US20080096900A1 - Methods for treating atherosclerosis - Google Patents

Methods for treating atherosclerosis Download PDF

Info

Publication number
US20080096900A1
US20080096900A1 US11/823,251 US82325107A US2008096900A1 US 20080096900 A1 US20080096900 A1 US 20080096900A1 US 82325107 A US82325107 A US 82325107A US 2008096900 A1 US2008096900 A1 US 2008096900A1
Authority
US
United States
Prior art keywords
carbonitrile
ylthio
pyrazine
lcat
thiadiazol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/823,251
Other languages
English (en)
Inventor
Frank Kayser
Marc Labelle
Bei Shan
Jian Zhang
Mingyue Zhou
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Amgen Inc
Original Assignee
Amgen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amgen Inc filed Critical Amgen Inc
Priority to US11/823,251 priority Critical patent/US20080096900A1/en
Assigned to AMGEN INC. reassignment AMGEN INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LABELLE, MARC, KAYSER, FRANK, SHAN, BEI, ZHANG, JIAN, ZHOU, MINGYUE
Publication of US20080096900A1 publication Critical patent/US20080096900A1/en
Priority to US13/087,261 priority patent/US8426358B2/en
Priority to US13/851,019 priority patent/US20130273024A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)

Definitions

  • This invention relates generally to the field of medicine and, more specifically, to compositions and methods for treating coronary heart disease and atherosclerosis.
  • Atherosclerosis is a polygenic complex disease of mammals characterized by the deposits or plaques of lipids and other blood derivatives in the arterial walls (aorta, coronary arteries, carotid). These plaques can be calcified to a greater or lesser extent according to the progression of the process. They are also associated with the accumulation of fatty deposits consisting mainly of cholesterol esters in the arteries. Cholesterol accumulates in the foam cells of the arterial wall, thereby narrowing the lumen and decreasing the flow of blood. This is accompanied by a thickening of the arterial wall, with hypertrophy of the smooth muscle, the appearance of foam cells and the accumulation of the fibrous tissue. Hypercholesterolemia can therefore result in very serious cardiovascular pathologies such as infarction, peripheral vascular disease, stroke, sudden death, cardiac decompensation, cerebral vascular accidents and the like.
  • the cholesterol is carried in the blood by various lipoproteins including the low-density lipoproteins (LDL) and the high-density lipoproteins (HDL).
  • LDL low-density lipoproteins
  • HDL high-density lipoproteins
  • the LDL is synthesized in the liver and makes it possible to supply the peripheral tissues with cholesterol.
  • the HDL captures cholesterol molecules from the peripheral tissues and transports them to the liver where they are converted to bile acids and excreted.
  • the development of atherosclerosis and the risk of coronary heart disease (CHD) inversely correlate to the levels of HDL in the serum.
  • CHDD coronary heart disease
  • statins inhibitors of the cholesterols biosynthesis enzyme 3-hydroxyl-3-methylglutary Coenzyme A reductase and thereby this treatment has been used as a successful approach for reducing the risk for atherosclerosis where the primary indication is high LDL level.
  • statins are beneficial for patients whose primary lipid abnormality is low HDL cholesterol.
  • Lecithin-cholesterol acyltransferase is an enzyme which catalyzes the esterification of free cholesterol by the transfer of an acyl group from phosphatidylcholine onto 3-hydroxyl group of the cholesterol, forming cholesteryl ester and lysophosphatidylcholine.
  • LCAT is synthesized in the liver and released into the plasma, where it is combined with HDL, called anti-atherogenic lipoproteins.
  • HDL particles have the capacity to accept the excess cholesterol, which is then esterified by LCAT.
  • the cholesteryl ester molecules in the HDL particles are either transported to the liver directly through SR-BI receptor, or transferred to apoB-containing lipoproteins, including very low density lipoproteins (VLDL) and LDL, mediated by CETP, and then transported to the liver through LDL-receptor pathway.
  • VLDL very low density lipoproteins
  • LDL very low density lipoproteins
  • CETP very low density lipoproteins
  • This mechanism called reverse cholesterol transport (Glomset (1968) J. Lipid Res. 9:155), allows the removal of excess cholesterol from the body, and therefore is involved in the prevention of atherogenesis.
  • LCAT plays a significant role in this process by creating a gradient of free cholesterol between the plasma membranes and the circulating lipoproteins.
  • compositions comprising LCAT modified to increase enzymatic activity and/or stability and methods for treatment and prevention of atherosclerosis, CHD, and other conditions, including inflammation, thrombosis, and disorders associated with these conditions using the compounds and compositions of the invention.
  • the present invention provides methods for treating atherosclerosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of Formula I wherein all substituents are as indicated in Detailed Description below, or a pharmaceutically acceptable salts thereof.
  • the invention provides methods for treating atherosclerosis in a subject wherein X and Y are each —N ⁇ .
  • Z can be —S—.
  • L can be —S—.
  • R 1 can be CN.
  • R 2 can be SR 3 .
  • R 3 can be C 1 -C 4 alkyl, for example, methyl.
  • the invention provides methods for treating atherosclerosis, inflammation, thrombosis, and conditions associated with these disorders in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a compound selected from the group consisting of 3-(5-(methylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile, 3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile, 3-(5-(allylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile, 3-(5-(propylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile, 3-(5-(butylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile, 3-(5-(isobutylthio)-1,3,4-thiadia
  • the invention further provides methods for treating atherosclerosis, inflammation, thrombosis, and conditions associated with these disorders in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a modified LCAT comprising a replacement of the amino acid residue 31 by a cysteine residue, wherein the cysteine residue is modified by replacing the thiol hydrogen with 3-pyrazinyl-2-carbonitrile.
  • the modified LCAT can be administered intravenously, for example, by bolus.
  • the invention provides methods for treating an LCAT-mediated disease comprising administering to a subject in need thereof an effective amount of a modified LCAT comprising a replacement of the amino acid residue 31 by a cysteine residue, wherein the cysteine residue is modified by replacing the thiol hydrogen with 3-pyrazinyl-2-carbonitrile.
  • the LCAT-mediated disease can be atherosclerosis, thrombosis, coronary heart disease, high blood pressure, LCAT deficiency syndrome, Alzheimer's disease, corneal opacity, metabolic syndrome, dyslipidemia, myocardial infarction, stroke, critical limb ischemia or angina, inflammation, and conditions associated with these disorders.
  • the invention further provides methods for increasing HDL cholesterol in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a modified LCAT comprising a replacement of the amino acid residue 31 by a cysteine residue, wherein the cysteine residue is modified by replacing the thiol hydrogen with 3-pyrazinyl-2-carbonitrile.
  • the invention provides methods for preventing accumulation of cholesterol in a subject comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a modified LCAT comprising a replacement of the amino acid residue 31 by a cysteine residue, wherein the cysteine residue is modified by replacing the thiol hydrogen with 3-pyrazinyl-2-carbonitrile, and a pharmaceutically acceptable carrier or excipient.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a modified LCAT comprising a replacement of the amino acid residue 31 by a cysteine residue, wherein the cysteine residue is modified by replacing the thiol hydrogen with 3-pyrazinyl-2-carbonitrile, and a pharmaceutically acceptable carrier.
  • the subject can be mammalian. In another aspect, the subject can be human.
  • FIG. 1 schematically represents sequences of human (A, SEQ ID NO:1), mouse (B, SEQ ID NO:2), rat (C, SEQ ID NO:3), and consensus LCAT polypeptide (D, SEQ ID NO:4).
  • FIG. 2 (A) demonstrates activity and specificity of the compounds of the invention on LCAT enzyme.
  • FIG. 2 (B) illustrates the mechanism of action of compounds of the invention on LCAT enzyme.
  • FIG. 3 summarizes data showing that compounds of the invention increase LCAT enzyme activity in a dose dependent manner in BALB/c mice.
  • FIG. 4 demonstrates that treatment with the compounds of the invention increases HDL cholesterol levels in CD1 mice.
  • FIG. 5 illustrates the time course of LCAT activation and the levels of HDL in mice following a single doze of the compounds of the invention.
  • FIG. 6 demonstrates that treatment with the compounds of the invention increases HDL levels and decreases apoB-containing lipoprotein in vivo.
  • FIG. 7 provides elution profiles indicating that treatment with the compounds of the invention increases HDL-Ch levels, increases HDL particle size, and decrease TG levels in the VLDL fraction in vivo.
  • pharmaceutically active means that a substance so described is determined to have activity that affects a medical parameter or disease state.
  • substantially homogenous as used herein with reference to an LCAT preparation means that the preparation includes a single species of a therapeutic LCAT compound detectable in the preparation of total therapeutic molecules in the preparation, unless otherwise stated at a specific percentage of total therapeutic molecules.
  • a substantially homogenous preparation is homogenous enough to display the advantages of a homogenous preparation, e.g., ease in clinical application in predictability of lot to lot pharmacokinetics.
  • Bioefficacy refers to the capacity to produce a desired biological effect. Bioefficacy of different compounds, or different dosages of the same compound, or different administrations of the same compound are generally normalized to the amount of compound(s) to permit appropriate comparison.
  • LCAT lecithin-cholesterol acyltransferase
  • LCAT refers to a glycoprotein enzyme that catalyzes the synthesis of cholesterol esters and lysolecithin from phosphatidylcholine and unesterified cholesterol present in lipoproteins. This enzyme is produced primarily by the liver and circulates in blood reversibly bound to lipoproteins.
  • Human LCAT has a polypeptide mass of 49 kDa, or around 67 kDa with added carbohydrate mass. Any polypeptide variants or fragments of mammalian LCAT that have the LCAT enzymatic activity as described above and in more detail below are useful as compounds and in methods of the instant invention.
  • a polypeptide fragment is a stretch of amino acid residues of at least 12 contiguous amino acids from a particular sequence.
  • modified LCAT refers to lecithin-cholesterol acyltransferase as defined above, with either increased enzymatic activity, wherein the enzymatic activity of the modified LCAT is increased compared to the wild type LCAT as measured in the same assay conditions; or increased plasma stability or half-life time, wherein LCAT stability is improved compared to wild type LCAT plasma stability as measured in the same assay conditions.
