Patent Application
20220233624
This application claims priority from Indian Provisional Application No. 202041009290 (IN), filed Mar. 4, 2020, which is incorporated by reference herein in its entirety.
The present invention relates to compositions and methods for treating cancer and/or inhibiting the proliferation of cancer cells and/or cancer-associated cells with compositions comprising Terminalia chebula (e.g. AyuFlex®), Terminalia bellerica (e.g. Ayuric®), Phyllanthus emblica (e.g. Capros®), Withania somnifera (e.g. Sensoril®), Shilajit (e.g. PrimaVie®), Azadirachta indica (e.g. PhytoBGS®), and combinations thereof, including for instance a trivalent chromium complex with extracts of Shilajit and P. emblica (e.g. Crominex-3+®). Combinations of the extracts, co-administration with anti-cancer drugs, and related methods are also described.
Terminalia chebula has been extensively used in Ayurveda, Unani and Homoeopathic systems of medicine for improvement of different health conditions. T. chebula may be rich in tannoids and may contain a variety of other constituents. Chemical constituents isolated from T. chebula may vary considerably in type and/or concentration due to number of factors, e.g., ecological variation, soil variation, nutrient variation, as well as variations in the process of extraction.
Terminalia bellerica (a.k.a. belerica, belirica, bellirica) is grown widely throughout India, Sri Lanka, and South East Asia. T. bellerica has been used for centuries in Ayurveda and may contain several chemical constituents in common with T. chebula.
Phyllanthus emblica, the Indian gooseberry, is also widely used in Indian medicine for treatment of various diseases.
Withania somnifera, commonly known as Ashwagandha, has been used in herbal formulations of the Ayurvedic or Indian system of medicine, for instance to help to ward off stress and act as an adaptogen.
Shilajit, also known as “Moomiyo,” is found in high altitudes, for instance of the Himalayan Mountains, and is considered one of the “wonder medicines” of Ayurveda, the traditional Indian system of medicine dating back to 3500 B.C.E. Shilajit is physiologically active organic matter, composed of rock humus, rock minerals, and organic substances that have been compressed by layers of rock mixed with marine organisms and microbial metabolites. Shilajit oozes out of the rocks as a black mass in the Himalayas at higher altitudes ranging from 1000 to 5000 meters, as the rocks become warm during summer. Shilajit contains fulvic acids (“FAs”) as its main components, along with dibenzo-α-pyrones (“DBPs”) and DBP chromoproteins, humic acid, and more than forty (40) minerals. DBPs are also known as Urolithins (e.g. Urolithin A, Urolithin B).
Azadirachta indica, commonly known as neem, nitree, or Indian lilac, is a tree in the mahogany family Meliaceae. It is one of two species in the genus Azadirachta, and is native to the Indian subcontinent, i.e., India, Nepal, Pakistan, Bangladesh, Sri Lanka, and Maldives. It is typically grown in tropical and semi-tropical regions. Neem trees also grow in islands located in the southern part of Iran. Its fruits and seeds are the source of neem oil.
Products made from neem trees have been used in India for over two millennia for their medicinal properties. Neem products are considered a major component in Siddha medicine and Ayurvedic and Unani medicine and are particularly prescribed for skin diseases. Neem oil is also used to promote healthy hair, to improve liver function, detoxify the blood, and balance blood sugar levels. Neem leaves have also been used to treat skin diseases like eczema and psoriasis.
Primary patient-derived cancer cells (PDCs), cultured directly from tissue obtained from surgery in cancer patients, provide a physiologically relevant platform for testing an individual's cancer cells and cell type responses to a specific drug or composition. Information derived from PDC testing may be useful in personalized as well as generalized cancer patient treatments.
In addition, in-vitro studies using standardized cancer cell lines provide valuable information for animal and human treatments.
The present invention is directed to a method of inhibiting the proliferation of cancer cells and/or cancer-associated cells comprising the steps of providing a composition comprising Terminalia chebula (for instance, AyuFlex®), Terminalia bellerica (for instance, Ayuric®), Phyllanthus emblica (for instance, Capros®), Withania somnifera (for instance, Sensoril®), Shilajit (for instance, PrimaVie®), and/or Azadirachta indica (for instance, PhytoBGS®), and combinations thereof, including for instance a trivalent chromium complex with extracts of Shilajit and P. emblica (e.g. Crominex-3+®), and applying the composition to cancer cells in an amount effective to cause an anti-proliferative effect. In an embodiment, cancer cells inhibited according to the present invention include glioma cells, breast cancer cells (ER/PR+ Her2 equivocal (“HR+”), ER/PR− Her2+(“Her2+”), and Triple Negative (“TN”)), chronic lymphocytic leukemia cells (“CLL”), acute myeloid leukemia cells (“AML”), small cell lung cancer cells, non-small lung cancer cells, colon cancer cells, pancreatic cancer cells, prostate cancer cells, and/or ovarian cancer cells.
Also, the present invention is directed to a method of treating cancer in a subject in need of such treatment, comprising the steps of providing a composition comprising Terminalia chebula (for instance, AyuFlex®), Terminalia bellerica (for instance, Ayuric®), Phyllanthus emblica (for instance, Capros®), Withania somnifera (for instance, Sensoril®), Shilajit (for instance, PrimaVie®), and/or Azadirachta indica (for instance, PhytoBGS®), and combinations thereof, including for instance a trivalent chromium complex with extracts of Shilajit and P. emblica (e.g. Crominex-3+®), and administering the composition to the subject in an amount effective to treat the cancer, for instance by slowing the progression or growth of the cancer, stopping the progression or growth of the cancer, shrinking the cancerous tumor, reducing the number of cancer cells and/or cancer-associated cells in the subject, and/or rendering the cancer almost undetectable or undetectable in the subject. In an embodiment, a cancer treated according to the present application includes glioma, breast cancer (including ER/PR+ Her2 equivocal, ER/PR− Her2+, and Triple Negative), chronic lymphocytic leukemia, acute myeloid leukemia, colon cancer, small-cell lung cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer, and/or ovarian cancer. In an embodiment, the composition is an extract, for instance a standardized aqueous extract, of the plants identified above. In an embodiment, a method of treatment according to this invention includes administering compositions such as extracts of the above with anti-cancer drugs to enhance the effects of the drugs and, in an embodiment, reduce side, adverse, or toxic effects of the drugs, in an embodiment by reducing the necessary dose of the traditional drug.
The below definitions and discussion are intended to guide understanding but are not intended to be limiting with regard to other disclosures in this application. References to percentage (%) in compositions of the present invention refers to the % by weight of a given component to the total weight of the composition being discussed, also signified by “w/w”, unless stated otherwise.
In the present invention, “anti-cancer” generally refers to preventing, treating and/or otherwise halting cancer in a subject for instance by reducing viability of cancer cells and/or cancer-associated cells, including for instance slowing the progression or growth of the cancer, stopping the progression or growth of the cancer, shrinking the cancerous tumor, reducing the number of cancer cells and/or cancer-associated cells in the subject, and/or rendering the cancer almost undetectable or undetectable in the subject. In an embodiment, cancer in a subject is diagnosed by a medical provider. In an embodiment, cancer in a subject is not diagnosed by a medical provider.
In the present invention, “anti-proliferative” and the like refers to inhibiting the proliferation of cancer cells, including for instance inhibiting the proliferation of cancer cells in a subject or for instance inhibiting the proliferation of cancer cells such as PDCs in a cell culture outside of a subject's body, by rendering the cancer cells temporarily or permanently non-viable (not able to grow or develop) and metabolically inactive. Reference to cells “inhibited by” a composition of this invention, or other substance, is to the inhibition of proliferation of cancer cells by the composition, for instance as shown by the anti-proliferation assays described in the Examples below, unless stated otherwise. In an embodiment, inhibiting the proliferation of cancer cells refers to killing cancer cells. In an embodiment, the proliferation of cancer cells may be inhibited by a composition of the present invention by 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or for instance by 1%-100%, or any range of numbers therein, such as for instance 33-37% or 62%-75%. In an embodiment, the number of viable cancer cells may be reduced by 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, by 1%-100%, or any range of numbers therein, such as for instance 2-8% or 79-93%. Known anti-cancer agents GDC-0941 (a class I phosphatidylinositol 3 kinase (PI3K) inhibitor; pictilisib) and doxorubicin are shown as anti-proliferative drug controls in Example I below, inhibiting the proliferation of cancer cells by about 50% to about 100%, depending e.g. on the sensitivity of the PDC type. An “anti-cancer drug” may be pictilisib, doxorubicin, temozolomide, docetaxel, 5-fluorouracil (5-FU), ibrutinib, arsenic trioxide, and/or cytarabine, and/or any other control or standard of care drug identified in this application. Comparisons of different concentrations of standardized aqueous extracts of the present invention with the inhibition of proliferation of PDCs by GDC-0941 and doxorubicin are also shown in the Figures.
