WO2009054001A1 - Composition pharmaceutique et procédé correspondant - Google Patents

Composition pharmaceutique et procédé correspondant Download PDF

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Publication number
WO2009054001A1
WO2009054001A1 PCT/IN2008/000688 IN2008000688W WO2009054001A1 WO 2009054001 A1 WO2009054001 A1 WO 2009054001A1 IN 2008000688 W IN2008000688 W IN 2008000688W WO 2009054001 A1 WO2009054001 A1 WO 2009054001A1
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WIPO (PCT)
Prior art keywords
cancer
combination
egfr
immunosuppressant
rapamycin
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PCT/IN2008/000688
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English (en)
Inventor
Ramakrishnan Melarkode
Pradip Nair
Indira Venkata Chivukula
Jose Enrique Montero Casimiro
Rolando Perez Rodriguez
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Biocon Limited
Centro De Inmunologia Molecular
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Application filed by Biocon Limited, Centro De Inmunologia Molecular filed Critical Biocon Limited
Publication of WO2009054001A1 publication Critical patent/WO2009054001A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the invention is directed towards novel synergistic combinations of anti-EGFR antibodies and immunosuppressive agents used to treat a mammalian host by administration of a therapeutically effective amount of the drug in the course of a treatment for cancer or suspected tumor cell growth.
  • the drug combination that is cytotoxic to tumor cells comprises an EGFR inhibiting antibody that competitively inhibits native EGF ligand binding to the EGFR receptor.
  • the combination also comprises atleast one further immunosuppressant selected from the group comprising rapamycin, tacrolimus, everolimus, pimecrolimus in the free form or in the form of a pharmaceutically acceptable salt.
  • the combination drug used is preferably an immunosuppressive agent and most preferably rapamycin.
  • Novel anti-EGFR-drug conjugates and compositions optionally containing at least one pharmaceutically acceptable carrier for simultaneous use is contemplated.
  • the above described agents may be used for the prophylaxis and/or treatment of tumors by increasing cytotoxicity of the tumor cells towards the described combinatorial therapeutic agent.
  • cancer recurrence still remains a problem. Issues concerning relapse and non-responsiveness to chemotherapeutic agents in cancer patients are a major problem improperly addressed. Further, overdoses of targeted antibody therapies results in increased blood toxicities such as neutropenia and thrombocytopenia. There remains a need to identify new methods of treatment that would alleviate and/or prevent relapse of tumors, whilst keeping the effective drug doses as low as possible
  • Combination therapies are among the current experimental regimens focusing on combining chemotherapy with immunotherapy in an attempt to overcome such resistance issues.
  • Combination therapy refers to simultaneous administration of two or more medications or treatments, such as chemotherapy (CT), radiation therapy (RT) or a drug, to treat a single disease.
  • CT chemotherapy
  • RT radiation therapy
  • the rationale for this tactic is to use drugs that work on different signal transduction pathways, thereby increasing the likelihood that more cells will be affected. For example, antibodies and RT treat cancer locally, while CT also kills metastatic cancer cells.
  • immunoconjugates which are antibody-drug conjugates used to deliver drugs specifically to target cells, as opposed to being freely available systemically.
  • These immunoconjugates show an augmented combined efficacy in vivo. It has been found that therapeutic monoclonal antibodies (mAb) might be more effective when conjugated with cytotoxic drugs.
  • mAb therapeutic monoclonal antibodies
  • Herceptin-geldanamycin chemical conjugate showed a greater anti-tumor effect than Herceptin alone in tumor xenograft mouse models.
  • the present invention provides a rationally designed synergistic combination that offers clinically meaningful survival advantages over a single monotherapy.
  • the combinations described in the invention helps achieve this clinical benefit with a manageable toxicity profile, making them more attractive options in the metastatic setting.
  • the invention is directed towards novel synergistic combinations of anti-EGFR antibodies antibodies binding to EGFR and immunosuppressive agents used to treat a mammalian host by administration of a therapeutically effective amount of the drug in the course of a treatment for cancer or suspected of tumor cell growth.
  • the drug combination cytotoxic to tumor cells comprise an EGFR inhibiting agent that competitively inhibits EGF ligand binding to the EGFR receptor such as nimotuzumab (hR3).
  • the combination also comprises at least one further immunosuppressive agent selected from the group comprising rapamycin, tacrolimus, everolimus, pimecrolimus in the free form or in the form of a pharmaceutically acceptable salt.
  • the combination drug used is preferably an immunosuppressive agent and most preferably rapamycin.
