Title of Invention	METHOD OF POTENTIATING THE ACTIONS OF THERAPEUTIC MONOCLONAL AND POLYCLONAL ANTIBODIES
Abstract	A method of potentiating the action of monoclonal and polyclonal antibodies  against growth factors and their receptors by using in coupling conjugation with polyunsaturated fatty acids (PUF As) in the prevention and/or treatment of cancer. and inflammatory conditions such as rheumatoid arthritis (RA). systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), mixed connective tissue disorder (MCTD), vasculitis; and disorders due to uncontrolled angiogenic activity such as proliferative diabetic retinopathy; other eye disorders such as macular degeneration; skin problems such as psoriasis, renal conditions such as proliferative glomerulonephritis, lymphoma and leukemias. More particularly, the invention is directed to the efficacious use of therapeutic monoclonal and polyclonal antibodies by using them in combination with polyunsaturated fatty acids chosen from linoleic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, alpha- linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, cis-parinaric acid, docosapentaenoic acid and conjugated linoleic acid in predetermined quantities. The invention also provides methods of selectively reducing the blood supply to a neoplastic region, such as a tumor region, by causing selectively occlusion of blood vessels feeding the neoplastic region. The invention also provides solutions of PUF As, or salts of PUF As, in combination with a lymphographic agent.

Title of Invention

METHOD OF POTENTIATING THE ACTIONS OF THERAPEUTIC MONOCLONAL AND POLYCLONAL ANTIBODIES

Abstract

A method of potentiating the action of monoclonal and polyclonal antibodies  against growth factors and their receptors by using in coupling conjugation with polyunsaturated fatty acids (PUF As) in the prevention and/or treatment of cancer. and inflammatory conditions such as rheumatoid arthritis (RA). systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), mixed connective tissue disorder (MCTD), vasculitis; and disorders due to uncontrolled angiogenic activity such as proliferative diabetic retinopathy; other eye disorders such as macular degeneration; skin problems such as psoriasis, renal conditions such as proliferative glomerulonephritis, lymphoma and leukemias. More particularly, the invention is directed to the efficacious use of therapeutic monoclonal and polyclonal antibodies by using them in combination with polyunsaturated fatty acids chosen from linoleic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, alpha- linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, cis-parinaric acid, docosapentaenoic acid and conjugated linoleic acid in predetermined quantities. The invention also provides methods of selectively reducing the blood supply to a neoplastic region, such as a tumor region, by causing selectively occlusion of blood vessels feeding the neoplastic region. The invention also provides solutions of PUF As, or salts of PUF As, in combination with a lymphographic agent.

