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WO2004026252A2 - Traitements du gliome - Google Patents

Traitements du gliome Download PDF

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Publication number
WO2004026252A2
WO2004026252A2 PCT/US2003/029582 US0329582W WO2004026252A2 WO 2004026252 A2 WO2004026252 A2 WO 2004026252A2 US 0329582 W US0329582 W US 0329582W WO 2004026252 A2 WO2004026252 A2 WO 2004026252A2
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WO
WIPO (PCT)
Prior art keywords
glioma
tumor
antithrombic
argatroban
group
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PCT/US2003/029582
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English (en)
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WO2004026252A3 (fr
Inventor
Ya Hua
Guohua Xi
Richard F. Keep
Julian T. Hoff
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University of Michigan System
University of Michigan Ann Arbor
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University of Michigan System
University of Michigan Ann Arbor
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Priority to AU2003272591A priority Critical patent/AU2003272591A1/en
Publication of WO2004026252A2 publication Critical patent/WO2004026252A2/fr
Publication of WO2004026252A3 publication Critical patent/WO2004026252A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof

Definitions

  • the present invention relates to novel therapies and therapeutic targets for treating and reducing the symptoms associated with tumors of the central nervous system (CNS).
  • CNS central nervous system
  • the present invention provides novel glioma therapies comprising administering agents that inhibit the coagulation process and, more particularly, inhibiting one or more of the various factors involved therein.
  • the present invention provides methods of treating gliomas comprising administering a therapeutically effective amount of an antithrombin agent (e.g., ARGATROBAN) to a subject.
  • an antithrombin agent e.g., ARGATROBAN
  • Brain tumors are among the most devastating and medically intractable forms of cancer. The diagnosis of a brain or spinal cord tumor often comes as a shock, leaving confusion, uncertainty, fear, or even anger in its wake. Brain and spinal cord tumors are abnormal growths of tissue found inside the skull or the bony spinal column. The word tumor is used to describe both abnormal growths that are new (neoplasms) and those present at birth (congenital tumors). No matter where a tumor is located, they are usually called benign (or non-cancerous) if the tumor cells are similar to normal cells, grow relatively slowly, and are confined to one location. Tumors are called malignant (or cancerous) when the cells are very different from normal cells, grow relatively quickly, and can spread easily to other locations.
  • a benign tumor growing next to an important blood vessel in the brain does not have to grow very large before it can block blood flow. Additionally, if a benign tumor is found deep inside the brain, surgery to remove it maybe very risky because of the chances of damaging vital brain centers.
  • Primary CNS tumors rarely grow from neurons (i.e., nerve cells that perform the nervous system's important functions) because once neurons are mature they no longer divide and multiply. Instead, most tumors are caused by out-of-control growth among cells that surround and support neurons.
  • Primary CNS tumors such as gliomas and meningiomas, are named by the types of cells they contain, their location, or both.
  • primary brain tumors may result from specific genetic diseases, such as neurofibromatosis and tuberous sclerosis, or exposure to radiation or cancer-causing chemicals.
  • specific genetic diseases such as neurofibromatosis and tuberous sclerosis, or exposure to radiation or cancer-causing chemicals.
  • smoking, alcohol consumption, and certain dietary habits are associated with some types of cancers, they have not been linked to primary brain and spinal cord tumors.
  • the cause of most primary brain and spinal cord tumors remains a mystery. Brain and spinal cord tumors are largely not preventable at this time and many tumors are associated with poor prognoses.
  • brain tumors arise in more than 40,000 Americans each year. About half of these tumors are primary, and the remainder are metastatic secondary. Individuals of any age can develop brain tumors. In fact, they are the second most common cause of cancer-related death in people up to the age of 35, with a slight peak in occurrence among children between the ages of 6 and 9. However, brain tumors are most common among middle-aged and older adults. People in their 60s face the highest risk. Each year 1 of every 5,000 people in this age group develops a brain tumor. Spinal cord tumors are less common than brain tumors with about 10,000 Americans developing primary or metastatic spinal cord tumors each year. Although spinal cord tumors affect people of all ages, they are most common in young and middle-aged adults.
  • Gliomas tumors of the brain's glial cells, are particularly damaging form of brain and CNS tumors.
  • Malignant gliomas are the most common form of brain cancer and are one of the most rapidly progressive and universally fatal of all cancer types. Each year, approximately 20,000 Americans are diagnosed with gliomas. More than half of those afflicted will die within 18 months.
  • the brain's left and right hemispheres jointly control hearing and vision; the front part of each hemisphere controls voluntary movements, like writing, for the opposite side of the body; and the brainstem is responsible for basic life-sustaining functions, including blood pressure, heartbeat, and breathing.
  • Brain tumors can cause a bewildering array of symptoms depending on their size, type, and location. Certain symptoms are quite specific because they result from damage to particular brain areas. Other, more general symptoms are triggered by increased pressure within the skull as the growing tumor encroaches on the brain's limited space or blocks the flow of cerebrospinal fluid (fluid that bathes the brain and spinal cord). Initial symptoms of brain tumors may include headache, vomiting, seizures, behavioral and cognitive changes, and motor and balance problems. More advanced brain tumors may cause weakness on one side of the body, difficulty with speech and changes in vision, hearing, and sensation perception. Whatever specific symptoms a patient has, the symptoms generally develop slowly and worsen over time.
  • the tumor In radiation therapy, the tumor is bombarded with energy that kills tumor cells.
  • Traditional radiation therapy delivers radiation from outside the patient's body, is usually begun a week or two after surgery, and continues for about six weeks. The dosage is fairly uniform throughout the treated areas, making it especially useful for tumors that are large or have infiltrated into surrounding tissue.
  • traditional radiation therapy is given to the brain, it may also cause damage to surrounding healthy tissue.
  • stereotactic radiosurgery techniques that use a combination of surgical and radiation methods have become available. Hyperfractionation radiation therapies have been tried, especially in children, in an effort to increase treatment effectiveness and decrease side effects.
  • Chemotherapy uses tumor-killing drugs that are given orally or injected into the bloodstream. Because not all tumors are vulnerable to the same anticancer drugs, physicians often use a combination of drugs for chemotherapy. Chemotherapeutic drugs generally kill cells that are actively growing or dividing, making these compounds relatively more effective against malignant tissue, which contains a high proportion of growing and dividing cells, than to most normal cells. Because a high proportion of the cells in the skin, gastrointestinal tract, and other areas are also growing and dividing at any given time, the side effects of chemotherapy commonly include skin reactions, hair loss, and digestive disturbances. The drugs BCNU (sometimes called carmustine) and CCNU (or lomustine) commonly used treating CNS tumors.
  • BCNU sometimes called carmustine
  • CCNU or lomustine
  • the present invention relates to novel therapies and therapeutic targets for treating and reducing the symptoms associated with tumors of the central nervous system (CNS).
  • the present invention provides novel glioma therapies comprising administering agents that inhibit the coagulation process and, more particularly, inhibiting one or more of the various factors involved therein.
  • the present invention provides methods of treating gliomas comprising administering a therapeutically effective amount of an antithrombin agent (e.g., ARGATROBAN) to a subject.
  • an antithrombin agent e.g., ARGATROBAN
  • the present invention provides methods of treating cancer in a subject, comprising: providing: a subject suspected of having a glioma; and an anticoagulant; administering the anticoagulant to the subject under conditions wherein the anticoagulant inhibits the growth of the glioma.
  • the anticoagulant inhibits thrombin.
  • anticoagulant comprises ARGATROBAN. It is understood, however, that in some aspects of the present invention anticoagulants encompass antithrombic agents such as ARGATROBAN.
  • the present invention is not limited however to providing novel therapeutic methods comprising administering ARGATROBAN to a subject.
  • derivatives, prodrugs, and pharmaceutically acceptable salts of ARGATROBAN are specifically contemplated, as are methods comprising co-administration of additional antithrombin agents (e.g., MELAGATRAN, H376/95, EFEGATRAN, boroarginine, boropeptides, hirudin, and derivatives and pharmaceutically acceptable salts thereof).
  • low molecule weight antithrombin agents contemplated for use include those described, for example, by C. Taparelli et al., Trends Pharmacol. Sci., 14(10):366-376 (1993) and D. N ⁇ teberg et al, J. Med. Chem., 43(9):1705-1713 (2000).
  • Still other embodiments of the present invention provide methods for the co-administration of at least one antithrombin agent (e.g., ARGATROBAN) with anti-platelet or platelet inhibitory agents (e.g., aspirin, piroxicam, ticlopidine, or clopidogrel, and factor Xa inhibitors, and the like), or anti-coagulant agents (e.g., acenocoumarol, anisindione, and dicumarol and the like), hi still other embodiments, the methods of the present invention further comprise co- administration of antisense, or siRNA molecules, or antibodies.
  • antithrombin agent e.g., ARGATROBAN
  • anti-platelet or platelet inhibitory agents e.g., aspirin, piroxicam, ticlopidine, or clopidogrel, and factor Xa inhibitors, and the like
  • anti-coagulant agents e.g., acenocoumarol, anisindione, and dicumarol and the like
  • the therapeutic agents administered in the methods of the present invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) a biologically active metabolite or residue thereof.
  • the present invention also includes prodrugs and pharmaceutically acceptable salts of such prodrugs, and other bioequivalents of the compounds used in the methods described herein.
  • the methods of the present invention comprise co- administration of an antithrombic and at least one additional cancer therapy, including, but not limited to, radiation therapy, surgical resection, chemotherapy (e.g., anticancer agents daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin, diethylstilbestrol (DES) etc.); anti-inflammatory drugs (e.g., nonsteroidal anti-inflammatory drugs, and corticosteroids etc.); and antiviral drugs (e.g., ribivirin, vidarabine, a
  • administrations of the present compositions provide effective methods of treating (e.g., ameliorating) or arresting (e.g., prophylaxis) disease states (e.g., cancer) in a subject.
  • the drug transported by the present compositions is gelonin.
  • compositions and methods for targeting and delivering many other therapeutic agents and molecules including, but not limited to: agents that induce apoptosis (e.g., Geranylgeraniol [3,7,1 l,15-tetramethyl-2,6,10,14-hexadecatraen-l-ol], pro-apoptotic Bcl-2 family proteins including Bax, Bak, Bid, and Bad); polynucleotides (e.g., DNA, RNA, ribozymes, RNAse, siRNAs, etc); polypeptides (e.g., enzymes); photodynamic compounds (e.g., Photofrin (IT), ruthenium red compounds [e.g., Ru-diphenyl-phenanthroline and Tris(l-10- phenanthroline)ruthenium(II) chloride], tin ethyl etiopurpurin, protoporphyrin IX, chloroaluminum phthalo
  • the present methods comprises use anticancer agents such as agents that induce or stimulate apoptosis including, but not limited to: kinase inhibitors (e.g., epidermal growth factor receptor kinase inhibitor [EGFR]); vascular growth factor receptor kinase inhibitor [VGFR]; fibroblast growth factor receptor kinase inhibitor [FGFR] ; platelet-derived growth factor receptor kinase inhibitor [PGFR] ; and Bcr-Abl kinase inhibitors such as STI-571, Gleevec, and Glivec); antisense molecules; antibodies (e.g., Herceptin and Rituxan); anti-estrogens (e.g., Raloxifene and Tamoxifen); anti- androgens (e.g., flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazo
  • kinase inhibitors e.g., epidermal growth factor receptor kinase inhibitor [EGFR
  • compositions of the present invention provide treatments for a number of conditions including, but not limited to, brain and CNS system cancers, breast cancer, prostate cancer, lung cancer, lymphomas, skin cancer, pancreatic cancer, colon cancer, melanoma, ovarian cancer, brain cancer, head and neck cancer, liver cancer, bladder cancer, non-small lung cancer, cervical carcinoma, leukemia, neuroblastoma and glioblastoma, and T and B cell mediated autoimmune diseases and the like.