  • Assays for measuring LCAT enzyme activity include, e.g., use of apoAI-liposome assay and use of plasma LCAT activity assay, which determine cholesterol esterification rate in an artificial system and in a physiologically relevant system, respectively.
  • Assays for measuring LCAT stability in vivo includes ELISA, which determines the half-life of recombinant LCAT protein in the blood after LCAT protein administration.
  • Atheromatous plaque refers to a condition characterized by the hardening and/or narrowing of the arteries caused by the buildup of athermatous plaque inside the arterial walls.
  • the atheromatous plaque is divided in three components, (1) the atheroma, a nodular accumulation of a soft flaky material at the center of large plaques, composed of macrophages nearest the lumen of the artery; (2) underlying areas of cholesterol crystals; (3) calcification at the outer base of more advanced lesions.
  • Indicators of atherosclerosis include, for example, the development of plaques in the arteries, their calcification, the extent of which can be determined by Sudan IV staining, or the development of foam cells in arteries.
  • the narrowing of the arteries can be determined by coronary angioplasty, ultrafast CT, or ultrasound.
  • Inflammation or “inflammatory disorder” refers to a localized, protective response elicited by injury or destruction of tissues, which serves to destroy, dilute or wall off (sequester) both the injurious agent and the injured tissue.
  • inflammatory disease or “inflammatory condition” as used herein, means any disease in which an excessive or unregulated inflammatory response leads to excessive inflammatory symptoms, host tissue damage, or loss of tissue function.
  • autoimmune disease means any group of disorders in which tissue injury is associated with humoral or cell-mediated responses to the body's own constituents.
  • allergic disease as used herein, means any symptoms, tissue damage, or loss of tissue function resulting from allergy.
  • arthritic disease means any of a large family of diseases that are characterized by inflammatory lesions of the joints attributable to a variety of etiologies.
  • skintitis means any of a large family of diseases of the skin that are characterized by inflammation of the skin attributable to a variety of etiologies.
  • transplant rejection means any immune reaction directed against grafted tissue (including organ and cell (e.g., bone marrow)), characterized by a loss of function of the grafted and surrounding tissues, pain, swelling, leukocytosis and thrombocytopenia.
  • Thrombosis and “thrombosis-related disorder” refer to abnormal thrombus formation that causes obstruction of blood vessels and conditions associated with such obstruction.
  • Blood vessels operate under significant shear stresses that are a function of blood flow shear rate. Frequently, there is damage to small blood vessels and capillaries. When these vessels are damaged, hemostasis is triggered to stop the bleeding. Under typical circumstances, such an injury is dealt with through a sequence of events commonly referred to as the “thrombus formation”. Thrombus formation is dependent upon platelet adhesion, activation and aggregation and the coagulation cascade that culminates in the conversion of soluble fibrinogen to insoluble fibrin clot. Thrombus formation at site of wound prevents extravasation of blood components. Subsequently, wound healing and clot dissolution occurs and blood vessel integrity and flow is restored.
  • HDL refers to the high-density lipoproteins.
  • LDL low-density lipoproteins
  • VLDL refers to the very low density lipoproteins.
  • treatment includes the administration, to a subject in need, of an amount of a compound of the invention which will inhibit, decrease or reverse development of, for example, a pathological atherosclerosis, inflammatory, or thrombosis-related condition as disclosed herein without limitation.
  • treatment as used herein means the administration, to a subject in need, of an amount of a compound of the invention, which will increase HDL cholesterol levels.
  • “Inhibiting,” in connection with inhibiting atherosclerosis, is intended to mean preventing, retarding, stabilizing, or reversing formation or growth of atheromatous plaques, inflammatory condition, or thrombosis-related indication.
  • Treatment of diseases and disorders herein is intended to also include therapeutic administration of a compound of the invention (or a pharmaceutical salt, derivative or prodrug thereof) or a pharmaceutical composition containing the compound to a subject (i.e., an animal, for example a mammal, such as a human) believed to be in need of treatment for diseases and disorders, such as, for example, inflammation, thrombosis, coronary heart disease, high blood pressure, LCAT deficiency syndrome, Alzheimer's disease, corneal opacity, metabolic syndrome, dyslipidemia, myocardial infarction, stroke, critical limb ischemia, angina and the like.
  • a subject i.e., an animal, for example a mammal, such as a human
  • diseases and disorders such as, for example, inflammation, thrombosis, coronary heart disease, high blood pressure, LCAT deficiency syndrome, Alzheimer's disease, corneal opacity, metabolic syndrome, dyslipidemia, myocardial infarction, stroke, critical limb ischemia
  • Treatment also encompasses administration of the compound or pharmaceutical composition to subjects not having been diagnosed as having a need thereof, i.e., prophylactic administration to the subject, such as prevention of accumulation of cholesterol.
  • the subject is initially diagnosed by a licensed physician and/or authorized medical practitioner, and a regimen for prophylactic and/or therapeutic treatment via administration of the compound(s) or compositions of the invention is suggested, recommended or prescribed.
  • the phrase “therapeutically effective amount” is the amount of the compound of the invention that will achieve the goal of improvement in disorder severity and the frequency of incidence.
  • the improvement in disorder severity includes, for example, prevention of accumulation of cholesterol in vessel walls increasing of blood levels of HDL cholesterol, the reversal of atherosclerosis, as well as slowing down the progression of atherosclerosis, prevention or treatment of inflammatory disorders, and prevention or treatment of thrombosis-relating conditions.
  • the term “subject” is intended to mean a human or other mammal, exhibiting, or at risk of developing, atherosclerosis, an inflammatory condition or thrombosis.
  • Such an individual can have, or be at risk of developing, for example, atherosclerosis associated with conditions such as thrombosis, coronary heart disease, high blood pressure, LCAT deficiency syndrome, Alzheimer's disease, corneal opacity, metabolic syndrome, dyslipidemia, myocardial infarction, stroke, critical limb ischemia, angina and the like.
  • atherosclerosis associated with conditions such as thrombosis, coronary heart disease, high blood pressure, LCAT deficiency syndrome, Alzheimer's disease, corneal opacity, metabolic syndrome, dyslipidemia, myocardial infarction, stroke, critical limb ischemia, angina and the like.
  • the prognostic and clinical indications of these conditions are known in the art.
  • the invention provides compounds, pharmaceutical compositions and methods for treating atherosclerosis and for decrease or prevention of accumulation of cholesterol in a subject by modifying LCAT polypeptide.
  • the modified LCAT can be obtained by activation using small molecule compounds of the invention.
  • modified LCAT can be obtained by modification at amino acid residue 31, for example, via the covalent binding to 3-pyrazinyl-2-carbonitrile.
  • LCAT activity can be determined by measuring the conversion of radiolabeled cholesterol to cholesterol ester after incubation of LCAT and radiolabeled LCAT substrates containing Apo A-I.
  • Cholesterol esterification rate (nmol CE/mL per hour) can be measured by determining the rate of conversion of labeled cholesterol to cholesteryl ester after incubation of plasma that is radiolabeled with a trace amount of radioactive cholesterol by equilibration with a [ 14 C] cholesterol-albumin mixture at 4° C.
  • the endogenous cholesterol esterification rate (as determined with plasma LCAT activity assay) reflects not only on mass of LCAT, but also the nature and amount of LCAT substrate and cofactor present in the serum, and therefore provides a better measure of the therapeutic LCAT activity.
  • LCAT stability half-life
  • recombinant LCAT protein levels in the plasma can be determined by using ELISA described by JR Crowther (ELISA theory and practice, methods in molecular Biology Volume 42).
  • Reagents for measuring LCAT stability and protein concentration include anti-LCAT antibodies, which are commercially available from several vendors. Examples of use of this assay to determine activity and/or stability of the modified LCAT are given below.
  • references to a certain element such as hydrogen or H is meant to include all isotopes of that element.
  • an R group is defined to include hydrogen or H, it also includes deuterium and tritium.
  • Compounds comprising radioisotopes such as tritium, 14 C, 32 P and 35 S are thus within the scope of the invention. Procedures for inserting such labels into the compounds of the invention will be readily apparent to those skilled in the art based on the disclosure herein.
  • substituted refers to a group, such as those defined below, in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms such as, but not limited to, a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides.
  • a halogen atom such as F, Cl, Br, and I
  • an oxygen atom in groups such as hydroxyl groups
  • a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides.
  • Substituents may be either monovalent or polyvalent depending on the context of their usage. For example, if description contained the group R 1 -R 2 -R 3 and R 2 was defined as C 1-6 alkyl, then the R 2 alkyl would be considered polyvalent because it must be bonded to at least R 1 and R 3 . Alternatively, if R 1 was defined as C 1-6 alkyl, then the R 1 alkyl would be monovalent (excepting any further substitution language).
  • unsubstituted as used herein with reference to a group, means that the group does not have one or more bonds to a hydrogen or carbon atom contained therein replaced by a bond to non-hydrogen or non-carbon atom, as described above.
  • alkyl as used herein either alone or within other terms such as “haloalkyl”, “alkylamino” and “cycloalkyl”, refers to linear, branched or cyclic radicals having one to about twelve carbon atoms. “Cycloalkyl” is also used exclusively herein to refer specifically to fully or partially saturated cyclic alkyl radicals.
  • alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, hexyl, cyclopropyl, cyclopentyl, cyclohexyl and the like.
  • C a-b alkyl refers to an alkyl group comprising from a to b carbon atoms in a branched, cyclical or linear relationship or any combination of the three.
  • the alkyl groups described in this section may also contain double or triple bonds. Examples of C 1-8 alkyl include, but are not limited to the following:
  • halogen and “halo” as used herein, refers to a halogen atoms selected from F, Cl, Br and I.
  • haloalkyl refers to radicals wherein any one or more of the alkyl carbon atoms is substituted with halo as defined above.
  • monohaloalkyl, dihaloalkyl and polyhaloalkyl radicals including perhaloalkyl may have either an iodo, bromo, chloro or fluoro atom within the radical.