Full Text	Field oflnvention The invention generally relates to a strategy or method of stabilizing and potentiating thr therapeutic actions of monoclonal and polyclonal ^antibodies against various growth factors and other proteins by polyunsaturated fatty acids (PUFAs) and their use thereof in the prevention and/or treatment of various cancers, and inflammatory conditions such as atherosclerosis, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), mixed connective tissue disorder (MCTD), vasculitis; and other disorders caused by uncontrolled angiogenic activity such as proliferative diabetic retinopathy; other eye disorders such as macular degeneration; and skin problems such as psoriasis, and various forms of proliferative glomerulonephritis and other disorders in which cell proliferation and angiogenesis plays a dominant role. More particularly, the invention is directed to the efficacious use of therapeutic monoclonal and polyclonal antibodies. Background of the Invention Growth factors and cancer Growth factors are proteins secreted by several types of cells in the body that have potent actions on cell proliferation, migration, and differentiation. These growth factors bind to their respective receptors that in turn lead to the activation of transmembrane receptor tyrosine kinases. Growth factors that are particularly relevant to the study of cancer are: epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF'), hepatocytes growth factor (HGF), placental growth factor (PIGF), and tyrosine kinase receptor erbB2, also known in humans as Her2. These growth factors also stimulate angiogenesis that facilitates tumor growth and metastasis. Thus, inhibition of the action of these growth factors or blocking the expression of their receptors interferes with angiogenesis and suppresses tumor growth and metastasis. These growth factors are believed to have a play a role in many inflammatory conditions such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) also called as lupus, scleroderma, vasculitis, and other collagen vascular diseases; and in atherosclerosis, glomerulonephritis both in primary and secondary types of nephritis, psoriasis, and in Alzheimer's diseases, and proliferative disorders seen in diabetes niellitus (DM) such as diabetic retinopathy, vasculopathy, nephropathy, neuropathy, and atherosclerosis. In addition, it was also observed that some of the complications such as retinopathy, neuropathy, nephropathy, etc., seen in diabetes mellitus are due to enhanced production of various growth factors such as EGF, VEGF, and IGF (insulin-like growth factor in the target tissues (1-3). Epidermal Growth Factor (EOF) Studies showed that EGF stimulates cells to divide by activating members of the EGFR (EGF receptor), EGFR activation also plays an important role in tumor survival. Several types of human cancer present sustained activation of EGFRs by secreted growth factors. Amplification and rearrangement of the gene encoding EGFR occur in a significant fraction of glioblastomas and squamous-cell carcinomas and correlate with reduced patient survival. Similarly, amplification of the gene encoding HER2 is associated with shorter time of breast cancer relapse, reduced overall patient survival and resistance to hormonal therapy and chemotherapy. Consistent with their pivotal role in stimulating cell proliferation, blocking EGFR function results in retarded tumor growth. Examples include the clinically approved anti-HER2 monoclonal antibody trastuzumab (Herceptin®), similar antagonistjs of the EGFR such as the monoclonal antibody cetuximab, and low-molecular weight inhibitors of tyrosine kinases. Erbitux® is a chimeric monoclonal antibody specific for the EGFR. Over expression of EGFR is common in many solid tumors, such as colorectal and lung carcinomas as well as cancers of the head and neck. It correlates with increased metastasis, decreased survival and a poor prognosis. EGFR protects malignant tumor cells from the cytotoxic effects of chemotherapy and radiotherapy, making these treatments less effective. Erbitux^ binds to the extracellular domain of EGFR on the tumor cell, thereby inhibiting receptor-associated tyrosine kinase. This inhibition blocks the intracellular pathways associated with tumor cell proliferation, so preventing tumor growth and dissemination as well as inducing tumor cell death or apoptosis. There is evidence to suggest that EGF and EGFR have angiogenic actions and that Erbitux^ prevents angiogenesis (4-6). Vascular Endothelial Growth Factor (VEGF) VEGF is a protein that is secreted by hypoxic cells, including those that are cancerous. VEGF stimulates new blood vessel formation or angiogenesis by binding to specific receptors on nearby blood vessels to stimulate extensions of existing blood vessels. Angiogenesis plays an important role in both tumor growth and metastasis. Monoclonal antibodies designed to bind to VEGF preventing it from binding to its receptors and therefore potentially inhibiting tumor growth. Bevacizumab is a humanized monoclonal antibody to VEGF developed by Genentech and is called as Avastin®. By inhibiting VEGF, Avastin interferes with the blood supply to tumors, a process that is critical to tumor growth and metastasis. Several clinical studies showed that both Erbitux'' and Avastin'^\ which are humanized monoclonal antibodies to EGFR and VEGF respectively, are useful in the treatment of colon cancer. Erbitux® shrank tumors in 22.9% of advanced colon cancer patients when combined with chemotherapy. Avastin^ in combination with chemotherapy extended colon cancer patients' lives by 5 months in a trial of 900 patients. EGF and VEGF are mentioned here only as examples on the role of various growth factors and their receptors in cancer. Several studies indicated that blocking or neutralizing the actions of various growth factors and their receptors suppresses cancer (7-11). Polyunsaturated fatty acids « (( The polyunsaturated fatty acids (PUFAs) are fatty acids having at least two carbon-to-carbon double bonds in a hydrophobic hydrocarbon chain, which typically includes X-Y carbon atoms and terminates in a carboxylic acid group. The PUFAs are classified in accordance with a short hand nomenclature, which designates the number of carbon atoms present (chain length), the number of double bonds in the chain and the position of the double bonds nearest to the terminal methyl group. The notation a:b" is used to denote the chain length and number of double bonds, and the notation n:x" is used to describe the position of the double bond nearest to the methyl group. There are 4 independent families of PUFAs, depending on the parent fatty acid from which they are synthesized. They are: (1) The "n-3" series derived from alpha-linolenic acid (ALA, 18:3, n-3). (2) The "n-6" series derived from cis-linoleic acid (LA, 18:2, n-6). (3) The "n-9" series derived from oleic acid (OA, 18:1, n-9). (4) The 'n-7" series derived from palmitoleic acid (PA, 16:1, n-7). The mammals cannot synthesize the parent fatty acids of the n-3 and n-6 series, and hence they are often referred to as "essential fatty acids" (EFAs). Because these compounds are necessary for normal health but cannot be synthesized by the human body, they must be obtained through the diet (12, 13). It is believed that both LA and ALA are metabolized by the same set of enzymes. LA is converted to gamma-linolenic acid (GLA, 18:3, n-6) by the action of the enzyme delta-6-desaturase (d-6-d) and GLA is elongated to form dihomo-GLA (DGLA, 20:3^-6), the precursor of the 1 series of prostaglandins (PGs). DGLA can also be converted to arachidonic acid (AA, 20:4, n-6) by the action of the enzyme delta-5-desaturase (d-5-d). AA forms the precursor of 2 series of prostaglandins, thromboxanes and the 4 series of leukotrienes. ALA is converted to eicosapentaenoic acid (EPA, 20:5, n-3) by d-6-d and d-5-d. EPA forms the precursor of the 3 series of prostaglandins and the 5 series of leukotrienes. LA, GLA, DGLA, AA, ALA, EPA and docosahexaenoic acid (DHA, 22:6, n-3) are all PUFAs, but only LA and ALA are EFAs (see Figure 1 for metabolism of essential fatty acids). Several studies showed that EFAs/PUFAs play a significant role in the pathobiology of inflammatory conditions such as RA, SLE, PSS; skin disorders such as psoriasis, eczema, atopic and non-atopic dermatitis; atherosclerosis, and cancer (13-16). Further, various PUFAs have also been shown to be of bene tit in all these conditions (13-16). PUFAs and Cancer Tumor cells are not only deficient in PUFAs but also have low rate(s) of lipid peroxidation, contain relatively large amounts of antioxidants such as vitamin E and superoxide dismutase (SOD). It is also believed that low rates of lipid peroxidation and consequent low amounts of lipid peroxides in the cells can contribute to an increase in the mitotic process which ultimately leads to an increase in cell proliferation. Thus, a deficiency of PUFAs, high amounts of antioxidants and the presence of low amounts of lipid peroxides in the tumor cells can contribute to the growth of tumor cells. This is supported by studies by the inventor wherein it was noted that PUFAs such as GLA, DGLA, AA, EPA and DHA could decrease tumor cell proliferation. In addition, it was also observed that when apprbpriate amounts of GLA, DGLA, AA, EPA and DHA were administered to tumor cells and normal cells. obtained from American Type Culture Collection, only tumor cells were killed without having any significant action on the survival of normal cells in vitro. In mixed culture experiments, in which both normal and tumor cells were grown together, GLA showed more selective tumoricidal action compared to AA, EPA and DHA though, these latter fatty acids were also effective to some extent. This indicated that selective delivery of GLA, DGLA, AA, EPA and DHA to tumor cells might offer a new therapeutic approach in the treatment of cancer (17-22). These in vitro results are supported by in vivo studies performed in animal tumor models. For example, it was noted that GLA, DGLA, AA, EPA and DHA when used either in the form of pure fatty acid alone or in the form of fatty acid rich oils could inhibit the growth of skin papilloma in mice, formation and growth of hepatoma in rats and ascitic tumor cells in the peritoneum of experimental animals. These results indicate that these fatty acids can inhibit the growth of a variety of tumors even in vivo. In further studies, it was noted that these fatty acids are able to enhance free radical generation and the lipid peroxidation process selectively in the tumor cells but not so much in the normal cells and thus, are able to bring about their cancer killing action (23-28). Inventor's studies have shown that PUFAs can be exploited as possible anti-cancer agents either alone or in combination with traditional anti-cancer drugs. In a series of investigations by the inventor, it was also observed that the cytotoxic action of anti-cancer drugs such as doxorubicin, vincristine and cis-platinum can be augmented by various PUFAs such as GLA, DGLA, AA, EPA and DHA. In addition, these fatty acids could also enhance the cellular uptake of these anti-cancer drugs by the tumor cells and thus, are able to potentiate the anti-cancer actions of these drugs (27). It is to be noted in this context that PUFAs can bind to albumin and other proteins and hence, if given intravenously may not be available to be taken up by the tumor cells and consequently may not be able to bring about their cell killing action on the tumor cells. In view of this, it is desirable that PUFAs including GLA should be delivered to the patients in such a manner that it is easily available to the tumor (tumor cells) and is delivered selectively to the tumor cells. It is highly desirable that PUFAs including GLA be given intra-tumorally as was experimentally done in the case of human gliomas, or, intra-arterially by selective intra-arterial infusion as was done experimentally in the case of hepatoma and giant cell tumor of the bone. But, it is also possible that in some cases of