelets, red cells and leukocytes. The effect of Extract A was evaluated at two doses, i.e. 5 mg/kg and 1.25 mg/kg, by the intra-peritoneal administration to mice. The administration schedule was five administrations a week for five consecutive weeks resulting in a total injection number of twenty-five. The animals were sacrificed three days after the last injection. There were ten mice per group.
As compared to control (
General Toxicity
The general toxicity of Extract A was histologically tested on mice by means of conventional histopathological analyses of hematoxilin-eosin-stained histological slides obtained from different types of organs and tissues. The effect of Extract A was evaluated at 5 mg/kg and at 1.25 mg/kg by intra-peritoneal administration. The administration schedule was five administrations a week for five consecutive weeks resulting in a total injection number of twenty-five. The animals were sacrificed three days after the last injection. The brain, the heart, the liver, the pancreas, the stomach, the intestines and the ovaries were collected. There were ten mice per group.
Examination of the collected organs showed that control (untreated) group did not show any particular modification in the organs. Also in treated mice the brain, the heart, the liver, the pancreas, the stomach, the intestines and the ovaries were not affected by Extract A treatments at doses of 5 mg/kg or 1.25 mg/kg, indicating that Extract A does not have a general toxic effect.
This example illustrates the anti-poisonous effect of the extracts according to the invention. Female mice of 4 to 5 weeks of age were injected with two or four times the maximal tolerable dose of two well-known and clinically used anti-tumor drugs (MTDx4) for adriamycine and (MTDx2) for vincristine. The mice were given a single intraperitoneal injection with 40 mg/kg body weight adriamycine or a single intraperitoneal injection of 20 mg/kg body weight vincristine at Day 0. Lot 1 in
Extract A according to the invention was injected intraperitoneally at a dose of 10 mg/kg body weight prior to the injection of the anti-tumor drugs. The following different schedules were applied.
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A second example illustrates the anti-poisonous effect of Extract A with another administration schedule. The mice were given a single intraperitoneal injection with 20 mg/kg body weight vincristine at Day 0. Lot 1 in
Extract A was injected intraperitoneally at a dose of 10 mg/kg body weight prior to or in the same time than the injection of anti-tumor drug. The following different schedules were applied.
Extract A was injected on the same day (i.e. at Day 0), but 6 hours prior to the injection of the therapeutic compound vincristine. Results of this treatment are represented by lot 2, in
The parameter, which was applied for determining the protective, i.e. anti-poisonous effect of Extract A, on the toxic effects induced by the anti-tumor drugs consisted of the “prolongation of survival”. Statistical analysis was performed by means of Kaplan-Meier statistics. The level of significance was p<0.05.
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It can thus be concluded that Extract A enables to significantly protect mice from the toxic effects of a mortal dose of frequently used anti-tumor compound such as adriamycine or vincristine. Extract A according to the invention has thus an anti-poisonous effect, by which enables to reduce the toxic effects of well-known anti-tumor compound.
A third example illustrates the anti-poisonous effect of Extract B Female mice of 4 to 5 weeks of age were injected of four well-known and clinically used anti-tumor compound: adriamycine, vincristine, oxaliplatine and camptothecine. The mice were given a single intraperitoneal injection with 20 mg/kg body weight adriamycine or a single intraperitoneal injection of 20 mg/kg body weight vincristine or a single intraperitoneal injection of 40 mg/kg body weight oxaliplatine or a single intraperitoneal injection of 20 mg/kg body weight camptothecine at Day 0.
Lot 1 in
Lot 2 in FIGS. 30 to 33 shows the “survival point” of the mice injected with Extract B at 10 mg/kg.
Lot 3 in FIGS. 30 to 33 show the survival curve of the mice injected intraperitoneally with Extract B at 10 mg/kg body weight on the same day (i.e. at Day 0) but 4 hours prior to the injection of the therapeutic compound.
As can be seen in FIGS. 30 to 33, Extract B used in combination with a therapeutic compound significantly prolongs the survival of the mice as compared to injection of the therapeutic compound as single agents. Extract B didn't show cytotoxic properties. Indeed, no mouse died when it was injected with Extract B as single agent. Statistical analysis was performed by means of Kaplan-Meier statistics. The level of significance was p<0.05 for each combination Extract B and therapeutic compound.
It can thus be concluded that Extract B provides significant protection to mice from the toxic effects of a mortal dose of frequently used anti-tumor drugs. Extract B has thus an anti-poisonous effect, which enables the reduction of the toxic effects of well-known anti-tumor drugs. The anti-poisonous activity was preserved in Extract B as in Extract A.
A fourth example illustrates the anti-poisonous effect of Extract C. Extract C was used in combination of adriamycine at 20 mg/kg body weight.
Lot 1 in
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A fifth example illustrates the anti-poisonous effect of Extract D. Female mice of 4 to 5 weeks of age were injected with adriamycine. The mice were given a single intraperitoneal injection with 20 mg/kg body weight adriamycine at Day 0 (Lot 1 in
As can be seen in
In conclusion, these experiments illustrate that the purified extracts according to the invention prolong significantly the survival of mice as compared to the mice injected with therapeutic compound as single agent. Extract D, the most purified extract, showed an anti-poisonous properties on the toxic effects induced by the anti-tumor drugs.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/EP03/11192 | Oct 2002 | WO | international |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP03/11192 | 10/9/2003 | WO | 4/8/2005 |