  • the methods of the present invention inhibit tumor growth and/or reduce the symptoms associated with gliomas (e.g., brain edema and tumor- related neurological deficits, including, but not limited to, increased intracranial pressure, headaches, seizures, weakness in the extremities, vision loss, loss of spatial orientation, diminished sensation sensory perception, difficulty with speech, memory loss, behavioral changes, and personality changes).
  • gliomas e.g., brain edema and tumor- related neurological deficits, including, but not limited to, increased intracranial pressure, headaches, seizures, weakness in the extremities, vision loss, loss of spatial orientation, diminished sensation sensory perception, difficulty with speech, memory loss, behavioral changes, and personality changes.
  • the present invention is directed to providing novel methods of treating cancers, and preferably for treating gliomas.
  • Gliomas suitable for treatment using the methods of the present invention include, but are not limited to, astrocytomas, ependymomas, oligodendrogliomas, and mixed gliomas.
  • the present invention provides methods of treating glioma in a subject, comprising: providing: a subject suspected of having a glioma; and an antithrombic; administering the antithrombic to the subject under conditions wherein the antithrombic inhibits the growth of the glioma.
  • the antithrombic comprises ARGATROBAN.
  • Additional embodiments provide methods of treating glioma in a subject, comprising: providing: a subject suspected of having a glioma; and ARGATROBAN; administering the ARGATROBAN to the subject under conditions wherein the ARGATROBAN inhibits the grow of the glioma.
  • the present invention also provides novel methods of treating glioma in a subject, comprising: providing: a subject suspected of having a glioma; an antithrombic; and at least one additional glioma treatment selected from the group consisting of radiation therapy, chemotherapy, and surgical resection; and administering the antithrombic and the at least one additional glioma treatment to the subject under conditions wherein the antithrombic and the at least on additional glioma treatment inhibit the grow of the glioma.
  • kits for treating cancer in a subject comprising: an antithrombic wherein the antithrombic inhibits the growth of glioma; instructions for using the antithrombic to inhibit the growth of the glioma in the subject, i some of these embodiments, the kits comprise instructions for selecting particular agents (e.g., one or more antithrombics), agent dosing considerations, agent administering considerations, guidelines or requirements for selecting suitable subjects for treatment, techniques for evaluating treatment safety and efficacy, among other routine considerations in the therapeutic and pharmacological arts.
  • the instructions meet the requirements, legal or otherwise, of one any agency such as the Food and Drug Administration, or similar domestic or international agency.
  • kits comprising two or more antithrombic agents, and instructions
  • the present invention provides kits comprising one or more antithrombic agent and one or more additional agent (e.g., pro-apoptotic agent), and optionally instructions.
  • additional agent e.g., pro-apoptotic agent
  • compositions and methods of the present invention are contemplated for treatment with certain embodiments of the compositions and methods of the present invention.
  • the subjects are mammals.
  • the present methods are optimized to treat humans, however, the present invention is not limited to treating humans.
  • the present invention contemplates effective treatment methods for a variety of vertebrate animals including, but not limited to, cows, pigs, sheep, goats, horses, cats, dogs, rodents, birds, fish, and the like.
  • Figure 1A shows coronal brain section of a glioma (day 12).
  • Figure IF shows forelimb placing scored using a vibrissae-elicited forelimb-placing test. Values are mean ⁇ SEM.
  • Figures 3 A, and 3B show coronal sections of rat brain at day 9 with vehicle (Figure 3A) or ARGATROBAN (Figure 3B) treatment.
  • Figures 3C, and 3D show tumor size measured by serial brain sections with hematoxylin and eosin staining ( Figure 3C).
  • Figures 4A-4J show laminin iimnunostaining used to examine blood vessels in the glioma with vehicle in 5 rats ( Figures 4A, 4B, 4C, 4D, and 4E), and ARGATROBAN in 5 rats ( Figures 4F, 4G, 4H, 41, and 4J).
  • Scale bar 200 ⁇ m.
  • Figure 6 shows tumor size was measured at day 12 after C6 glioma cell implantation.
  • Figures 8A, 8B, and 8C show forelimb asymmetry (Figure 8A) at days 3, 6 and 9, forelimb placing score (Figure 8B), and corner turn (Figure 8C) score at days 3, 6, 9 and 12 after intracerebral infusion of F98 glioma cells with systemic treatment of ARGATROBAN or vehicle.
  • anticoagulant refers to inhibitors of the coagulation cascade.
  • inhibitors include, but are not limited to, agents (e.g., proteins and/or small molecules and the like) that inhibit fibrin/fribrinogen, factors X/Xa, LX/LXa, V/Va, ND/V ⁇ a, Xl/XIa, XU/X ⁇ a, Xm/X ⁇ ia, etc.).
  • thrombin inhibitor As used herein, the term "thrombin inhibitor,” “antithrombin agent,” or
  • antithrombic(s) refers to inhibitors of the serine protease thrombin.
  • various thrombin-mediated processes such as thrombin-mediated platelet activation (e.g., the aggregation of platelets, and/or the granular secretion of plasminogen activator inhibitor- 1 and/or serotonin), and/or fibrin formation are disrupted.
  • thrombin-mediated platelet activation e.g., the aggregation of platelets, and/or the granular secretion of plasminogen activator inhibitor- 1 and/or serotonin
  • fibrin formation include, but are not limited to, ARGATROBAN, boroarginine derivatives and boropeptides, hirudin, MELAGATRAN, H376/95 and EFEGATRAN, including pharmaceutically acceptable salts and prodrugs thereof.
  • thrombolytics refers to agents that lyse blood clots (thrombi). Such agents include, but are not limited to, tissue plasminogen activator, anistreplase, urokinase or streptokinase, including pharmaceutically acceptable salts or prodrugs thereof.
  • tissue plasminogen activator tPA is commercially available from
  • non-antithrombic therapeutic or “non-antithrombic therapeutic agent” refer in the broadest sense to any biologically active (or made to be active), or otherwise therapeutically, diagnostically, or pharmacologically useful compound not possessing antithrombic activity.
  • drug refers to a pharmacologically active substance or substances that are used to diagnose, treat, or prevent diseases or conditions. Drugs act by altering the physiology of a living organism, tissue, cell, or in vitro system that they are exposed to. It is intended that the term encompass antimicrobials, including, but not limited to, antibacterial, antifungal, and antiviral compounds. It is also intended that the term encompass antibiotics, including naturally occurring, synthetic, and compounds produced by recombinant DNA technology.
  • prodrug refers to a pharmacologically inactive derivative of a parent drug molecule that requires biotransformation (e.g., either spontaneous or enzymatic) within the organism to release, or to convert (e.g., enzymatically, mechanically, electromagnetically, etc) the prodrug into the active drug.
  • Prodrugs are designed to overcome problems associated with stability, toxicity, lack of specificity, or limited bioavailability.
  • the prodrug comprises the active drug compound itself and a beneficial chemical masking group (e.g., one that reversible suppresses activity and/or appreciably reduces toxicity).
  • prodrugs are variations or derivatives of the compounds that have groups cleavable under metabolic conditions.
  • prodrugs become pharmaceutically active in vivo when they undergo solvolysis under physiological conditions or undergo enzymatic degradation or other biochemical transformation (e.g., phosphorylation, hydrogenation, dehydrogenation, glycosylation etc).
  • Prodrugs often offer advantages of solubility, tissue compatibility, or delayed release in the mammalian organism. (See e.g., Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam [1985]; and Silverman, The Organic Chemistry of Drug Design and Drug Action,/?/?. 352-401, Academic Press, San Diego, CA [1992]).
  • Common prodrugs include acid derivatives such as, esters prepared by reaction of parent acids with a suitable alcohol, amides prepared by reaction of the parent acid compound with an amine, or basic groups reacted to form an acylated base derivative.
  • the prodrug derivatives of this invention may be combined with other commonly known pharmacological molecules and reaction schemes to enhance bioavailability.
  • Coadministration refers to administration of at least one antithrombic agent and one or more additional pharmacological agent (e.g., drug, prodrug, or small molecule), or therapy to a subject. Coadministration may be concurrent or, alternatively, the chemical compounds described herein may be administered in advance of or following the administration of the other agent(s). One skilled in the art can readily determine the appropriate dosage for coadministered agents. In some embodiments, the coadministered agents are administered in doses that are lower than indicated for either agent when administered alone.
  • additional pharmacological agent e.g., drug, prodrug, or small molecule
  • a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vivo, in vitro or ex vivo.
  • the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and an emulsion, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see Martin, Remington's Pharmaceutical Sciences, Gennaro AR ed. 20th edition, 2000: Williams & Wilkins PA, USA.
  • “Pharmaceutically acceptable salt” as used herein, relates to any pharmaceutically acceptable salt (acid or base) of a compound of the present invention which, upon administration to a recipient, is capable of providing a compound of this invention or an active metabolite or residue thereof.
  • “salts” of the compounds of the present invention maybe derived from inorganic or organic acids and bases.
  • acids examples include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2- sulfonic and benzenesulfonic acid.
  • Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid.
  • the term "therapeutically effective amount” refers to an amount of a compound sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, tumor growth or at least one symptom associated with a tumor, including, but not limited to, brain edema and tumor- related neurological deficits (e.g., increased intracranial pressure, headaches, seizures, weakness in the extremities, vision loss, loss of spatial orientation, diminished sensation sensory perception, difficulty with speech, memory loss, behavioral changes, and personality changes).
  • brain edema and tumor- related neurological deficits (e.g., increased intracranial pressure, headaches, seizures, weakness in the extremities, vision loss, loss of spatial orientation, diminished sensation sensory perception, difficulty with speech, memory loss, behavioral changes, and personality changes).
  • Neoplasm and “tumor” refer to a tissue growth which is characterized, in part, by the uncontrolled abnormal growth (e.g., hyperproliferation) of the cells comprising the tissue growth.
  • Neoplasms may be benign and are exemplified, but not limited to, a hemangioma, glioma, teratoma, and the like. Neoplasms may alternatively be malignant, for example, a carcinoma, sarcoma, glioblastoma, astrocytoma, neuroblastoma, retinoblastoma, and the like.
  • malignant neoplasm and "malignant tumor” refer to a neoplasm which contains at least one cancer cell.
  • a “cancer cell” refers to a cell undergoing early, intermediate or advanced stages of multi-step neoplastic progression as previously described [H.C. Pitot (1978) in “Fundamentals of Oncology,” Marcel Dekker (Ed.), New York pp 15-28]. The features of early, intermediate and advanced stages of neoplastic progression have been described using microscopy. Cancer cells at each of the three stages of neoplastic progression generally have abnormal karyotypes, including translocations, inversion, deletions, isochromosomes, monosomies, and extra chromosomes.
  • a cell in the early stages of malignant progression is referred to as "hyperplastic cell” and is characterized by dividing without control and/or at a greater rate than a normal cell of the same cell type in the same tissue. Proliferation may be slow or rapid but continues unabated.
  • a cell in the intermediate stages of neoplastic progression is referred to as a "dysplastic cell.”
  • a dysplastic cell resembles an immature epithelial cell, is generally spatially disorganized within the tissue and loses its specialized structures and functions. During the intermediate stages of neoplastic progression, an increasing percentage of the epithelium becomes composed of dysplastic cells. "Hyperplastic” and “dysplastic” cells are referred to as "pre-neoplastic” cells.
  • Neoplastic cells are typically invasive i.e., they either invade adjacent tissues, or are shed from the primary site and circulate through the blood and lymph to other locations in the body where they initiate one or more secondary cancers, i.e., "metastases.”
  • cancer is used herein to refer to a malignant neoplasm, which may or may not be metastatic.
  • cancers of the nervous tissues refer to any cancers associated with the central nervous system (e.g., the brain and spinal chord). Such cancers include both primary and secondary tumors.
  • Brain tumors include, but are not limited to astrocytoma, craniopharyngioma, glioma, ependymoma, neuroglioma, oligodendroglioma, glioblastoma multiforme, meningioma, and meduUoblastoma.