cancer such as Hodgkin's and non-Hodgkin's lymphoma wherein the tumor cells are extremely sensitive to the cytotoxic actions of PUFAs, even oral administration may be sufficient as was observed in certain patients. Since, PUFAs can potentiate the cell killing effect of anti-cancer drugs and cytokines, it is desirable to administer a combination of PUFAs, anti-cancer drugs, or specific monoclonal or polyclonal antibodies to growth factors or a combination thereof so that fatty acids are carried to the tumor cells. The present invention in one aspect resides in a method of selectively delivering PUFAs that are toxic to cancer cells by conjugating thehi or mixing them with monoclonal antibodies or specific polyclonal antibodies to growth factors that are known to enhance tumor growth such that tumoricidal PUFAs are delivered to the cancer cells very selectively. In addition, such a combination of PUFAs and monoclonal and/or specific polyclonal antibodies to growth factors will also inhibit blood supply to a tumor such that ultimately the tumor cells will die since by using two types of substances: one a lipid and the other a protein or a peptide both of which have very potent anti-angiogenic action. SUMMARY OF THE INVENTION All the above factors and observations attest to the fact that malignant tumors are dependent on various growth factors for their growth and are also angiogenesis-dependent diseases. Growth factors also behave as angiogenic factors and thus, promote tumor growth. But, it should be mentioned here that tumor-associated angiogenesis is a complex, multi-step process which can be controlled by both positive and negative factors. It appears, as though, angiogenesis is necessary, but not sufficient, as the single event for tumor growth (29). But, it is evident from several experimental results that angiogenesis may be a common pathway for tumor growth and progression. Though several anti-angiogenic agents, antibodies to growth factors are being tried to arrest tumor growth, these are not without problems. Since the majority of these agents are proteins/peptides, their long-term use may lead to the development of antibodies, which can neutralize their action. These anti-angiogenic and anti-growth substances need to be given repeatedly and some of them are unstable and are difficult to produce in large amounts. In view of this, it is desirable and necessary to ntake efforts to stabilize and potentiate the actions of,known anti-angiogenic molecules, and antibodies against various growth factors. The present invention teaches the efficacious use of anti-growth factor and anti-angiogenic substances, which can inhibit endothelial cell proliferation, growth of tumor cells and coupling them to cis-unsaturated fatty acids, which also have anti-angiogenic and cytotoxic actions on tumor cells, such that the actions of these substances are potentiated by each other. Further, as angiogenesis is involved in other disease processes such as inflammation, tumor metastasis, etc., it is envisaged that the conjugate(s) of anti-angiogenic substances and PUFAs will be useful in these diseases also. In this context, it is important to note that the inventor has found that polyunsaturated fatty acids (PUFAs) such as gamma-linolenic acid (GLA), dihomo-GLA (DGLA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can selectively kill the tumor cells (as described above) and under specific conditions and in conjugation with salts such as lithium and a lymphographic agent these fatty acids can actually behave as anti-angiogenic substances, i.e. they block all the blood supply to the tumor and also prevent generation of new blood vessels. Using these fatty acids in this particular combination, the inventor has successfully treated human hepatocellular carcinoma and giant cell tumor of bone with few or no side effects. Described hereinafter is a novel combination of a protein and a lipid and method(s) foriJs use. The protein referred to herein is a potent and specific antagonist of growth factor(s) and inhibitor of endothelial proliferation and angiogenesis. The lipid may be one or more of the polyunsaturated fatty acids: LA (linoleic acid), GLA, DGLA, AA, ALA (alpha-linolenic acid), EPA, DHA and cis-parinaric acid. In this instance or method the polyunsaturated fatty acid need to be given only once or at the most twice within a period of 1 to 2 months. This invention teaches that unlike anti-growth factor antibodies, these fatty acids are not only cytotoxic to the tumor cells but are also able to function as anti-angiogenic agents. Further, polyunsaturated fatty acids when given in the formulated form, are more potent than monoclonal antibodies or polyclonal antibodies against growth factors in their anti-angiogenic and anticancer actions. The invention in one aspect teaches a method of killing tumor cells by using a combination of monoclonal antibodies /polyclonal antibodies against growth factors that have been conjugated to polyunsaturated fatty acids to cause necrosis or apoptosis of tumor cells. The invention also provides a method of causing anti-angiogenic action in the tumor region with the result that new blood vessels and collaterals are not formed to sustain the tumor. The present invention in another aspect tackles the issue of drug delivery to the target tissue and provides the most efficacious method of administering an admixture of selected PUFAs with other elements such as anti-growth factor antibodies as will be described hereinafter. The invention in yet another aspect teaches a method of regressing the tumor /or interrupting blood supply to the tumor using a pre-determined admixture of at least I a PUFA and an anti-growth factor antibody to produce necrosis with very desirable results. Both the PUFAs and anti-growth factor antibodies being similar in function, the invention also provides a method of causing anti-tumor and anti-angiogenic action in the tumor region with the result that new blood vessels and collaterals are not formed to sustain the tumor in the tumor region treated according to the invention. The present invention in another aspect tackles the issue of drug delivery to the target tissue and provides the most efficacious method of administering an admixture of selected PUFAs along with an anti-growth factor antibody as will be described hereinafter. Modification of monoclonal and polyclonal antibodies with EFAs/PUFAs/LCPUFAs Mixing or conjugating specific monoclonal and polyclonal antibodies such as Erbitux® and Avastin® with EFAs/PUFAs such that they form stable complexes. The conjugation between monoclonal antibodies of EGFR and VEGF and EFAs/PUFAs can be covaient bond preferably is an amide bond, which survives the conditions in the stomach. Such anti-EGFR antibody-EFAs/PUFAs complex and anti-VEGF antibody-EFAs/PUFAs complex will be stable without interfering the actions of monoclonal antibodies of EGFR and VEGF such that they will be able to suppress tumor cell proliferation, tumor metastasis and angiogenesis. These anti-EGFR antibody-EFAs/PUFAs and anti-VEGF antibody-EFAs/PUFAs complexes can be given orally, parentarally (including but not limited to subcutaneoirs, intravenous, intra-arterial, rectal, submucosal) aqd as aerosols for administration through nose, mouth and intra-tracheal routes. These preparations could also be given rectally. It is expected that these anti-EGFR antibody-EFAs/PUFAs and anti-VEGF antibody-EFAs/PUFAs complexes will have significant inhibitory action on tumor cell proliferation and inhibit angiogenesis and also occlude specifically tumor feeding blood vessels irrespective of the route of administration. The ratio between anti-EGFR antibody and EFAs/PUFAs can vary from 1:1 to 1:1000 and 1:1 to 1000:1. The anti-EGFR and anti-VEGF antibodies can be conjugated with any one or a combination of fatty acids. For example, anti-EGFR antibody can be conjugated with LA, GLA, DGLA, AA, ALA, EPA and/or DHA and similarly anti-VEGF antibody is conjugated with LA, GLA, DGLA, AA, ALA, EPA and/or DHA. In certain instances, EFAs/PUFAs may be conjugated with both anti-EGFR and anti-VEGF antibodies simultaneously. The EFAs/PUFAs can be in the form of pure acid, sodium salt, lithium salt, meglumine salt, magnesium salt, iodized salt and/or any other type of stable salt. For intra-arterial injection the preferred conjugate is in the form of an amide or complex between Li-LA, Li-GLA, Li-DGLA, Li-AA, Li-ALA,Li-EPA or Li-DHA and iodized oily lymphographic oil such as Lipiodol, and anti-EGFR antibody and/or anti-VEGF antibody. The amount of these anti-EGFR and anti-VEGF antibodies to be given orally or parentarally can vary from I mg per dose to 100 gm. These anti-EGFR antibody-EFAs/PUFAs complexes can be given daily as a single injection or as a continuous infusion in a day and/or daily for a period of one week or as frequently as needed depending on the response. Administration of these complexes can be repeated daily, weekly or monthly as the situation demands. In the same fashion as described for anti-EGF and anti-VEGF antibodies and their conjugation with various EFAs/PUFAs, similar complexes can also be prepared between anti-TNF-a, anti-IL-1, anti-IL-2, anti-IL-6, etc., antibodies and various EFAs/PUFAs. Summary of the Invention All the above factors and observations attest to the fact that both EFAs/PUFAs and growth factors have important roles in many clinical conditions. In view of the significant role of growth factors in the growth and progression of cancer including metastasis, several attempts have been made and are being made to develop specific monoclonal and polyclonal antibodies that specifically inhibit or block the actions of these growth factors. Such attempts are expected to inhibit the growth of the tumor cells. It is known that these monoclonal and polyclonal antibodies against various growth factors also inhibit angiogenesis that ultimately leads to the regression of the tumors. But, it should be mentioned here that in majority of the instances these monoclonal and polyclonal antibodies against various growth factors have not been very effective in inhibiting the growth of various tumors. The best examples of this failure on the part of monoclonal and polyclonal antibodies developed against various growth factors to effectively prevent the growth of various tumors are: Erbitux® and Avastin'^, which are humanized monoclonal antibodies to EGFR and VEGF respectively. Brbitux** shrank tumors in only 22.9% of advanced cplon cancer patients when combined with chemotherapy, whereas Avastin"® in combination with chemotherapy extended colon cancer patients' lives only by 5 months in a trial of 900 patients. Furthermore, both Erbitux® and Avastin® have many side effects. This clearly shows that monoclonal and polyclonal antibodies developed against various growth factors are not very effective in preventing the growth of tumors and are ineffective in preventing angiogenesis. In the same manner other monoclonal and polyclonal antibodies developed against several other growth factors also failed to be effective in the treatment of other diseases such as psoriasis, inflammatory conditions such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), mixed connective tissue disorder (MCTD), vasculitis; and other disorders caused by uncontrolled angiogenic activity such as proliferative diabetic retinopathy; other eye disorders such as macular degeneration; and skin problems such as psoriasis, various forms of proliferative glomerulonephritis, and other disorders in which cell proliferation and angiogenesis plays a dominant role. It may be mentioned here that specific monoclonal and polyclonal antibodies are also being developed against various receptors on the cell surfaces and other markers with the hope that these antibodies will be useful in the management of various clinical conditions mentioned above. The present invention specifically teaches the efficacious use of monoclonal and polyclonal antibodies developed against various growth factors, receptors, and other cell surface and