In the present invention, “cancer cells” refers to cells of, or taken from or derived from, a cancerous source, such as a solid cancerous tumor or hematopoietic cancerous tissue or cells. An example of “cancer cells” of the present invention is a mixed culture of primary patient-derived cancer cells (PDC), taken directly from an individual human subject's cancerous tissue post-surgery. PDCs may include cells from any cancer, including for instance cells from a glioma, such as a glioblastoma Grade IV; cells from a breast cancer tumor, such as breast cancer cells that are ER/PR+ Her2 equivocal, ER/PR-Her2+, or triple negative; leukemia cells from a subject having chronic lymphocytic leukemia; and/or leukemia cells from a subject having acute myeloid leukemia (e.g. M 4 subtype), and so forth. See for instance Table 1. Also, cancer cells of this invention include cells of a standard cell line, such as the cell lines discussed in the below Examples. In an embodiment, cancer cells of the present invention are part of a tumor or other cancerous cell source in a mammalian subject, such as in a human body. In an embodiment, cancer cells of the present invention include characteristics attributed to cells of a cancerous tumor or malignancy or the like, for instance as known in the art.
In the present invention, “applying” (and the like) a composition of the present invention refers to making the composition physically available to the cancer cells, for instance by administering the extract to a subject having cancer so that the extract or at least its active components reach the cancer cells in the subject's body. Applying the extract in an “effective amount” to cancer cells refers to applying the extract in an amount that will inhibit proliferation of the cancer cells, for instance as described in Example I and other Examples below.
In the present invention, “administering”, “administration”, and the like refer to providing a composition of the present invention to a subject so that the composition (or components thereof) reaches the subject's bloodstream and/or tissues and thus reaches cancer cells, and acts on the cancer cells to slow and/or stop their proliferation and render them non-viable. Administration may be by the subject or by another. Administration to the subject may be oral, for instance in the form of a dietary supplement, and/or in a solid pharmaceutical dosage form, preferably in a discrete dose unit, such as a table, hard gelatin capsule, soft gelatin capsule, etc. Administration may also be through parenteral, intramuscular, transdermal, topical, sublingual, intravenous, and other physiologically acceptable routes.
In an embodiment, references to active components and the like throughout this application are not intended as being bound by theory, as it is the administration of the full composition of the present invention which is shown to provide the remarkable inhibition of cancer cells of the present invention.
“Co-administration” refers to administering two or more compositions such as extract(s) of the present invention, or one or more such compositions with another composition or with an anti-cancer drug. Such co-administration may be at different times, so long as both extract and anti-cancer drug are available in the bloodstream and/or tissues of the subject in an effective amount to slow and/or stop the proliferation of cancer cells and in an embodiment render them non-viable. Doses of anti-cancer drug may be per the normal standard of care or at the lower end or in a lower dose, for instance in view of a subject's intolerance of adverse effects from the medication.
In the present invention, “treating cancer” and the like refers to halting the progression of cancer in a subject and/or causing the cancer including e.g. cancer cells to decrease, shrink, and if possible become or remain undetectable in the subject, for instance by reducing the size of a cancer tumor and/or number of cancerous cells. In an embodiment, the number of viable cancer cells and/or size of the tumor may be reduced by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, by 1%-100%, or any range of numbers therein. In an embodiment, cancer being treated according to this invention is diagnosed in a subject by a medical professional, such that the subject has been diagnosed as having cancer. In an embodiment, cancer being treated according to this invention has not been diagnosed in a subject by a medical professional, such that the subject has not been diagnosed as having cancer, however cancer cells exist in the subject.
In an embodiment, a “subject” according to this invention is a human; in another embodiment, the subject is a mouse, rat, dog, horse, or any mammal including for instance a mammal serving as a model for cancer research, or a human. In an embodiment, treating the cancer with an “effective amount” of a composition of this invention includes administering an amount effective to slow the progression or growth of the cancer, stop the progression or growth of the cancer, shrink the cancerous tumor, reduce the number of cancer cells in the subject, and/or render the cancer almost undetectable or undetectable in the subject, and/or act against the cancer for instance as discussed elsewhere in this application or in the state of the art. “Enhancing” the treatment of cancer refers to administering a composition of this invention to a subject undergoing treatment with a different anti-cancer drug, to improve treatment outcome, as discussed throughout this application.
A “composition” of the present invention comprises Terminalia chebula (e.g. AyuFlex®), Terminalia bellerica (e.g. Ayuric®), Phyllanthus emblica (e.g. Capros®), Withania somnifera (e.g. Sensoril®), Shilajit (e.g. PrimaVie®), Azadirachta indica (e.g. PhytoBGS®), or a combination thereof, including for instance a trivalent chromium complex with extracts of Shilajit and P. emblica (e.g. Crominex-3+®). In an embodiment, a composition consists essentially of, or consists of, one or more of the above.
In an embodiment, a composition of this invention is an extract of Terminalia chebula (e.g. AyuFlex®), Terminalia bellerica (e.g. Ayuric®), Phyllanthus emblica (e.g. Capros®), Withania somnifera (e.g. Sensoril®), Shilajit (e.g. PrimaVie®), Azadirachta indica (e.g. PhytoBGS®), or a combination thereof, including for instance a trivalent chromium complex with extracts of Shilajit and P. emblica (e.g. Crominex-3+®). In an embodiment, an extract of this invention refers to a natural substance prepared from Terminalia chebula fruit, Terminalia bellerica fruit, Phyllanthus emblica fruit, Withania somnifera roots and leaves, Shilajit, Azadirachta indica twigs and leaves, or a combination thereof, that has been disrupted from its natural state and treated with water or aqueous solution such as phosphate buffered saline (PBS), alcohol such as methanol or ethanol, or a hydroalcoholic mixture. In an embodiment a hydroalcoholic mixture may include alcohol and water together, for instance in a ratio of 9:1 to 1:9; in another embodiment, an extract may be prepared by first treating the plant or e.g. herbomineral material with one substance, such as ethanol, and then with a second substance, such as water or an aqueous solution. See for instance Example IX. In an embodiment, extracted substances may be pooled. In an embodiment, an extract of this invention is a standardized extract.
In an embodiment, an aqueous standardized extract of this invention is AyuFlex®, Ayuric®, Capros®, Sensoril®, PrimaVie®, and/or PhytoBGS®. A composition of the present invention may be a combination, for instance, a blend of one or more extracts such as AyuFlex®, Ayuric®, Capros®, Sensoril®, PrimaVie®, and/or PhytoBGS®, and/or a trivalent chromium complex such as Crominex+3®. In an embodiment Ayuflex® and Ayuric® are combined. In another embodiment, AyuFlex® and Capros® are combined. In an embodiment, Ayuric® and Capros® are combined. In an embodiment, AyuFlex®, Ayuric®, and Capros® are all combined in a composition of the present invention. In an embodiment, Sensoril® and PrimaVie® are combined. In an embodiment, Sensoril® and Phyto-BGS® are combined. In an embodiment, PrimaVie® and Phyto-BGS® are combined. In an embodiment, Sensoril®, PrimaVie®, and Phyto-BGS® are combined. Other combinations of the present extracts may be combined into a composition of the present invention. A composition of the present invention may comprise, consist essentially of, or consist of, any extract of the present invention or combination thereof.
In an embodiment, an extract such as an aqueous standardized extract is in powdered form and may be blended together with another extract or trivalent chromium complex such as Crominex+3® or another substance, for instance in powdered form, or other solid or form. In another embodiment, the extract(s) may be for instance blended or dissolved into a composition in liquid form, or combined in another form. A composition of the present invention may further comprise one or more excipients, additives, and/or other substances, including for instance microcrystalline cellulose, croscarmellose sodium, magnesium stearate, and/or silicon dioxide; and/or a suitable aqueous solution such as a buffer solution. A composition of the present invention may be formulated into nutraceutical or pharmaceutical dosage forms comprising for instance tablets, capsules, powders, liquids, chews, gummies, transdermals, injectables, dietary supplements, topical creams or gels, lozenges, pills, and so forth. In an embodiment, a composition of the present invention is a solution comprising an extract and applied to cancer cells as in the Examples below, or used to prepare the applied solutions.
In an embodiment, a composition comprising one or more standardized aqueous extracts of this invention is administered in an effective amount to a mammal, including a daily dose for a human being of at least 1-2000 mg of at least one extract, in an embodiment at least 50 mg, 100 mg, 250 mg, 500 mg, 750 mg, 1000 mg, 1500 mg, or 2000 mg; and optionally 1-20000 mg of each and any other extract included in the composition. In a mammal, a dose may be about the same as in a human, and may be adjusted per kilogram of weight of the mammal. A powdered blend of one or more extracts and optionally excipients or other substances such as fillers, disintegrants, flow enhancers, and lubricants, for instance, microcrystalline cellulose, croscarmellose sodium, silicon dioxide, and magnesium stearate, may be blended using standard powder blending techniques. In an embodiment, a composition of this invention may be marked as organic by an appropriate agency or organization.
Bioactive Components of Extracts of this Invention
AyuFlex® of this invention is a standardized aqueous extract of the fruits of Terminalia chebula plant, off white to brown color powder and soluble in water with astringent taste. It contains not less than 39% w/w low molecular weight hydrolysable tannins as bioactives with not less than 27% w/w chebulinic acid and chebulagic acid combined and not less than 12% w/w of other unindentified low molecular weight hydrolysable tannins. Ellagic acid and gallic acid are also present in the extract, for which the analytical results may be reported without any specification, but specifications for these bioactives may also be identified, for instance in an embodiment, each may be present in amounts of less than 10% of the extract or composition. In an embodiment, an aqueous extract of this invention, Ayuflex®, contains about 65-70% w/w low molecular weight hydrolysable tannins including about 45-50% w/w chebulinic acid and chebulagic acid combined. In an embodiment, said aqueous extract may have a measured amount of 8-9% each of gallic acid and ellagic acid. In an embodiment, a composition of this invention may be a preparation of Terminalia chebula fruit, dried and powdered and in an embodiment, standardized for pharmaceutical or nutraceutical usage. In an embodiment, an extract of this invention may be prepared from such a fruit preparation.