  • hR3 is an anti-EGFR (epidermal growth factor receptor) humanized monoclonal antibody (mAb).
  • EGFR activation stimulates cell proliferation, angiogenesis, dedifferentiation, and migration, as well as protects from apoptosis,which are all unregulated in cancer.
  • hR3 binds to EGFR, preventing EGF-EGFR binding and inhibiting downstream signaling through the reduction of cytoplasmic tyrosine phosphorylation thereby blocking the signal transduction for cell proliferation.
  • hR3 is the subject of product patents US 5,891,996 and US6,506,883.
  • Rapamycin is a macrolide antibiotic, produced from the bacterium Streptomyces hygroscopicus, with potent immunosuppressive and anti-proliferative effects, used mainly to prevent organ transplant rejection. Rapamycin binds to the cytosolic protein FK-binding protein 12 (FKBP 12) and this complex inhibits the mammalian target of Rapamycin (mTOR) pathway, specifically the mTOR-mediated S6K1 and 4E-BP1 arms by directly binding to mTOR Complex- 1 (mTORCl).
  • FKBP 12 cytosolic protein FK-binding protein 12
  • mTOR inhibition is selectively blocking the transcriptional activation of interleukin-2 (IL-2), making IL-2 unavailable to bind to IL-2 receptors expressed by lymphocytes, thereby blocking T- and B-cell activation.
  • IL-2 interleukin-2
  • Rapamycin was first described in US 3,929,992.
  • Goudar R, et al (2005) 1 describe a combination therapy of inhibitors of epidermal growth factor receptor/vascular endothelial growth factor receptor 2 (AEE788) and the mammalian target of rapamycin (RADOOl) everolimus offering improved glioblastoma tumor growth inhibition.
  • AEE788 epidermal growth factor receptor/vascular endothelial growth factor receptor 2
  • RADOOl mammalian target of rapamycin
  • Buck E, et al (2006) describes rapamycin synergizing with the epidermal growth factor receptor inhibitor erlotinib in non-small-cell lung, pancreatic, colon and breast tumors.
  • hR3 and rapamycin would be novel to the above approaches because it involves an anti-EGFR monoclonal antibody, as opposed to a small molecule, to be combined with rapamycin for an epidermoid carcinoma target.
  • a monoclonal antibody has a significantly longer half-life than a small molecule.
  • the dosing and therapeutic method would be significantly different from a small molecule inhibitor of the EGFR tyrosine kinase domain.
  • the advantage of the present invention over the cited prior art is the synergistic benefit achieved by the concurrent administration of the EGFR inhibiting agent and a chemotherapeutic drug such as rapamycin. It has now surprisingly been found that a synergistic combination of hR3 and rapamycin shows increased cytoxicity towards tumors.
  • the main objective of the present invention is to obtain a synergistic combination of a therapeutic drug comprising a therapeutically effective mixture of an anti-EGFR antibody and an immunosuppressant.
  • Another main objective of the present invention is to obtain a pharmaceutical composition for therapeutic or prophylactic treatment of cancer
  • Yet another main objective of the present invention is to obtain a process of manufacturing a pharmaceutical composition for therapeutic or prophylactic treatment of cancer comprising a therapeutically effective mixture of an anti-EGFR antibody and an immunosuppressant, comprising step of mixing anti-EGFR antibody and an immunosuppressant along with pharmaceutically acceptable excipients.
  • Still another main objective of the present invention is to obtain a method of detecting a presence of the EGFR antigen or a cell expressing EGFR comprising contacting the sample with the diagnostic composition of claim 13 to allow a formation of a complex and detecting the formation of the complex.
  • Still another main objective of the present invention is to obtain a kit comprising the diagnostic combination.
  • the present invention relates to a synergistic combination of a therapeutic drug comprising a therapeutically effective mixture of an anti-EGFR antibody and an immunosuppressant; a pharmaceutical composition for therapeutic or prophylactic treatment of cancer comprising administration of a therapeutically effective mixture of an anti-EGFR antibody and an immunosuppressant and a pharmaceutically acceptable carrier; a process of manufacturing a pharmaceutical composition for therapeutic or prophylactic treatment of cancer comprising a therapeutically effective mixture of an anti-EGFR antibody and an immunosuppressant, comprising step of mixing anti-EGFR antibody and an immunosuppressant along with pharmaceutically acceptable excipients; a method of detecting a presence of the EGFR antigen or a cell expressing EGFR comprising contacting the sample with the diagnostic composition of claim 13 to allow a formation of a complex and detecting the formation of the complex; and a kit comprising the diagnostic combination.