  • Secondary brain tumors occur from the spread of cancer into the brain from a distant cancerous organ (metastasis).
  • Gliomas are a diverse group of brain tumors that arise from the normal "glial” cells of the brain. These cells outnumber the “neurons” that conduct impulses and serve to provide metabolic support to the neurons. The most important determinant of survival for gliomas is the “grade” of the glioma. The low-grade gliomas have a protracted natural history, while the high grade gliomas (anaplastic astrocytoma and glioblastoma multiforme) are much more difficult to successfully treat. The gliomas are associated with specific signs and symptoms that are primarily related to the location of the glioma. Temporal lobe gliomas, for example may cause epilepsy, difficulty with speech or loss of memory.
  • the frontal lobe gliomas may cause behavioral changes, weakness in the extremities (e.g., arms and/or legs) and/or speech difficulty.
  • Occipital gliomas may cause loss of vision and parietal gliomas may cause loss of spatial orientation, diminished sensation on the opposite side of the body, and/or inability to recognize once familiar objects or persons.
  • the term "symptoms associated with glioma" refers to abnormal neurological conditions and brain pathologies commonly experienced by those affected with brain tumors (e.g., gliomas).
  • abnormal neurological conditions and brain pathologies include, but are not limited to, brain edema, and tumor-related neurological deficits (including, but not limited to, increased intracranial pressure, headaches, seizures, weakness in the extremities, vision loss, loss of spatial orientation, diminished sensation sensory perception, difficulty with speech, memory loss, behavioral changes, and personality changes).
  • tumor-related neurological deficits including, but not limited to, increased intracranial pressure, headaches, seizures, weakness in the extremities, vision loss, loss of spatial orientation, diminished sensation sensory perception, difficulty with speech, memory loss, behavioral changes, and personality changes.
  • the term "host” refers to any animal (e.g., warm blooded mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment.
  • the terms “host” and “patient” are used interchangeably herein in reference to a human subject.
  • non-human animals refers to all non-human animals.
  • Such non-human animals include, but are not limited to, vertebrates such as rodents, non- human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • the term “gene targeting” refers to the alteration of genes through molecular biology techniques. Such gene targeting includes, but is not limited to, generation of mutant genes and knockout genes through recombination. When a gene is altered such that its product is no longer biologically active in a wild-type fashion, the mutation is referred to as a "loss-of-function” mutation. When a gene is altered such that a portion or the entirety of the gene is deleted or replaced, the mutation is referred to as a “knockout” mutation.
  • the term “gene transfer system” refers to any means of delivering a composition comprising a nucleic acid sequence to a cell or tissue.
  • gene transfer systems include, but are not limited to vectors (e.g., retroviral, adenoviral, adeno- associated viral, and other nucleic acid-based delivery systems), microinjection of naked nucleic acid, polymer-based delivery systems (e.g., liposome-based and metallic particle- based systems), biolistic injection, and the like.
  • viral gene transfer system refers to gene transfer systems comprising viral elements (e.g., intact viruses and modified viruses) to facilitate delivery of the sample to a desired cell or tissue.
  • adenovirus gene transfer system refers to gene transfer systems comprising intact or altered viruses belonging to the family A denoviridae.
  • site-specific recombination target sequences refers to nucleic acid sequences that provide recognition sequences for recombination factors and the location where recombination takes place.
  • antisense is used in reference to RNA sequences that are complementary to a specific RNA sequence (e.g., mRNA). Included within this definition are antisense RNA (“asRNA”) molecules involved in gene regulation by bacteria. Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter that permits the synthesis of a coding strand. Once introduced into an embryo, this transcribed strand combines with natural mRNA produced by the embryo to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. In this manner, mutant phenotypes may be generated.
  • asRNA antisense RNA
  • antisense strand is used in reference to a nucleic acid strand that is complementary to the "sense” strand.
  • the designation (-) i.e., “negative” is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e., "positive") strand.
  • siRNAs refers to small interfering RNAs. hi some embodiments, siRNAs comprise a duplex, or double-stranded region, of about 18-25 nucleotides long; often siRNAs contain from about two to four unpaired nucleotides at the 3' end of each strand.
  • At least one strand of the duplex or double-stranded region of a siRNA is substantially homologous to, or substantially complementary to, a target RNA molecule.
  • the strand complementary to a target RNA molecule is the "antisense strand;" the strand homologous to the target RNA molecule is the "sense strand,” and is also complementary to the siRNA antisense strand.
  • siRNAs may also contain additional sequences; non-limiting examples of such sequences include linking sequences, or loops, as well as stem and other folded structures. siRNAs appear to function as key intermediaries in triggering RNA interference in invertebrates and in vertebrates, and in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants.
  • RNA interference refers to the silencing or decreasing of gene expression by siRNAs. It is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by siRNA that is homologous in its duplex region to the sequence of the silenced gene.
  • the gene maybe endogenous or exogenous to the organism, present integrated into a chromosome or present in a transfection vector that is not integrated into the genome.
  • the expression of the gene is either completely or partially inhibited.
  • RNAi may also be considered to inhibit the function of a target RNA; the function of the target RNA may be complete or partial.
  • biologically active refers to a protein or other biologically active molecules (e.g., catalytic RNA) having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • agonist refers to a molecule which, when interacting with a biologically active molecule, causes a change (e.g., enhancement) in the biologically active molecule, which modulates the activity of the biologically active molecule.
  • Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules that bind or interact with biologically active molecules.
  • agonists can alter the activity of gene transcription by interacting with RNA polymerase directly or through a transcription factor.
  • Antagonist refers to a molecule which, when interacting with a biologically active molecule, blocks or modulates the biological activity of the biologically active molecule.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecules that bind or interact with biologically active molecules.
  • Inhibitors and antagonists can affect the biology of entire cells, organs, or organisms (e.g., an inhibitor that slows tumor growth).
  • modulate refers to a change in the biological activity of a biologically active molecule. Modulation encompasses increases and decreases in activity, a change in binding characteristics, or any other change in the biological, functional, or immunological properties of biologically active molecules.
  • nucleic acid molecule refers to any nucleic acid containing molecule including, but not limited to DNA or RNA.
  • the term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-
  • the term "gene” refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor (e.g., prothrombin).
  • the polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the full-length or fragment are retained.
  • the term also encompasses the coding region of a structural gene and the including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full- length mRNA.
  • sequences that are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences.
  • sequences that are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences.
  • the term "gene” encompasses both cDNA and genomic forms of a gene.
  • a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions” or “intervening sequences.” Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers.
  • Introns are removed or "spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
  • mRNA messenger RNA
  • the mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
  • heterologous gene refers to a gene encoding a factor that is not in its natural environment.
  • a heterologous gene includes a gene from one species introduced into another species.
  • a heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to non-native regulatory sequences, etc).
  • Heterologous genes are distinguished from endogenous genes in that the heterologous gene sequences are typically joined to DNA sequences that are not found naturally associated with the gene sequences in the chromosome or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).
  • Heterologous genes may be introduced into hematopoietic stem cells through molecular biology manipulation.
  • the coding sequence of the heterologous gene is operatively linked to an expression control sequence.
  • a heterologous gene is first placed into a vector.
  • RNA expression refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene (i. e. , via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through “translation” of mRNA.
  • Gene expression can be regulated at many stages in the process.
  • Up-regulation” or “activation” refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while “down-regulation” or “repression” refers to regulation that decrease production.
  • Molecules e.g., transcription factors
  • activators e.g., transcription factors
  • nucleic acid molecule encoding As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
  • antigenic determinant refers to that portion of an antigen that makes contact with a particular antibody (i.e., an epitope).
  • a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants.
  • An antigenic determinant may compete with the intact antigen (i.e., the "immunogen” used to elicit the immune response) for binding to an antibody.
  • the term “vector” is used in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another.
  • the term “vehicle” is sometimes used interchangeably with “vector.” Vectors are often derived from plasmids, bacteriophages, or plant or animal viruses.
  • expression vector refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.
  • Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences.
  • Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
  • overexpression and “overexpressing” and grammatical equivalents are used in reference to levels of mRNA to indicate a level of expression approximately 3-fold higher than that typically observed in a given tissue in a control or non-transgenic animal.
  • Levels of mRNA are measured using any of a number of techniques known to those skilled in the art including, but not limited to Northern blot analysis. Appropriate controls are included on the Northern blot to control for differences in the amount of RNA loaded from each tissue analyzed (e.g., the amount of 28S rRNA, an abundant RNA transcript present at essentially the same amount in all tissues, present in each sample can be used as a means of normalizing or standardizing the mRNA-specific signal observed on Northern blots).
  • the amount of mRNA present in the band corresponding in size to the correctly spliced transgene RNA is quantified; other minor species of RNA which hybridize to the transgene probe are not considered in the quantification of the expression of the transgenic mRNA.
  • transfection refers to the introduction of foreign DNA into eukaryotic cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
  • stable transfection or “stably transfected” refers to the introduction and integration of foreign DNA into the genome of the transfected cell.
  • stable transfectant refers to a cell that has stably integrated foreign DNA into the genomic DNA.
  • transient transfection or “transiently transfected” refers to the introduction of foreign DNA into a cell where the foreign DNA fails to integrate into the genome of the transfected cell.
  • the foreign DNA persists in the nucleus of the transfected cell for several days. During this time the foreign DNA is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes.
  • transient transfectant refers to cells that have taken up foreign DNA but have failed to integrate this DNA.
  • cell culture refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro.
  • in vitro refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture.
  • in vivo refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.
  • test compound refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function.
  • Test compounds comprise both known and potential therapeutic compounds.
  • a test compound can be determined to be therapeutic by screening using the screening methods of the present invention.
  • a "known therapeutic compound” refers to a therapeutic compound that has been shown (e.g. , through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.
  • sample is used in its broadest sense, hi one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples.
  • Biological samples maybe obtained from animals (including humans) and encompass fluids, solids, tissues, and gases.
  • Biological samples include blood products, such as plasma, serum and the like.
  • Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples.
  • the present invention relates to novel methods for treating tumors of the CNS, and especially for treating gliomas.
  • the three types of glial cells, astrocytes, oligodendrocytes, and ependymal cells, provide a scaffold structure and metabolic support for the neurons and other tissues of the CNS.
  • Gliomas arise in, and are primarily named after, the various types of CNS glial cells.
  • Other gliomas are named after the center of the brain or CNS where they arise, for example, brainstem and optic gliomas.
  • gliomas contemplated for treatment by administration of the methods of the present invention are described below.
  • the present invention is not limited, however, to the enumerated types of gliomas. Indeed, certain embodiments of the present invention contemplate treating a number of mixed gliomas and other types of cancers of the head and neck as well as other types of primary and secondary cancers in other tissues (e.g., skin, bone, breast, ovaries, testicles, liver, colon, prostate, etc.).
  • Astrocytomas are tumors of astrocyte cells. About 60% of all primary brain tumors are astrocytomas. Pathologists grade astrocytomas on a four-point scale to indicate how drastically the cancerous astrocyte cells differ from normal non-cancerous cells. Grade I astrocytomas have slightly unusual looking cells, while grade IV astrocytomas are very abnormal in appearance. Brain tumors are categorized by the highest grade (most abnormal) cell seen in the tumor, however, astrocytomas often contain a mixture of cells and grades. Certain types of astrocytoma take their name from their general morphological appearance (e.g., butterfly glioma) or from their location (e.g., cerebellar astrocytoma).
  • cerebellar astrocytomas The majority, about 80%, of cerebellar astrocytomas are grade I, localized, cystic (fluid filled) tumors. Malignant cerebellar astrocytomas of this type of tumor are rare. Cerebellar astrocytomas are more common in children than in adults.
  • Desmoplastic infantile astrocytoma are rare grade I astrocytomas that tend to arise in the supratentorium area located above the membrane that separates the cerebral hemispheres from the posterior fossa of the brain. These large cystic tumors are usually diagnosed in infants under the age of two, although they are also occasionally seen in older children and young adults. Desmoplastic infantile astrocytomas often grow quite large and spread through more than one lobe of the brain.