intracellular markers and proteins by coupling them to PUFAs such that the actions of these antibodies are potentiated. It is suggested that the beneficial actions of compounds formed as a result of such coupling of monoclonal and polyclonal antibodies to PUFAs will be more than the sum effect seen when these antibodies and PUFAs are given separately. The monoclonal and polyclonal antibodies referred to herein is a growth factor(s), their receptor(s), cell surface receptors or markers, and intracellular proteins that have various physiological and pathological actions. The lipid may be one or more of the PUFAs: LA, GLA, DGLA, AA, ALA, EPA and DHA. The objective of the invention is to provide a covalently coupled or complex containing a monoclonal and polyclonal antibody (dies) and one or more of PUFAs containing between 16 and 26 carbon atoms. Another objective of the invention is to provide pharmacological compositions comprising amides of the PUFAs combined with a monoclonal antibody or polyclonal antibody against growth factors and various proteins (either extracellular or intracellular) such that it is stable enough to pass through the acidic environment of the stomach, in the blood stream, and also enter the brain crossing the blood brain barrier. Another objective of the invention is to provide an amide derivative of monoclonal and polyclonal antibodies against various growth factors with biological activities useful in many clinical conditions that have been enumerated above. It should be mentioned here that PUFAs are not used as carriers (though they may serve as carriers under certain circumstances when combined, complexed or covalently linked to/ with the antibodies) but themselves serve as effective agents to treat the clinical condition in question and also potentiate the actions of various monoclonal and polyclonal antibodies. The compounds of the invention containing a monoclonal and polyclonal antibody (dies) against a growth factor(s) and its receptor(s), and various intra and extracellular proteins and one or more of PUFAs can be prepared in pharmaceutical preparations containing the compounds themselves or their suitable derivatives in appropriate or suitable proportions. Administration may be made by any method, which allows the compound (containing the monoclonal and polyclonal antibody (dies) and one or more of PUFAs) to reach the site of desired action including brain. The-compound(s) can be administered orally in the form of dragees, tablets, and syrups or by inhalation or ampules. When compounds are administered rectally the composition can be in the form of a suppository. When the compounds of the invention are to be administered by topical application for instance for various skin conditions, they can be in the form of pomade or a gel. Another example of preparation can be as an intra-tumoral preparation in appropriate doses for the treatment of human brain gliomas or any other accessible tumor (eg. urinary bladder cancer, carcinoma of the esophagus, carcinoma of the lung, breast cancer, etc.) by any route by using flexible fiber optic scopes such as bronchoscope, etc. Another example of administration of the preparation can be as selective intra-arterial infusion or injection into the tumor-feeding vessel by femoral, brachial or carotid routes or any other suitable route or in a combination of routes or any other suitable agent all in a mixture or in conjugated form(s) (like GLA or lithium GLA+ monoclonal and polyclonal antibody (dies), LA/GLA/DGLA/AA/ALA/ EPA/DHA/cis-parinaric acid/docosapentaenoic acid or their salts including lithium salts all individually or in combination thereof-- mjonoclonal and polyclonal antibody (dies), LA/GLA/DGLA/AA/ALA/ EPA/DHA/cis-parinaric acid/docosapentaenoic acid or their salts including lithium salt in combination with or conjugated to monoclonal and polyclonal antibody (dies), which show selective occlusion of tumor feeding vessels and/or anti-angiogenic actions and anti-cancer action. Further the compound(s) can be delivered using suitable devices, or a slow releasing capsule/tablet at an appropriate site or organ of the body. This preparation can be administered daily, weekly, or monthly or at various intervals as deemed appropriate. This preparation can be given as an intravenous infusion continuously or intermittently in a day, daily, weekly or monthly or several times a day or as frequently as necessary depending on the necessity. It is also within the purview of this invention, as stated supra to administer an admixture of PUFAs, anti-cancer drugs, and selected anti-growth factor, and anti-angiogenic substance(s) at the same time, administering predetermined doses of PUFAs orally. All such variations are envisaged to be within the ambit of this invention. Application to mammals: Even though the examples described supra relate to humans, it is envisaged that the method of suppressing or reducing the growth of the cancer and/or anti-angiogenic action using admixture of this invention including an anti-growth faclor(s)/anti-cytokine(s) antibodies and/or angiogenic substances are equally applicable to other mammals. Equivalents While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. For example, anti-growth factors referred to herein include EGF and VEGF but other agents with actions on cancer cells and angiogenesis. Also sodium and potassium salts are considered equivalents of each other. 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Exp Cell Res 2004; 300:379-387. 12..Das Ut^, et al. Clinical significance of essential fatty acidsi Nutrition 1988; 4: 13. Das UN. Essential fatty acids: Biology and their clinical implications. Asian Pacific J Pharmacol 1991;6:317-330. 14. Das UN. Tumoricida! action of cis-unsaturated fatty acids and its relationship to free radicals and lipid peroxidation. Cancer Lett 1991; 56: 235-243. 15. Das UN. Beneficial action(s) of eicosapentaenoic acid/docosahexaenoic acid and nitric oxide in systemic lupus erythematosus. Med Sci Res. 1995; 23: 723-726. 16. Das UN, Mohan IK, Raju TR. Effect of corticosteroids and eicosapentaenoic ■ acid/docosahexaenoic acid on pro-oxidant and anti-oxidant status and metabolism of essential fatty acids in patients with glomerular disorders. Prostaglandins Leukotrienes Essen Fatty Acids 2001; 65: 197-203. 17. Begin ME, Das UN, et al. Selective killing of tumor cells by polyunsaturated fatty acids. Prostaglandins Leukotrienes Med 1985; 19: 177. 18. Begin ME, Ells G. Das UN. Horrobin DF. Differential killing of human carcinoma cells supplemented with n-3 and n-6 polyunsaturated fatty acids. J Natl Cancer Inst 1986; 77: 105. 19. Begin ME, Das UN, Ells G. Cytotoxic effects of essential fatty acids (EFA) in mixed cultures of normal and malignant human cells. Prog Lipid Res 1986; 25:573. 20. Das UN, et al. Polyunsaturated fatty acids augment free radical generation in tumor cells in vitro. Biochem Biophys Res Commun 1987; 145: 15. 21. Das UN. Gamma-linolenic acid, arachidonic acid and eicosapentaenoic acid as potential anti-cancer drugs. Nutrition 1990;6:429-434. 22. Das UN. Tumoricidal action of cis-unsaturated fatty acids and its relationship to free radicals and lipid peroxidation. Cancer Lett 1991; 56: 235-243. 23. Ramesh G and Das UN, Effect of dietary fat on diethylnitrosamine induced hepatocarcinogenesis in Wistar rats. Cancer Lett 1995; 95: 237-245. 24. Ramesh G and Das UN. Effect of free fatty acids on two-stage skin carcinogenesis in mice. Cancer Lett 1996; 100: 199-209. 25. Ramesh G and Das UN. Effect of cis-unsaturated fatty acids on Meth-A ascitic tumor cells in vitro and in vivo. Cancer Letters 1998; 123: 207-214. 26. Das UN, Padma M. Sangeetha P, et aL Stimulation of free radical generation in human leukocytes by various stimulants including tumor necrosis factor is a calmodulin dependent process. Biochem Biophys Res Commun 1990; 167: 1030. 27. Sangeetha PS and Das UN. Gamma-linolenic acid and eicosapentaenoic acid potentiate the cytotoxicity of anti-cancer drugs on human cervical carcinoma (HeLa) cells in vitro. Med Sci Res 1993;21:457-459. 28. Madhavi N and Das UN. Reversal of KB-3-1 and KB-Ch-8-5 tumor cell drug-resistance by cis-unsaturated fatty acids in vitro. Med Sci Res 1994; 22: 689- 692. 29. Chen Q R, Kumar D, Stass S A, Mixson A J. Liposomes complexed to plasm ids encoding angiostatin and endostatin inhibit breast cancer in nude mice. Cancer Res 1999;59:3308-3312. I claim: 1. A drug comprising, a monoclonal or specific polyclonal antibody to growth factors and/or their specific receptors conjugated using a water-soluble synthetic polymers or covalently coupled to a straight-chained fatty acid molecule containing 18 to 22 carbon atoms, wherein the antibodies are directed against growth factors, their receptors selected from, but not limited to, EGF (epidermal growth factor), VEGF (vascular endothelial growth factor), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), hepatocytes growth factor (HGF), placental growth factor (PIGF), and tyrosine kinase receptor erbB2, also known in humans as Her2, wherein the fatty acid molecule is selected from the group consisting of CI 8:2, C18:3, C20:3, C20:4, C20:5, C22:5, C22:6, cis-parinaric acid, conjugated linoleic acid and in the form of a salt selected from the group consisting of a lithium salt, a sodium salt, a potassium salt, a magnesium salt, a calcium salt, a manganese salt, an iron salt, a copper salt, an aluminum salt, a zinc salt, a chromium salt, a cobalt salt, a nickel salt and an iodide and/or in the form of a fatty acid derivative selected from the group consisting of glycerides, esters, free acids, amides, phospholipids and salts. 2. A pharmaceutical preparation comprising the drug of claim I and a pharmaceutically acceptable carrier. 3. An oral and parentaral composition of claim 1 wherein the weight ratio of antibody to growth factor(s) and/or growth factor receptor(s) to fatty acid in the composition and the weight ratio of antibody to growth factor and/or its receptor to fatty acid ranges from 1:10 to 10:1. 4. An oral composition of claim 1, wherein the quantity of antibody to growth factor(s) and/or growth factor receptor(s) varies from 0.5 mg to 500 gm and that of fatty acid ranges from 0.5 mg to 500 gm. 5. A parentaral preparation of claim 1 that is sterile and injectable administered subcutaneously, intravenously, intramuscularly or intra-arterially and additionally comprising an osmolyte and in a buffer at pH from 5 to 8. 6. A parentaral composition of claim 7 that is administered as a single injection or as a continuous infusion. 7. A parentaral composition of claim 6 wherein the quantity of antibody to growth factor(s) and/or growth factor receptor(s) varies from 0.5 mg to 500 gm and that of fatty acid ranges from 0.5 mg to 500 gm additionally comprising an osmolyte and a stabilizer. 8. A pharmaceutical composition comprising the drug of claim 1 and a pharmaceutically acceptable carrier for treating proliferative diseases associated with angiogenesis. 9. A method of treating diseases as in claim 8, wherein the disease is a malignant tumor. 10. A method in claim 9, wherein the malignant tumor is cancer selected from hepatoma, bronchogenic cancer of the lung, colon cancer, breast cancer, ovarian cancer, cancer of the kidney, skin cancer, Kaposi's sarcoma, cancer of the esophagus, cancer of the stomach, leukemias, lymphomas, multiple myeloma. 11. A method in claim 10, wherein the drug is administered as an injection, continuous infusion, orally or by inhalation. 12. A method of treating diseases associated with inflammation using the composition of drug as in claim 1. 13. A method in claim 12, wherein the diseases associated with inflammation are rheumatoid arthritis, lupus, progressive systemic sclerosis, mixed connective tissue disease, vasculitis, proliferative glomerulonephritis, psoriasis, diabetic retinopathy, and macular degeneration. 14. A method in claim 13 of treating diseases associated inflammation, wherein the drug is administered as an injection, continuous infusion, orally or by inhalation.