  • FIG 1 SRB Assay: Percentage cytotoxicity of A431 cells exposed for 72 hours to hR3 in combination with Rapamycin for a serially diluted concentration range from 5.2nM to 83nM for hR3 and 1.6nM to 25nM for Rapamycin.
  • FIG 2 MTS Assay: Percentage cytotoxicity of A431 cells exposed for 72 hours to hR3 in combination with Rapamycin for a serially diluted concentration range from 5.2nM to 83nM for hR3 and 1.6nM to 25nM for Rapamycin.
  • FIG 3 SRB Assay: Percentage cytotoxicity of A431 cells exposed for 72 hours to hR3 in combination with Rapamycin for a serially diluted concentration range from 2.6nM to 1328nM for hR3 and 0.78nM to 40OnM for Rapamycin.
  • the analyses of Figures 3-A and 3 -B correspond to Figures 4-A and 4-B (below), respectively.
  • FIG 4 Bliss Independence Analysis of the hR3-Rapamycin combination
  • FIG 5 Western blot image composite.
  • the blots show key signal transduction molecules downstream to hR3 and rapamycin and evaluates the combination of these drugs to these molecules.
  • the figure also has quantification of the blot as compared to TFIID or Akt control.
  • the values shown are relative to the Cells + EGF or Cells+EGF+DMSO control.
  • FIG 6 (A) Data illustrates the daily tumor volumes of the tumor xenografts in nude mice during the treatment course, starting from Day 12 (dose commencement) until Day 28 after cell implantation (date of sacrifice).
  • Error bars express SD of mean tumor measurements for mice in each drug arm.
  • FIG7 Data represents relative values assigned according to mitotic activity frequency in H&E (Hematoxylin and eosin) stained tumor sections. Data shows a decrease in mitotic activity in the 606.5 hR3 + 12.5nM Rapamycin combination group, as compared to the placebo group.
  • the present invention relates to a synergistic combination of a therapeutic drug comprising a therapeutically effective mixture of an anti-EGFR antibody and an immunosuppressant.
  • the antibody competitively inhibits native EGF ligand binding to the EGFR receptor.
  • the anti-EGFR antibody is hR3.
  • the immunosuppressant is selected from the group comprising macrolides selected from rapamycin, tacrolimus, everolimus, pimecrolimus or cyclosporins.
  • the anti-EGFR antibody is conjugated to the immunosuppressant.
  • the drug elicits increased cytoxicity against tumor cells.
  • the combination can be administered in the form of solid, semi-solid, or liquid, tablets, pills, powders, capsules, gels, ointments, liquids, suspensions or aerosols.
  • the present invention relates to a pharmaceutical composition for therapeutic or prophylactic treatment of cancer comprising administration of a therapeutically effective mixture of an anti-EGFR antibody and an immunosuppressant and a pharmaceutically acceptable carrier.
  • the therapeutically effective mixture of an anti-EGFR antibody and an immunosuppressant further comprises pharmaceutically acceptable excipients.
  • composition is administered intravenously, intramuscularly, subcutaneously, intrasynovially, by infusion, sublingually, transdermally, orally, topically or by inhalation.
  • the combination drug is used in the treatment of breast cancer, bladder cancer, colon cancer, esophageal cancer, pancreatic cancer, stomach cancer, lung cancer, uterine cancer, cervical cancer, kidney cancer, ovarian cancer, prostrate cancer, renal cancer and head and neck cancer.
  • the present invention relates to a process of manufacturing a pharmaceutical composition for therapeutic or prophylactic treatment of cancer comprising a therapeutically effective mixture of an anti-EGFR antibody and an immunosuppressant, comprising step of mixing anti-EGFR antibody and an immunosuppressant along with pharmaceutically acceptable excipients.
  • the immunosuppressant is selected from the group comprising macrolides selected from rapamycin, tacrolimus, everolimus, pimecrolimus or cyclosporins.
  • the present invention relates to the use of the combination of an anti-EGFR antibody and an immunosuppressant according to any of preceding claims for the preparation of a diagnostic composition for the immunohistological detection of cancers.
  • a method of detecting a presence of the EGFR antigen or a cell expressing EGFR comprising contacting the sample with the diagnostic composition of claim 13 to allow a formation of a complex and detecting the formation of the complex.
  • the present invention relates to a kit comprising the diagnostic combination.