  • Pilocytic astrocytoma also called juvenile pilocytic astrocytoma, are usually non- infiltrating grade I astrocytomas. Pilocytic astrocytomas are the most common gliomas in children. They are generally diagnosed in children and young adults under the age of 20, and are rarely seen in older adults. Many optic gliomas and cerebellar astrocytomas are pilocytic astrocytomas. These tumors generally form cysts or are enclosed within cysts.
  • Subependymal giant cell astrocytomas are ventricular tumors associated with 5-7% of patients with tuberous sclerosis (BoumeviUe's disease).
  • Tuberous sclerosis disease is an autosomal dominant genetic disorder that occurs near the foramen of Monro (interventricular foramen).
  • Diffuse astrocytoma are yet another type of astrocyte tumor. Diffuse astrocytomas are grouped by appearance and their behavior into one of three types: fibrillary, gemistocytic, and protoplasmic astrocytomas. Diffuse astrocytomas are usually of grade ⁇ relatively slow growing infiltrating tumors. The nuclei of fibrillary astrocytoma cells are cigar-shaped, while protoplasmic astrocytoma nuclei are round or oval in shape. Both fibrillary and protoplasmic astrocytoma cells tend to contain microcysts and are filled with a mucous-like fluid. Gemistocytic astrocytomas are plump, glassy, and angular shaped cells. Low grade diffuse astrocytoma tumors often reoccur as a higher-grade tumors.
  • Gliomas of the brainstem are usually comprised mainly of cancerous astrocyte cells. About 10-20% of brain tumors in children are brainstem gliomas. In children, these tumors are most often associated with children between 5 and 10 years old. These tumors can however also be found in adults between 30 and 40 years old. Brainstem tumors can range from grade I astrocytomas (mostly in children) to aggressive infiltrating grade II and IV tumors. Brainstem tumors are further classified based upon location, for example, upper brainstem (midbrain or tectum), middle brainstem (pons), and lower brainstem (cervico-medullary).
  • brainstem tumors can be localized or circumscribed, diffusely infiltrating, or exophytic.
  • the majority of brainstem tumors occur in the pons and are diffusely infiltrating, thus they are not amenable to surgical resection.
  • brainstem gliomas depend largely upon the location of the tumor. Common symptoms are related to eye movement abnormalities such as diplopia (double vision). Other symptoms include sensation changes of the face, swallowing difficulty and hoarseness. Weakness, loss/changes in sensation, or poor coordination on one side of the body may also occur. These tumors may also block cerebrospinal fluid circulation resulting in hydrocephalus that causes headache, nausea, vomiting and gait unsteadiness.
  • Treatment of brainstem gliomas is dictated by the tumor location, grade and the symptoms present. Surgical resection may be attempted on circumscribed or exophytic tumors. Shunts are often implanted if there is blockage of cerebrospinal fluid circulation. Localized brainstem gliomas are often treated with radiation therapy including focal radiosurgery. Hyperfractionation radiation therapy has been used in children to increase effectiveness and to decrease side effects. Unfortunately, hyperfractionation radiation therapy has not provided significant advantages over standard radiation therapies.
  • Chemotherapy using drugs similar to those used to treat glioblastomas are typically used if the tumor progresses following radiation therapy.
  • Optic gliomas can occur in any part of the optic pathway including the optic nerve, the optic chiasm, the optic tracts, or the optic radiations within the brain. Optic gliomas can spread along any of these pathways. These tumors may be slow growing, or quiet, causing few or no symptoms. The occurrence of the tumors along the nerves involved in vision can cause loss of vision in one or both eyes depending on tumor location. Hormonal disturbances associated with optical gliomas may also cause developmental delays or early onset puberty and other problems. Most optic glioma tumors occur in children under the age of 10.
  • Grade I pilocytic astrocytoma and grade II fibrillary astrocytoma are the most common types of optic gliomas, although higher-grade gliomas are possible in these tissues as well.
  • Adults with NF-1 generally do not develop optic gliomas.
  • Neurofibromatosis 2 (NF-2) is often associated with nervous system tumors and may include ependymomas of the spinal cord or brain.
  • optical gliomas Suggested treatment for optical gliomas is based on long-term observation and disease progression considering specific symptoms and recurring MRI scans. Surgical resection is available for optic gliomas involving only the optic nerve. Radiation therapies are used for tumors of the optic chiasm and other optic pathways.
  • Anaplastic astrocytomas and malignant astrocytomas are typically referred to as grade III astrocytomas.
  • Anaplastic astrocytoma tumors tend to have tentacle-like projections that grow into surrounding tissues, making them difficult to completely remove during surgery.
  • the invasive tentacle-like cells of anaplastic astrocytomas are very difficult to surgically remove from a subject's brain.
  • Treatment of anaplastic astrocytomas is usually more aggressive than treatment for lower grade gliomas and astrocytoma tumors.
  • anaplastic astrocytomas tend to recur, and when they do, they often regrow as higher-grade tumors.
  • Glioblastomas are more common in older adults, and affect more men than women. About 9% of childhood brain tumors are glioblastomas. Glioblastomas may arise from lower grade astrocytomas (grade II or Hi) or start directly as grade IV tumors. These tumors are generally found in the cerebral hemispheres of the brain, but can occur anywhere in the brain or spinal cord.
  • glioblastomas are referred to as being grade IV tumors because they have several features of rapidly growing tumors including abnormal and numerous blood vessels, as well as dead tissue called necrosis. Glioblastomas can easily intermingle with normal brain tissues and invade and migrate away from the main tumor site, however, glioblastomas rarely spread elsewhere in the body. Because glioblastomas are capable of very rapid growth, the first tumor symptoms are usually due to increased pressure in the brain with headaches, seizures, memory loss, and changes in behavior being the most common symptoms. Glioblastomas commonly contain a mixture of cell types. It is not unusual for these types of tumors to contain cystic material, calcium deposits, blood vessels, or a mixed grade of cells. The lack of uniformity in these types of tumors makes them very difficult brain tumors to treat since one cell type in the tumor mass may respond to treatment, while others may not. hi preferred embodiments, the antithrombic treatments of the present invention can be combined with previously described therapies.
  • the first step in treating glioblastomas is usually to surgically resect as much of the tumor as possible. Radiation therapy almost always follows surgery or biopsy. Several different forms of radiation therapy are currently used to treat glioblastomas including conventional external beam radiation, stereotactic radiosurgery, and conformal radiation therapy. Other types of radiation therapy, such as implanted liquid radiation or monoclonal antibodies tagged with radioactive particles, are also possible. Chemotherapy can be given before, during, or after radiation therapies. The most commonly used drugs for treating glioblastomas in adults include BCNU, CCNU, procarbazine, and temozolomide. In one existing treatment, biodegradable wafers containing BCNU are placed in the cavity created by tumor removal. However, clusters of glioblastoma cells commonly become resistant to radiation and chemotherapy.
  • protease inhibitors such as marimastat and tamoxifen, block the ability of tumor cells to make the proteins needed for tumor cell reproduction.
  • Angiogenesis inhibitors are used to interrupt blood supply to the tumor, thus controlling tumor growth.
  • Cytostatic agents are used to inhibit tumor cells from communicating with each other or with their environment (e.g., tamoxifen), invading and migrating (e.g., marimastat), undergoing angiogenesis (e.g., thalidomide, endostatin), and undergoing dedifferentiation (e.g., 13-cis-retinoic acid).
  • immunotherapy techniques are adapted to treat glioblastomas.
  • immunotoxins such as diptheria toxin or toxins from pseudomonas, attached to highly specific antibodies are used to carrier cellular toxins to tumor cells.
  • Additional treatments include attaching radioactive compounds or cytotoxic drugs to cancer cell specific antibodies.
  • Cytokine therapies e.g., interferons, interleukins, TNF
  • TNF interferons, interleukins, TNF
  • Vaccines using either tumor cells and/or immune cells are also available.
  • Gene therapies can also be used to restore the normal function of tumor suppressing genes that have been switched off by tumor cells.
  • antisense therapies are used to block the primary transcripts from malignant cells thus altering the cancer cell's ability to interfere with the normal growth of surrounding cells and tissues, hi still other embodiments, the present invention provides antisense therapies that block the production of prothrombin (e.g., target transcripts from the prothrombin gene).
  • prothrombin e.g., target transcripts from the prothrombin gene
  • Ependymoma arise from the ependymal glial cells that line the ventricles of the brain and the center of the spinal cord. Ependymoma tumors are relatively rare and account for only about 3-6% of all primary brain tumors in adults. Ependymomas are however the most common brain tumors in children. About one-third of pediatric ependymomas are found in children under the age three.
  • Ependymomas are soft, grayish or red colored tumors that often form cysts or mineral calcifications. There are four major types of ependyomas: ependymomas, anaplastic ependymomas, myxopapillary ependymomas, and subependymomas. A grading system similar to that used in astrocytoma pathology is often used to describe the appearance and pathological state of ependymomas.
  • Myxopapillary ependymomas tend to occur in the lower part of the spinal column, while subependymomas usually occur near a ventricle. Both of these types of ependymomas are slow growing and are usually considered low-grade or grade I tumors. Ependymomas are the most common form of this type of tumor and are considered grade II. Anaplastic ependymomas are difficult high-grade tumors (grades m or IV) that tend to be faster growing than low-grade ependymoma tumors. fritramedullary spinal cord tumors can include ependymomas, astrocytomas, and less commonly metastases .
  • oligodendrogliomas Glial tumors occurring in the oligodendrocytes are called oligodendrogliomas. Microscopic inspection of oligodendrocytes show that these glial cells have short appendages as compared to astrocytes that have long star-like appendages.
  • Oligodendrogliomas occur as low-grade (grade 11) tumors or high-grade (grade TR also called anaplastic oligodendrogliomas) tumors. Grading of oligodendrogliomas is based on microscopic observation of the appearance of the tumor cell's nucleus, the number of blood vessels, and presence or absence of dead tissue called necrosis. Higher-grades of oligodendrogliomas indicate more rapidly growing and aggressive tumors. Oligodendrogliomas are occasionally present in mixed type gliomas.
  • Oligodendrogliomas occur most frequently in young and middle-aged adults, but they can also found in children. The most common location for oligodendrogliomas is in the cerebral hemisphere, with about half of these tumors occurring in the frontal lobe. Seizures are the most common initial symptoms of oligodendrogliomas, particularly low- grade tumors.
  • Surgical resection is the standard treatment for accessible low-grade oligodendrogliomas. Notable, the prognosis for recovery from an oligodendroglioma tumor is generally better than that for same grade astrocytomas.
  • Treatment of anaplastic oligodendroglioma typically comprises a combination of radiation and chemotherapy using agents such as PCV (procarbazine, CCNU, and vincristine) or temozolomide.
  • Recurrent low-grade oligodendrogliomas are treated with surgery, radiation therapy and/or chemotherapy.
  • Recurrent anaplastic oligodendrogliomas are typically treated by surgical resection and/or chemotherapy.
  • Mixed oligoastrocytomas usually contain a high proportion of more than one type of cancerous glial cells. Most often these tumors contain astrocytes and oligodendrocytes, however, ependymal cells are also occasionally found in these tumors. The behavior of a mixed oligoastrocytomas is similar to other same
  • Treatment for mixed oligoastrocytomas is similar to that for astrocytomas and oligodendrogliomas of the same grade.
  • Treatment plans often include surgical resection followed by radiation therapy, particularly, if the tumor is high-grade (grade in or IV).
  • Chemotherapy is also generally used in high-grade tumors. Since these tumors usually have a component of oligodendroglioma, they often respond well to chemotherapy.
  • Ghomatosis cerebri is an uncommon primary brain tumor characterized by the diffuse spread of glial tumor cells in the brain. Ghomatosis cerebri tumors are distinguished from other types of gliomas by their scattered and widespread nature, typically involving two or more lobes of the brain. These tumors are often called "widespread low-grade glioma.” They occur most often in the cerebral hemispheres, and less often in the cerebellum and brainstem.