Full Text

Field oflnvention
The invention generally relates to a strategy or method of stabilizing and potentiating thr therapeutic actions of monoclonal and polyclonal ^antibodies against various growth factors and other proteins by polyunsaturated fatty acids (PUFAs) and their use thereof in the prevention and/or treatment of various cancers, and inflammatory conditions such as atherosclerosis, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), mixed connective tissue disorder (MCTD), vasculitis; and other disorders caused by uncontrolled angiogenic activity such as proliferative diabetic retinopathy; other eye disorders such as macular degeneration; and skin problems such as psoriasis, and various forms of proliferative glomerulonephritis and other disorders in which cell proliferation and angiogenesis plays a dominant role. More particularly, the invention is directed to the efficacious use of therapeutic monoclonal and polyclonal antibodies.
Background of the Invention
Growth factors and cancer
Growth factors are proteins secreted by several types of cells in the body that have potent actions on cell proliferation, migration, and differentiation. These growth factors bind to their respective receptors that in turn lead to the activation of transmembrane receptor tyrosine kinases. Growth factors that are particularly relevant to the study of cancer are: epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF'), hepatocytes growth factor (HGF), placental growth factor (PIGF), and tyrosine kinase receptor erbB2, also known in humans as Her2. These growth factors

also stimulate angiogenesis that facilitates tumor growth and metastasis. Thus, inhibition of the action of these growth factors or blocking the expression of their receptors interferes with angiogenesis and suppresses tumor growth and metastasis.
These growth factors are believed to have a play a role in many inflammatory conditions such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) also called as lupus, scleroderma, vasculitis, and other collagen vascular diseases; and in atherosclerosis, glomerulonephritis both in primary and secondary types of nephritis, psoriasis, and in Alzheimer's diseases, and proliferative disorders seen in diabetes niellitus (DM) such as diabetic retinopathy, vasculopathy, nephropathy, neuropathy, and atherosclerosis. In addition, it was also observed that some of the complications such as retinopathy, neuropathy, nephropathy, etc., seen in diabetes mellitus are due to enhanced production of various growth factors such as EGF, VEGF, and IGF (insulin-like growth factor in the target tissues (1-3).
Epidermal Growth Factor (EOF)
Studies showed that EGF stimulates cells to divide by activating members of the EGFR (EGF receptor), EGFR activation also plays an important role in tumor survival. Several types of human cancer present sustained activation of EGFRs by secreted growth factors. Amplification and rearrangement of the gene encoding EGFR occur in a significant fraction of glioblastomas and squamous-cell carcinomas and correlate with reduced patient survival. Similarly, amplification of the gene encoding HER2 is associated with shorter time of breast cancer relapse, reduced overall patient survival and resistance to hormonal therapy and chemotherapy. Consistent with their pivotal role in stimulating cell proliferation, blocking EGFR function results in retarded tumor growth. Examples include the clinically approved anti-HER2

monoclonal antibody trastuzumab (Herceptin®), similar antagonistjs of the EGFR such as the monoclonal antibody cetuximab, and low-molecular weight inhibitors of tyrosine kinases. Erbitux® is a chimeric monoclonal antibody specific for the EGFR. Over expression of EGFR is common in many solid tumors, such as colorectal and lung carcinomas as well as cancers of the head and neck. It correlates with increased metastasis, decreased survival and a poor prognosis. EGFR protects malignant tumor cells from the cytotoxic effects of chemotherapy and radiotherapy, making these treatments less effective. Erbitux*^ binds to the extracellular domain of EGFR on the tumor cell, thereby inhibiting receptor-associated tyrosine kinase. This inhibition blocks the intracellular pathways associated with tumor cell proliferation, so preventing tumor growth and dissemination as well as inducing tumor cell death or apoptosis. There is evidence to suggest that EGF and EGFR have angiogenic actions and that Erbitux*^ prevents angiogenesis (4-6).
Vascular Endothelial Growth Factor (VEGF)
VEGF is a protein that is secreted by hypoxic cells, including those that are cancerous. VEGF stimulates new blood vessel formation or angiogenesis by binding to specific receptors on nearby blood vessels to stimulate extensions of existing blood vessels. Angiogenesis plays an important role in both tumor growth and metastasis. Monoclonal antibodies designed to bind to VEGF preventing it from binding to its receptors and therefore potentially inhibiting tumor growth. Bevacizumab is a humanized monoclonal antibody to VEGF developed by Genentech and is called as Avastin®. By inhibiting VEGF, Avastin interferes with the blood supply to tumors, a process that is critical to tumor growth and metastasis.

Several clinical studies showed that both Erbitux'*' and Avastin'^\ which are humanized monoclonal antibodies to EGFR and VEGF respectively, are useful in the treatment of colon cancer. Erbitux® shrank tumors in 22.9% of advanced colon cancer patients when combined with chemotherapy. Avastin**^ in combination with chemotherapy extended colon cancer patients' lives by 5 months in a trial of 900 patients.
EGF and VEGF are mentioned here only as examples on the role of various growth factors and their receptors in cancer. Several studies indicated that blocking or neutralizing the actions of various growth factors and their receptors suppresses cancer (7-11).