  • Dihalo and polyhaloalkyl radicals may have two or more of the same halo atoms or a combination of different halo radicals.
  • haloalkyl radicals include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl.
  • C a-b haloalkyl refers to an alkyl group, as described above, wherein any number, but at least on of the hydrogen atoms attached to the alkyl chain are replaced by F, Cl, Br or I.
  • haloalkyl includes, without limitation, trifluoromethyl, pentafluoroethyl and the like.
  • heteroalkyl refers to an alkyl having one or more of the carbon atoms replaced by a heteroatom, selected from nitrogen, oxygen and sulfur.
  • a heteroalkyl would include an ether or a thioether chain, or an alkoxide moiety, wherein the heteroatom is in the linear region of the moeity.
  • the term also includes moieties where the heteroatom is in a branched region. For example, the term includes 2-amino-n-hexane or 5-hydroxy-pentane.
  • hydroxyalkyl refers to linear or branched alkyl radicals having one to about ten carbon atoms any one of which may be substituted with one or more hydroxyl radicals.
  • examples of such radicals include hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl and hydroxyhexyl.
  • alkoxy refers to linear or branched oxy-containing radicals each having alkyl portions of one to about ten carbon atoms. Examples of such radicals include methoxy, ethoxy, propoxy, butoxy and tert-butoxy. Alkoxy radicals may be further substituted with one or more halo atoms, such as fluoro, chloro or bromo, to provide “haloalkoxy” radicals. Examples of lower haloalkoxy radicals having one to three carbon atoms include fluoromethoxy, chloromethoxy, trifluoromethoxy, trifluoroethoxy, fluoroethoxy and fluoropropoxy.
  • sulfonyl refers to divalent radicals —SO 2 —.
  • aryl refers to a carbocyclic aromatic system containing one, two or three rings wherein such rings may be attached together in a fused manner.
  • aryl includes, without limitation, aromatic radicals such as phenyl, naphthyl, indenyl, tetrahydronaphthyl, and indanyl.
  • the “aryl” group may have 1 to 3 substituents such as alkyl, hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy and alkylamino.
  • Aryl also includes the moiety wherein the aromatic carbocycle is fused with a C 3-6 cycloalkyl bridge, wherein the bridge optionally includes 1, 2 or 3 heteroatoms selected from N, O and S.
  • the bridge optionally includes 1, 2 or 3 heteroatoms selected from N, O and S.
  • phenyl substituted with —O—CH 2 —O— forms the aryl benzodioxolyl substituent.
  • “Saturated or unsaturated” means a substitutent that is completely saturated, completely unsaturated, or has any degree of unsaturation in between. Examples of a saturated or unsaturated 6-membered ring carbocycle would include phenyl, cyclohexyl, cyclohexenyl and cyclohexadienyl.
  • salt refers to a salt form of a free base compound of the present invention, as appreciated by persons of ordinary skill in the art. Salts may be prepared by conventional means, known to those skilled in the art.
  • pharmaceutically-acceptable when used in reference to a salt, refers to salt forms of a given compound, which are within governmental regulatory safety guidelines for ingestion and/or administration to a subject.
  • pharmaceutically-acceptable salts embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases.
  • the nature of the salt is not critical, provided that it is pharmaceutically acceptable. Some specific examples are acetate; trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide; sulfate; citrate; tartrate; glycolate; and oxalate.
  • Suitable pharmaceutically-acceptable acid addition salts of compounds of Formula I may be prepared from an inorganic acid or from an organic acid.
  • inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid.
  • organic acids may be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, example of which are formic, acetic, adipic, butyric, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, ethanedisulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, camphoric, camphorsulfonic,
  • Suitable pharmaceutically acceptable base addition salts of compounds of Formula I include metallic salts, such as salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, or salts made from organic bases including primary, secondary and tertiary amines, substituted amines including cyclic amines, such as caffeine, arginine, diethylamine, N-ethyl piperidine, aistidine, glucamine, isopropylamine, lysine, morpholine, N-ethyl morpholine, piperazine, piperidine, triethylamine, trimethylamine.
  • metallic salts such as salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc
  • organic bases including primary, secondary and tertiary amines, substituted amines including cyclic amines, such as caffeine, arginine, diethylamine, N-ethyl piperidine, aistidine, glucamine, isopropylamine, ly
  • the basic nitrogen-containing groups of compounds of Formula I can be quaternized with such agents as lower alkyl halides including, without limitation, methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates including dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides, and others. Water or oil-soluble or dispersible products may be obtained by quaternizing such basic nitrogen groups in compounds of Formula I.
  • lower alkyl halides including, without limitation, methyl, ethyl, propyl, and butyl chloride, bromides and iodides
  • dialkyl sulfates including dimethyl, diethyl, dibut
  • derivative refers to simple modifications, readily apparent to those of ordinary skill in the art, on the parent core structure of Formula I, which does not significantly affect (generally decrease) the activity of the compound in-vitro as well as in vivo, in a subject.
  • derivative as used herein, is contemplated to include pharmaceutically acceptable derivatives of compounds of Formula I.
  • leaving group refers to groups readily displaceable by a nucleophile, such as an amine, a thiol or an alcohol nucleophile.
  • a nucleophile such as an amine, a thiol or an alcohol nucleophile.
  • Such leaving groups are well known in the art. Examples of such leaving groups include, but are not limited to, N-hydroxysuccinimide, N-hydroxybenzotriazole, halides, triflates, tosylates and the like. Exemplary leaving groups are indicated herein where appropriate.
  • protecting group refers to groups well known in the art which are used to prevent selected reactive groups, such as carboxy, amino, hydroxy, mercapto and the like, from undergoing undesired reactions, such as nucleophilic, electrophilic, oxidation, reduction and the like. Protecting groups are indicated herein where appropriate. Examples of amino protecting groups include, but are not limited to, aralkyl, substituted aralkyl, cycloalkenylalkyl and substituted cycloalkenyl alkyl, allyl, substituted allyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, silyl and the like.
  • aralkyl examples include, but are not limited to, benzyl, ortho-methylbenzyl, trityl and benzhydryl, which can be optionally substituted with halogen, alkyl, alkoxy, hydroxy, nitro, acylamino, acyl and the like, and salts, such as phosphonium and ammonium salts.
  • aryl groups include phenyl, naphthyl, indanyl, anthracenyl, 9-(9-phenylfluorenyl), phenanthrenyl, durenyl and the like.
  • cycloalkenylalkyl or substituted cycloalkylenylalkyl radicals include, but are not limited to, cyclohexenyl methyl and the like.
  • Suitable acyl, alkoxycarbonyl and aralkoxycarbonyl groups include benzyloxycarbonyl, t-butoxycarbonyl, iso-butoxycarbonyl, benzoyl, substituted benzoyl, butyryl, acetyl, tri-fluoroacetyl, tri-chloro acetyl, phthaloyl and the like.
  • a mixture of protecting groups can be used to protect the same amino group, such as a primary amino group can be protected by both an aralkyl group and an aralkoxycarbonyl group.
  • Amino protecting groups can also form a heterocyclic ring with the nitrogen to which they are attached, for example, 1,2-bis(methylene)benzene, phthalimidyl, succinimidyl, maleimidyl and the like and where these heterocyclic groups can further include adjoining aryl and cycloalkyl rings.
  • the heterocyclic groups can be mono-, di- or tri-substituted, such as nitrophthalimidyl.
  • Amino groups may also be protected against undesired reactions, such as oxidation, through the formation of an addition salt, such as hydrochloride, toluenesulfonic acid, trifluoroacetic acid and the like.
  • an addition salt such as hydrochloride, toluenesulfonic acid, trifluoroacetic acid and the like.
  • Many of the amino protecting groups including aralkyl groups for example, are also suitable for protecting carboxy, hydroxy and mercapto groups.
  • Alkyl groups are also suitable groups for protecting hydroxy and mercapto groups, such as tert-butyl.
  • Silyl protecting groups are groups containing silicon atoms which are optionally substituted by one or more alkyl, aryl and aralkyl groups. Suitable silyl protecting groups include, but are not limited to, trimethylsilyl, triethylsilyl, tri-isopropylsilyl, tert-butyldimethylsilyl, dimethylphenylsilyl, 1,2-bis(dimethylsilyl)benzene, 1,2-bis(dimethylsilyl)ethane and diphenylmethylsilyl. Silylation of an amino groups provide mono- or di-silylamino groups. Silylation of aminoalcohol compounds can lead to a N,N,O-tri-silyl derivative.
  • silyl function from a silyl ether function is readily accomplished by treatment with, for example, a metal hydroxide or ammonium fluoride reagent, either as a discrete reaction step or in situ during a reaction with the alcohol group.
  • Suitable silylating agents are, for example, trimethylsilyl chloride, tert-butyl-dimethylsilyl chloride, phenyldimethylsilyl chloride, diphenylmethyl silyl chloride or their combination products with imidazole or DMF.
  • Methods for silylation of amines and removal of silyl protecting groups are well known to those skilled in the art.
  • Methods of preparation of these amine derivatives from corresponding amino acids, amino acid amides or amino acid esters are also well known to those skilled in the art of organic chemistry including amino acid/amino acid ester or aminoalcohol chemistry.
  • Protecting groups are removed under conditions which will not affect the remaining portion of the molecule. These methods are well known in the art and include acid hydrolysis, hydrogenolysis and the like.
  • One method involves removal of a protecting group, such as removal of a benzyloxycarbonyl group by hydrogenolysis utilizing palladium on carbon in a suitable solvent system such as an alcohol, acetic acid, and the like or mixtures thereof.
  • a t-butoxycarbonyl protecting group can be removed utilizing an inorganic or organic acid, such as HCl or trifluoroacetic acid, in a suitable solvent system, such as dioxane or methylene chloride.
  • the resulting amino salt can readily be neutralized to yield the free amine.
  • Carboxy protecting group such as methyl, ethyl, benzyl, tert-butyl, 4-methoxyphenylmethyl and the like, can be removed under hydrolysis and hydrogenolysis conditions well known to those skilled in the art.