  • This invention is directed towards novel synergistic combinations of EGFR inhibiting agents specific to the EGFR receptor and immunosuppressive drugs or agents, which can be used to treat a mammalian host, usually a human, suffering from cancer by administering the combination in a therapeutically-effective amount exhibiting cytotoxicity.
  • Preferred EGFR inhibiting agents are molecules which can competitively inhibit the binding of the native ligands for the receptor, the binding of which elicits signal transduction pathways that regulate cell proliferation, differentiation and apoptosis.
  • Preferred immunosuppressive agents may be selected from the group comprising rapamycin, tacrolimus, everolimus, pimecrolimus in the free form or in the form of a pharmaceutically acceptable salt.
  • the combination drug used is preferably an immunosuppressive agent and most preferably rapamycin.
  • the effective dosage of each of the combination partners employed in the method of the invention may vary depending on the particular compound or pharmaceutical composition employed, the mode of administration, the severity of the condition being treated.
  • Another aspect of the present invention relates to anti-EGFR drug- conjugates, and compositions optionally containing at least one pharmaceutically acceptable carrier for simultaneous use.
  • Another aspect of the present invention relates to anti-EGFR drug- conjugates, and compositions optionally containing at least one pharmaceutically acceptable carrier for simultaneous use.
  • the synergistic drug combination of the present invention comprises at least two components, an immunosuppressant drug or agent and an EGFR inhibiting molecule that is characterized as a tyrosine kinase inhibitors displaying cytoxicity towards tumor cells.
  • the present invention provides novel combinatorial therapies for treatment of cancer and methods as set forth within this specification.
  • all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs, unless clearly indicated otherwise.
  • listed below are definitions for certain terms used herein to describe the present invention. These definitions apply to the terms as they are used throughout this specification, unless otherwise clearly indicated.
  • EGF receptor is a transmembrane glycoprotein which has a molecular weight of 175 kDa and is found on many epithelial cell types. It is activated by at least three ligands, EGF (epidermal growth factor), TGF-.alpha. (transforming growth factor alpha) and amphiregulin. Both EGF and TGF-.alpha. have been demonstrated to bind to EGFR and lead to cellular proliferation and tumor growth.
  • EGFR inhibiting agent may be any monoclonal antibody directed against the epidermal growth factor receptor (EGFR) with potential antineoplastic activity.
  • the antibody binds to the extracellular domain of EGFR, thereby preventing the activation and subsequent dimerization of the receptor; the decrease in receptor activation and dimerization that may result in signal transduction inhibition and anti-proliferative effects.
  • the anti-EGFR agent is Nimotuzumab that binds to and inhibits EGFR, resulting in growth inhibition of tumor cells that overexpress EGFR.
  • the antibodies may be murine, chimeric or humanized.
  • hR3 is an anti-EGFR (epidermal growth factor receptor) humanized monoclonal antibody (mAb). EGFR activation stimulates cell proliferation, angiogenesis, dedifferentiation, and migration, as well as protects from apoptosis,which are all unregulated in cancer. hR3 binds to EGFR, preventing EGF-EGFR binding and inhibiting downstream signaling through the reduction of cytoplasmic tyrosine phosphorylation thereby blocking the signal transduction for cell proliferation.
  • mAb humanized monoclonal antibody
  • immunosuppressant as used herein is meant to include compounds or compositions which suppress immune responses.
  • exemplary immunosuppressants include macrolides and cyclosporins, in particular macrolides (such as pimecrolimus, tacrolimus and sirolimus) and cyclosporins (such as phosphorine A) in the free form or in the form of a pharmaceutically acceptable salt or solvates.
  • the immunosuppressant is a macrolide, in particular rapamycin.
  • the anti-EGFR antibody is conjugated to the immunosuppressant.
  • the immunosuppressant is preferably hR3 in context of the present invention.
  • Preferred EGFR inhibiting agents are molecules which can competitively inhibit the binding of the native ligands for the receptor, the binding of which elicits signal transduction pathways that regulate cell proliferation, differentiation and apoptosis.
  • the immunosuppressive drugs or agents contemplated for use in this invention are selected from the group comprising rapamycin, tacrolimus, everolimus, pimecrolimus in the free form or in the form of a pharmaceutically acceptable salt.
  • the combination drug used is preferably an immunosuppressive agent and most preferably rapamycin.
  • dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects.
  • Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
  • a preferred means is to measure the physiological potency of a given compound.
  • the invention provides for a kit comprising a combination of antibodies comprising anti-EGFR antibody and an immunosuppressant or a diagnostic composition of the invention.