Polyunsaturated fatty acids
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The polyunsaturated fatty acids (PUFAs) are fatty acids having at least two carbon-to-carbon double bonds in a hydrophobic hydrocarbon chain, which typically includes X-Y carbon atoms and terminates in a carboxylic acid group. The PUFAs are classified in accordance with a short hand nomenclature, which designates the number of carbon atoms present (chain length), the number of double bonds in the chain and the position of the double bonds nearest to the terminal methyl group. The notation a:b" is used to denote the chain length and number of double bonds, and the notation n:x" is used to describe the position of the double bond nearest to the methyl group. There are 4 independent families of PUFAs, depending on the parent fatty acid from which they are synthesized. They are:
(1) The "n-3" series derived from alpha-linolenic acid (ALA, 18:3, n-3).
(2) The "n-6" series derived from cis-linoleic acid (LA, 18:2, n-6).
(3) The "n-9" series derived from oleic acid (OA, 18:1, n-9).
(4) The *'n-7" series derived from palmitoleic acid (PA, 16:1, n-7).
The mammals cannot synthesize the parent fatty acids of the n-3 and n-6 series, and hence they are often referred to as "essential fatty acids" (EFAs). Because these compounds are necessary for normal health but cannot be synthesized by the human body, they must be obtained through the diet (12, 13).
It is believed that both LA and ALA are metabolized by the same set of enzymes. LA is converted to gamma-linolenic acid (GLA, 18:3, n-6) by the action of the enzyme delta-6-desaturase (d-6-d) and GLA is elongated to form dihomo-GLA (DGLA, 20:3^-6), the precursor of the 1 series of prostaglandins (PGs). DGLA can

also be converted to arachidonic acid (AA, 20:4, n-6) by the action of the enzyme delta-5-desaturase (d-5-d). AA forms the precursor of 2 series of prostaglandins, thromboxanes and the 4 series of leukotrienes. ALA is converted to eicosapentaenoic acid (EPA, 20:5, n-3) by d-6-d and d-5-d. EPA forms the precursor of the 3 series of prostaglandins and the 5 series of leukotrienes. LA, GLA, DGLA, AA, ALA, EPA and docosahexaenoic acid (DHA, 22:6, n-3) are all PUFAs, but only LA and ALA are EFAs (see Figure 1 for metabolism of essential fatty acids).
Several studies showed that EFAs/PUFAs play a significant role in the pathobiology of inflammatory conditions such as RA, SLE, PSS; skin disorders such as psoriasis, eczema, atopic and non-atopic dermatitis; atherosclerosis, and cancer (13-16). Further, various PUFAs have also been shown to be of bene tit in all these conditions (13-16).
PUFAs and Cancer
Tumor cells are not only deficient in PUFAs but also have low rate(s) of lipid peroxidation, contain relatively large amounts of antioxidants such as vitamin E and superoxide dismutase (SOD). It is also believed that low rates of lipid peroxidation and consequent low amounts of lipid peroxides in the cells can contribute to an increase in the mitotic process which ultimately leads to an increase in cell proliferation. Thus, a deficiency of PUFAs, high amounts of antioxidants and the presence of low amounts of lipid peroxides in the tumor cells can contribute to the growth of tumor cells. This is supported by studies by the inventor wherein it was noted that PUFAs such as GLA, DGLA, AA, EPA and DHA could decrease tumor cell proliferation. In addition, it was also observed that when apprbpriate amounts of GLA, DGLA, AA, EPA and DHA were administered to tumor cells and normal cells.

obtained from American Type Culture Collection, only tumor cells were killed without having any significant action on the survival of normal cells in vitro. In mixed culture experiments, in which both normal and tumor cells were grown together, GLA showed more selective tumoricidal action compared to AA, EPA and DHA though, these latter fatty acids were also effective to some extent. This indicated that selective delivery of GLA, DGLA, AA, EPA and DHA to tumor cells might offer a new therapeutic approach in the treatment of cancer (17-22).
These in vitro results are supported by in vivo studies performed in animal tumor models. For example, it was noted that GLA, DGLA, AA, EPA and DHA when used either in the form of pure fatty acid alone or in the form of fatty acid rich oils could inhibit the growth of skin papilloma in mice, formation and growth of hepatoma in rats and ascitic tumor cells in the peritoneum of experimental animals. These results indicate that these fatty acids can inhibit the growth of a variety of tumors even in vivo. In further studies, it was noted that these fatty acids are able to enhance free radical generation and the lipid peroxidation process selectively in the tumor cells but not so much in the normal cells and thus, are able to bring about their cancer killing action (23-28). Inventor's studies have shown that PUFAs can be exploited as possible anti-cancer agents either alone or in combination with traditional anti-cancer drugs.
In a series of investigations by the inventor, it was also observed that the cytotoxic action of anti-cancer drugs such as doxorubicin, vincristine and cis-platinum can be augmented by various PUFAs such as GLA, DGLA, AA, EPA and DHA. In addition, these fatty acids could also enhance the cellular uptake of these anti-cancer drugs by the tumor cells and thus, are able to potentiate the anti-cancer actions of

these drugs (27).
It is to be noted in this context that PUFAs can bind to albumin and other proteins and hence, if given intravenously may not be available to be taken up by the tumor cells and consequently may not be able to bring about their cell killing action on the tumor cells. In view of this, it is desirable that PUFAs including GLA should be delivered to the patients in such a manner that it is easily available to the tumor (tumor cells) and is delivered selectively to the tumor cells. It is highly desirable that PUFAs including GLA be given intra-tumorally as was experimentally done in the case of human gliomas, or, intra-arterially by selective intra-arterial infusion as was done experimentally in the case of hepatoma and giant cell tumor of the bone. But, it is also possible that in some cases of cancer such as Hodgkin's and non-Hodgkin's lymphoma wherein the tumor cells are extremely sensitive to the cytotoxic actions of PUFAs, even oral administration may be sufficient as was observed in certain patients. Since, PUFAs can potentiate the cell killing effect of anti-cancer drugs and cytokines, it is desirable to administer a combination of PUFAs, anti-cancer drugs, or specific monoclonal or polyclonal antibodies to growth factors or a combination thereof so that fatty acids are carried to the tumor cells.
The present invention in one aspect resides in a method of selectively delivering PUFAs that are toxic to cancer cells by conjugating thehi or mixing them with monoclonal antibodies or specific polyclonal antibodies to growth factors that are known to enhance tumor growth such that tumoricidal PUFAs are delivered to the cancer cells very selectively. In addition, such a combination of PUFAs and monoclonal and/or specific polyclonal antibodies to growth factors will also inhibit

blood supply to a tumor such that ultimately the tumor cells will die since by using two types of substances: one a lipid and the other a protein or a peptide both of which have very potent anti-angiogenic action. SUMMARY OF THE INVENTION
All the above factors and observations attest to the fact that malignant tumors are dependent on various growth factors for their growth and are also angiogenesis-dependent diseases. Growth factors also behave as angiogenic factors and thus, promote tumor growth. But, it should be mentioned here that tumor-associated angiogenesis is a complex, multi-step process which can be controlled by both positive and negative factors. It appears, as though, angiogenesis is necessary, but not sufficient, as the single event for tumor growth (29). But, it is evident from several experimental results that angiogenesis may be a common pathway for tumor growth and progression. Though several anti-angiogenic agents, antibodies to growth factors are being tried to arrest tumor growth, these are not without problems. Since the majority of these agents are proteins/peptides, their long-term use may lead to the development of antibodies, which can neutralize their action. These anti-angiogenic and anti-growth substances need to be given repeatedly and some of them are unstable and are difficult to produce in large amounts. In view of this, it is desirable and necessary to ntake efforts to stabilize and potentiate the actions of,known anti-angiogenic molecules, and antibodies against various growth factors.
The present invention teaches the efficacious use of anti-growth factor and anti-angiogenic substances, which can inhibit endothelial cell proliferation, growth of tumor cells and coupling them to cis-unsaturated fatty acids, which also have anti-angiogenic and cytotoxic actions on tumor cells, such that the actions of these

substances are potentiated by each other. Further, as angiogenesis is involved in other disease processes such as inflammation, tumor metastasis, etc., it is envisaged that the conjugate(s) of anti-angiogenic substances and PUFAs will be useful in these diseases also.
In this context, it is important to note that the inventor has found that polyunsaturated fatty acids (PUFAs) such as gamma-linolenic acid (GLA), dihomo-GLA (DGLA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can selectively kill the tumor cells (as described above) and under specific conditions and in conjugation with salts such as lithium and a lymphographic agent these fatty acids can actually behave as anti-angiogenic substances, i.e. they block all the blood supply to the tumor and also prevent generation of new blood vessels. Using these fatty acids in this particular combination, the inventor has successfully treated human hepatocellular carcinoma and giant cell tumor of bone with few or no side effects.
Described hereinafter is a novel combination of a protein and a lipid and method(s) foriJs use. The protein referred to herein is a potent and specific antagonist of growth factor(s) and inhibitor of endothelial proliferation and angiogenesis. The lipid may be one or more of the polyunsaturated fatty acids: LA (linoleic acid), GLA, DGLA, AA, ALA (alpha-linolenic acid), EPA, DHA and cis-parinaric acid. In this instance or method the polyunsaturated fatty acid need to be given only once or at the most twice within a period of 1 to 2 months. This invention teaches that unlike anti-growth factor antibodies, these fatty acids are not only cytotoxic to the tumor cells but are also able to function as anti-angiogenic agents. Further, polyunsaturated fatty