  • LCAT LCAT modifications described above, it is contemplated that other “derivatives” of LCAT may be substituted for an LCAT protein described above. Such derivatives may improve the solubility, absorption, biological half life, and the like of the compounds. The moieties may alternatively eliminate or attenuate any undesirable side-effect of the compounds and the like.
  • Such derivative LCATs include compounds in which:
  • the compound or some portion thereof is cyclic.
  • the peptide portion may be modified to contain two or more cysteine residues (e.g., in the linker), which could cyclize by disulfide bond formation.
  • the compound is cross-linked or is rendered capable of cross-linking between molecules.
  • the peptide portion may be modified to contain one cysteine residue and thereby be able to form an intermolecular disulfide bond with a like molecule.
  • the compound may also be cross-linked through its C-terminus.
  • One or more peptidyl [—C(O)NR—] linkages (bonds) is replaced by a non-peptidyl linkage.
  • Exemplary non-peptidyl linkages are —CH 2 -carbamate [—CH 2 —OC(O)NR—], phosphonate, —CH 2 -sulfonamide [—CH 2 —S(O) 2 NR—], urea [—NHC(O)NH—], —CH 2 -secondary amine, and alkylated peptide [—C(O)NR 6 — wherein R 6 is lower alkyl].
  • the N-terminus is derivatized. Typically, the N-terminus may be acylated or modified to a substituted amine.
  • Exemplary N-terminal derivative groups include —NRR1-(other than —NH2), —NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR1, succinimide, or benzyloxycarbonyl-NH— (CBZ-NH—), wherein R and R1 are each independently hydrogen or lower alkyl with the proviso that R and R1 are not both hydrogen and wherein the phenyl ring may be substituted with 1 to 3 substituents selected from the group consisting of C 1 -C 4 alkyl, C 1 -C 4 alkoxy, chloro, and bromo; to a succinimide group; to a benzyloxycarbonyl-NH— (CBZ-NH—) group; and peptides wherein the free C terminus is
  • the free C-terminus is derivatized. Typically, the C-terminus is esterified or amidated. For example, one may use methods described in the art to add (NH—CH2-CH2-NH2)2 to compounds of this invention at the C-terminus. Likewise, one may use methods described in the art to add —NH2 to compounds of this invention at the C-terminus.
  • Exemplary C-terminal derivative groups include, for example, —C(O)R2 wherein R2 is lower alkoxy or —NR3R4 wherein R3 and R4 are independently hydrogen or C 1 -C 8 alkyl (preferably C 1 -C 4 alkyl).
  • a disulfide bond is replaced with another, preferably more stable, cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am. Pep. Symp., 357-9.
  • another, preferably more stable, cross-linking moiety e.g., an alkylene
  • One or more individual amino acid residues is modified.
  • Various derivatizing agents are known to react specifically with selected side chains or terminal residues, as described in detail below.
  • modifications of individual amino acids may be introduced into the LCAT amino acid sequence by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • Lysinyl and amino terminal residues may be reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine guanidino group.
  • Carboxyl side groups may be selectively modified by reaction with carbodiimides (R′—N ⁇ C ⁇ N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.
  • carbodiimides R′—N ⁇ C ⁇ N—R′
  • aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues may be deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • Cysteinyl residues at a position other than residue 31 can be replaced by amino acid residues or other moieties either to eliminate disulfide bonding or, conversely, to stabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9.
  • Derivatization with bifunctional agents is useful for cross-linking the peptides or their functional derivatives to a water-insoluble support matrix or to other macromolecular carriers.
  • Commonly used cross-linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis (succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane.
  • Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 may be employed for protein immobilization.
  • Such derivatized moieties preferably improve one or more characteristics including thrombopoietic activity, solubility, absorption, biological half life, and the like of the inventive compounds.
  • derivatized moieties result in compounds that have the same, or essentially the same, characteristics and/or properties of the compound that is not derivatized.
  • the moieties may alternatively eliminate or attenuate any undesirable side effect of the compounds and the like.
  • Carbohydrate (oligosaccharide) groups may conveniently be attached to sites that are known to be glycosylation sites in proteins.
  • O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline.
  • X is preferably one of the 19 naturally occurring amino acids other than proline.
  • the structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different.
  • sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycosylated compound.
  • site(s) may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites may further be glycosylated by synthetic or semi-synthetic procedures known in the art.
  • Compounds of the present invention may be changed at the DNA level, as well.
  • the DNA sequence of any portion of the compound may be changed to codons more compatible with the chosen host cell.
  • optimized codons are known in the art. Codons may be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell.
  • the vehicle, linker and peptide DNA sequences may be modified to include any of the foregoing sequence changes.
  • Isotope- and toxin-conjugated derivatives are the above-described molecules conjugated to toxins, tracers, or radioisotopes. Such conjugation is especially useful for molecules comprising peptide sequences that bind to tumor cells or pathogens. Such molecules may be used as therapeutic agents or as an aid to surgery (e.g., radioimmunoguided surgery or RIGS) or as diagnostic agents (e.g., radioimmunodiagnostics or RID).
  • conjugated derivatives possess a number of advantages. They facilitate use of toxins and radioisotopes that would be toxic if administered without the specific binding provided by the peptide sequence. They also can reduce the side-effects that attend the use of radiation and chemotherapy by facilitating lower effective doses of the conjugation partner.
  • Useful conjugation partners include:
  • the molecule of the present invention would comprise a benign capture molecule.
  • This capture molecule would be able to specifically bind to a separate effector molecule comprising, for example, a toxin or radioisotope.
  • Both the vehicle-conjugated molecule and the effector molecule would be administered to the patient.
  • the effector molecule would have a short half-life except when bound to the vehicle-conjugated capture molecule, thus minimizing any toxic side-effects.
  • the vehicle-conjugated molecule would have a relatively long half-life but would be benign and non-toxic.
  • the specific binding portions of both molecules can be part of a known specific binding pair (e.g., biotin, streptavidin) or can result from peptide generation methods such as those described herein.
  • conjugated derivatives may be prepared by methods known in the art.
  • protein effector molecules e.g., Pseudomonas endotoxin
  • Radioisotope conjugated derivatives may be prepared, for example, as described for the BEXA antibody (Coulter).
  • Derivatives comprising cytotoxic agents or microbial toxins may be prepared, for example, as described for the BR96 antibody (Bristol-Myers Squibb).
  • Molecules employed in capture systems may be prepared, for example, as described by the patents, patent applications, and publications from NeoRx.
  • Molecules employed for RIGS and RID may be prepared, for example, by the patents, patent applications, and publications from NeoProbe.
  • the compounds of the invention may also be covalently or noncovalently associated with a carrier molecule, such as a linear polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), a branched-chain polymer (see, for example, U.S. Pat. No. 4,289,872 to Denkenwalter et al., issued Sep. 15, 1981; U.S. Pat. No. 5,229,490 to Tam, issued Jul. 20, 1993; WO 93/21259 by Frechet et al., published 28 Oct. 1993); a lipid; a cholesterol group (such as a steroid); or a carbohydrate or oligosaccharide.
  • a carrier molecule such as a linear polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), a branched-chain polymer (see, for example, U.S. Pat. No. 4,289,872 to Denkenwalter et al
  • Other possible carriers include one or more water soluble polymer attachments such as polyoxyethylene glycol, or polypropylene glycol as described U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and 4,179,337.
  • Still other useful polymers known in the art include monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of these polymers.
  • the carrier is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG group may be of any convenient molecular weight and may be straight chain or branched.
  • the average molecular weight of the PEG will range from about 2 kDa to about 100 kDa, or from about 5 kDa to about 50 kDa, or from about 5 kDa to about 10 kDa.
  • the PEG groups will generally be attached to the compounds of the invention via acylation, reductive alkylation, Michael addition, thiol alkylation or other chemoselective conjugation/ligation methods through a reactive group on the PEG moiety (e.g., an aldehyde, amino, ester, thiol, ⁇ -haloacetyl, maleimido or hydrazino group) to a reactive group on the target compound (e.g., an aldehyde, amino, ester, thiol, ⁇ -haloacetyl, maleimido or hydrazino group).
  • a reactive group on the PEG moiety e.g., an aldehyde, amino, ester, thiol, ⁇ -haloacetyl, maleimido or hydrazino group
  • a reactive group on the target compound e.g., an aldehyde, amino, ester, thiol, ⁇ -halo
  • Prodrugs of the compounds of this invention are also contemplated by this invention.
  • a “prodrug” is a compound, which when administered to the body of a subject (such as a mammal), breaks down in the subject's metabolic pathway to provide an active compound of Formula I. More specifically, a prodrug is an active or inactive “masked” compound that is modified chemically through in vivo physiological action, such as hydrolysis, metabolism and the like, into a compound of this invention following administration of the prodrug to a subject or patient.
  • the suitability and techniques involved in making and using prodrugs are well known by those skilled in the art. For a general discussion of prodrugs involving esters see Svensson and Tunek Drug Metabolism Reviews 165 (1988) and Bundgaard Design of Prodrugs, Elsevier (1985).
  • a prodrug is a masked carboxylic acid group.
  • a masked carboxylate anion include a variety of esters, such as alkyl (for example, methyl, ethyl), cycloalkyl (for example, cyclohexyl), aralkyl (for example, benzyl, p-methoxybenzyl), and alkylcarbonyloxyalkyl (for example, pivaloyloxymethyl).
  • esters such as alkyl (for example, methyl, ethyl), cycloalkyl (for example, cyclohexyl), aralkyl (for example, benzyl, p-methoxybenzyl), and alkylcarbonyloxyalkyl (for example, pivaloyloxymethyl).
  • Amines have been masked as arylcarbonyloxymethyl substituted derivatives which are cleaved by esterases in vivo releasing the free drug and formaldehy
  • drugs containing an acidic NH group such as imidazole, imide, indole and the like, have been masked with N-acyloxymethyl groups (Bundgaard Design of Prodrugs, Elsevier (1985)). Hydroxy groups have been masked as esters and ethers.
  • EP 039,051 (Sloan and Little, Apr. 11, 1981) discloses Mannich-base hydroxamic acid prodrugs, their preparation and use.