  • ghomatosis cerebri tumors lack many of the malignant features (e.g., abnormal blood vascularization and necrotic tissue) associated with high-grade gliomas.
  • the diffuse nature of ghomatosis cerebri often causes distension of the part of the brain where the tumor is located.
  • ghomatosis cerebri Symptoms of ghomatosis cerebri are often nonspecific but usually include personality and behavioral changes, memory disturbances, and increased intracranial pressure with headaches and seizures. Treatment of these tumors is less well defined than for other gliomas given their rarity and diffuse nature. Accordingly, surgical resection is generally not attempted because of their diffuse nature. Radiation and chemotherapies are often considered.
  • Ganglioneuromas are the rarest form of glioma; these tumors contain both glial cells and mature neurons. These tumors grow relatively slowly and may occur in the brain or spinal cord.
  • Thrombin cleaves fibrinogen ultimately leading to a hemostatic plug (clot formation), potently activates platelets through a unique proteolytic cleavage of the cell surface thrombin receptor (Coughlin, Seminars in Hematology 31(4):270-277 [1994]), and autoamplifies its own production through a feedback mechanism.
  • thrombin As a multifactorial protein, thrombin induces a number of effects on platelets, endothelial cells, smooth muscle cells, leukocytes, the heart, and neurons (Tapparelli et al, Trends in Pharmacological Sci., 14:366-376 [1993]; Church and Hoffman, Trends in Cardio. Med., 4(3): 140-146 [1993]). Platelet activation leads to shape change and aggregation as well as the synthesis release and secretion of vasoactive substances and lysosomal enzymes.
  • Endothelial cell activation results in the secretion of stimulatory agents leading to increased vascular permeability and adhesiveness for mononuclear cells, one consequence of which is extravasation of leukocytes at the site of thrombin generation.
  • Thrombin induces fibroblast and smooth muscle cell proliferation suggesting that thrombin plays a key role in lesion development following vascular damage.
  • Enhanced automaticity and prolongation of repolarization have been observed in cardiac myocytes showing sensitivity to thrombin.
  • Thrombin has been shown to influence normal neural development.
  • inhibitors of thrombin function have therapeutic potential in a host of cardiovascular and non-cardiovascular diseases, including: myocardial infarction; unstable angina; stroke; restenosis; deep vein thrombosis; disseminated intravascular coagulation caused by trauma, sepsis or tumor metastasis; hemodialysis; cardiopulmonary bypass surgery; adult respiratory distress syndrome; endotoxic shock; rheumatoid arthritis; ulcerative colitis; induration; metastasis; hypercoaguability during chemotherapy; Alzheimer's disease; and Down's syndrome.
  • the discovery and cloning of series of thrombin receptors See e.g., S. R.
  • thrombin may play a role in a range of other important biological processes. For example, evidence exists for its role in inducing brain edema formation, angiogenesis, and cell proliferation. Edema formation, angiogenesis, and cell proliferation heavily influence the poor prognosis associated with certain types of tumors and especially the poor prognosis in gliomas.
  • CNS tumors e.g., gliomas
  • CNS tumors e.g., gliomas
  • the vasculature of glioma tumors is typically highly permeable with the potential for prothrombin entry from blood into the tumor.
  • Brain edema in and around gliomas contributes to the high mortality (within months) that is seen in patients with malignant gliomas by causing herniation-related death.
  • thrombin is essential for rapid glioma growth because of the need for oxygen and metabolites. Although many factors regulate angiogenesis, the present invention contemplates that thrombin may play a key role.
  • Thrombin is a potent mitogen, that appears to enhance the synthesis and secretion of nerve growth factors (NGFs) in glial cells. It also appears to stimulate astrocyte and tumor cell proliferation (See e.g., I. Neveu et al, J. Neurochem, 60:858-867 [1993] and K. P. Cavanaugh et al, J. Neurochemistry, 54:1735- 1743 [1990]).
  • Thrombin also appears to play a major role in edema foraiation in the brain after intracerebral hemorrhage (K. R. Lee, et al, J. Neurosurg, 84:91-96 [1996]). Thrombin is produced immediately in the brain after intracerebral hemonhage or blood-brain barrier breakdown following many kinds of brain injury (M. B. Gingrich and S. F. Traynelis, Trends in Neurosciences, 23:399-407 [2000]). Direct infusion of large doses of thrombin into the brain causes inflammatory cell infiltration, mesenchymal cell proliferation and brain edema formation (K. R. Lee, et al, J.
  • Thrombin-induced brain edema results from a direct opening of the blood-brain barrier or direct neurotoxicity (K. R. Lee et al, J. Neurosurg, 86:272-278 [1997]). Thrombin, at high concentrations, also kills neurons and astrocytes in vitro (P. J. Vaughan et al, J. Neurosci.,
  • thrombin and other factors associated with the coagulation cascade provide a novel treatment for gliomas and other types of CNS tumors.
  • a rat glioma model was used to examine the role of thrombin in the development of gliomas. Experiments show that thrombin activity is elevated and that thrombin positive cells are present in rat gliomas.
  • Administration of antithrombin treatments reduced brain edema, tumor growth, and tumor-related neurological deficits.
  • one or more anticoagulants e.g., fibrin/fribrinogen, factors X/Xa, IX/IXa, V/Va, YWVJIa, XE/XIa, XH/XIIa, Xm/X ⁇ ia, etc.
  • the present invention comprises administering anticoagulant or thrombolytic agents that inhibit (e.g., prevent) the conversion of fibrinogen to fibrin by thrombin.
  • the present invention is not limited however to administering antithrombin agents to subjects suffering from gliomas.
  • the coagulation process involves numerous factors and profactors several that are suitable targets for inhibition and in the present glioma treatments.
  • the present invention relates to novel therapies and therapeutic targets for treating and reducing the symptoms associated with tumors of the central nervous system (CNS).
  • the present invention provides novel glioma therapies comprising administering agents that inhibit the coagulation process and, more particularly, inhibiting one or more of the various factors involved therein.
  • the present invention provides methods of treating gliomas comprising administering a therapeutically effective amount of an antithrombin agent (e.g., ARGATROBAN) to a subject.
  • an antithrombin agent e.g., ARGATROBAN
  • Figure 1 A shows a coronal section of the brain tumor model (Day 12 after C6 cell infusion). Ipsilateral basal ganglia water and sodium ion contents stay at normal range at day 3, but increase markedly at day 12 after C6 cell infusion ( Figures IB and IC). Potassium ion contents are still in the normal range even at day 12 ( Figure ID) suggesting that glioma-induced edema is predominantly vasogenic edema. Weight differences between the ipsilateral (tumor side) and contralateral hemisphere were used to estimate tumor mass. These weights include both tumor mass and brain edema. There is a significant difference between day 3 and day 12 after C6 cell infusion ( Figure IE).
  • test animals were placed in cylindrical enclosures to record preferential use of the non-impaired forelimb for weight shifting movements during spontaneous vertical exploration.
  • thrombin-positive cells are localized in the tumor edge rather than in the tumor center ( Figure 2). This result was confirmed by immunohistochemistry using sheep anti-human thrombin antibody (Affinity Biologicals Inc., Ancaster, Ontario, Canada).
  • Thrombomodulin an endogenous thrombin inhibitor, is expressed in normal brain tissue at low levels, but is increased markedly in the glioma ( Figures 2E-2F). Antithrombin in levels were increased around the tumor tissue (Western blot analysis, 5788 ⁇ 2624 pixels versus 2865 ⁇ 1529 pixels in the control, PO.05).
  • ARGATROBAN a thrombin inhibitor.
  • ARGATROBAN is a small molecular (MW 508.7) direct thrombin inhibitor that inhibits both free and fibrin-bound thrombin (M. J. Hursting, et al, Seminars in Thrombosis & Hemostasis 23:503-516 [1997]).
  • rats were implanted with an osmotic minipump to deliver ARGATROBAN or saline into the right caudate.
  • the pump was preloaded with or without ARGATROBAN (1 ⁇ g/ ⁇ l) and delivers 0.5 ⁇ l/h.
  • Neurological deficits were tested at 3, 6 and 9 days after C6 cells infusion. After 9 days, the rats were reanesthetized with pentobarbital and decapitated. The brains were then removed for determination of brain water content, glioma mass, and glioma volume.
  • Antithrombin treatment with ARGATROBAN reduced tumor mass and had a trend towards a smaller tumor volume determined histologically ( Figures 3A-3D). Functional outcome is an essential endpoint for glioma treatment. Antithrombin treatment improves both forelimb placing score and forelimb use asymmetry ( Figures 3E-3F).
  • ARGATROBAN treatment also reduced brain edema (Figure 3G).
  • the improvement in functional outcome reflects both a reduced glioma mass and reduced edema.
  • thrombin enhances the synthesis and secretion of nerve growth factor in glial cells (I. Neveu et al, J. Neurochem, 60:858-867 [1993]) and stimulates astrocyte proliferation (K. P. Cavanaugh et al, J. Neurochemisrry, 54:1735-1743
  • thrombin may act as a growth factor for tumor cells (C. Chinni, et al, J. Biol. Chem., 274:9169-9174 [1999]) and induces proliferative response in T-47D mammary tumor cells (E. E. Medrano et al, Exp. Cell Res., 172:354-364 [1987]). In T98G and TM-1 human glioma cells, thrombin also induced proliferation. This mitogenic effect was abolished by birudin, a specific thrombin inhibitor (T. Ogiicbi, et al, J. Neuro- Oncology, 46:1-9 [2000]).
  • thrombin may reduce tumor mass by affecting angiogenesis.
  • administration of ARGATROBAN reduces vessel size in the gliomas.
  • rabbit anti-laminin Sigma, St. Louis, MO
  • Figure 4 shows the blood vessels in the tumor with or without ARGATROBAN treatment (five rats each group).
  • Thrombin is a potent promoter of angiogenesis. Thrombin activates an angiogenic cascade through, at least in part, modulating hypoxia inducible factor- 1, matrix metalloproteinases, vascular endothelial growth factor (VEGF) and the receptors of VEGF (M. E. Maragoudakis and N. E.
  • Hypoxia inducible factor- 1 (HIF-1)
  • HIF-1 Hypoxia inducible factor- 1
  • PAI-1 Plasminogen activator inhibitor-1
  • PAI-1 one of the serine protease inhibitors (serpins)
  • fflF-l ⁇ upregulation in the tumor can be induced by thrombin rather than hypoxia.
  • Richard et al. reported that FflF-l ⁇ levels are upregulated in vascular smooth muscle cells by thrombin (D. E. Richard et al, J. Biol. Chem., 275:26765-26771 [2000]) i.e. a non- hypoxic pathway.
  • Gorlach et al. also found that thrombin activates HIF-1 ⁇ (A. Gorlach, et al, Circulation Res., 89:47-54 [2001]).
  • smooth muscle cells recent data shows that thrombin causes HIF-1 ⁇ accumulation in the brain (Y.
  • VEGF Vascular endothelial growth factor
  • Thrombin stimulates tumor cells to secrete VEGF and upregulates VEGF receptors in endothelial cells (M. E. Maragoudakis and N. E. Tsopanoglou, Adv. Exp. Med. Biol., 476:47-55 [2000]).
  • Matrix metalloproteinases contribute to tumor angiogenesis and invasion (S. Zucker et al, Oncogene, 19:6642-6650 [2000]).
  • Thrombin activates gelatinase A (metalloproteinases 2, MMP-2) in endothelial cells (M. Nguyen et al, Lab. Invest., 79:467-475 [1999]).
  • thrombin receptors Three protease-activated receptors (PARs), PAR-1, PAR-3 and PAR-4, have been identified as thrombin receptors (S. R. Coughlin, Nature, 407:258-164 [2000]). Thrombin receptor mRNA expression is found in C6 glioma cells (J. J. Ubl et al, Neuroscience, 86:597-609 [1998]). Thrombin receptors are activated by proteolytic cleavage rather than by ligand binding and thrombin receptor-activated peptides are able to mimic many cellular activities of thrombin. Recent studies indicate that PARs mediate some of the pathophysiological effects of thrombin.