  • the kit of the present invention further comprises, optionally (a) buffer (s), storage solutions and/or remaining reagents or materials required for the conduct of medical, scientific or diagnostic assays and purposes.
  • parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units.
  • the kit of the present invention may be advantageously used, inter alia, for carrying out the (diagnostic) methods of the invention and could be employed in a variety of applications referred herein, e. g. , as diagnostic kits, as research tools or medical tools. Additionally, the kit of the invention may contain means for detection suitable for scientific, medical and/or diagnostic purposes. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.
  • the susceptibility of a particular tumor cell to killing with the combined therapy may be determined by in vitro testing. For example, a culture of the tumor cell is combined with combinations at varying concentrations for a period of time sufficient to allow the active agents to inhibit cell proliferation. For in vitro testing, cultured cells from a biopsy sample of the tumor may be used.
  • compositions herein are preferably administered to human patients via oral, intravenous or parenteral administrations and other systemic forms.
  • the pharmaceutical formulations or compositions of this invention may be in the dosage form of solid, semi-solid, or liquid such as, e.g., tablets, pills, powders, capsules, gels, ointments, liquids, suspensions, aerosols or the like.
  • the compositions are administered in unit dosage forms suitable for single administration of precise dosage amounts.
  • the compositions may also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the EGFR inhibiting agent and the immunosuppressant may be administered alone or in combination with adjuvants that enhance stability of the ingredients, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase activity, provide adjunct therapy, and the like, including other active ingredients that may further lower toxic dosage levels of the immunosuppressants.
  • the components of the therapy and pharmaceutical compositions containing them may be administered to a patient in any conventional manner and in any pharmaceutically acceptable dosage form, including, but not limited to, intravenously, intramuscularly, subcutaneously, intrasynovially, by infusion, sublingually, transdermal ⁇ orally, topically or by inhalation.
  • Preferred modes of administration are oral and intravenous.
  • the total amount of therapeutic compound, hR3 + Rapamycin combination as per the instant invention to be used can be determined by those skilled in the art.
  • the amount of therapuetic agent is an amount effective to accomplish the purpose of the particular active agents.
  • the amount in the composition is a therapeutically effective dose, i.e., a pharmacologically or biologically effective amount.
  • the amount can be less than a pharmacologically or biologically effective amount when the composition is used in a dosage unit form, such as a capsule, a tablet or a liquid, because the dosage unit form may contain a multiplicity of delivery agent/biologically or chemically active agent compositions or may contain a divided pharmacologically or biologically effective amount.
  • the total effective amounts can then be administered in cumulative units containing, in total, pharmacologically or biologically or chemically active amounts of biologically or pharmacologically active agent.
  • the composition may include one or more adjuvants, one or more carriers, one or more excipients, one or more stabilizers, one or more imaging reagents, one or more effectors; one or more photodynamic agents; and/or physiologically acceptable saline.
  • composition of the invention can additionally comprise inert constituents including carriers, diluents, fillers, salts, and other materials well-known in the art, the selection of which depends upon the particular purpose to be achieved and the properties of such additives which can be readily determined by one skilled in the art.
  • the composition includes pharmaceutically acceptable carriers.
  • Pharmaceutically accepted carriers include but are not limited to saline, sterile water, phosphate buffered saline, and the like. Other buffering agents, dispersing agents, and inert non-toxic substances suitable for delivery to a patient may be included in the compositions of the present invention.
  • the compositions may be solutions suitable for administration, and are typically sterile and free of undesirable particulate matter.
  • compositions may be sterilized by conventional sterilization techniques. It may be desirable to add a preservative to the formulation of the present invention.
  • the polypeptide or antibodies of the present invention may be used to treat a mammal.
  • the antibody is administered to a nonhuman mammal for the purposes of obtaining preclinical data, for example.
  • exemplary nonhuman mammals to be treated include nonhuman primates, dogs, cats, rodents and other mammals in which preclinical studies are performed. Such mammals may be established animal models for a disease to be treated with the antibody or may be used to study toxicity of the antibody of interest. In each of these embodiments, dose escalation studies may be performed on the mammal to ascertain the optimal dosage.
  • dosages for any one patient depend on many factors, including the general health, sex, weight, body surface area, and age of the patient, as well as the particular compound to be administered, the time and route of administration, and other drugs being administered concurrently.
  • an effective amount means an amount required to achieve a desired end result.
  • the amount required to achieve the desired end result will depend on the nature of the disease or disorder being treated, and can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed will also depend on the route of administration and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the cell line A431 was maintained in Dulbecco's modified eagle medium (DMEM) containing 10% FBS, 1% Penicillin-Streptomycin, and 2OmM HEPES.