Terminalia bellerica (for instance, Ayuric®)—Ayuric® of this invention is a standardized aqueous extract of the fruits of Terminalia bellerica plant, brown color powder and soluble in water with astringent taste. It contains not less than 15% w/w of low molecular weight hydrolysable tannins as bioactives, including chebulinic acid and chebulagic acid and other unidentified low molecular weight hydrolysable tannins. Ellagic acid and gallic acid are also present in the extract, for which the analytical results are only reported without any specification, but specifications for these bioactives may also be identified. In an embodiment, Ayuric® is a composition of this invention and contains 33-38% w/w low molecular weight hydrolysable tannins, including chebulinic acid and chebulagic acid and other unidentified low molecular weight hydrolysable tannins, and in a further embodiment, also contains 6-8% w/w gallic acid and 0.5-2% w/w ellagic acid. In an embodiment, a composition of this invention may be a preparation of Terminalia bellerica fruit, dried and powdered and in an embodiment, standardized for pharmaceutical or nutraceutical usage. In an embodiment, an extract of this invention may be prepared from such a fruit preparation.
Phyllanthus emblica (for instance, Capros®)—Capros® of this invention is a standardized aqueous extract of the fruits of Phyllanthus emblica plant, light yellow color powder and soluble in water with astringent taste. It contains not less than 60% w/w of low molecular weight hydrolysable tannins, comprising of emblicanin-A, emblicanin-B, punigluconin and pedunculagin, and not more than 4% w/w of gallic acid. In an embodiment, Capros® as a composition of this invention contains about 75-80% w/w low molecular-weight hydrolysable tannins, and about 0.5-1.5% w/w gallic acid. In an embodiment, a composition of this invention may be a preparation of Phyllanthus emblica fruit, dried and powdered and in an embodiment, standardized for pharmaceutical or nutraceutical usage. In an embodiment, an extract of this invention may be prepared from such a fruit preparation.
Withania somnifera (for instance, Sensoril®)—Sensoril® of this invention is a standardized aqueous extract of the roots and leaves of Withania somnifera plant, brown color powder and soluble in water with bitter taste. It contains not less than 10% w/w of withanolide glycosides, less than 0.5% w/w of withanolide aglycones (as Withaferin-A) and not less than 32% w/w oligosaccharides as bioactives. Withanolides, such as withastromolide, withanolide A, withanolide B, 27-hydroxy withanone, withanone, etc. may also be present in this extract. In an embodiment, Sensoril® as a composition of this invention contains about 10-12% w/w of withanolide glycosides, about 0-0.5% w/w of withanolide aglycones (as Withaferin-A), and 35-40% w/w oligosaccharides.
In an embodiment of the present invention, an extract of Withania somnifera is made by using ethanol extraction followed by water extraction. This extract is referred to as Sensoril®-AWE (“Alcohol-Water-Extract”). Other alcohols, such as methanol, isopropyl alcohol, etc. may also be used as extraction solvents instead of ethanol. Such a hydro-alcoholic Sensoril®-AWE extract of this invention contains not less than 10% w/w withanolide glycosides, not less than 2%, preferably not less than 3.0% w/w withanolide aglycones (as Withaferin-A) and not less than 20% w/w oligosaccharides as bioactives. Withanolides, such as withastromolide, withanolide A, withanolide B, 27-hydroxy withanone, withanone, etc. may also be present in this extract. In an embodiment, a composition of this invention is a hydroethanolic Withania somnifera (Sensoril®-AWE) extract containing 12-20% w/w withanolide glycosides (including as mentioned elsewhere in this application ranges therein, such as 12% w/w, 13% w/w, and so on), 2-8% w/w withanolide aglycones (as Withaferin-A), and 24-35% w/w oligosaccharides.
Shilajit (for instance, PrimaVie®)—PrimaVie® of this invention is a standardized aqueous extract of Shilajit, an herbo-mineral exudate from the Himalayan, Altai and other mountains, brown color powder and soluble in water with earthy taste. It contains not less than 50% w/w of fulvic acid and not less than 10.3% w/w of free dibenzo-α-pyrones and dibenzo-α-pyrones conjugated with chromoproteins combined. Dibenzo-α-pyrones are also known as Urolithins, for example Urolithin A and Urolithin B. In an embodiment, PrimaVie® as a composition of this invention is a standardized aqueous extract containing 58-63% fulvic acids with a dibenzo pyrone core nucleus and 14-18% of free dibenzo-α-pyrones and dibenzo-α-pyrones conjugated with chromoproteins combined.
Azadirachta indica (for instance, PhytoBGS®)—PhytoBGS® of this invention is a standardized aqueous extract of the leaves and twigs of Azadirachta indica plant, brown color powder and soluble in water with bitter taste. It contains not less than 2% w/w of flavonoids (comprising of quercetin-3-O-glucoside, quercetin-3-O-rutinoside, apigenin rutionoside and other rutin derivatives) and not less than 5% w/w and up to 20% w/w of myoinositol monophosphate as bioactives, and is devoid of possibly toxic compounds such as azadirachtone, azadiradione, nimbolide, nimbin. In an embodiment, a PhytoBGS® composition of this invention contains about 3% w/w flavonoids and 6-8% w/w myoinositol monophosphate.
Trivalent chromium complex (for instance, Crominex-3+®)—Crominex-3+® of this invention is a complex of trivalent chromium chloride, Phyllanthus emblica extract and Shilajit extract. It is a light brown color powder, soluble in water with astringent taste. It contains not less than 2% w/w of trivalent chromium as the bioactive. In an embodiment, a composition of this invention is Crominex-3+® containing 2-3% of trivalent chromium.
The bioactive compositions of the extracts described above are only representative and may differ depending on the analytical method used, especially so with natural products, as natural products contain multiple bioactives for which reference standards are not readily available in the market.
In an embodiment, a method of manufacturing of a T. chebula fruit extract is described in U.S. Pat. No. 10,500,240, which is incorporated by reference herein for this purpose, to the extent allowed by law. The same method is used for manufacturing T. bellerica extract also.
In an embodiment, the extraction process of the current invention includes the steps of providing dried fruits of T. chebula or T. bellerica, de-seeding the fruits, pulverizing or grinding the pulp to a powder, extracting the pulp powder with an extraction solvent or solvent mixture, optionally, with heating, to provide a T. chebula or T. bellerica enriched liquid extract, optionally concentrating the liquid extract and drying the concentrated liquid extract to provide a hydrolyzable tannoid enriched T. chebula or T. bellerica extract powder. Aqueous solvent is preferred. A particularly preferred solvent is water. Useful extraction temperatures can range from about 25° C. (ambient) to about 90° C. Particularly useful extraction temperatures can range from about 25° C. to about 80° C.
In an embodiment, AyuFlex® and Ayuric® are prepared in keeping with the methods described above.
In another embodiment, ethanol or methanol or a hydro-alcoholic mixture may be used as the solvent system for extraction.
In an embodiment, useful extraction times in conjunction with maintaining useful temperatures can range from about 2 hours to about 16 hours. A particularly useful extraction time range at about 25°±5° C. is from about 12 hours to about 16 hours, and at a temperature of 40°±5° C. is from about 2 hours to about 6 hours. Length and temperature of extraction may be varied at atmospheric pressure (i.e., approx. 1 atm). It is contemplated that pressure can be varied in the extraction process, for example, by use of a commercial pressure reactor apparatus.
The extraction process can also include drying the liquid extract to a powder form. Suitable drying methods include spray drying, lyophilization (freeze drying), vacuum drying (with or without heating), evaporation (with or without heating), and concentration under vacuum. Once isolated or obtained, the hydrolyzable tannoid enriched T. chebula or T. bellerica extract powder may be processed by any suitable means, including grinding, milling, sieving, sizing, blending and the like. The obtained hydrolyzable tannoid enriched T. chebula or T. bellerica extract powder may be prepared in any suitable particle size or particle size range.
Process additives such as microcrystalline cellulose, starch, maltodextrin and the like as carrier materials, anti-adherents such as silicone dioxide, rice bran powder and the like, and preservatives such as sodium benzoate, methyl paraben, propyl paraben, natural preservatives and the like may be added during the extraction process or during the final blending of the dried extract powder.
In the case of Phyllanthus emblica extract, seeds are removed from the fresh fruits after washing, the juice is separated from the pulp by pressing and/or centrifugation and the juice is dried by spray drying or another suitable drying technique, such as microwave drying, freeze drying, etc. Additives, such as preservatives, such as sodium chloride and sodium benzoate, carrier materials such as maltodextrin, microcrystalline cellulose, anti-adherent such as silicon dioxide and rice bran powder may also be added, optionally.
In the cases of Withania somnifera, Shilajit and Azadirachta indica, the dried roots and/or leaves of the plant, the shilajit stone and the dried leaves and twigs respectively, are milled and subjected to extraction and drying. Extraction is done using water only as the solvent or an alcohol, preferably ethanol or methanol, followed by water. The liquid extract is then optionally concentrated by evaporation and then dried by spray drying, freeze drying, microwave drying or another suitable drying technique. Extraction temperatures may range from 40° C.±5° C. to 90° C.±5° C., preferably 60° C.±5° C. to 80° C.±5° C. Extraction times may vary from 1 hour to 12 hours, preferably from 2 hours to 6 hours. Several cycles of extraction may also be done. In an embodiment of Azadirachta indica, only leaves may be used as the starting raw material.