  • stereoisomer refers to a compound having one or more asymmetric centers. Chiral centers in a compound generally cause that compound to exist in many different conformations or stereoisomers.
  • stereoisomers includes enantiomers, diastereomers, atropisomers and geometric isomers. Stereoisomers generally possess different chemical properties and/or biological activity, as appreciated by those skilled in the art. For example, one stereoisomer may be more active and/or may exhibit beneficial effects in comparison to other stereoisomer(s) or when separated from the other stereoisomer(s). However, it is well within the skill of the ordinary artisan to separate, and/or to selectively prepare said stereoisomers. Accordingly, “stereoisomers” of the present invention necessarily include mixtures of stereoisomers, including racemic mixtures, individual stereoisomers, and optically active forms.
  • solvate when used with reference to a compound refers to a compound, which is associated with one or more molecules of a solvent, such as an organic solvent, inorganic solvent, aqueous solvent or mixtures thereof.
  • a solvent such as an organic solvent, inorganic solvent, aqueous solvent or mixtures thereof.
  • the compounds of Formula I may also be solvated, especially hydrated. Hydration may occur during manufacturing of the compounds or compositions comprising the compounds, or the hydration may occur over time due to the hygroscopic nature of the compounds.
  • Compounds of the invention may exist as organic solvates as well, including DMF, ether, and alcohol solvates among others. The identification and preparation of any particular solvate is within the skill of the ordinary artisan of synthetic organic or medicinal chemistry.
  • an LCAT protein of the invention includes at least one vehicle attached to the protein through the N-terminus, C-terminus or a side chain of one of the amino acid residues.
  • an Fc domain is a vehicle.
  • an Fc domain may be fused to the N or C termini of the peptides or at both the N and C termini.
  • Multiple vehicles, as exemplified herein, may also be used; e.g., Fc's at each terminus or an Fc at a terminus and a PEG group at the other terminus or a side chain.
  • the Fc component is either a native Fc or an Fc variant.
  • the Fc component is an Fc region of the human immunoglobulin IgG1 heavy chain or a biologically active fragment, derivative, or dimer thereof, see Ellison, J. W. et al., Nucleic Acids Res. 10:4071-4079 (1982). It is understood, however, that an Fc region for use in the invention may be derived from an IgG, IgA, IgM, IgE or IgD from any species.
  • Native Fc domains are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and/or non-covalent association.
  • the number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2).
  • class e.g., IgG, IgA, IgE
  • subclass e.g., IgG1, IgG2, IgG3, IgA1, IgGA2
  • One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9).
  • Fc sequence contemplated include those known in the art such as, for example, Fc IgG1 (GenBank Accession No. P01857), Fc IgG2 (GenBank Accession No. P01859), Fc IgG3 (GenBank Accession No. P01860), Fc IgG4 (GenBank Accession No. P01861), Fc IgA1 (GenBank Accession No. P01876), Fc IgA2 (GenBank Accession No. P01877), Fc IgD (GenBank Accession No. P01880), Fc IgM (GenBank Accession No. P01871), and Fc IgE (GenBank Accession No. P01854).
  • an Fc variant is incorporated which comprises a molecule or sequence that is humanized from a non-human native Fc.
  • an Fc variant comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC), each of which is described in detail in U.S. Patent Application No. 20040087778, the disclosure of which is incorporated by reference in its entirety.
  • ADCC antibody-dependent cellular cytotoxicity
  • Variant (or analog) polypeptides include insertion variants, wherein one or more amino acid residues supplement an Fc amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the Fc amino acid sequence. Insertion variants, with additional residues at either or both termini, can include for example, fusion proteins and proteins including amino acid tags or labels.
  • the Fc molecule may optionally contain an N-terminal Met, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli.
  • Fc deletion variants one or more amino acid residues in an Fc polypeptide are removed.
  • Deletions can be effected at one or both termini of the Fc polypeptide, or with removal of one or more residues within the Fc amino acid sequence.
  • Deletion variants therefore, include all fragments of an Fc polypeptide sequence.
  • Fc substitution variants one or more amino acid residues of an Fc polypeptide are removed and replaced with alternative residues.
  • substitutions are conservative in nature and conservative substitutions of this type are well known in the art.
  • the invention embraces substitutions that are also non-conservative.
  • cysteine residues can be deleted or replaced with other amino acids to prevent formation of some or all disulfide crosslinks of the Fc sequences.
  • Each cysteine residue can be removed and/or substituted with other amino acids, such as Ala or Ser.
  • modifications may also be made to introduce amino acid substitutions to (1) ablate the Fc receptor binding site; (2) ablate the complement (C1q) binding site; and/or to (3) ablate the antibody dependent cell-mediated cytotoxicity (ADCC) site.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • one or more tyrosine residues can be replaced by phenylalanine residues.
  • other variant amino acid insertions, deletions and/or substitutions are also contemplated and are within the scope of the present invention.
  • Conservative amino acid substitutions will generally be preferred.
  • alterations may be in the form of altered amino acids, such as peptidomimetics or D-amino acids.
  • native Fcs and Fc variants are suitable Fc domains for use within the scope of this invention.
  • a native Fc may be extensively modified to form an Fc variant provided binding to the salvage receptor is maintained; see, for example WO 97/34631 and WO 96/32478.
  • One may remove these sites by, for example, substituting or deleting residues, inserting residues into the site, or truncating portions containing the site.
  • the inserted or substituted residues may also be altered amino acids, such as peptidomimetics or D-amino acids.
  • Fc variants may be desirable for a number of reasons, several of which are described below.
  • Exemplary Fc variants include molecules and sequences in which:
  • cysteine-containing segment at the N-terminus may be truncated or cysteine residues may be deleted or substituted with other amino acids (e.g., alanyl, seryl).
  • cysteine residues may be deleted or substituted with other amino acids (e.g., alanyl, seryl).
  • one may truncate the N-terminal 20-amino acid segment of SEQ ID NO: 3 or delete or substitute the cysteine residues at positions 7 and 10 of SEQ ID NO: 3.
  • cysteine residues are removed, the single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently.
  • a native Fc is modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fc, which may be recognized by a digestive enzyme in E. coli such as proline iminopeptidase. One may also add an N-terminal methionine residue, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli .
  • the Fc domain of SEQ ID NO: 3 is one such Fc variant.
  • a portion of the N-terminus of a native Fc is removed to prevent N-terminal heterogeneity when expressed in a selected host cell. For this purpose, one may delete any of the first 20 amino acid residues at the N-terminus, particularly those at positions 1, 2, 3, 4 and 5.
  • Residues that are typically glycosylated may confer cytolytic response. Such residues may be deleted or substituted with unglycosylated residues (e.g., alanine).
  • Sites involved in interaction with complement such as the C1q binding site, are removed. For example, one may delete or substitute the EKK sequence of human IgG1. Complement recruitment may not be advantageous for the molecules of this invention and so may be avoided with such an Fc variant.
  • a native Fc may have sites for interaction with certain white blood cells that are not required for the fusion molecules of the present invention and so may be removed.
  • ADCC site is removed.
  • ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgG1. These sites, as well, are not required for the fusion molecules of the present invention and so may be removed.
  • the native Fc When the native Fc is derived from a non-human antibody, the native Fc may be humanized. Typically, to humanize a native Fc, one will substitute selected residues in the non-human native Fc with residues that are normally found in human native Fc. Techniques for antibody humanization are well known in the art.
  • Fc monomers will spontaneously dimerize when the appropriate cysteine residues are present, unless particular conditions are present that prevent dimerization through disulfide bond formation. Even if the cysteine residues that normally form disulfide bonds in the Fc dimer are removed or replaced by other residues, the monomeric chains will generally form a dimer through non-covalent interactions.
  • the term “Fc” herein is used to mean any of these forms: the native monomer, the native dimer (disulfide bond linked), modified dimers (disulfide and/or non-covalently linked), and modified monomers (i.e., derivatives).
  • Fc sequences may also be derivatized, i.e., bearing modifications other than insertion, deletion, or substitution of amino acid residues.
  • the modifications are covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties.
  • non-covalent modifications are also contemplated.
  • Derivatives of the invention may be prepared to increase circulating half-life, or may be designed to improve targeting capacity for the polypeptide to desired cells, tissues, or organs.
  • Fc part of a compound of the invention such as described in WO 96/32478, entitled “Altered Polypeptides with Increased Half-Life.”
  • Additional members of the class of molecules designated as Fc herein are those that are described in WO 97/34631, entitled “Immunoglobulin-Like Domains with Increased Half-Lives.”
  • the Fc fusions may be at the N or C terminus of a TMP of the invention, or at both the N and C termini of the TMP. It has been previously been shown that peptides in which an Fc moiety is ligated to the N terminus of the TMP group is more bioactive than the other possibilities. When the Fc is fused at the N-terminus of the TMP or linker, such fusion will generally occur at the C-terminus of the Fc chain, and vice versa.
  • PCT Patent Cooperation Treaty
  • WO 96/11953 entitled “N-Terminally Chemically Modified Protein Compositions and Methods,” herein incorporated by reference in its entirety.
  • This PCT publication discloses, among other things, the selective attachment of water soluble polymers to the N-terminus of proteins.
  • WSP water-soluble polymer
  • Suitable, clinically acceptable, WSP include without limitation, PEG, polyethylene glycol propionaldehyde, copolymers of ethylene glycol/propylene glycol, monomethoxy-polyethylene glycol, carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, poly (.beta.-amino acids) (either homopolymers or random copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers (PPG) and other polyakylene oxides, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (POG) (e.g., glycerol) and other poly
  • any of the forms of PEG that have been used to derivatize other proteins such as and without limitation mono-(C 1-C10) alkoxy- or aryloxy-polyethylene glycol, are provided.
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the PEG group may be of any convenient molecular weight and may be linear or branched.
  • the average molecular weight of PEG contemplated for use in the invention ranges from about 2 kDa to about 100 kDa, from about 5 kDa to about 50 kDa, from about 5 kDa to about 10 kDa.