  • DMEM Dulbecco's modified eagle medium
  • the principle of this assay is based on the ability of the protein dye sulforhodamine B to bind electrostatically and pH dependently on amino acid residues of trichloroacetic acid (TCA)-fixed cells. Under mild acidic conditions SRB binds to the protein of cells and under mild basic conditions it can be eluted from cells for measurement. Results of the SRB assay are linear with respect to cell number and cellular protein amount.
  • A431 human epidermoid carcinoma cells overexpressing EGFR
  • A431 human epidermoid carcinoma cells overexpressing EGFR
  • Plates were incubated in a humidified CO 2 incubator at 37 0 C for 24 hours.
  • One 96-well plate was fixed using 10% TCA to be used as the 0-hour baseline value.
  • Serial dilutions of Rapamycin (R) and hR3 were performed using 96-well round-bottom plates. Control per plate: twelve wells as background reference and twelve wells as negative controls.
  • R + 83 hR3 shows a significantly higher percentage of cytotoxicity as compared with R alone.
  • hR3 + 25 R also shows significantly higher cytotoxicity as compared with hR3 alone.
  • the cell line A431 was maintained in DMEM containing 10% FBS, 1% Penicillin- Streptomycin, and 2OmM HEPES.
  • This colorimetric assay uses a soluble form of MTT, known as MTS, to determine the cell viability in a sample.
  • MTT a soluble form of MTT
  • Metabolically active cells bioreduce MTS into a soluble formazan product, so this conversion is directly related to the number of viable cells. It is the absorbance of the formazan product that is measured spectrophotometrically.
  • the cell line A431 was maintained in DMEM containing 10% FBS, 1% Penicillin- Streptomycin, and 2OmM HEPES.
  • This colorimetric assay uses a soluble form of MTT, known as MTS, to determine the cell viability in a sample.
  • MTT a soluble form of MTT
  • Metabolically active cells bioreduce MTS into a soluble formazan product, so this conversion is directly related to the number of viable cells. It is the absorbance of the formazan product that is measured spectrophotometrically.
  • A431 cells were seeded at a concentration of 10,000 cells per well in four 96-well flat- bottom plates, leaving twelve wells per plate without cells as a background reference. Plates were incubated in a humidified CO 2 incubator at 37 0 C for 24 hours. The identical plate set-up and dilutions as the SRB assay were used, but in this MTS-based assay, the total volume per well was lOO ⁇ L. Plates were incubated in a humidified CO 2 values normalized. Here, Rapamycin alone displays greater cytotoxicity than hR3 in its lower doses and begins to show similar cytotoxicity as the dose concentration increases. The Bliss curve shows the dose response of R + hR3 if the combination effect was exactly additive.
  • Cell extracts were prepared by Radiolmmuno Precipitation Assay (RIPA) Buffer containing PMSF, protease inhibitor cocktail and sodium orthovanadate (sc-24948, Santa Cruz Biotechnology, Santa Cruz, CA). The soluble protein concentration was determined by the Bradford method as a macro-bovine serum albumin assay (#105570, Bangalore Genei, Bangalore, India). Protein immuno-detection was done by electrophoretic transfer of 10% SDS-PAGE separated proteins to Polyvinylidene Fluoride (PVDF #162-0177, Biorad Laboratories,Hercules,CA), incubation with primary/secondary antibody, and chemiluminescent substrate ECL plus (RPN 2132,GE Healthcare, Buckinghamshire,UK).
  • RVDF #162-0177 Polyvinylidene Fluoride
  • the primary antibodies included phospho-Tyrosine[#9411] (pTYR), total EGFR[#2232], phospho-Akt(S473)[#9271] (pAKT), Akt panl lE7[#4685], phospho-MAPK (p42/p44)[#9101] (pMAPK), and phospho-Stat3(Y705)[#9131] (pSTAT3), mTOR[#2972], phosphor-S6 Ribosomal Protein(S235/236) [#2211] (pS6RP). All these primary antibodies were obtained from Cell Signaling Technologies (Danvers, MA). TFIID (TBP) (SI-I) [sc-273] was used as loading control which was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
  • A431 cells were seeded at 1x10 6 cells per well in a 6- well plate in DMEM 10%FBS, 1% Penicillin-Streptomycin, 2OmM HEPES. Plates were incubated in a humidified CO 2 incubator at 37 0 C for 24 hours. Following which they were washed once with 2mL IXDPBS (Gibco #14190).