Trivalent chromium complex is made by dissolving trivalent chromium chloride in water, with or without heat, adding Phyllanthus emblica extract and mixing for a suitable length of time to form a complex, then adding shilajit and mixing for a suitable length of time, then adding a carrier material such as microcrystalline cellulose, maltodextrin, starch, etc. and mixing for a suitable length of time, followed by spray drying, freeze drying, microwave drying or another suitable drying method. The temperature of the complexation step may be from 30° C.±5° C. to 80° C.±5° C., preferably from 40° C.±5° C. to 60° C.±5° C.
In an embodiment, a “combination” of this invention refers to a mixture of an extract of the present invention with another extract, and/or a mixture of one or more extract(s) with a known anti-cancer drug, for instance those drugs used as controls and/or identified as standard of care drugs in the Examples. In an embodiment, a combination is in the same composition. In an embodiment, a composition of the present invention includes one or more extracts as described, and the combination of extract with an anti-cancer drug occurs in a subject's body. The term “combination” is not intended to be limiting in the context of this invention. Combination therapy refers to a regimen for administering an extract including a combination of extracts of this invention with an anti-cancer drug, for instance to improve treatment outcome, and/or to increase anti-cancer effects as compared with the extract(s) alone and the anti-cancer drug alone. In an embodiment, a combination according to the present invention provides a synergistic effect, greater than the effect achieved by its individual components. While references to synergy and synergistic effects may be made throughout this application, all instances of such may not be expressly pointed out.
A “dietary supplement” according to the present invention refers to a composition of this invention which is orally administered as an addition to a subject's diet, which is not a natural or conventional food, which when administered inhibits the proliferation of cancer cells in the subject's body. In an embodiment, the dietary supplement is administered daily; in an embodiment, the dietary supplement is administered daily for instance for at least 3 days, 5 days, 1 week, 1 day to 1 week, 1 week to 26 weeks, or chronically for at least 1 day to 3 months, 6 months, 9 months, or 1 year or more, or for another period of time according to the present invention. A dietary supplement may be formulated into various forms, as discussed throughout this application. In an embodiment, a dietary supplement of this invention comprises one or more extracts of this invention, such as one or more aqueous standardized extracts or one or more hydroalcoholic extracts of the present invention.
The present invention may be further understood in connection with the below Examples and with embodiments described throughout this application. The following non-limiting Examples and embodiments described below and throughout this application are provided to illustrate the invention. Materials and methods used in Example I were also used with regard to the other Examples, unless indicated or expressly stated otherwise.
Anti-proliferation assays were performed on 6 types of primary patient-derived cancer cells (PDCs) from 6 different human donors, as described in Table 1:
Tests were performed in 384-well plates (24×16), using 1250 to 10,000 cells/well depending on the size and proliferation rate of different PDCs, in 30 ul of culture media (DMEM/F12 with serum and growth factors), with control wells set aside on each plate for media alone (4 wells), untreated PDCs (4 wells), and for PDCs treated with 10 ul of a known anti-cancer drug as a control. For instance, 10 ul of 40 uM GDC-0941 (pictilisib)), or 40 uM doxorubicin (2 wells each), was used, for a final concentration of 10 uM each well. The outermost rows/columns of the plates were filled with PBS (pH 7.4) to avoid an edge-effect and were considered as blanks.
Standardized aqueous extracts identified in Table 2 were prepared in main stock solutions by dissolving 1 mg of extract in 1 ml PBS (phosphate-buffered saline, pH 7.4).
Terminalia
chebula fruits
Terminalia
bellerica fruits
Phyllanthus
emblica fruits
Withania
somnifera roots + leaves
Azadirachta
indica leaves + twigs
All extracts used in this Example were provided by Natreon, Inc. (New Brunswick, N.J.).
Anti-Proliferation Assay Protocol: 30 ul of cells were plated at the density of 1250 to 10,000 cells/well. The plate was incubated in a CO2 incubator for 0/N incubation for 24 hours. Next day, 10 ul of 40 uM GDC-0941 (pictilisib) or 10 ul of 40 uM doxorubicin were added to incubated cells in the specified control drug wells, and 10 ul of 4× extract solutions were added to incubated cells in 3 concentrations: 400 ug/ml (2 wells), 120 ug/ml (3 wells), 40 ug/ml (3 wells). The final assay concentration of control drug was 10 uM for DGC-0941 (pictilisib) and doxorubicin. Final assay concentrations of made from stock solutions of standardized aqueous extracts identified in Table 2 were 100 ug/ml, 30 ug/ml, and 10 ug/ml. Since 10 ul extract was added to 30 ul of cell suspension, it diluted 4 times, so to maintain the final assay concentrations, 4× concentrated extract solutions (400 ug/ml, 120 ug/ml, and 40 ug/mo) were prepared from main stock (1 mg/ml) and added to the plate. In all experiments, 10 ul of 4 times concentrated stock (4×) was added to wells containing 30 ul of cell suspension to achieve final assay concentrations at 1X. Other anti-cancer drugs used as controls throughout this Example include temozolomide, docetaxel, 5-fluorouracil (5-FU), ibrutinib, arsenic trioxide, and cytarabine, and were applied to PDCs generally as described.
Once extracts were added to incubated cells and mixed gently, the plate was given a brief spin and incubated in the CO2 incubator for 72 hours. The plate was removed from the incubator and 10 ul of detection reagent (CellTiterGlo®; Promega Corporation, Wisconsin, USA) added to each well of the 384 well plate. The plate was incubated (10 min) and luminescence counts measured, with luminescence produced proportional to the number of viable (living, metabolically active) cells for each assay. The assay described above is a two-dimensional (2D) assay.
References to synergy below are not intended as limiting; other instances of synergy may be present in view of the data provided below.
1. Terminalia chebula Fruit Extract (AyuFlex®)
Table 3 shows the percentage of PDCs inhibited by AyuFlex® (±Standard Deviation) in the anti-proliferation assay described above. The results are pictured in
AyuFlex® showed dose-dependent, high anti-cancer activity against CLL (Chronic lymphocytic leukemia) primary cells, with 50% of CLL cells inhibited by AyuFlex® extract at the 30 ug/ml concentration applied to the CLL cells, and 95% inhibition of CLL proliferation at the 100 ug/ml concentration. AyuFlex® inhibited about 33% of AML (acute myeloid leukemia) cells at the 100 ug/ml concentration.
AyuFlex® showed potent anti-cancer activity against all solid cancer PDCs. The most potent anti-cancer activity was seen against Breast Cancer PDCs (Triple Negative (“TN”), and ER/PR− Her2+(“Her2+”), with AyuFlex® showing more than 50% inhibition of these cells at the concentration of 10 ug/ml. Anti-cancer activity against the other Breast Cancer PDCs (“HR+”) was also high, showing 50% inhibition at greater than/equal to the 30 ug/ml concentration. Anti-cancer activity against glioma cells were also high, with 35% inhibition at 30 ug/ml and 68% inhibition at the 100 ug/ml concentration.
2. Terminalia bellerica Fruit Extract (Ayuric®)
Table 4 shows the percentage of PDCs inhibited by Ayuric® (±Standard Deviation) in the anti-proliferation assay described above. The results are pictured in
Ayuric® showed the highest anti-cancer activity against CLL primary cells, with dose-dependent inhibitory activity and 70% inhibition observed with the 100 ug/ml concentration applied to the CLL PDCs. In AML cells, Ayuric® exhibited moderate anti-cancer activity.
Ayuric® showed good to moderate anti-cancer activity for all solid cancer PDCs. Ayuric® demonstrated dose-response inhibition and about 50% inhibition of breast cancer Triple Negative PDCs. The anti-cancer activity was found to be more moderate against HR+ breast cancer PDCs and glioma cells.
3. Phyllanthus emblica Fruit Extract (Capros®)
Table 5 shows the percentage of PDCs inhibited by Capros® (±Standard Deviation) in the anti-proliferation assay described above. The results are pictured in
Capros® showed dose-response and highest anti-cancer activity against CLL primary cells, showing 75% inhibition at the 100 ug/ml concentration applied to the cells. In AML cells, Capros® inhibited about 25% of AML (acute myeloid leukemia) cells at the 30 ug/ml concentration.
For all solid cancer PDCs, Capros® showed good to moderate anti-cancer activity. Capros® inhibited Breast Cancer Triple Negative and Her2+ cells by 50% at the 30 ug/ml concentration, and inhibited Glioma and Breast Cancer HR+ cells by 45% and 60% at the 100 ug/ml concentration.
4. Withania somnifera Roots+Leaves (Sensoril®), Aqueous Extract
Table 6 shows the percentage of PDCs inhibited by Sensoril® (±Standard Deviation) in the anti-proliferation assay described above. The results are pictured in
Sensoril® showed very potent activity against AML primary cells, inhibiting ≥50% at 10 ug/ml concentration. Sensoril® did not show much anti-cancer activity for CLL.
Sensoril® inhibited the proliferation of breast cancer “TN” cells by 50% at the 100 ug/ml concentration. Sensoril® did not show substantial anti-cancer activity for glioma or the other breast cancer cells tested.