  • PAR-1 activation by thrombin receptor activating peptide results in angiogenesis (M. E. Maragoudakis and N. E. Tsopanoglou, Adv. Exp. Med. Biol., 476:47-55 [2000]).
  • Thrombin receptors have been found in many types of tumor cells (M. Z. Wojtukiewicz, et al, Cancer Res., 55:698-704 [1995]).
  • PAR-1, PAR-3 and PAR-4 are found in the rat C6 glioma model tested.
  • the present invention comprises methods of administering a therapeutically effective amount of ARGATROBAN (Mitsubishi-Tokyo Pharmaceuticals, Inc., Tokyo, Japan) to treat glioma.
  • ARGATROBAN Mitsubishi-Tokyo Pharmaceuticals, Inc., Tokyo, Japan
  • the chemical name for the compound known as ARGATROBAN is l-[5- [(aminoiminomethyl)amino]-l-oxo-2-[[(l,2,3,4- tetrahydro-3 -methyl-8-quinolinyl)sulfonyl] aminojpentyl] -4-methyl-2- piperidinecarboxylic acid, monohydrate; this compound is synthetic and it is derived from L-arginine. While not being limited to any particular mechanism, the present invention contemplates that
  • ARGATROBAN is a direct thrombin inhibitor.
  • the compound has 4 asymmetric carbons.
  • One of the asymmetric carbons has an R configuration (stereoisomer Type 1) and an S configuration (stereoisomer Type 2).
  • Commercially available ARGATROBAN comprise a mixture of about 65 :35 R to S stereoisomers. It is to be understood that the present invention encompasses stereoisomers as well as optical isomers, e.g. mixtures of enantiomers as well as individual enantiomers and diastereomers, which arise as a consequence of structural asymmetry in selected compounds (e.g., ARGATROBAN).
  • ARGATROBAN is a white, odorless crystalline powder that is freely soluble in glacial acetic acid, slightly soluble in ethanol, and insoluble in acetone, ethyl acetate and ether.
  • Injectable ARGATROBAN is typically formulated as a sterile colorless to pale yellow slightly viscous solution.
  • ARGATROBAN is commercially available in 250 mg (in 2.5 mL) single-use vials. Preferably each mL of sterile nonpyrogenic solution contains 100 mg ARGATROBAN.
  • the compound known as ARGATROBAN, and derivatives thereof, are described in one or more U.S.
  • suitable derivatives of l-[5- [(aminoiminomethyl)amino]-l-oxo-2-[[(l,2,3,4-tetrahydro-3- methyl-8-quinolinyl)sulfonyl]amino]pentyl]-4-methyl-2- piperidinecarboxylic acid, monohydrate i.e., the compound known as ARGATROBAN
  • ARGATROBAN derivatives contemplated for use in methods of the present invention are described in U.S. Pat.
  • Low molecule weight antithrombin agents contemplated for use in certain embodiments also include those described, for example, by C. Taparelli et al, Trends Pharmacol. Sci., 14(10):366-376 (1993) and D. N ⁇ teberg et al, J. Med. Chem., 43(9):1705-1713 (2000).
  • Still other embodiments of the present invention provide co-administration of at least one antithrombin agent (e.g., ARGATROBAN) with anti-platelet or platelet inhibitory agents (e.g., aspirin, piroxicam, ticlopidine, or clopidogrel, and factor Xa inhibitors), or anti-coagulant agents (e.g., acenocoumarol, anisindione, and dicumarol).
  • antithrombin agent e.g., ARGATROBAN
  • anti-platelet or platelet inhibitory agents e.g., aspirin, piroxicam, ticlopidine, or clopidogrel, and factor Xa inhibitors
  • anti-coagulant agents e.g., acenocoumarol, anisindione, and dicumarol.
  • the therapeutic agents administered in the methods of the present invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • the present invention also includes prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
  • prodrug indicates a therapeutic agent that is prepared in an inactive fonn that is converted to an active form (i.e., drag) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention (i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto).
  • Pharmaceutically acceptable base addition salts are fonned with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine.
  • the base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
  • the free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner.
  • the free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.
  • a "pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines.
  • Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates.
  • Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraorbital, intracapsular, intraspinal, intrasternal, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular etc., administration.
  • Oligonucleotides with at least one 2'-O-methoxyethyl modification are believed to be particularly useful for oral administration.
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Aqueous suspensions may further contain substances that increase tide viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
  • compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • the compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • the therapeutic agents used in the methods of the present invention are formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.
  • compositions may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the therapeutic agent(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the therapeutic agent(s) of the formulation.
  • the present invention employs compositions comprising oligomeric antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding prothrombin, ultimately modulating the amount of prothrombin produced. This is accomplished by providing antisense compounds that specifically hybridize with one or more nucleic acids encoding prothrombin.
  • target nucleic acid and “nucleic acid encoding prothrombin” encompass DNA encoding prothrombin, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid.
  • This modulation of function of a target nucleic acid by compounds that specifically hybridize to it is generally refened to as "antisense.”
  • the functions of DNA to be interfered with include replication and transcription.
  • the functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity that may be engaged in or facilitated by the RNA.
  • the overall effect of such interference with target nucleic acid function is modulation of the expression of prothrombin.
  • modulation means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.
  • inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.
  • Targeting an antisense compound to a particular nucleic acid is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding prothrombin.
  • the targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result.
  • a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also refened to as the "AUG codon,” the "start codon” or the "AUG start codon".
  • translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo.
  • translation initiation codon and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes).
  • Eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions, hi the context of the present invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding prothrombin, regardless of the sequence(s) of such codons.
  • Translation termination codon (or "stop codon") of a gene may have one of three sequences (i.e., 5'-UAA, 5'-UAG and 5'-UGA; the conesponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively).
  • the tenns "start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon.
  • stop codon region and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon.
  • Other target regions include the 5' untranslated region (5' UTR), referring to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3' untranslated region (3' UTR), referring to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding nucleotides on the gene.
  • 5' UTR 5' untranslated region
  • 3' UTR 3' untranslated region
  • the 5' cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5 '-most residue of the mRNA via a 5'-5' triphosphate linkage.
  • the 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap.
  • the cap region may also be a prefened target region.
  • mRNA splice sites may also be prefened target regions, and are particularly useful in situations where abenant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Abenant fusion junctions due to rearrangements or deletions are also prefened targets. It has also been found that introns can also be effective, and therefore prefened, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.
  • oligonucleotides are chosen that are sufficiently complementary to the target (i.e., hybridize sufficiently well and with sufficient specificity) to give the desired effect.
  • antisense oligonucleotides are targeted to or near the start codon.
  • “hybridization,” with respect to antisense compositions and methods means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
  • an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
  • An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired (i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed).
  • Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with specificity, can be used to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway.
  • antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides are useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues, and animals, especially humans.
  • antisense oligonucleotides are a prefened form of antisense compound
  • the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below.
  • the antisense compounds in accordance with this invention preferably comprise from about 8 to about 30 nucleobases (i.e., from about 8 to about 30 linked bases), although both longer and shorter sequences may find use with the present invention.
  • Particularly prefened antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 25 nucleobases.
  • prefened antisense compounds useful with the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages.
  • oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • oligonucleosides include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thiofonnacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos.:
  • both the sugar and the internucleoside linkage (i.e., the backbone) of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties is refened to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein inco ⁇ orated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science 254:1497 (1991).
  • oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones and in particular -CH 2 , ⁇ NH ⁇ O-CH 2 -, ⁇ CH 2 --N(CH 3 )--O--CH 2 -- [known as a methylene (methylimino) or MMI backbone], -CH 2 ⁇ O ⁇ N(CH 3 ) ⁇ CH 2 ⁇ , -CH2-N(CH 3 )-N(CH 3 )-CH 2 ⁇ , and -O-N(CH 3 )-CH2 ⁇ CH 2 ⁇ [wherein the native phosphodiester backbone is represented as — O--P— O ⁇ CH 2 ⁇ ] of the above referenced U.S.
  • oligonucleotides having mo ⁇ holino backbone structures of the above-referenced U.S. Pat. No. 5,034,506. Modified oligonucleotides may also contain one or more substituted sugar moieties.
  • Prefened oligonucleotides comprise one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted d to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • oligonucleotides comprise one of the following at the 2' position: Ci to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a preferred modification includes 2'-methoxyethoxy (2'-O ⁇ CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al, Helv. Chim. Acta, 78:486 [ 1995]) i. e. , an alkoxyalkoxy grou .
  • a further prefened modification includes
  • 2'-dimethylaminooxyethoxy i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group
  • 2'-DMAOE also known as 2'-DMAOE
  • 2'-dimethylaminoethoxyethoxy also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE
  • Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat.
  • Oligonucleotides may also include nucleobase (often refened to in the art simply as “base”) modifications or substitutions.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substitute
  • nucleobases include those disclosed in U.S. Pat. No. 3,687,808. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree °C and are presently prefened base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
  • oligonucleotides of the present invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, (e.g., hexyl-S-tritylthiol), a thiocholesterol, an aliphatic chain, (e.g., dodecandiol or undecyl residues), a phospholipid, (e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or a polyethylene glycol chain or adamantane acetic acid, a palmityl mo
  • the present invention also includes antisense compounds that are chimeric compounds.
  • "Chimeric” antisense compounds or “chimeras,” in the context of the present invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.
  • oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • RNaseH is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression.
  • RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
  • Chimeric antisense compounds of the present invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above.
  • Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein inco ⁇ orated by reference in its entirety.
  • the compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or abso ⁇ tion.
  • Representative United States patents that teach the preparation of such uptake, distribution and/or abso ⁇ tion assisting formulations include, but are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844;
  • prefened examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalactur
  • cationic lipids such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 97/30731), also enhance the cellular uptake of oligonucleotides.
  • Certain embodiments of the invention provide therapeutic methods comprising the administration (a) one or more antisense compounds and (b) one or more anticoagulants (e.g. , ARGATROBAN) or thrombolytic agents that function by a non-antisense mechanism, h some embodiments, the therapeutic methods of the present invention further the comprise co-administration of one or more chemotherapeutic agents.
  • chemotherapeutic agents include, but are not limited to, anticancer drags such as daunorabicin, dactinomycin, doxorabicin, bleomycin, mitomycin, nitrogen mustard, ' chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridme (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin and diethylstilbestrol (DES).
  • anticancer drags such as daunorabicin, dactinomycin, doxorabicin, bleomycin, mitomycin, nitrogen mustard, ' chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine
  • Anti-inflammatory drugs including but not limited to nonsteroidal anti-inflammatory drags and corticosteroids, and antiviral drags, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention.
  • Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.
  • RNAi represents an evolutionary conserved cellular defense for controlling the expression of foreign genes in most eukaryotes, including humans. RNAi is triggered by double-stranded RNA (dsRNA) and causes sequence-specific mRNA degradation of single- stranded target RNAs homologous in response to dsRNA.
  • the mediators of mRNA degradation are small interfering RNA duplexes (siRNAs), which are normally produced from long dsRNA by enzymatic cleavage in the cell.
  • siRNAs are generally approximately twenty-one nucleotides in length (e.g., 21-23 nucleotides in length), and have a base-paired structure characterized by two nucleotide 3 '-overhangs.
  • RISC RNA-induced silencing complex
  • siRNAs Chemically synthesized siRNAs have become powerful reagents for genome-wide analysis of mammalian gene function in cultured somatic cells. Beyond their value for validation of gene function, siRNAs also hold great potential as gene-specific therapeutic agents (Tuschl and Borkhardt, Molecular Intervent., 2(3): 158-67 [2002], herein inco ⁇ orated by reference).