  • TIh a non-specific IgG antibody was used here as a negative control. Plates were incubated in a humidified CO 2 incubator at 37 0 C for an additional 24 hours without EGF. Subsequently 10ng/mL of EGF was added and kept in humified CO 2 incubator for lOmins to stimulate the cells. The medium was removed, the plate wells were washed once with IX DPBS and cells were lysed as previously described. The total protein in the lysate was estimated by Bradfords method as mentioned above. 50ug of total protein was loaded and separated using 10%SDS-PAGE and it was transferred to a PVDF membrane by a overnight wet transfer method at 4 0 C.
  • the membrane was blocked with 3% Bovine Serum Albumin (# RM' 3151, Himedia Laboratories,Mumbai,India) for one hour at room temperature.
  • the blots were then incubated overnight at 4 0 C with respective primary antibody diluted as per the manufacturer's dilution recommendation. Subsequently the blots were washed thrice with IX Tris buffered saline-Tween20 (TBS-T).
  • TBS-T IX Tris buffered saline-Tween20
  • the secondary antibody was added and incubated for one hour at room temperature.
  • the blots were washed again in IX TBS-T and to the membrane ECL plus reagent was added.
  • the blots were exposed to scientific imaging film (BiomaxTMMS film (#829 4985 Kodak, Rochester,NY) and the film was developed using hyperprocessor ( SRX-IOlA, GE Amersham Biosciences). The blots were quantified using Alpha view software (Alpha Innotech, San Leandro, CA).
  • Figure 5 shows a relative expression of proteins downstream to hR3 and rapamycin by western blotting.
  • the samples 1-8 have been normalized to control 3 (cells + TIh + EGF) and samples 9-15 have been normalized to the sample 9 (cells + DMSO + EGF).
  • the numbers below the blot are relative values to their respective controls. TFIID values were observed to be almost equivalent and hence not mentioned.
  • the proteins like pTyr (17OkDa), pMAPK and pSTAT3 that are downstream effectors of EGFR were downregulated by BioMab EGFR (hR3) while, rapamycin treatment did not have any effect on these proteins except for pTyr at the highest concentration of rapamycin.
  • hR3 was prepared in PBS and Rapamycin was prepared in DMSO as separate injection solutions.
  • the formalin fixed tumor tissues from the mice xenografts were processed and mounted in wax.
  • Microtome sections at 5 micron thick were taken on poly L lysine coated slides. The sections were deparaffmized in three changes Xylene (Merck) of 15 minutes, 10 minutes and finally 5 minutes.
  • the slides are subsequently transferred in to alcohol grades of 100, 90 and 80% for three minutes each. Finally the sections are washed in running tap water for around two minutes.
  • the slides are then transferred to Hematoxyline (Harris Hematoxlyin solution Merck) for 7 - 10 minutes.
  • the sections are then brought to tap water and washed till the sections are blue.
  • the section is then differentiated using 1% acid alcohol with 1-2 dips.
  • the sections are subsequently dipped in ammonia water and washed in tap water till the sections are blue.
  • the sections are then counter stained with Eosin (Eosin Yellowish Merck) for 30 seconds and washed in running tap water for two minutes.
  • the sections are dehydrated in ascending grades of alcohol cleared in Xylene and coverslip mounted with DPX (Merck).
  • Figure 7 shows the representation of the scores in each group, with one of the combinations showing substantially lower mitotic activity.

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Abstract

Cette invention concerne une polythérapie qui consiste en un agent d'inhibition du EGFR qui inhibe à la fois le facteur EGF natif se liant au récepteur et un immunosuppresseur pour traiter un hôte mammifère, de préférence, un humain, par administration d'une quantité thérapeutiquement efficace du médicament de manière à prévenir et/ou à traiter des tumeurs. L'immunosuppresseur sélectionné dans le groupe comprenant la rapamycine, le tacrolimus, l'évérolimus, le pimécrolimus sous une forme libre ou sous la forme d'un sel pharmaceutiquement acceptable ou de solvates pharmaceutiquement acceptables. Cette invention concerne également des conjugués de médicaments anti-EGFR, ainsi que des compositions contenant éventuellement au moins un excipient pharmaceutiquement acceptable destinées à une utilisation simultanée.