  • the PEG moiety has a molecular weight from about 6 kDa to about 25 kDa.
  • PEG groups generally are attached to peptides or proteins via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the target peptide or protein (e.g., an aldehyde, amino, or ester group).
  • a reactive group on the PEG moiety e.g., an aldehyde, amino, thiol, or ester group
  • a reactive group on the target peptide or protein e.g., an aldehyde, amino, or ester group.
  • a mixture of polymer/peptide conjugate molecules can be prepared, and the advantage provided herein is the ability to select the proportion of polymer/peptide conjugate to include in the mixture.
  • a mixture of peptides with various numbers of polymer moieties attached i.e., zero, one or two
  • a useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a WSP (PEG) moiety, each bearing a special functionality that is mutually reactive toward the other.
  • the peptides can be easily prepared with conventional solid phase synthesis.
  • the peptides are “preactivated” with an appropriate functional group at a specific site.
  • the precursors are purified and fully characterized prior to reacting with the PEG moiety.
  • Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC.
  • the PEGylated peptides can be easily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry.
  • Polysaccharide polymers are another type of WSP which may be used for protein modification.
  • Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by ⁇ 1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kD to about 70 kD.
  • Dextran is a suitable water soluble polymer for use in the present invention as a vehicle by itself or in combination with another vehicle (e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456, which is hereby incorporated by reference. Dextran of about 1 kD to about 20 kD is preferred when dextran is used as a vehicle in accordance with the present invention.
  • the WSP moiety of the molecule may be branched or unbranched.
  • the polymer is pharmaceutically acceptable.
  • a desired polymer is selected based on such considerations as whether the polymer conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations.
  • the average molecular weight of each WSP is between about 2 kDa and about 100 kDa, between about 5 kDa and about 50 kDa, between about 12 kDa and about 40 kDa and between about 20 kDa and about 35 kDa.
  • the molecular weight of each polymer is between about 6 kDa and about 25 kDa.
  • the term “about” as used herein and throughout, indicates that in preparations of a water soluble polymer, some molecules will weigh more, some less, than the stated molecular weight. Generally, the higher the molecular weight or the more branches, the higher the polymer/protein ratio. Other sizes may be used, depending on the desired therapeutic profile including for example, the duration of sustained release; the effects, if any, on biological activity; the ease in handling; the degree or lack of antigenicity and other known effects of a water soluble polymer on a therapeutic protein.
  • the WSP should be attached to the protein with consideration given to effects on functional or antigenic domains of the peptide or protein.
  • chemical derivatization may be performed under any suitable condition used to react a protein with an activated polymer molecule.
  • Activating groups which can be used to link the water soluble polymer to one or more proteins include without limitation sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane and 5-pyridyl. If attached to the peptide by reductive alkylation, the polymer selected should have a single reactive aldehyde so that the degree of polymerization is controlled.
  • Alternative vehicles include a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule (e.g., a peptidomimetic compound) capable of binding to a salvage receptor.
  • a polypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al.
  • Peptides could also be selected by phage display for binding to the FcRn salvage receptor.
  • salvage receptor-binding compounds are also included within the meaning of “vehicle” and are within the scope of this invention.
  • Such vehicles should be selected for increased half-life (e.g., by avoiding sequences recognized by proteases) and decreased immunogenicity (e.g., by favoring non-immunogenic sequences, as discovered in antibody humanization).
  • X, Y and Z are independently selected from the group consisting of —N ⁇ , —S—, —CH ⁇ and provided that at least two of X, Y and Z are not —S—, and provided that no more than one of X, Y and Z is —CH ⁇ ;
  • L is —S—, —S(O)—, or —S(O) 2 —;
  • R 1 is selected from the group consisting of CN, COOR 5 , SO 2 R 6 and halogen;
  • R 2 is selected from the group consisting of H, optionally substituted C 1 -C 12 alkyl, optionally substituted C 1 -C 12 alkenyl, optionally substituted C 1 -C 8 alkynyl, optionally substituted aryl, and optionally substituted heteroaryl, and SR 3 , wherein the substituents are selected from the group consisting of C 1 -C 4 alkyl, NH 2 , halo and CN; and wherein R 3 is selected from the group consisting of H, optionally substituted C 1 -C 12 alkyl, optionally substituted C 1 -C 8 alkenyl, optionally substituted C 1 -C 8 alkynyl, optionally substituted aryl and optionally substituted heteroaryl, wherein the substituents are selected from the group consisting of NH 2 , halo and CN;
  • R 4 is H or C 1 -C 8 alkyl
  • R 5 and R 6 are each independently C 1 -C 4 alkyl.
  • X and Y are each —N ⁇ .
  • Z is —S—.
  • L is —S—.
  • R 1 is CN.
  • R 2 is SR 3 .
  • R 3 can be C 1 -C 4 alkyl, for example, methyl.
  • the invention contemplates the use of the following compounds:
  • the invention provides the modified LCAT polypeptide, wherein the amino acid residue 31 is replaced by a cysteine residue, wherein the cysteine residue is modified by replacing the thiol hydrogen with 3-pyrazinyl-2-carbonitrile.
  • the invention contemplates the use of a pharmaceutical composition comprising the modified LCAT polypeptide, wherein the amino acid residue 31 is replaced by a cysteine residue, wherein the cysteine residue is modified by replacing the thiol hydrogen with 3-pyrazinyl-2-carbonitrile, and a pharmaceutically acceptable carrier.
  • Scheme 1 illustrates methods for the preparation of compounds of structural formula (III).
  • One of skill in the art will understand that similar methods can be used for the synthesis of compounds in the other structural classes.
  • compounds of the present invention can be prepared beginning with the commercially available 2-chloropyrazinecarbonitrile (I).
  • a thiol such as II in the presence of base such as NaH, K 2 CO 3 or CsCO 3 in a suitable solvent such as THF, DMF or DMSO provides the adduct (III).
  • Oxidation of the thio group in III with for example H 2 O 2 , oxone, or MnO 2 will give the sulfone or sulfoxide derivative.
  • other oxidizing agents may be employed as described in March, J; Advanced Organic Chemistry, 5th ed., John Wiley & Sons, New York, pp. 1541 (2001).
  • compositions Comprising Modified LCAT and Methods of Administration
  • a pharmaceutical composition comprising a compound of this invention in combination with a pharmaceutically acceptable carrier.
  • Acceptable pharmaceutical carriers generally include diluents, excipients, adjuvants and the like as described herein.
  • a pharmaceutical composition of the invention may comprise an effective amount of a compound of the invention or an effective dosage amount of a compound of the invention.
  • An effective dosage amount of a compound of the invention includes an amount less than, equal to, or greater than an effective amount of the compound.
  • a pharmaceutical composition in which two or more unit dosages, such as in tablets, capsules and the like, are required to administer an effective amount of the compound or alternatively, a multi-dose pharmaceutical composition, such as powders, liquids and the like, in which an effective amount of the compound may be administered by administering a portion of the composition.
  • “Unit dosage” is defined as a discrete amount of a therapeutic composition dispersed in a suitable carrier. Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient.
  • compositions may generally be prepared by mixing one or more compounds of Formula I including stereoisomers or tautomers, solvates, pharmaceutically acceptable salts, derivatives or prodrugs thereof, with pharmaceutically acceptable carriers, excipients, binders, adjuvants, diluents and the like, to form a desired administrable formulation to treat or ameliorate a variety of disorders related to atherosclerosis or cardiovascular diseases.
  • the pharmaceutical compositions may generally be prepared by mixing one or more LCAT compounds with one or more pharmaceutically acceptable carriers, excipients, binders, adjuvants, diluents, preservatives, solubilizers, emulsifiers and the like, to form a desired administrable formulation to treat or ameliorate a variety of diseases.
  • compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes.
  • buffer content e.g., Tris-HCl, acetate, phosphate
  • additives e.g., Tween 80, Polysorbate 80
  • anti-oxidants e.g., ascorbic acid, sodium metabisulfite
  • preservatives e.g., Thimersol, benzyl alcohol
  • Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation.
  • Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference.
  • the compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal formulations.
  • compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.
  • conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.
  • the pharmaceutically active compounds of this invention can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals.
  • compositions can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, emulsifying or levigating processes, among others.
  • the compositions can be in the form of, for example, granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions.
  • compositions can be formulated for various routes of administration, for example, by oral administration, by transmucosal administration (including pulmonary and nasal administration), parenteral administration (including subcutaneous administration), transdermal (topical) administration or by rectal administration, as well as intrathecal, intravenous, intramuscular, intraperitoneal, intranasal, intraocular or intraventricular injection.
  • transmucosal administration including pulmonary and nasal administration
  • parenteral administration including subcutaneous administration
  • transdermal (topical) administration or by rectal administration, as well as intrathecal, intravenous, intramuscular, intraperitoneal, intranasal, intraocular or intraventricular injection.
  • rectal administration as well as intrathecal, intravenous, intramuscular, intraperitoneal, intranasal, intraocular or intraventricular injection.
  • the compound or compounds of the instant invention can also be administered in a local rather than a systemic fashion, such as injection as a sustained release formulation.
  • excipients and carriers are generally known to those skilled in the art and are thus included in the instant invention.
  • excipients and carriers are described, for example, in “Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (2000); and “Pharmaceutics The Science of Dosage Form Design, 2 nd Ed. (Aulton, ed.) Churchill Livingstone (2002).
  • the following dosage forms are given by way of example and should not be construed as limiting the invention.
  • Solid dosage forms also include liposomal or proteinoid encapsulation (for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Pat. No.
  • the formulation includes the LCAT compound, and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.
  • the compounds are chemically modified to enhance bioefficacy of oral delivery.
  • the chemical modification contemplated is the attachment of at least one moiety to the compound molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine.
  • the increase in overall stability of the compound and increase in circulation time in the body are also be used for this purpose. Examples of such moieties include: PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline as well as other moieties described herein.
  • a salt of a modified aliphatic amino acid such as sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC)
  • SNAC sodium N-(8-[2-hydroxybenzoyl]amino) caprylate
  • the compounds of this invention can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm.