  • siRNAs are effective at lowering the amounts of targeted RNA, and by extension proteins, frequently to undetectable levels.
  • the silencing effect can last several months, and is highly specific, because one nucleotide mismatch between the target RNA and the central region of the siRNA is frequently sufficient to prevent silencing Brummelkamp et al, Science, 296:550-553 [2001]; and Holen et al, Nucleic Acids Res., 30:1757-1766 [2002], both of which are herein inco ⁇ orated by reference.
  • the present invention contemplates the use of any genetic manipulation for use in modulating prothrombin expression.
  • genetic manipulation include, but are not limited to, expression of antisense constructs with or without inducible promoters, and the like.
  • Delivery of nucleic acid construct to cell in vitro or in vivo may be conducted using any suitable method.
  • a suitable method is one that introduces the nucleic acid construct into the cell such that the desired event occurs (e.g., expression of an antisense construct).
  • Introduction of molecules carrying genetic information into cells is achieved by any of various methods including, but not limited to, directed injection of naked DNA constracts, bombardment with gold particles loaded with said constructs, and macromolecule mediated gene transfer using, for example, liposomes, biopolymers, dendrimers, and the like.
  • Prefened methods use gene delivery vehicles derived from virases, including, but not limited to, adenovirases, refrovirases, vaccinia virases, and adeno-associated virases. Because of the higher efficiency as compared to retroviruses, vectors derived from adenovirases are the prefened gene delivery vehicles for transferring nucleic acid molecules into host cells in vivo. Adenoviral vectors have been shown to provide very efficient in vivo gene transfer into a variety of solid tumors in animal models and into human solid tumor xenografts in immune-deficient mice.
  • Vectors may be administered to subjects in a variety of ways. For example, in some embodiments of the present invention, vectors are administered into tumors or tissue associated with tumors using direct injection.
  • administration is via the blood or lymphatic circulation (See e.g., PCT publication 99/02685 herein inco ⁇ orated by reference in its entirety).
  • exemplary dose levels of adenoviral vector are preferably 10 to 10 11 vector particles added to the perfusate.
  • the present invention provides isolated antibodies, preferably monoclonal antibodies, that specifically bind to an isolated polypeptide comprised of at least five amino acid residues of thrombin. It is understood that in other embodiments, antibodies to prothrombin are within the scope of the present invention.
  • An antibody against the thrombin may be any monoclonal or polyclonal antibody, as long as it can recognize thrombin.
  • the antibody against thrombin can be produced by using thrombin protein as the antigen according to a conventional antibody or antiserum preparation process. For example, for preparation of a monoclonal antibody, pro/thrombin, as such, or together with a suitable carrier or diluent is administered to an animal (e.g., mammal) under conditions that permit the antibody production.
  • complete or incomplete Freund's adjuvant may be administered.
  • the protein is administered once every 2 weeks to 6 weeks, in total, about 2 times to about 10 times.
  • the animals to be used include, but are not limited to, primates, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, etc.
  • an individual whose antibody titer has been confirmed is selected from an animal immunized with the antigen (e.g., a mouse) and, 2 days to 5 days after the final immunization, its spleen or lymph node is harvested and antibody-producing cells contained therein are fused with myeloma cells to prepare the desired monoclonal antibody producer hybridoma.
  • Measurement of the antibody titer in an antiserum can be carried out, for example, by reacting the labeled protein, as described hereinafter and an antiserum and then measuring the activity of the labeling agent bound to the antibody.
  • the cell fusion can be carried out according to known methods, for example, the method described by Koehler and Milstein (Nature 256:495, [1975]).
  • a fusion promoter for example, polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.
  • myeloma cells examples include NS-1, P3U1, SP2/0, AP-1 and the like.
  • the proportion of the number of antibody producer cells (spleen cells) and the number of myeloma cells to be used is preferably about 1 :1 to about 20:1 and PEG (preferably PEG 1000-PEG 6000) is added in concentration of about 10% to about 80%.
  • Cell fusion can be carried out efficiently by incubating a mixture of both cells at about 20 °C to about 40 °C, preferably about 30 °C to about 37 °C for about 1 minute to 10 minutes.
  • Various methods may be used for screening for a hybridoma producing the antibody against thrombin.
  • a supernatant of the hybridoma is added to a solid phase (e.g., microplate) to which thrombin antibody is adsorbed directly or together with a carrier and then an anti-immuno globulin antibody (if mouse cells are used in cell fusion, anti-mouse immunoglobulin antibody is used) or Protein A labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase.
  • a solid phase e.g., microplate
  • an anti-immuno globulin antibody if mouse cells are used in cell fusion, anti-mouse immunoglobulin antibody is used
  • Protein A labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase.
  • a supernatant of the hybridoma is added to a solid phase to which an anti-immunoglobulin antibody or Protein A is adsorbed and then the thrombin protein labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase.
  • Selection of the monoclonal antibody can be carried out according to any known method or its modification. Normally, a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) are added is employed. Any selection and growth medium can be employed as long as the hybridoma can grow.
  • RPMI 1640 medium containing 1% to 20%, preferably 10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetal bovine serum, a serum free medium for cultivation of a hybridoma (SFM-101, Nissui Seiyaku) and the like can be used.
  • the cultivation is carried out at 20 °C to 40 °C, preferably 37 °C for about 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% CO 2 gas.
  • the antibody titer of the supernatant of a hybridoma culture can be measured according to the same manner as described above with respect to the antibody titer of the anti-protein in the antiserum.
  • Separation and purification of a monoclonal antibody against thrombin can be carried out according the same manner as those of conventional polyclonal antibodies such as separation and purification of immunoglobulins, for example, salting-out, alcoholic precipitation, isoelectric point precipitation, electrophoresis, adso ⁇ tion and deso ⁇ tion with ion exchangers (e.g., DEAE), ultracentrifugation, gel filtration, or a specific purification method wherein only an antibody is collected with an active adsorbent such as an antigen-binding solid phase, Protein A or Protein G and dissociating the binding to obtain the antibody.
  • Polyclonal antibodies may be prepared by any known method or modifications of these methods including obtaining antibodies from patients.
  • a complex of an immunogen (an antigen against the protein) and a carrier protein is prepared and an animal is immunized by the complex according to the same manner as that described with respect to the above monoclonal antibody preparation.
  • a material containing the antibody against thrombin is recovered from the immunized animal and the antibody is separated and purified.
  • any carrier protein and any mixing proportion of the carrier and a hapten can be employed as long as an antibody against the hapten, which is crosslinked on the carrier and used for immunization, is produced efficiently.
  • bovine serum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. may be coupled to an hapten in a weight ratio of about 0.1 part to about 20 parts, preferably, about 1 part to about 5 parts per 1 part of the hapten.
  • various condensing agents can be used for coupling of a hapten and a carrier.
  • glutaraldehyde, carbodiimide, maleimide activated ester, activated ester reagents containing thiol group or dithiopyridyl group, and the like find use with the present invention.
  • the condensation product as such or together with a suitable carrier or diluent is administered to a site of an animal that permits the antibody production.
  • complete or incomplete Freund's adjuvant may be administered. Normally, the protein is administered once every 2 weeks to 6 weeks, in total, about 3 times to about 10 times.
  • the polyclonal antibody is recovered from blood, ascites and the like, of an animal immunized by the above method.
  • the antibody titer of antithrombin in the antiserum can be measured according to the same manner as that described above with respect to the supernatant of the hybridoma culture. Separation and purification of the antibody can be carried out according to the same separation and purification method of immunoglobulin as that described with respect to the above monoclonal antibody.
  • the thrombin protein used herein as the immunogen is not limited to any particular type of immunogen.
  • the thrombin protein that is encoded by a prothrombin gene can be used as the immunogen.
  • fragments of the thrombin protein may be used. Fragments may be obtained by any methods including, but not limited to expressing a fragment of the prothrombin gene, enzymatic processing of the prothrombin protein, chemical synthesis, and the like.
  • a group of monoclonal antibodies to different regions and/or domains of thrombin are used (i.e., "polymonoclonal antibody therapy") to increase the effectiveness of targeting glioma cells that show antigenic heterogeneity.
  • polymonoclonal antibody therapy i.e., "polymonoclonal antibody therapy”
  • the selection of suitable antibodies with the desired effect can be identified using cell migration assays as described herein (See e.g., Bjerkvig et al, J. Neurosurg., 72:463 [1990]).
  • the present invention provides novel methods of treating tumors of the CNS, and particularly gliomas, comprising administering pharmaceutical compositions comprising at least one antithrombin agent administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the methods of the present comprising administering the antithrombin agent known as ARGATROBAN in a suitable pharmaceutical composition.
  • the pharmaceutical compositions contain a mixture of at least two agents (e.g., two antithrombics) of similar or dissimilar type co-administered to a subject, hi still further embodiments, the pharmaceutical compositions of the present invention contain at least two agents (e.g., two antithrombics) that are administered to a patient under one or more of the following conditions: at different periodicities, different durations, different concentrations, different administration routes, etc.
  • the compositions and methods of the present invention find use in treating diseases or altering physiological states characterized by undesirable cell migration, angiogenesis, or loss of apoptotic control (e.g., cancers).
  • compositions may be formulated and administered systemically or locally.
  • Techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences” (Mack Publishing Co, Easton Pa.).
  • the invention contemplates administering therapeutic compounds in accordance with acceptable pharmaceutical delivery methods and preparation techniques.
  • some therapeutic compounds of the present invention are administered to a subject intravenously in a pharmaceutically acceptable carrier such as physiological saline.
  • the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Standard methods for intracellular delivery of pharmaceutical agents are used in other embodiments (e.g., delivery via liposome). Such methods are well known to those of ordinary skill in the art.
  • therapeutic agents are formulated for parenteral administration, such as, intravenous, subcutaneous, intramuscular, intraperitoneal and the like.
  • the therapeutic agents in for parenteral administration include aqueous solutions of the active compounds in water-soluble form.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipopbilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions of the present invention can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, capsules, dragees, liquids, gels, syrups, slurries, suspensions and the like, for oral or nasal ingestion by a patient to be treated.
  • the therapeutic compounds are administered orally to a patient orally.
  • compositions for oral use can be obtained by combining the active compounds (e.g., antithrombics) with a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, etc. ; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.
  • h gestible formulations of the present compositions may further include any material approved by the United States Department of Agriculture for inclusion in foodstuffs and substances that are generally recognized as safe (GRAS), such as, food additives, flavorings, colorings, vitamins, minerals, and phytonutrients.
  • phytonutrients refers to organic compounds isolated from plants that have a biological effect, and includes, but is not limited to, compounds of the following classes: isoflavonoids, oligomeric proanthcyanidins, indol-3-carbinol, sulforaphone, fibrous ligands, plant phytosterols, ferulic acid, anthocyanocides, trite ⁇ enes, omega 3/6 fatty acids, polyacetylene, quinones, te ⁇ enes, cathechins, gallates, and quercitin.
  • Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, (i.e., dosage).
  • compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers, hi soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • therapeutic agents are administered to a patient alone, or in combination with one or more other drags or therapies (e.g., conventional anticancer agents, including, but not limited to, nucleotide sequences, drugs, hormones, etc.) or in pharmaceutical compositions where it is mixed with excipient(s) or other pharmaceutically acceptable carriers, hi one embodiment of the present invention, the pharmaceutically acceptable carrier is pharmaceutically inert.
  • Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • an effective amount of therapeutic compound(s) may be that amount that inhibits hype ⁇ roliferation, angiogenesis, cell migration, cell adhesion, and/or cell survival in a cell as compared to control cells.
  • prefened pharmaceutical compositions optionally comprise pharmaceutically acceptable carriers, such as, excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the pharmaceutical compositions used in the methods of the present invention are manufactured according to well known and standard pharmaceutical manufacturing techniques (e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes).
  • compositions comprising a compound of the invention formulated in a pharmaceutical acceptable carrier may be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • the pharmaceutical compositions are provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.
  • the prefened preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.
  • Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual therapeutic agents, and can generally be estimated based on EC 50 s found to be effective in in vitro and in vivo animal models or based on the examples described herein.
  • dosage is from 0.01 ⁇ g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly.
  • the treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
  • the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the therapeutic agent is administered in maintenance doses, ranging from 0.01 ⁇ g to 100 g per kg of body weight, once or more daily, to once about every 20 years.
  • the therapeutically effective dose can be estimated initially from cell culture assays. Then, preferably, dosage can be formulated in animal models (particularly murine or rat models) to achieve a desirable circulating concentration range that results in increased PKA activity in cells/tissues characterized by undesirable cell migration, angiogenesis, cell migration, cell adhesion, and/or cell survival.
  • a therapeutically effective dose refers to that amount of therapeutic compound(s) that ameliorate symptoms of the disease state (e.g., hype ⁇ roliferation, unregulated angiogenesis, cell migration, and/or loss of apoptotic control). Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in
  • the dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50/ED50.
  • Compounds that exhibit large therapeutic indices are prefened.
  • the data obtained from cell culture assays and additional animal studies can be used in formulating a range of dosage, for example, mammalian use (e.g., humans, Equus caballus, Felis catus, and Canisfamiliaris, etc.).
  • the dosage of such compounds lies preferably, however the present invention is not limited to this range, within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • Rats were anesthetized with pentobarbital (50 mg/kg ip). Rats were then placed in a Kopf stereotaxic frame. The tip of a 26-gauge stainless steel cannula was lowered into the right caudate (0.2 mm anterior, 5.5 mm ventral, 3.5 mm lateral to bregma) and 6xl0 5 C6 rat glioma cells (from American Type Culture Collection) were infused.
  • Figure 1A shows a coronal brain section of the glioma (day 12).
  • Figures IB, IC and ID respectively, show brain water, sodium ion and potassium ion contents in the ipsilateral and contralateral caudate.
  • a bar graph in Figure 2D shows thrombin activity at day 12 in the contralateral and ipsilateral of caudate after infusion of C6 glioma cells.
  • Rat brain samples were homogenized and thrombin activities were measured using the thrombin-specific chromogenic substrate S2238 (Chromogenix, Milan, Italy).
  • PPACK phenyl-propyl-arginyl-chloromethyl ketone
  • Rats were anesthetized with pentobarbital (60 mg/kg, i.p.) and perfused with 4% paraformaldehyde in 0.1 M pH 7.4 phosphate-buffered saline (PBS). Brains were removed. The removed brains were kept in 4% paraformaldehyde for six hours, then immersed in 25% sucrose for three to four days at 4 °C. The brains were then embedded in O.C.T compound (Sakura Finetek, Inc., Tonance, CA) and sectioned on a cryostat (18- ⁇ m thick). Sections were incubated using avidin-biotin complex technique.
  • PBS pH 7.4 phosphate-buffered saline
  • Laminin immunostaining was used to examine blood vessels in the glioma with vehicle in 5 rats (See, Figures 4A, 4B, 4C, 4D, and 4E) and ARGATROBAN in 5 rats (See, Figures 4F, 4G, 4H, 41, and 4J) treatment as described in Example 3.
  • Scale bar 200 ⁇ m.
  • mice were anesthetized with pentobarbital (50 mg/kg respectively). Anesthetized animals were then placed in a Kopf stereotaxic frame. Body temperature was maintained at
  • ARGATROBAN Treatment was provided by Mitsubishi Pharma Co ⁇ oration (Osaka, Japan). ARGATROBAN (0.3 mg/h/rat) or vehicle was delivered by osmotic minipumps intraperitoneally immediately after glioma cell implantation.
  • C6 glioma cells (passage number 36 to 42) were obtained from American Type Culture Collection, ATCC (Manassas, VA). C6 cells were grown at 37 °C in air with 5% CO 2 in Ham's F-10 medium (Gibco Laboratories, Grand Island, NY) with 2.5% fetal bovine serum and 15% horse serum. F98 (passage number 9 to 11) glioma cells were purchased from ATCC and grown in Dulbecco's Modified Eagle's medium from ATCC with 10% fetal bovine serum at 37 °C in air with 5% CO 2 .
  • Tumor Mass The weight difference between the ipsilateral (tumor side) and contralateral hemisphere was used to estimate tumor mass.
  • Behavioral Tests Three behavioral tests were used: 1) a forelimb placing test, 2) a forelimb use asymmetry (cylinder) test, and 3) a corner turn test, as described more fully below.
  • the first behavioral test was a vibrissae-elicited forelimb placing test. Animals were held by their torsos allowing the forelimb to hang free. The animals were gently moved up and down prior to the placing testing to facilitate muscle relaxation and eliminate any struggling movements. Trials during which extreme muscle tension, straggling or placing of any of the limbs onto the experimenter's hand occurred were not counted. Independent testing of each forelimb was induced by brushing the respective vibrissae on the corner edge of a countertop. Intact animals place the forelimb ipsilateral to the stimulated vibrissae quickly onto the countertop.
  • the behavior was scored using the following criteria: 1) independent use of the left or right forelimb for contacting the wall during a full rear to initiate a weight shifting movement or to regain center of gravity while moving laterally in a vertical posture; and 2) simultaneous use of both the left and right forelimb for contacting the cylinder wall during a full rear and for alternating lateral stepping movements along the wall.
  • Behavior was quantified by determining the occasions when the non-impaired (Ipsilateral) forelimb was used as a percentage of total number of limb use observations on the wall (I).
  • rats were allowed to proceed into a corner, the angle of which was 30 degrees. To the exit the corner, a rat could turn either to the left or the right and this was recorded. This was repeated 10 to 15 times, with at least 30 seconds between trial, and the percentage of right turns calculated. Only turns involving full rearing along either wall were included (e.g., ventral tucks or horizontal turns were excluded). Rats were not picked up immediately following each turn so that they did not develop an aversion for their prepotent turning response.
  • Rats were anesthetized with pentobarbital (60 mg/kg, i.p.) and perfused with 4% paraformaldehyde in 0.1 M pH 7.4 phosphate-buffered saline (PBS). Removed brains were kept in 4% paraformaldehyde for six hrs, then immersed in 25% sucrose for three to four days at 4 °C. The brains were embedded in O.C.T compound (Sakura Finetek USA, Inc., Torrance, CA) and sectioned on a cryostat (18 ⁇ m thick). , Tumor size was measured by serial brain sections (360 ⁇ m apart) with hematoxylin and eosin staining.
  • PBS pH 7.4 phosphate-buffered saline
  • Low dose (0.3 mg/h/rat) of ARGATROBAN or vehicle was administrated intraperitoneally immediately after glioma cell implantation. The weight difference between the ipsilateral (tumor side) and the contralateral hemisphere was used to estimate tumor mass.
  • Figure 5 shows a bar graph showing glioma mass at day 9 after C6 glioma cell implantation in the ARGATROBAN treated group (0.3 mg/h/rat) and vehicle-treated group.
  • ARGATROBAN treatment also improved forelimb placing score at day 3, 6 and 9 ( Figures 7A and 7B).
  • C6 glioma cells were injected into the caudate. This takes advantage of the fact that a number of behavioral tests have been devised to assess caudate damage induced by ischemia, hemorrhage or neurotoxins.
  • this C6 glioma model is stable and reproducible for edema, tumor mass, and neurological deficit measurements.
  • the properties of C6 glioma cell line can be retained for several years. There are limitations on the survival studies, however, since C6 glioma can be immtinogenic in allogeneic hosts. For this reason, the present invention also developed the F98 glioma model in the rat for survival time studies.
  • Anti-thrombin treatment with ARGATROBAN improved glioma-related neurological deficits including forelimb use asymmetry, forelimb placing and corner turn.
  • the present invention tested the glioma rats to day 9 because the rats could not perform at day 12.
  • Systemic treatment of ARGATROBAN prolonged survival time.
  • ARGATROBAN 0.3 mg/h/rat, i.p.
  • thrombin inhibitors such as ARGATROBAN
  • ARGATROBAN are potential therapeutic agents and that such agents can be efficacious when given systemically as well as intracerebrally.

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Abstract

La présente invention concerne des nouvelles thérapies et des nouvelles cibles thérapeutiques permettant de traiter et de réduire les symptômes associés aux tumeurs du système nerveux central (CNS). Plus particulièrement, cette invention concerne des nouvelles thérapies du gliome consistant à administrer des agents qui inhibent le processus de coagulation, et plus spécifiquement, un ou plusieurs facteurs impliqués dans ce processus de coagulation. Cette invention concerne, entre autres, des méthodes permettant de traiter des gliomes, lesquelles méthodes consistent à administrer une quantité thérapeutiquement efficace d'un agent antithrombotique (par exemple, ARGATROBAN) à un sujet.
PCT/US2003/029582 2002-09-23 2003-09-23 Traitements du gliome Ceased WO2004026252A2 (fr)

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WO2008045410A3 (fr) * 2006-10-11 2008-06-12 Scidose Llc Préparation sans alcool d'argatroban
WO2008009639A3 (fr) * 2006-07-17 2008-06-26 Boehringer Ingelheim Int Nouvelles indications portant sur les inhibiteurs directs de la thrombine
US7589106B2 (en) 2006-09-27 2009-09-15 Eagle Pharmaceuticals, Inc. Alcohol free formulation of argatroban
US7687516B2 (en) 2006-09-27 2010-03-30 Eagle Pharmaceuticals, Inc. Alcohol free formulation of argatroban
WO2014152715A1 (fr) * 2013-03-15 2014-09-25 University Of Rochester Utilisation d'inhibiteurs de liaison entre un récepteur par-1 et ses ligands dans le traitement d'un gliome
WO2019035075A1 (fr) * 2017-08-17 2019-02-21 NantOmics, LLC. Changements dynamiques dans l'arn libre circulant de tumeurs neurales
USRE48164E1 (en) * 2006-08-31 2020-08-18 Emisphere Technologies Inc. Compounds and compositions for delivering active agents

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US4963655A (en) * 1988-05-27 1990-10-16 Mayo Foundation For Medical Education And Research Boron analogs of amino acid/peptide protease inhibitors
AU659432B2 (en) * 1991-03-08 1995-05-18 Novartis Ag A method for the inhibition or prevention of tumor cell metastasis with hirudin
IL129344A0 (en) * 1996-11-27 2000-02-17 Du Pont Pharm Co Novel integrin receptor antagonists

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008009639A3 (fr) * 2006-07-17 2008-06-26 Boehringer Ingelheim Int Nouvelles indications portant sur les inhibiteurs directs de la thrombine
JP2009543843A (ja) * 2006-07-17 2009-12-10 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング 直接トロンビン阻害剤に関する新規適応
USRE48164E1 (en) * 2006-08-31 2020-08-18 Emisphere Technologies Inc. Compounds and compositions for delivering active agents
US7589106B2 (en) 2006-09-27 2009-09-15 Eagle Pharmaceuticals, Inc. Alcohol free formulation of argatroban
US7687516B2 (en) 2006-09-27 2010-03-30 Eagle Pharmaceuticals, Inc. Alcohol free formulation of argatroban
WO2008045410A3 (fr) * 2006-10-11 2008-06-12 Scidose Llc Préparation sans alcool d'argatroban
WO2014152715A1 (fr) * 2013-03-15 2014-09-25 University Of Rochester Utilisation d'inhibiteurs de liaison entre un récepteur par-1 et ses ligands dans le traitement d'un gliome
US20160045506A1 (en) * 2013-03-15 2016-02-18 University Of Rochester Use of inhibitors of binding between a par-1 receptor and its ligands for the treatment of glioma
WO2019035075A1 (fr) * 2017-08-17 2019-02-21 NantOmics, LLC. Changements dynamiques dans l'arn libre circulant de tumeurs neurales
US11821043B2 (en) 2017-08-17 2023-11-21 Nantomics Llc Dynamic changes in circulating free RNA of neural tumors

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