5. Shilajit (PrimaVie®)
Table 7 shows the percentage of PDCs inhibited by PrimaVie® (±Standard Deviation) in the anti-proliferation assay described above. The results are pictured in
PrimaVie® showed the highest anti-cancer activity against AML primary cells, inhibiting ≥50% at the 30 ug/ml concentration applied to the PDCs. PrimaVie® showed a lower anti-cancer activity for CLL PDCs.
PrimaVie® showed a lower anti-cancer activity for all solid tumor PDCs.
6. Azadirachta indica Leaves+Twigs Extract (PhytoBGS®)
Table 8 shows the percentage of PDCs inhibited by Phyto-BGS® (±Standard Deviation) in the anti-proliferation assay described above. The results are pictured in
PhytoBGS® showed the highest anti-cancer activity against AML primary cells, showing 50% inhibition at the 30 ug/ml concentration applied to the PDCs. PhytoBGS® showed a lower anti-cancer activity for CLL PDCs.
PhytoBGS® showed a lower anti-cancer activity for all solid tumor PDCs.
Also,
Also,
Capros® (100 ug/ml) combined with 3 uM docetaxel inhibited proliferation of HR+ breast cancer cells by 75%, compared with 59% inhibition by docetaxel alone, 60% inhibition by Capros® alone, and 60% inhibition when combined with 3 uM 5-FU. 3 uM 5-FU alone inhibited cell proliferation by 29%.
Ayuric® (100 ug/ml) combined with 3 uM docetaxel inhibited proliferation of HR+ breast cancer cells by 83%, compared with 59% inhibition by docetaxel alone, 54% inhibition by Ayuric® alone, and 71% inhibition when combined with 3 uM 5-FU. 3 uM 5-FU alone inhibited cell proliferation by 29%.
Each of the 3 extracts studied—Ayuflex®, Capros®, Ayuric®—showed better inhibition when used in combination with marketed drugs docetaxel and 5-FU than compared to the marketed drugs alone. Also, when these extracts were tested in the antiproliferative assay in combination with each other, for any combination of 2 of the extracts (Ayuflex® 30 ug/ml, Capros® 100 ug/ml, Ayuric® 100 ug/ml), a very good inhibition of HR+ breast cancer cell proliferation was observed, at greater than 78%. See
As shown in
Overall, all the three extracts—Ayuflex®, Capros®, and Ayuric®—showed strong anti-cancer activity against HR+ breast cancer PDCs. The cells are highly aggressive in culture; the finding and confirmation that each extract can substantially inhibit the PDCs proliferation in significant percentages in 72 hours, alone or in combination, was surprising and unexpected.
As Ayuflex® showed the highest anti-cancer, anti-proliferative activity of the extracts tested in
In contrast, 5-FU in combination with Ayuflex® (30 ug/ml) and Capros® (100 ug/ml), inhibited proliferation by about 24% and 27% respectively, as compared with 18% by 5-FU alone.
Finally, combinations of the three extracts share similarities in their inhibition of Her2+ cells (57%, 47%, 46% respectively for Ayuflex®/Capros®, Ayuflex®/Ayuric®, and Capros®/Ayuric®). With the exception of the combination of Ayuric® and 5-FU, the extracts share similarities in their inhibition of Her2+ cells when used in combination with docetaxel and 5-FU. It is noted that the PDCs used in this experiment were slow-growing and differed in morphology. Also, a new stock of Ayuflex® was prepared for this study, which showed a little less potency than stock used in some previous experiments.
As Ayuflex® showed the highest anti-cancer, anti-proliferative activity of the extracts tested in
As previous testing showed Ayuflex®, Capros®, and Ayuric® shared good activity for inhibiting CLL PDC proliferation in the anti-proliferative assay discussed above, testing with the aim of revealing a dose-response curve and the IC50 of Ayuflex®, Capros®, and Ayuric® for CLL PDCs was performed.
Increasing the ibrutinib concentration to 10 uM increased the inhibition of CLL cell proliferation by ibrutinib alone by about 11%, to 41% inhibition of proliferation. In combination with Ayuflex® (30 ug/ml), Capros® (100 ug/ml) or Ayuric® (100 ug/ml), inhibition of proliferation of CLL cells changed to about 62% (Ayuflex®), 59% (Capros®), and 69% (Ayuric®). The highest level of inhibitory activity toward CLL cells shown in
As Phyto-BGS®, PrimaVie®, and Sensoril® showed the highest anti-cancer, anti-proliferative activity for AML PDCs of the extracts tested in
AML (Acute myeloid leukemia) is one of the most common types of leukemia in adults. AML is an aggressive disease in which numerous myeloblasts are found in the bone marrow and blood of a subject. AML can spread to other parts of the body including the lymph nodes, liver, spleen, central nervous system, and testicles.
Several drugs have been approved to treat AML by the US Food and Drug Administration and are available to subjects in need, however, these drugs may have less than desired efficacy and may produce undesirable side, adverse, or toxic effects. Examples of drugs currently available to treat AML include arsenic trioxide (As2O3), cytarabine, and doxorubicin. Current therapeutic targets for treating AML include inhibitors of FLT3, IDH, histone deacetylase (HDAC), and BCL-2 polo-like kinase (Plk).
Anti-cancer luminescence assays on AML primary cells included incubation with extract for 72 hours, as in Example I. Doxorubicin was used as an assay control. In this Example, AML drugs were used as disease specific controls.
As discussed above in Example I, 6 extracts (Ayuflex®, Capros®, Phyto-BGS®, PrimaVie®, Sensoril®, and Ayuric®) were tested on AML PDC-SB cells (33866, M-4 type, 76 year old subject) at 3 concentrations: 100 ug/ml, 30 ug/ml, and 10 ug/ml. Three extracts (Phyto-BGS®, PrimaVie®, and Sensoril®) showed desirable activity in those tests and were further tested on the same PDCs (patient-derived cells) for determination of their IC50 values and in combination with marketed AML drugs such as cytarabine, doxorubicin, or arsenic trioxide or in combination with other extracts. Sensoril® showed the best and most remarkable anti-cancer anti-proliferative activity alone with 60% inhibition at 10 ug/ml, tested at 3 graded doses, and with an IC50 of about 20 ug/ml (See
The data in Tables 52-59 and
Sensoril® was evaluated at 3 different concentrations (IC75, IC50, and IC25, derived from information such as shown in
In
Sensoril® shows potent anti-cancer anti-proliferative activity against different subtypes of AML, alone and in combination with drugs relevant to AML and APL treatment. Sensoril® may be administered with standard AML drugs, including at a low dose of the AML drugs, to achieve optimal efficacy and lower side effects of the drugs.
Anti-proliferative and anti-cancer activity of extracts of this invention on non-small cell lung cancer cells and colon cancer cells were assessed generally as described in Example I and are discussed below. Terminalia chebula (AyuFlex®) and Terminalia bellerica (Ayuric®) showed significant activity. As noted throughout this application, the below data is intended to describe the present invention but not to be limiting.
Non-small cell lung cancer cells (cell line NCI-H-358) and colon cancer cells (cell line HT-29) were plated in a 384-well plate in amounts of about 1.25 k cells/well. AyuFlex®, Capros®, Phyto-BGS®, Primavie®, Sensoril®, Ayuric®, and Crominex® were added to the cells in concentrations of 100 ug/ml, 30 ug/ml, and 10 ug/ml, in triplicate, and incubated for 72 hours, and CellTiterGlo (Promega Corporation, Wisconsin, USA) used as a detection reagent, as described in Example I. Drugs used to treat non-small cell lung cancer (Standard of Care (SOC) drugs) Docetaxel (10 uM) and Doxorubicin (10 uM) were used as controls, as were drugs used to treat colon cancer (Standard of Care (SOC) drugs) 5-FU (5-fluoro-uracil, 10 uM) and Doxorubicin (10 uM). The IC50 of docetaxel for non-small cell lung cancer H-358 cells has been published as 367 nM (Intl. J. Oncol. 31:241-252 (2007)). The IC50 of 5-FU for colon cancer H-29 cells has been published as 13 uM (J. Surg. Res. 111(1):63-69 (2003)). Wells with untreated cells and with plain media were also included as controls. Plates passed quality control parameters.
Tables 60-62 show data for controls, and
Of the 7 extracts tested and shown in
Tables 63-65 show data for controls and that the data passed quality control parameters.
Of the 7 extracts tested, Ayuric® showed the most potent anti-cancer anti-proliferative activity, providing 80% inhibition of colon cancer cell proliferation at a concentration of 100 ug/ml and 22% inhibition at 30 ug/ml. Ayuflex® inhibited colon cancer cell proliferation by 45% at a 100 ug/ml concentration. The remaining 5 extracts showed little to no effect on this colon cancer cell line.
As discussed with regard to
The anti-cancer activity of AyuFlex®, Capros®, and Ayuric® was further tested on two other glioma PDCs taken from 2-3 different subjects (SB 6129, SB 32833 (55 year old male)). These glioma PDCs had a different mix of cell types (neuronal, astrocytic, dendroglioma). Also, the anti-cancer activity of AyuFlex®, Capros®, and Ayuric® was further tested on existing glioblastoma cell line U87 MG, to allow comparisons with other compositions known in the art. AyuFlex®, Capros®, and/or Ayuric® were incubated with the new PDC lines and the U87 MG cell line for 72 hours, as described in Example I above.