  • the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets.
  • the therapeutic could be prepared by compression.
  • Oral pharmaceutical compositions contemplated can be prepared, for example, by mixing one or more compounds of the instant invention, or stereoisomers, solvates, prodrugs, pharmaceutically acceptable salts or tautomers thereof, with at least one additive or excipient such as a starch or other additive and tableted, encapsulated or made into other desirable forms for conventional administration.
  • additive or excipient such as a starch or other additive and tableted, encapsulated or made into other desirable forms for conventional administration.
  • Suitable additives or excipients are sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, sorbitol, starch, agar, alginates, chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins, collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides, methyl cellulose, hydroxypropylmethyl-cellulose, and/or polyvinylpyrrolidone.
  • oral dosage forms can contain other ingredients to aid in administration, such as an inactive diluent, or lubricants such as magnesium stearate, or preservatives such as paraben or sorbic acid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, a disintegrating agent, binders, thickeners, buffers, sweeteners, flavoring agents or perfuming agents. Additionally, dyestuffs or pigments may be added for identification. Tablets and pills may be further treated with suitable coating materials known in the art.
  • Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, slurries and solutions, which may contain an inactive diluent, such as water.
  • Pharmaceutical formulations may be prepared as liquid suspensions or solutions using a sterile liquid, such as, but not limited to, an oil, water, an alcohol, and combinations of these.
  • Pharmaceutically suitable surfactants, suspending agents, emulsifying agents, and the like may be added for oral or parenteral administration. More specifically, various aspects of oral pharmaceutical compositions include one or more of the following additives.
  • Colorants and flavoring agents may all be included.
  • the protein (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
  • diluents could include carbohydrates, especially mannitol, ⁇ -lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch.
  • Certain inorganic salts may also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride.
  • Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
  • Disintegrants may be included in the formulation of the therapeutic into a solid dosage form.
  • Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used.
  • Another form of the disintegrants are the insoluble cationic exchange resins.
  • Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
  • Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.
  • MC methyl cellulose
  • EC ethyl cellulose
  • CMC carboxymethyl cellulose
  • PVP polyvinyl pyrrolidone
  • HPMC hydroxypropylmethyl cellulose
  • Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.
  • the glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
  • surfactant might be added as a wetting agent.
  • Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents might be used and could include benzalkonium chloride or benzethonium chloride.
  • nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.
  • Additives may also be included in the formulation to enhance uptake of the compound. Additives potentially having this property are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
  • Controlled release formulation may be desirable.
  • the compound of this invention could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms e.g., gums.
  • Slowly degenerating matrices may also be incorporated into the formulation, e.g., alginates, polysaccharides.
  • Another form of a controlled release of the compounds of this invention is by a method based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect.
  • coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan.
  • the therapeutic agent could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups.
  • the first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols.
  • the second group consists of the enteric materials that are commonly esters of phthalic acid.
  • Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating.
  • the protein (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.
  • Adjei et al. Pharma. Res. (1990) 7: 565-9
  • Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
  • Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.
  • each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy.
  • the inventive compound should most advantageously be prepared in particulate form with an average particle size of less than 10 ⁇ m (or microns), most preferably 0.5 to 5 ⁇ m, for most effective delivery to the distal lung.
  • Pharmaceutically acceptable carriers for pulmonary delivery include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol.
  • Other ingredients for use in formulations may include DPPC, DOPE, DSPC and DOPC.
  • Natural or synthetic surfactants may be used.
  • PEG may be used (even apart from its use in derivatizing the protein or analog).
  • Dextrans such as cyclodextran, may be used.
  • Bile salts and other related enhancers may be used.
  • Cellulose and cellulose derivatives may be used.
  • Amino acids may be used, such as use in a buffer formulation.
  • liposomes are contemplated.
  • microcapsules or microspheres inclusion complexes, or other types of carriers.
  • Formulations suitable for use with a nebulizer will typically comprise the inventive compound dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution.
  • the formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure).
  • the nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.
  • Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive compound suspended in a propellant with the aid of a surfactant.
  • the propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof.
  • Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.
  • Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and may also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
  • a bulking agent such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
  • Nasal delivery of the inventive compound is also contemplated.
  • Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung.
  • Formulations for nasal delivery include those with dextran or cyclodextran. Delivery via transport across other mucous membranes is also contemplated.
  • the pharmaceutical formulations may be a spray or aerosol containing an appropriate solvent and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bioavailability modifiers and combinations of these.
  • a propellant for an aerosol formulation may include compressed air, nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.
  • the compound or compounds of the instant invention are conveniently delivered in the form of an aerosol spray presentation from a nebulizer or the like.
  • Injectable dosage forms for parenteral administration generally include aqueous suspensions or oil suspensions, which may be prepared using a suitable dispersant or wetting agent and a suspending agent. Injectable forms may be in solution phase or a powder suitable for reconstitution as a solution. Both are prepared with a solvent or diluent. Acceptable solvents or vehicles include sterilized water, Ringer's solution, or an isotonic aqueous saline solution. Alternatively, sterile oils may be employed as solvents or suspending agents. Typically, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.
  • the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these.
  • the compounds may be formulated for parenteral administration by injection such as by bolus injection or continuous infusion.
  • a unit dosage form for injection may be in ampoules or in multi-dose containers.
  • the pharmaceutical formulations may be in the form of a suppository, an ointment, an enema, a tablet or a cream for release of compound in the intestines, sigmoid flexure and/or rectum.
  • Rectal suppositories are prepared by mixing one or more compounds of the instant invention, or pharmaceutically acceptable salts or tautomers of the compound, with acceptable vehicles, for example, cocoa butter or polyethylene glycol, which is solid phase at room temperature but liquid phase at those temperatures suitable to release a drug inside the body, such as in the rectum.
  • acceptable vehicles for example, cocoa butter or polyethylene glycol, which is solid phase at room temperature but liquid phase at those temperatures suitable to release a drug inside the body, such as in the rectum.
  • acceptable vehicles for example, cocoa butter or polyethylene glycol, which is solid phase at room temperature but liquid phase at those temperatures suitable to release a drug inside the body, such as in the rectum.
  • Various other agents and additives may be used in
  • the formulations of the invention may be designed to be short-acting, fast-releasing, long-acting, and sustained-releasing as described below.
  • the pharmaceutical formulations may also be formulated for controlled release or for slow release.
  • the instant compositions may also comprise, for example, micelles or liposomes, or some other encapsulated form, or may be administered in an extended release form to provide a prolonged storage and/or delivery effect. Therefore, the pharmaceutical formulations may be compressed into pellets or cylinders and implanted intramuscularly or subcutaneously as depot injections or as implants such as stents. Such implants may employ known inert materials such as silicones and biodegradable polymers.
  • Specific dosages may be adjusted depending on conditions of disease, the age, body weight, general health conditions, sex, and diet of the subject, dose intervals, administration routes, excretion rate, and combinations of drugs. Any of the above dosage forms containing effective amounts are well within the bounds of routine experimentation and therefore, well within the scope of the instant invention.
  • a therapeutically effective dose may vary depending upon the route of administration and dosage form.
  • the compound or compounds of the instant invention are selected to provide a formulation that exhibits a high therapeutic index.
  • the therapeutic index is the dose ratio between toxic and therapeutic effects which can be expressed as the ratio between LD 50 and ED 50 .
  • the LD 50 is the dose lethal to 50% of the population and the ED 50 is the dose therapeutically effective in 50% of the population.
  • the LD 50 and ED 50 are determined by standard pharmaceutical procedures in animal cell cultures or experimental animals.
  • the dosage regimen for treating LCAT-mediated diseases and other diseases listed above with the compounds of this invention and/or compositions of this invention is based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. Dosage levels of the order from about 0.01 mg to 30 mg per kilogram of body weight per day, for example from about 0.1 mg to 10 mg/kg, or from about 0.25 mg to 1 mg/kg are useful for all methods of use disclosed herein. Generally, the daily regimen should be in the range of 0.1-1000 micrograms of the compound per kilogram of body weight, preferably 0.1-150 micrograms per kilogram.
  • the pharmaceutical composition may be in the form of, for example, a capsule, a tablet, a suspension, or liquid.
  • the pharmaceutical composition can be made in the form of a dosage unit containing a given amount of the active ingredient.
  • these may contain an amount of active ingredient from about 1 to 2000 mg, for example from about 1 to 500 mg, or from about 5 to 150 mg, or from 10 to 100 mg.
  • a suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but, once again, can be determined using routine methods.
  • the active ingredient may also be administered by injection as a composition with suitable carriers including saline, dextrose, or water.
  • suitable carriers including saline, dextrose, or water.
  • the daily parenteral dosage regimen will be from about 0.1 to about 30 mg/kg of total body weight, such as from about 0.1 to about 10 mg/kg, or from about 0.25 mg to 1 mg/kg.
  • Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose.
  • liquid or semi-liquid preparations suitable for penetration through the skin e.g., liniments, lotions, ointments, creams, or pastes
  • drops suitable for administration to the eye, ear, or nose e.g., liniments, lotions, ointments, creams, or pastes
  • a suitable topical dose of active ingredient of a compound of the invention is 0.1 mg to 150 mg administered one to four, for example one or two times daily.
  • the active ingredient may comprise from 0.001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but typically not more than 5% w/w.
  • the concentration is from 0.1% to 1% of the formulation.
  • compositions can be systemic or local, and may comprise a single site injection of a therapeutically-effective amount of the modified LCAT polypeptide composition.
  • Any route known to those of skill in the art for the administration of a therapeutic composition of the invention is contemplated including, for example, intravenous, intramuscular, subcutaneous or a catheter for long-term administration.
  • the therapeutic composition may be delivered to the patient at multiple sites.
  • the multiple administrations may be rendered simultaneously or may be administered over a period of several hours.
  • the modified LCAT polypeptide is provided locally to the site of reperfusion.
  • the method of treatment of the invention is therapeutic, and compounds and compositions of the invention are administered to a subject already suffering from atherosclerosis, cardiovascular disease or an associated disease.