acids when given in the formulated form, are more potent than monoclonal antibodies or polyclonal antibodies against growth factors in their anti-angiogenic and anticancer actions.
The invention in one aspect teaches a method of killing tumor cells by using a combination of monoclonal antibodies /polyclonal antibodies against growth factors that have been conjugated to polyunsaturated fatty acids to cause necrosis or apoptosis of tumor cells. The invention also provides a method of causing anti-angiogenic action in the tumor region with the result that new blood vessels and collaterals are not formed to sustain the tumor. The present invention in another aspect tackles the issue of drug delivery to the target tissue and provides the most efficacious method of administering an admixture of selected PUFAs with other elements such as anti-growth factor antibodies as will be described hereinafter.
The invention in yet another aspect teaches a method of regressing the tumor
/or interrupting blood supply to the tumor using a pre-determined admixture of at least
I a PUFA and an anti-growth factor antibody to produce necrosis with very desirable
results. Both the PUFAs and anti-growth factor antibodies being similar in function,
the invention also provides a method of causing anti-tumor and anti-angiogenic action
in the tumor region with the result that new blood vessels and collaterals are not
formed to sustain the tumor in the tumor region treated according to the invention.
The present invention in another aspect tackles the issue of drug delivery to the target
tissue and provides the most efficacious method of administering an admixture of
selected PUFAs along with an anti-growth factor antibody as will be described

hereinafter.
Modification of monoclonal and polyclonal antibodies with EFAs/PUFAs/LCPUFAs
Mixing or conjugating specific monoclonal and polyclonal antibodies such as Erbitux*® and Avastin® with EFAs/PUFAs such that they form stable complexes. The conjugation between monoclonal antibodies of EGFR and VEGF and EFAs/PUFAs can be covaient bond preferably is an amide bond, which survives the conditions in the stomach. Such anti-EGFR antibody-EFAs/PUFAs complex and anti-VEGF antibody-EFAs/PUFAs complex will be stable without interfering the actions of monoclonal antibodies of EGFR and VEGF such that they will be able to suppress tumor cell proliferation, tumor metastasis and angiogenesis.
These anti-EGFR antibody-EFAs/PUFAs and anti-VEGF antibody-EFAs/PUFAs complexes can be given orally, parentarally (including but not limited to subcutaneoirs, intravenous, intra-arterial, rectal, submucosal) aqd as aerosols for administration through nose, mouth and intra-tracheal routes. These preparations could also be given rectally.
It is expected that these anti-EGFR antibody-EFAs/PUFAs and anti-VEGF antibody-EFAs/PUFAs complexes will have significant inhibitory action on tumor cell proliferation and inhibit angiogenesis and also occlude specifically tumor feeding blood vessels irrespective of the route of administration.
The ratio between anti-EGFR antibody and EFAs/PUFAs can vary from 1:1 to 1:1000 and 1:1 to 1000:1. The anti-EGFR and anti-VEGF antibodies can be conjugated with any one or a combination of fatty acids. For example, anti-EGFR

antibody can be conjugated with LA, GLA, DGLA, AA, ALA, EPA and/or DHA and similarly anti-VEGF antibody is conjugated with LA, GLA, DGLA, AA, ALA, EPA and/or DHA. In certain instances, EFAs/PUFAs may be conjugated with both anti-EGFR and anti-VEGF antibodies simultaneously. The EFAs/PUFAs can be in the form of pure acid, sodium salt, lithium salt, meglumine salt, magnesium salt, iodized salt and/or any other type of stable salt. For intra-arterial injection the preferred conjugate is in the form of an amide or complex between Li-LA, Li-GLA, Li-DGLA, Li-AA, Li-ALA,Li-EPA or Li-DHA and iodized oily lymphographic oil such as Lipiodol, and anti-EGFR antibody and/or anti-VEGF antibody.
The amount of these anti-EGFR and anti-VEGF antibodies to be given orally
or parentarally can vary from I mg per dose to 100 gm. These anti-EGFR antibody-EFAs/PUFAs complexes can be given daily as a single injection or as a continuous infusion in a day and/or daily for a period of one week or as frequently as needed depending on the response. Administration of these complexes can be repeated daily, weekly or monthly as the situation demands.
In the same fashion as described for anti-EGF and anti-VEGF antibodies and their conjugation with various EFAs/PUFAs, similar complexes can also be prepared between anti-TNF-a, anti-IL-1, anti-IL-2, anti-IL-6, etc., antibodies and various EFAs/PUFAs.
Summary of the Invention
All the above factors and observations attest to the fact that both EFAs/PUFAs and growth factors have important roles in many clinical conditions. In view of the

significant role of growth factors in the growth and progression of cancer including metastasis, several attempts have been made and are being made to develop specific monoclonal and polyclonal antibodies that specifically inhibit or block the actions of these growth factors. Such attempts are expected to inhibit the growth of the tumor cells. It is known that these monoclonal and polyclonal antibodies against various growth factors also inhibit angiogenesis that ultimately leads to the regression of the tumors. But, it should be mentioned here that in majority of the instances these monoclonal and polyclonal antibodies against various growth factors have not been very effective in inhibiting the growth of various tumors. The best examples of this failure on the part of monoclonal and polyclonal antibodies developed against various growth factors to effectively prevent the growth of various tumors are: Erbitux® and Avastin'^, which are humanized monoclonal antibodies to EGFR and VEGF respectively. Brbitux*** shrank tumors in only 22.9% of advanced cplon cancer patients when combined with chemotherapy, whereas Avastin"® in combination with chemotherapy extended colon cancer patients' lives only by 5 months in a trial of 900 patients. Furthermore, both Erbitux® and Avastin® have many side effects. This clearly shows that monoclonal and polyclonal antibodies developed against various growth factors are not very effective in preventing the growth of tumors and are ineffective in preventing angiogenesis. In the same manner other monoclonal and polyclonal antibodies developed against several other growth factors also failed to be effective in the treatment of other diseases such as psoriasis, inflammatory conditions such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), mixed connective tissue disorder (MCTD), vasculitis; and other disorders caused by uncontrolled angiogenic activity such as proliferative diabetic retinopathy; other eye disorders such as macular degeneration; and skin

problems such as psoriasis, various forms of proliferative glomerulonephritis, and other disorders in which cell proliferation and angiogenesis plays a dominant role. It may be mentioned here that specific monoclonal and polyclonal antibodies are also being developed against various receptors on the cell surfaces and other markers with the hope that these antibodies will be useful in the management of various clinical conditions mentioned above.
The present invention specifically teaches the efficacious use of monoclonal and polyclonal antibodies developed against various growth factors, receptors, and other cell surface and intracellular markers and proteins by coupling them to PUFAs such that the actions of these antibodies are potentiated. It is suggested that the beneficial actions of compounds formed as a result of such coupling of monoclonal and polyclonal antibodies to PUFAs will be more than the sum effect seen when these antibodies and PUFAs are given separately.
The monoclonal and polyclonal antibodies referred to herein is a growth factor(s), their receptor(s), cell surface receptors or markers, and intracellular proteins that have various physiological and pathological actions. The lipid may be one or more of the PUFAs: LA, GLA, DGLA, AA, ALA, EPA and DHA.
The objective of the invention is to provide a covalently coupled or complex containing a monoclonal and polyclonal antibody (dies) and one or more of PUFAs containing between 16 and 26 carbon atoms. Another objective of the invention is to provide pharmacological compositions comprising amides of the PUFAs combined with a monoclonal antibody or polyclonal antibody against growth factors and various proteins (either extracellular or intracellular) such that it is stable enough to pass through the acidic environment of the stomach, in the blood stream, and also enter the