Tables 66-67 show data for controls and that the data passed quality control parameters. Table 68 relates to
In the three independent studies using 3 different gliomas (1 grade III and 2 grade IV gliomas), AyuFlex®, Capros®, and/or Ayuric® alone and in combination exhibited significant anti-cancer effects. Glioblastoma multiforme (GBM) is one of the most challenging brain tumor to treat, as patients generally do not live more than 1-2 years. Temozolomide is one of the only approved treatments, or the only treatment, for gliomas, however, 50-70% of patients treated with temozolomide have been reported as non-responders to temozolomide. Accordingly, the present invention includes treatment with AyuFlex®, Capros®, and/or Ayuric®, or other compositions of the present invention.
As shown in
Inhibition assays were generally run as described for other AML assays above, with 5000 cells per well in 384 well plates. Standard-of-care drugs cytarabine (3 uM), arsenic trioxide (As2O3) (60 uM) and doxorubicin (10 uM) were assayed as controls. Phyto-GBS®, PrimaVie®, and Crominex® were assayed at 100 ug/ml, 30 ug/ml, and 10 ug/ml concentrations, and Sensoril® tested with a 9-point, 2-fold dose response curve (DRC), starting from 200 ug/ml. CellTiterGlo detection reagent was used, as discussed for instance in Example I above, and the 4 compositions were incubated with the cells for 72 hours. Tables 69-70 show data for controls and that the data passed quality control parameters.
As shown in
As shown for instance in
In this Example, AyuFlex®, Capros®, and Ayuric® were each applied to different PDCs than previous tests, TN breast cancer PDCs SB 30750, and tested via the anti-proliferation assay described above. As shown in
Also, AyuFlex®, Capros®, and Ayuric® were tested in the anti-proliferative assay discussed above in the TN Breast Cancer cell line MDAMB-231. AyuFlex®, Capros®, and Ayuric® were incubated for 72 hours and 120 hours. The dose-response efficacy of AyuFlex®, Capros®, and Ayuric® after a 72-hour period of incubation was similar to that observed after a 120-hour period of incubation with the MDAMB-231 cells. Without being bound by theory, possibly this was due to saturation of cell growth at the 120-hour time-point.
Surprisingly, AyuFlex®, Capros®, and Ayuric® extracts showed better activity in both PDCs than in the MDAMB-231 cell line. The MDAMB-231 cell line was established from pleural effusion of a Caucasian female with a metastatic mammary adenocarcinoma 1, and has a high stem cell percentage. The TNBC PDCs used in earlier studies (shown in
In summary, with the results of this Example VI and previous studies described herein, AyuFlex®, Capros®, and Ayuric® extracts demonstrated reproducible anti-cancer effects in 3 independent experiments using two different TNVC cancer patient derived cells.
SHP-77 (small cell lung cancer) cells were incubated for 72 hours with 100 ug/ml, 30 ug/ml, and 10 ug/ml each of AyuFlex®, Capros® Phyto-BGS®, PrimaVie®, Sensoril®, Ayuric®, and Crominex®, and tested according to the anti-proliferative assay described above. Cells were plated at 1.25 k/well in 384 well plates. Standard of care drug docetaxel was also tested at a 10 uM concentration as a control. Anti-proliferative assays were generally performed as discussed above, in triplicate, with detection reagent CellTiterGlo® as discussed for instance in Example I. Tables 77-79 show data for controls and that the data passed quality control parameters.
Docetaxel, a drug currently used for the treatment of small cell lung cancer, exhibited 90% inhibition of cell proliferation at the 10 uM concentration tested. The assay control, doxorubicin, exhibited 93% inhibition at the 10 uM concentration tested.
Of the extracts tested, AyuFlex® and Ayuric® exhibited the most potent anti-cancer effect on the SHP-77 small cell lung cancer cell line, exhibiting 98% inhibition at 100 ug/ml. The anti-cancer activity of the extracts on SHP-77 cells is similar to that of H-358 cells (non-small cell cancer, NSCLC, shown in Example III), with Ayuric® and AyuFlex® also showing the best anti-cancer effect in those cells.
Prostate cancer (PC-3) cells and Ovarian cancer (OVCAR-3) cells were incubated for 72 hours with 100 ug/ml, 30 ug/ml, and 10 ug/ml each of AyuFlex®, Capros® Phyto-BGS®, PrimaVie®, Sensoril®, Ayuric®, and Crominex®, and tested according to the anti-proliferative assay described above. Cells were plated at 1.25 k/well in 384 well plates. Standard of care drug docetaxel and control doxorubicin were each also tested at a 10 uM concentration. Anti-proliferative assays were generally performed as discussed in Example I above, in triplicate, with detection reagent CellTiterGlo. Tables 80-82 show data for controls for assays on prostate cancer (PC-3 cells), and that the data passed quality control parameters. Tables 83-85 show data for controls for assays on ovarian cancer (OVCAR-3 cells), and that the data passed quality control parameters.
With regard to prostate cancer (PC-3) cells, docetaxel, a drug currently used for the treatment of prostate cancer, exhibited 58% inhibition of cell proliferation at the 10 uM concentration tested. The assay control, doxorubicin, exhibited 70% inhibition at the 10 uM concentration tested.
Of the extracts tested, Ayuric® exhibited the most potent anti-cancer effect on the prostate cancer (PC-3) cell line, exhibiting 79% inhibition at 100 ug/ml; followed by AyuFlex®, which exhibited 50% inhibition in the assay at 100 ug/ml.
With regard to ovarian cancer (OVCAR-3) cells, docetaxel, a drug currently used for the treatment of ovarian cancer, exhibited 66% inhibition of cell proliferation at the 10 uM concentration tested. The assay control, doxorubicin, exhibited 76% inhibition at the 10 uM concentration tested.
Of the extracts tested, Ayuric® exhibited the most potent anti-cancer effect on the ovarian cancer cell line, exhibiting 92% inhibition of cell proliferation at 100 ug/ml, and 23% inhibition at 30 ug/ml. The next effective extracts against the proliferation of the OVCAR-3 ovarian cancer cells were AyuFlex® and Sensoril®, exhibiting 46% and 35% inhibition respectively at 100 ug/ml.
Hydroethanolic extracts of Withania somnifera (Sensoril®-AWE) showed significant anti-cancer effects in AML cancer cells of the HL60 cell line. 2500 cells were plated per well in 384 well plates. Standard of care drug doxorubicin was included as a control at 10 uM. 6 different samples of Sensoril®-AWE, prepared with hydroethanolic extraction were tested in the anti-proliferative assay as generally described in Example I above. Extracts were prepared at 2 mg/ml concentrations in view of increased solubility and tested at 400 ug/ml, 2-fold serial dilutions, to prepare an 8-point Dose-Response Curve. CellTiter Glo was used as a detection reagent. Extracts were incubated with the AML HL60 cells for 72 hours. Table 86 provides information on hydroethanolic extract quantification and markers according to this invention. Full analysis of some Samples described below are as follows: Lot #WS09120: 19.34% w/w Withanolide glycosides, 7.52% w/w Withanolide Aglycones (as Withaferin A), 34.42% w/w Oligosaccharides; Lot #WS09820: 13.16% w/w Withanolide glycosides, 2.60% w/w Withanolide Aglycones (as Withaferin A), 26.52% w/w Oligosaccharides; Lot #WS09920: 12.68% w/w Withanolide glycosides, 2.71% w/w Withanolide Aglycones (as Withaferin A), 23.55% w/w Oligosaccharides; Lot #WS10020: 12.20% w/w Withanolide glycosides, 2.39% w/w Withanolide Aglycones (as Withaferin A), 24.64% w/w Oligosaccharides. Tables 87-90 show data for controls and that the data passed quality control parameters.
All hydroethanolic samples of Withania somnifera (Sensoril®-AWE) showed a sigmoidal saturated IC50 dose-response curve with significant inhibition of AML HL60 cell proliferation. The most potent sample was the “Nepal” sample (Sample 1 of Table 91 below, Lot #WS09120), which showed an IC50 of 17.5 ug/ml, with Sample 2, based solely on Withania somnifera leaves, following closely with an IC50 of 19.2 ug/ml. Sample 3, 4, and 6 showed higher IC50 values, ranging from 26-28 ug/ml, and Sample 5 showed lowest activity with an IC50 of 48.7 ug/ml. See Table 91 for further information on Sample preparation and a listing of IC50 s found for each Sample, Tables 92-97 for experimental data relating to the Samples, and
Withania somnifera (Ashwagandha) hydroethanolic extracts (Sensoril ®-AWE) of the present invention
Withania somnifera
Withania somnifera (Sensoril ®-AWE) Hydroethanolic
Withania somnifera (Sensoril ®-AWE) Hydroethanolic Extract Sample 2
Withania somnifera (Sensoril ®-AWE) Hydroethanolic Extract Sample 3
Withania somnifera (Sensoril ®-AWE) Hydroethanolic Extract Sample 4
Withania somnifera (Sensoril ®-AWE) Hydroethanolic Extract Sample 5
Withania somnifera (Sensoril ®-AWE) Hydroethanolic Extract Sample 6
Histiocytic lymphoma (U-937) cells and Pancreatic cancer (Panc-1) cells were incubated for 72 hours with 100 ug/ml, 30 ug/ml, and 10 ug/ml each of fresh samples of AyuFlex®, Capros®, Phyto-BGS®, 2 different samples of Shilajit (PrimaVie®) extracts, several different samples of hydroethanolic Withania somnifera (Sensoril®-AWE) extracts, and Ayuric®. Cells were plated at 1.25 k/well in 384 well plates. Standard of care drug doxorubicin was also tested at a 10 uM concentration. Anti-proliferative assays were generally performed as described in Example I above, in triplicate, with detection reagent CellTiterGlo. Tables 98-99 show data for controls for assays on histiocytic lymphoma (U-937) cells, and that the data passed quality control parameters. Tables 101-102 show data for controls for assays on pancreatic cancer (Panc-1) cells, and that the data passed quality control parameters.