PCT/IN2008/000688 2007-10-22 2008-10-20 Composition pharmaceutique et procédé correspondant WO2009054001A1 (fr)

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US8481498B1 (en) 2012-01-17 2013-07-09 Steven Hoffman Pharmaceutical compositions and methods
US9308188B2 (en) 2013-10-22 2016-04-12 Steven Hoffman Compositions and methods for treating intestinal hyperpermeability
US9549969B2 (en) 2012-01-17 2017-01-24 Tyme, Inc. Pharmaceutical compositions and methods
US9585841B2 (en) 2013-10-22 2017-03-07 Tyme, Inc. Tyrosine derivatives and compositions comprising them
US9763903B2 (en) 2013-10-22 2017-09-19 Steven Hoffman Compositions and methods for treating intestinal hyperpermeability
US10272068B2 (en) 2012-01-17 2019-04-30 Tyme, Inc. Pharmaceutical compositions and methods
US10646552B2 (en) 2012-01-17 2020-05-12 Tyme, Inc. Pharmaceutical compositions and methods
US10751313B2 (en) 2013-10-22 2020-08-25 Yamo Pharmaceuticals Llc Compositions and methods for treating autism
US10813901B2 (en) 2013-10-22 2020-10-27 Yamo Pharmaceuticals Llc Compositions and methods for treating autism

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WO2004056847A2 (fr) * 2002-12-16 2004-07-08 Genmab A/S Anticorps monoclonaux humains contre le recepteur de facteur de croissance epidermique (egfr)
WO2005056606A2 (fr) * 2003-12-03 2005-06-23 Xencor, Inc Proteines optimisees qui ciblent le recepteur du facteur de croissance epidermique
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Publication number Priority date Publication date Assignee Title
US10272068B2 (en) 2012-01-17 2019-04-30 Tyme, Inc. Pharmaceutical compositions and methods
US11103559B2 (en) 2012-01-17 2021-08-31 Tyme, Inc. Pharmaceutical compositions and methods
US11052068B2 (en) 2012-01-17 2021-07-06 Tyme, Inc. Pharmaceutical compositions and methods
US9549969B2 (en) 2012-01-17 2017-01-24 Tyme, Inc. Pharmaceutical compositions and methods
US10646552B2 (en) 2012-01-17 2020-05-12 Tyme, Inc. Pharmaceutical compositions and methods
US8481498B1 (en) 2012-01-17 2013-07-09 Steven Hoffman Pharmaceutical compositions and methods
US10507198B2 (en) 2012-01-17 2019-12-17 Tyme, Inc. Pharmaceutical compositions and methods
US9895425B2 (en) 2012-01-17 2018-02-20 Tyme, Inc. Pharmaceutical compositions and methods
US10010590B2 (en) 2012-01-17 2018-07-03 Tyme, Inc. Pharmaceutical compositions and methods
US10307465B2 (en) 2012-01-17 2019-06-04 Tyme, Inc. Pharmaceutical compositions and methods
US9757348B2 (en) 2013-10-22 2017-09-12 Steven Hoffman Composition and methods for treating intestinal hyperpermeability
US10085959B2 (en) 2013-10-22 2018-10-02 Steven Hoffman Compositions and methods for treating intestinal hyperpermeability
US9763903B2 (en) 2013-10-22 2017-09-19 Steven Hoffman Compositions and methods for treating intestinal hyperpermeability
US10517845B2 (en) 2013-10-22 2019-12-31 Steven Hoffman Compositions and methods for treating intestinal hyperpermeability
US9585841B2 (en) 2013-10-22 2017-03-07 Tyme, Inc. Tyrosine derivatives and compositions comprising them
US10751313B2 (en) 2013-10-22 2020-08-25 Yamo Pharmaceuticals Llc Compositions and methods for treating autism
US10813901B2 (en) 2013-10-22 2020-10-27 Yamo Pharmaceuticals Llc Compositions and methods for treating autism
US10857118B2 (en) 2013-10-22 2020-12-08 Yamo Pharmaceuticals Llc Compositions and methods for treating intestinal hyperpermeability
US9326962B2 (en) 2013-10-22 2016-05-03 Steven Hoffman Compositions and methods for treating intestinal hyperpermeability
US11058638B2 (en) 2013-10-22 2021-07-13 Tyme, Inc. Tyrosine derivatives and compositions comprising them
US9308188B2 (en) 2013-10-22 2016-04-12 Steven Hoffman Compositions and methods for treating intestinal hyperpermeability
US11633372B2 (en) 2013-10-22 2023-04-25 Yamo Pharmaceuticals Llc Compositions and methods for treating autism
US11786496B2 (en) 2013-10-22 2023-10-17 Yamo Pharmaceuticals Llc Composition and methods for treating intestinal hyperpermeability

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