brain crossing the blood brain barrier. Another objective of the invention is to provide an amide derivative of monoclonal and polyclonal antibodies against various growth factors with biological activities useful in many clinical conditions that have been enumerated above. It should be mentioned here that PUFAs are not used as carriers (though they may serve as carriers under certain circumstances when combined, complexed or covalently linked to/ with the antibodies) but themselves serve as effective agents to treat the clinical condition in question and also potentiate the actions of various monoclonal and polyclonal antibodies.
The compounds of the invention containing a monoclonal and polyclonal antibody (dies) against a growth factor(s) and its receptor(s), and various intra and extracellular proteins and one or more of PUFAs can be prepared in pharmaceutical preparations containing the compounds themselves or their suitable derivatives in appropriate or suitable proportions. Administration may be made by any method, which allows the compound (containing the monoclonal and polyclonal antibody (dies) and one or more of PUFAs) to reach the site of desired action including brain. The-compound(s) can be administered orally in the form of dragees, tablets, and syrups or by inhalation or ampules. When compounds are administered rectally the composition can be in the form of a suppository. When the compounds of the invention are to be administered by topical application for instance for various skin conditions, they can be in the form of pomade or a gel. Another example of preparation can be as an intra-tumoral preparation in appropriate doses for the treatment of human brain gliomas or any other accessible tumor (eg. urinary bladder cancer, carcinoma of the esophagus, carcinoma of the lung, breast cancer, etc.) by any route by using flexible fiber optic scopes such as bronchoscope, etc. Another example of administration of the preparation can be as selective intra-arterial infusion or

injection into the tumor-feeding vessel by femoral, brachial or carotid routes or any other suitable route or in a combination of routes or any other suitable agent all in a mixture or in conjugated form(s) (like GLA or lithium GLA+ monoclonal and polyclonal antibody (dies), LA/GLA/DGLA/AA/ALA/ EPA/DHA/cis-parinaric acid/docosapentaenoic acid or their salts including lithium salts all individually or in combination thereof-*- mjonoclonal and polyclonal antibody (dies), LA/GLA/DGLA/AA/ALA/ EPA/DHA/cis-parinaric acid/docosapentaenoic acid or their salts including lithium salt in combination with or conjugated to monoclonal and polyclonal antibody (dies), which show selective occlusion of tumor feeding vessels and/or anti-angiogenic actions and anti-cancer action. Further the compound(s) can be delivered using suitable devices, or a slow releasing capsule/tablet at an appropriate site or organ of the body. This preparation can be administered daily, weekly, or monthly or at various intervals as deemed appropriate. This preparation can be given as an intravenous infusion continuously or intermittently in a day, daily, weekly or monthly or several times a day or as frequently as necessary depending on the necessity.
It is also within the purview of this invention, as stated supra to administer an admixture of PUFAs, anti-cancer drugs, and selected anti-growth factor, and anti-angiogenic substance(s) at the same time, administering predetermined doses of PUFAs orally. All such variations are envisaged to be within the ambit of this invention.
Application to mammals: Even though the examples described supra relate to humans, it is envisaged that the method of suppressing or reducing the growth of the

cancer and/or anti-angiogenic action using admixture of this invention including an anti-growth faclor(s)/anti-cytokine(s) antibodies and/or angiogenic substances are equally applicable to other mammals.
Equivalents
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. For example, anti-growth factors referred to herein include EGF and VEGF but other agents with actions on cancer cells and angiogenesis. Also sodium and potassium salts are considered equivalents of each other. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the appended claims.
References:
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2. Watanabe D, et al. Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy. N Engl J Med 2005; 353: 782-792.

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acid/docosahexaenoic acid on pro-oxidant and anti-oxidant status and
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I claim:
1. A drug comprising,
a monoclonal or specific polyclonal antibody to growth factors and/or their specific receptors conjugated using a water-soluble synthetic polymers or covalently coupled to a straight-chained fatty acid molecule containing 18 to 22 carbon atoms,
wherein the antibodies are directed against growth factors, their receptors selected from, but not limited to, EGF (epidermal growth factor), VEGF (vascular endothelial growth factor), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), hepatocytes growth factor (HGF), placental growth factor (PIGF), and tyrosine kinase receptor erbB2, also known in humans as Her2,
wherein the fatty acid molecule is selected from the group consisting of CI 8:2, C18:3, C20:3, C20:4, C20:5, C22:5, C22:6, cis-parinaric acid, conjugated linoleic acid and in the form of a salt selected from the group consisting of a lithium salt, a sodium salt, a potassium salt, a magnesium salt, a calcium salt, a manganese salt, an iron salt, a copper salt, an aluminum salt, a zinc salt, a chromium salt, a cobalt salt, a nickel salt and an iodide and/or in the form of a fatty acid derivative selected from the group consisting of glycerides, esters, free acids, amides, phospholipids and salts.
2. A pharmaceutical preparation comprising the drug of claim I and a
pharmaceutically acceptable carrier.

3. An oral and parentaral composition of claim 1 wherein the weight ratio of antibody to growth factor(s) and/or growth factor receptor(s) to fatty acid in the composition and the weight ratio of antibody to growth factor and/or its receptor to fatty acid ranges from 1:10 to 10:1.
4. An oral composition of claim 1, wherein the quantity of antibody to growth factor(s) and/or growth factor receptor(s) varies from 0.5 mg to 500 gm and that of fatty acid ranges from 0.5 mg to 500 gm.
5. A parentaral preparation of claim 1 that is sterile and injectable administered subcutaneously, intravenously, intramuscularly or intra-arterially and additionally comprising an osmolyte and in a buffer at pH from 5 to 8.
6. A parentaral composition of claim 7 that is administered as a single injection or as a continuous infusion.
7. A parentaral composition of claim 6 wherein the quantity of antibody to growth factor(s) and/or growth factor receptor(s) varies from 0.5 mg to 500 gm and that of fatty acid ranges from 0.5 mg to 500 gm additionally comprising an osmolyte and a stabilizer.
8. A pharmaceutical composition comprising the drug of claim 1 and a pharmaceutically acceptable carrier for treating proliferative diseases associated with angiogenesis.
9. A method of treating diseases as in claim 8, wherein the disease is a malignant tumor.

10. A method in claim 9, wherein the malignant tumor is cancer selected from
hepatoma, bronchogenic cancer of the lung, colon cancer, breast cancer,
ovarian cancer, cancer of the kidney, skin cancer, Kaposi's sarcoma, cancer of
the esophagus, cancer of the stomach, leukemias, lymphomas, multiple
myeloma.
11. A method in claim 10, wherein the drug is administered as an injection, continuous infusion, orally or by inhalation.
12. A method of treating diseases associated with inflammation using the composition of drug as in claim 1.
13. A method in claim 12, wherein the diseases associated with inflammation are
rheumatoid arthritis, lupus, progressive systemic sclerosis, mixed connective
tissue disease, vasculitis, proliferative glomerulonephritis, psoriasis, diabetic
retinopathy, and macular degeneration.
14. A method in claim 13 of treating diseases associated inflammation, wherein
the drug is administered as an injection, continuous infusion, orally or by
inhalation.

Documents:

1159-che-2004 correspondence others 02-06-2011.pdf

1159-CHE-2004 FORM-13 10-05-2011.pdf

1159-che-2004 power of attorney 02-06-2011.pdf

1159-CHE-2004 ABSTRACT.pdf

1159-CHE-2004 CLAIMS.pdf

1159-CHE-2004 CORRESPONDENCE OTHERS 21-02-2011.pdf

1159-CHE-2004 DRAWINGS.pdf

1159-CHE-2004 POWER OF ATTORNEY 16-11-2009.pdf

1159-che-2004-abstract.pdf

1159-che-2004-claims.pdf

1159-che-2004-correspondnece-others.pdf

1159-che-2004-description(complete).pdf

1159-che-2004-description(provisional).pdf

1159-che-2004-drawings.pdf

1159-che-2004-form 1.pdf

1159-che-2004-form 26.pdf

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Patent Number

249934

Indian Patent Application Number

1159/CHE/2004

PG Journal Number

47/2011

Publication Date

25-Nov-2011

Grant Date

23-Nov-2011

Date of Filing

08-Nov-2004

Name of Patentee

UNDURTI NARASIMHA DAS

Applicant Address

H.NO.8-3-677/26,YELLAREDDYGUDA,HYDERABAD-500 073

Inventors:

#	Inventor's Name	Inventor's Address
1	UNDURITI NARASIMHA DAS,	H.NO.8-3-677/26,YELLAREDDYGUDA,HYDERABAD-500 073

PCT International Classification Number

A61K 39/395

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/598,366	2004-08-02	U.S.A.