Tables 100 and 103 show data relating to the inhibition of histiocytic lymphoma (U-937) cells and pancreatic cancer (Panc-1) cells by extracts of this invention. The assay is generally as described in Example I.
Compositions of the present invention provide anti-cancer activity, as shown by the remarkable inhibition of cancer cell proliferation by compositions of this invention in the above Examples. Anti-proliferative activity was seen with the present compositions in glioma cancer cells, breast cancer cells, chronic lymphocytic leukemia cancer cells, acute myeloid leukemia cancer cells, small cell lung cancer cells, non-small lung cancer cells, colon cancer cells, prostate cancer cells, pancreatic cancer cells, and ovarian cancer cells. Inhibition was seen with different extracts on difference cancer cell types, sometimes with similar or even synergistic effects with combinations of extracts and/or extract(s) with a known anti-cancer drug. See for instance
For instance, Ayuflex® and Ayuric® inhibited the proliferation of several cancer cell types, including pancreatic, ovarian, prostatic, small cell lung cancer, and colon cancer cells. Ayuflex® and Ayuric® may be administered for instance with standard non-small cell lung cancer and colon cancer drugs, and other drugs useful in treating cancer, including a low dose of said drugs to achieve optimal efficacy and lower side effects of the drugs. In an embodiment, a composition of the present invention may be used with a standard of care cancer drug to minimize the amount of standard of care drug given to a subject. Given the harsh adverse and toxic effects of many known cancer drugs, the present invention provides a useful method for co-administration with such drugs.
Also for instance Sensoril® showed potent anti-cancer and anti-proliferative activity against different subtypes of AML, alone and synergistically in combination with drugs relevant to AML (Acute Myeloid Leukemia) and subtype APL (Acute Promyelocytic Leukemia (APL, APML)) treatment such as arsenic trioxide and doxorubicin. Hydroethanolic Sensoril®-AWE compositions were shown to be effective against AML HL60 cell line cancer cells. Sensoril® or Hydroethanolic Sensoril® (Sensoril®-AWE) may be administered with standard AML drugs, including at a low dose of the AML drugs, to achieve optimal efficacy and lower side effects of the drugs.
Several embodiments of the present invention are set out herein. In addition to the below, embodiments supported by the above Examples are intended as general embodiments of the invention, similar to the below.
A method of inhibiting the proliferation of cancer cells and/or cancer-associated cells comprising the steps of providing a composition comprising an extract of Terminalia chebula fruits, and applying said composition to said cancer cells to inhibit proliferation of the cells. In an embodiment, said composition is a standardized aqueous extract of the Terminalia chebula fruits, and said extract is Ayuflex®. In an embodiment, said cancer cells are glioma cells, breast cancer cells, chronic lymphocytic leukemia cells, acute myeloid leukemia cells, non-small cell lung cancer cells, small cell lung cancer cells, prostate cancer cells, ovarian cancer cells, pancreatic cancer cells, and/or colon cancer cells. In an embodiment, said cancer cells are chronic lymphocytic leukemia cells, glioma cells, prostate cancer cells, ovarian cancer cells, pancreatic cancer cells, breast cancer “HR+” cells, breast cancer “Her2+” cells, or breast cancer “triple negative” cells. A method of treating cancer in a subject in need thereof, and/or enhancing the treatment of cancer in a subject in need thereof, comprising the steps of providing a composition comprising an extract of Terminalia chebula fruits, and administering an effective amount of said composition to treat and/or enhance treatment of cancer in the subject. In an embodiment, said composition is a standardized aqueous extract of the Terminalia chebula fruits, and said extract is Ayuflex®. In an embodiment, said cancer is glioma, breast cancer, chronic lymphocytic leukemia, acute myeloid leukemia, non-small cell lung cancer, prostate cancer cells, ovarian cancer cells, pancreatic cancer cells, and/or colon cancer. In an embodiment, said cancer is chronic lymphocytic leukemia, glioma, prostate cancer cells, ovarian cancer cells, pancreatic cancer cells, breast cancer “HR+”, breast cancer “Her2+”, or breast cancer “triple negative”.
A method of inhibiting the proliferation of cancer cells comprising the steps of providing a composition comprising an extract of Terminalia bellerica fruits, and applying said composition to said cancer cells to inhibit proliferation of the cells. In an embodiment, said composition is a standardized aqueous extract of the Terminalia bellerica fruits, and said extract is Ayuric®. In an embodiment, said cancer cells are glioma cells, breast cancer cells, chronic lymphocytic leukemia cells, acute myeloid leukemia cells, non-small cell lung cancer cells, prostate cancer cells, ovarian cancer cells, pancreatic cancer cells, and/or colon cancer cells. In an embodiment, said cancer cells are chronic lymphocytic leukemia cells, breast cancer “HR+” cells, breast cancer “Her2+” cells, breast cancer “triple negative” cells, acute myeloid leukemia cells, non-small cell lung cancer cells, prostate cancer cells, ovarian cancer cells, pancreatic cancer cells, and/or colon cancer cells. A method of treating cancer in a subject in need thereof, and/or enhancing the treatment of cancer in a subject in need thereof, comprising the steps of providing a composition comprising an extract of Terminalia bellerica fruits, and administering said composition in an effective amount to treat and/or enhance treatment of cancer in the subject. In an embodiment, said composition is a standardized aqueous extract of Terminalia bellerica fruits, and said extract is Ayuric®. In an embodiment, said cancer is glioma, breast cancer, chronic lymphocytic leukemia, acute myeloid leukemia, non-small cell lung cancer, prostate cancer cells, ovarian cancer cells, pancreatic cancer cells, and/or colon cancer. In an embodiment, said cancer is chronic lymphocytic leukemia, breast cancer “HR+”, breast cancer “Her2+”, breast cancer “triple negative”, or acute myeloid leukemia.
A method of inhibiting the proliferation of cancer cells comprising the steps of providing a composition comprising an extract of Phyllanthus emblica fruits, and applying said composition to said cancer cells to inhibit proliferation of the cells. The method of claim 17, wherein said composition is a standardized aqueous extract of Phyllanthus emblica fruits, and said extract is Capros®. In an embodiment, said cancer cells are glioma cells, breast cancer cells, chronic lymphocytic leukemia cells, and/or acute myeloid leukemia cells, non-small cell lung cancer cells, prostate cancer cells, ovarian cancer cells, pancreatic cancer cells, and/or colon cancer cells. In an embodiment, said cancer cells are chronic lymphocytic leukemia cells, breast cancer “Her2+” cells, or breast cancer “triple negative” cells. A method of treating cancer in a subject in need thereof and/or enhancing the treatment of cancer in a subject in need thereof, comprising the steps of providing a composition comprising an extract of Phyllanthus emblica fruits, and administering said composition in an effective amount to treat and/or enhance treatment of cancer in the subject. In an embodiment, said composition is a standardized aqueous extract of said Phyllanthus emblica fruits, and said extract is Capros®. In an embodiment, said cancer is glioma, breast cancer, chronic lymphocytic leukemia, acute myeloid leukemia, non-small cell lung cancer, prostate cancer cells, ovarian cancer cells, pancreatic cancer cells, and/or colon cancer. In an embodiment, said cancer is chronic lymphocytic leukemia, breast cancer “Her2+”, or breast cancer “triple negative.”
A method of inhibiting the proliferation of cancer cells comprising the steps of providing a composition comprising an extract of Withania somnifera leaves, roots, or roots plus leaves, and applying said composition to said cancer cells to inhibit proliferation of the cells. In an embodiment, said composition is a standardized aqueous extract or is a standardized hydroalcoholic extract of said leaves, roots, or roots plus leaves, and said extract is Sensoril®. In an embodiment, said cancer cells are glioma cells, breast cancer cells, chronic lymphocytic leukemia cells, acute myeloid leukemia cells including APL cells, non-small cell lung cancer cells, prostate cancer cells, ovarian cancer cells, pancreatic cancer cells, and/or colon cancer cells. In an embodiment, said cancer cells are acute myeloid leukemia cells or breast cancer “triple negative” cells. A method of treating cancer in a subject in need thereof, and/or enhancing the treatment of cancer in a subject in need thereof, comprising the steps of providing a composition comprising an extract of Withania somnifera leaves, roots, or roots plus leaves, and administering said composition in an effective amount to treat and/or enhance treatment of cancer in the subject. In an embodiment, said composition is a standardized aqueous extract or a standardized hydroalcoholic extract of the leaves, roots, or roots plus leaves, and said extract is Sensoril® or Sensoril®-AWE. In an embodiment, said cancer is glioma, breast cancer, chronic lymphocytic leukemia, acute myeloid leukemia including APL, non-small cell lung cancer, prostate cancer, ovarian cancer, pancreatic cancer, and/or colon cancer. In an embodiment, said cancer is acute myeloid leukemia or breast cancer “triple negative”.
A method of inhibiting the proliferation of cancer cells comprising the steps of providing a composition comprising an extract of Shilajit and applying said composition to said cancer cells to inhibit proliferation of the cells. In an embodiment, said extract is a standardized aqueous extract of Shilajit, and said extract is Primavie®. In an embodiment, said cancer cells are glioma cells, breast cancer cells, chronic lymphocytic leukemia cells, acute myeloid leukemia cells, non-small cell lung cancer cells, and/or colon cancer cells. In an embodiment, said cancer cells are acute myeloid leukemia cells. A method of treating cancer in a subject in need thereof, and/or enhancing the treatment of cancer in a subject in need thereof, comprising the steps of providing a composition comprising an extract of Shilajit, and administering said composition in an effective amount to treat and/or enhance treatment of cancer in the subject. In an embodiment, said extract is a standardized aqueous extract of Shilajit, and said extract is Primavie®. In an embodiment, said cancer is glioma, breast cancer, chronic lymphocytic leukemia, acute myeloid leukemia, non-small cell lung cancer, and/or colon cancer. In an embodiment, said cancer is acute myeloid leukemia.
A method of inhibiting the proliferation of cancer cells comprising the steps of providing a composition comprising an extract of Azadirachta indica leaves and twigs, and applying said composition to said cancer cells to inhibit proliferation of the cells. In an embodiment, said composition is a standardized aqueous extract, and said extract is PhytoBGS®. In an embodiment, said cancer cells are glioma cells, breast cancer cells, chronic lymphocytic leukemia cells, acute myeloid leukemia cells, non-small cell lung cancer cells, and/or colon cancer cells. In an embodiment, said cancer cells are acute myeloid leukemia cells or breast cancer “Her2+” cells. A method of treating cancer in a subject in need thereof, and/or enhancing the treatment of cancer in a subject in need thereof, comprising the steps of providing a composition comprising an extract of Azadirachta indica leaves and twigs, and administering said composition in an effective amount to treat and/or enhance treatment of cancer in the subject. In an embodiment, said composition is a standardized aqueous extract, and said extract is PhytoBGS®. In an embodiment, said cancer is glioma, breast cancer, chronic lymphocytic leukemia, acute myeloid leukemia, non-small cell lung cancer, and/or colon cancer. In an embodiment, said cancer is acute myeloid leukemia or breast cancer “Her2+”.
Other embodiments of this invention include a composition comprising a at least one of Terminalia chebula fruits, Terminalia bellerica fruits, Phyllanthus emblica fruits, Withania somnifera roots and leaves, Shilajit, Azadirachta indica leaves and twigs, or a standardized alcohol-water extract of the leaves, roots, or roots plus leaves of Withania somnifera. In an embodiment, said Terminalia chebula fruit extract is AyuFlex®, said Terminalia bellerica fruit extract is Ayuric®, said Phyllanthus emblica fruit extract is Capros®, said Withania somnifera roots and/or leaves extract is Sensoril®, said Shilajit extract is PrimaVie®, and said Azadirachta indica leaves and twigs extract is PhytoBGS®. In an embodiment, said composition comprises a Terminalia chebula fruit extract and a Terminalia bellerica fruit extract, and optionally where said T. chebula extract is AyuFlex® and said T. bellerica extract is Ayuric®. In an embodiment, said composition comprises a Terminalia chebula fruit extract and a Phyllanthus emblica fruit extract, and optionally where said T. chebula extract is AyuFlex® and said P. emblica extract is Capros®. In an embodiment, said composition comprises a Terminalia chebula fruit extract and a Withania somnifera roots and leaves extract, or a standardized alcohol-water extract of the leaves, roots, or roots plus leaves of Withania somnifera, and optionally where said T. chebula extract is AyuFlex® and said W. somnifera extract is Sensoril®. In an embodiment, said composition comprises a Terminalia chebula fruit extract and a Shilajit extract, and optionally where said T. chebula extract is AyuFlex® and said Shilajit extract is PrimaVie®. In an embodiment, said composition comprises a Terminalia chebula fruit extract and an Azadirachta indica leaves and twigs extract, and optionally where said T. chebula extract is AyuFlex® and said A. indica extract is PhytoBGS®. In an embodiment, said composition comprises a Terminalia bellerica fruit extract and a Phyllanthus emblica fruit extract, and optionally where said T. bellerica extract is Ayuric® and said P. emblica extract is Capros®. In an embodiment, said composition comprises a Terminalia bellerica fruit extract and a Withania somnifera roots and/or leaves extract, and optionally where said T. bellerica extract is Ayuric® and said W. somnifera extract is Sensoril®. In an embodiment, said composition comprises a Terminalia bellerica fruit extract and a Shilajit extract, and optionally where said T. bellerica extract is Ayuric® and said Shilajit extract is PrimaVie®. In an embodiment, said composition comprises a Terminalia bellerica fruit extract and an Azadirachta indica leaves and twigs extract, and optionally where said T. bellerica extract is Ayuric® and said A. indica extract is PhytoBGS®. In an embodiment, said composition comprises a Phyllanthus emblica fruit extract and a Withania somnifera roots and/or leaves extract, and optionally where said P. emblica extract is Capros® and said W. somnifera extract is Sensoril®. In an embodiment, said composition comprises a Phyllanthus emblica fruit extract and a Shilajit extract, and optionally where said P. emblica extract is Capros® and said Shilajit extract is PrimaVie®. In an embodiment, said composition comprises a Phyllanthus emblica fruit extract and an Azadirachta indica leaves and twigs extract, and optionally where said P. emblica extract is Capros® and said A. indica extract is PhytoBGS®. In an embodiment, said composition comprises a Withania somnifera roots and leaves extract and a Shilajit extract, and optionally where said W. somnifera extract is Sensoril® and said Shilajit extract is PrimaVie®. In an embodiment, said composition comprises a Withania somnifera roots and/or leaves extract and an Azadirachta indica leaves and twigs extract, and optionally where said W. somnifera extract is Sensoril® and said A. indica extract is PhytoBGS®. In an embodiment, said composition comprises a Shilajit extract and an Azadirachta indica leaves and twigs extract, and optionally where said Shilajit extract is PrimaVie® and said A. indica extract is PhytoBGS®. In an embodiment, said composition comprising Ayuflex® and Ayuric®. In an embodiment, said composition comprising Ayuflex® and Capros®. In an embodiment, said composition comprising Ayuric® and Capros®. In an embodiment, said composition comprising Ayuflex®, Ayuric®, and Capros®. In an embodiment, said composition comprising Sensoril® and PrimaVie®. In an embodiment, said composition comprising Sensoril® and Phyto-BGS®. In an embodiment, said composition comprising PrimaVie® and Phyto-BGS®. In an embodiment, said composition comprising Sensoril®, PrimaVie®, and Phyto-BGS®. In an embodiment, said composition comprises Crominex+3®. In an embodiment, said composition further comprises an anti-cancer drug; in an embodiment, said anti-cancer drug is pictilisib, doxorubicin, temozolomide, docetaxel, 5-fluorouracil (5-FU), ibrutinib, arsenic trioxide, and/or cytarabine, and/or any other anti-cancer drug, preferably identified in this application. In an embodiment, said composition comprises co-administration with an anti-cancer drug. In an embodiment, the present invention is directed to a method of treating cancer in a subject in need thereof, and/or enhancing the treatment of cancer in a subject in need thereof, comprising the steps of providing a composition comprising at least one extract, optionally a standardized aqueous extract that is Ayuflex®, Ayuric®, Capros®, Sensoril®, PrimaVie®, and/or PhytoBGS® or a hydroalcoholic extract that is Sensoril-AWE or a trivalent chromium complex that is Crominex+3®, and administering said composition in combination with an anti-cancer drug in an effective amount to treat and/or enhance treatment of cancer in the subject. In an embodiment, said anti-cancer drug is pictilisib, doxorubicin, temozolomide, docetaxel, 5-fluorouracil (5-FU), ibrutinib, arsenic trioxide, and/or cytarabine, and/or any other anti-cancer drug identified in this application. In an embodiment, a composition of the present invention comprises a combination of an anti-cancer drug, and in a separate composition, an extract. In an embodiment, the present invention is directed to a method of inhibiting the proliferation of cancer cells comprising the steps of providing a composition comprising at least one extract, preferably a standardized aqueous extract, preferably Ayuflex®, Ayuric®, Capros®, Sensoril®, PrimaVie®, and/or PhytoBGS®, and administering said composition in combination with an anti-proliferation drug in an effective amount to inhibit the proliferation of the cancer cells.
The use of the terms “a,” “an,” “the,” and similar referents in the context of describing the present invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Use of the term “about” is intended to describe values either above or below the stated value in a range of approximately ±20%; in other embodiments, the values may range in value above or below the stated value in a range of approximately ±5%; in other embodiments, the values may range in value above or below the stated value in a range of approximately ±2%; in other embodiments, the values may range in value above or below the stated value in a range of approximately ±1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All method steps described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
While in the foregoing specification the present invention has been described in relation to certain embodiments thereof, and many details have been put forth for the purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202041009290 | Mar 2020 | IN | national |
| Number | Date | Country | |
|---|---|---|---|
| 20210315959 A1 | Oct 2021 | US |