  • methods of treatment are prophylactic and compounds and compositions are administered to those subjects at risk for developing atherosclerosis.
  • an atherogenic lipoprotein profile can be assessed. For example, a ratio of serum cholesterol to HDLs of 5:1 or above indicates a higher than average risk of developing atherosclerosis.
  • a serum cholesterol level of 240 mg/dL or above an HDL level 35 mg/dL or below, or an LDL level 190 mg/dL or above, a plasma LCAT protein level lower than normal ( ⁇ 5 ug/ml), and a decreased plasma cholesterol esterification rate ( ⁇ 60 nmol/ml/hr).
  • the amount of the modified LCAT effective to decrease accumulation of cholesterol depends on several factors, including the species, the manner of administration, the general health of the subject, the desired result (e.g., prophylaxis or therapeutic treatment) and the judgment of the prescribing physician. For example, the practitioner may decide what risk levels for heart disease indicate prophylactic treatment, and what target level of the modified LCAT is indicated for the treatment of a person already suffering from atherosclerosis.
  • the normal cholesterol esterification rate ranges from about 60 nmol/ml/hr to about 130 nmol/mL per hour.
  • the effective treatment of atherosclerosis in humans can involve administration of the compositions of the invention to achieve a cholesterol esterification rate of about 200 nmol/ml/hr.
  • cardiovascular diseases refers to diseases of the heart and circulatory system.
  • Cardiovascular diseases which the compositions of the present invention are useful for preventing or treating include but are not limited to arteriosclerosis; atherosclerosis; stroke; ischemia; endothelium dysfunctions, in particular those dysfunctions affecting blood vessel elasticity; peripheral vascular disease; coronary heart disease; myocardial infarction, cerebral infarction and restenosis, thrombosis, high blood pressure and angina.
  • Other diseases which the compositions of the present invention are useful for preventing or treating include LCAT deficiency syndrome, Alzheimer's disease, corneal opacity, metabolic syndrome, dyslipidemia, myocardial infarction, stroke, critical limb ischemia.
  • inflammatory cell activation means the induction by a stimulus (including, but not limited to, cytokines, antigens or auto-antibodies) of a proliferative cellular response, the production of soluble mediators (including but not limited to cytokines, oxygen radicals, enzymes, prostanoids, or vasoactive amines), or cell surface expression of new or increased numbers of mediators (including, but not limited to, major histocompatability antigens or cell adhesion molecules) in inflammatory cells (including but not limited to monocytes, macrophages, T lymphocytes, B Lymphocytes, granulocytes, polymorphonuclear leukocytes, mast cells, basophils, eosinophils, dendritic cells, and endothelial cells). It will be appreciated by persons skilled in the art that the activation of one or a combination of these phenotypes in these cytokines, antigens or auto-antibodies) of a proliferative cellular response, the production
  • Methods, compounds and compositions of the invention are useful in treating such diseases as arthritic diseases (such as rheumatoid arthritis), osteoarthritis, gouty arthritis, spondylitis, thyroid-associated opthalmopathy, Behcet disease, sepsis, septic shock, endotoxic shock, gram negative sepsis, gram positive sepsis, toxic shock syndrome, asthma, chronic bronchitis, allergic rhinitis, allergic conjunctivitis, vernal conjunctivitis, eosinophilic granuloma, adult (acute) respiratory distress syndrome (ARDS), chronic pulmonary inflammatory disease (such as chronic obstructive pulmonary disease), silicosis, pulmonary sarcoidosis, reperfusion injury of the myocardium, brain or extremities, brain or spinal cord injury due to minor trauma, fibrosis including cystic fibrosis, keloid formation, scar tissue formation, atherosclerosis, autoimmune diseases, such as systemic
  • GvH host
  • CLL chronic lymphocytic leukemia
  • inflammatory bowel diseases such as Crohn's disease and ulcerative colitis
  • proliferative lymphocytic diseases such as leukemias (e.g. chronic lymphocytic leukemia; CLL) (see Munoz et al., J. Exp. Med. 172:95-103 (1990); Mentz et al., Blood 88:2172-2182 (1996)
  • inflammatory dermatoses such as atopic dermatitis, psoriasis, or urticaria.
  • the compounds, compositions and methods of the present invention are used in the treatment of a variety of disorders in which there is a need to prevent or treat thrombosis and subsequent decrease or loss of blood flow.
  • thromobotic disorders include but not limited to atherosclerosis, myocardial infarction, stroke, and kidney ischemia, and thrombosis in any part of the mammalian body.
  • the composition of the present invention will also be used in the prevention and treatment of microangiopathy in which formation of microthrombi or von Willebrand factor (VWF) binding to platelets causes excessive consumption of platelets and/or VWF leading to subsequent bleeding diathesis.
  • VWF von Willebrand factor
  • Examples of latter disorders include but not limited to thrombotic thrombocytopenic purpura, type II and platelet type von Willebrand disease (VWD).
  • the compounds or combination therapeutic methods of the present invention inhibit VWF-dependent platelet adhesion and aggregation.
  • the compounds, compositions and methods are also useful in prolonging bleed time in a mammal and as such, are useful as anti-thrombotic agents both in therapeutic and prophylactic methods. As such, these compounds, compositions and methods are useful as anticoagulant agents and/or anti-platelet agents.
  • the present invention provides compounds, compositions and methods for the treatment of thrombosis and other disorders of the cardiovascular circulatory system that require and increase in the flow or reducing blockage of the vessels.
  • Compounds, compositions and methods are also useful for the treatment of any disorder that is presently treated using anticoagulant therapy.
  • disorders include pulmonary embolism, unstable angina, myocardial infarction, deep vein thrombosis, atrial fibrillation with embolization, acute and chronic coagulopathies (disseminated intravascular coagulation), for prevention of clotting in arterial and cardiac surgery, for prophylaxis and treatment of peripheral arterial embolism.
  • the compounds, compositions and methods are also used to treat thrombotic thrombocytopic purpura, other types of microangiopathy that are mediated by spontaneous interaction between VWF and platelets, platelet type or type IIb von Willebrand diseases in which there is an increased binding of VWF to platelets (either caused by a defect in GPIb or in VWF).
  • the compounds, compositions and methods described herein are useful as anti-platelet agents in blood transfusions, extracorporeal circulation, dialysis procedures as well as blood sampling for laboratory procedures.
  • the compounds, compositions and methods are also used to maintain the patency of an indwelling venipucture device that is being used for intermittent injection or infusion therapy or blood sampling.
  • the compounds, compositions and methods are particularly useful in surgical procedures to prevent the formation of blood clots. Such indications are particularly desirable for patients undergoing abdominal surgery to reduce the risk of thromboemolic complications, patients undergoing knee or hip replacement therapy during and following the replacement procedure, as well as a general prophylactic to prevent clot formation at a later stage.
  • the compounds, compositions and methods are further useful in the treatment of subjects that are under risk of thromboembolic complications due to severely restricted mobility e.g., during acute illness. Any such disorders may be readily treated by the compositions described herein.
  • the therapeutic methods include both medical therapeutic and/or prophylactic administration, as appropriate.
  • the term “inhibits platelet aggregation” includes its generally accepted meaning which includes prohibiting, slowing, or reducing the severity or degree of platelet aggregation. Such an inhibition may be measured as a function of time taken for a given sample to coagulate. In other embodiments, animal models of thrombosis. Methods of determining the efficacy of the agents include coagulation testing, monitoring the time of bleeding, determining hemoglobin levels of an animal and the like.
  • the invention further provides combination therapy, wherein the compounds and/or compositions of the invention are administered with one or more additional agent(s)
  • additional agent(s) In general, the therapeutic methods, compositions and compounds may also be employed in combination with other therapeutics in the treatment of various disease states, with the additional agents being administered concurrently or sequentially with a composition of the invention.
  • Exemplary cytokines or hematopoietic factors for such co-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1), M-CSF, SCF, GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha (IFN-alpha), consensus interferon, IFN-beta, IFN-gamma, IFN-omega, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-31, IL-32 alpha, IL-33, thrombopoietin (TPO), angiopoietins, for example Ang-1, Ang-2
  • Additional active agents may act in complementary or synergistic ways with the modified LCAT when used to treat, and prevent atherosclerosis or manage cholesterol, or related disorders such as cardiovascular disease.
  • compounds of the invention can be used with statins.
  • Statins are drugs that competitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase “HMG-CoA reductase,” which is the enzyme that catalyzes an early, rate-limiting step in cholesterol biosynthesis. Hebert et al., JAMA 1997, 278: 313-21. This combination, in addition to raising HDL levels and lowering LDL levels may also lowers triglyceride and reduce inflammation.
  • the combination can have additional therapeutic effects, for example, the combination may lower blood pressure; protect against heart disease, for example, by reducing smooth muscle proliferation, reduce heart attacks, reduce platelet aggregation, and to reduce strokes as well as peripheral arterial disease (clogging of the arteries to the legs).
  • statins of the invention include, but are not limited to, mevastatin, pitavastatin, rosuvastatin, pentostatin (Nipent®), nystatin, lovastatin (Mevacor®), simvastatin (Zocor®), pravastatin (Pravachol®), fluvastatin (Lescol®), atorvastatin (Lipitor®), cerivastatin (Baycol®), or combinations thereof.
  • Statins suitable for use in the compositions and methods of the invention are also disclosed in U.S. Pat. Nos. 4,681,893; 5,273,995; 5,356,896; 5,354,772; 5,686,104; 5,969,156; and 6,126,971.
  • statins may exist in an inactive form, such as a lactone (e.g., simvastatin), the invention encompasses using the active form (e.g., b-hydroxy acid form) of them. See Physicians Desk Reference, 54.sup.th Ed. (2000) pp. 1917-1920.
  • active form e.g., b-hydroxy acid form
  • Fibrates or fibric acid derivatives are regarded as broad-spectrum lipid-modulating agents in that although their main action is to decrease serum triglycerides they also tend to reduce LDL-cholesterol and to raise HDL-cholesterol. It is believed that the combined use of compounds of the invention and a fibrate may reduce the risk of coronary heart disease events in those with low HDL-cholesterol or with raised triglycerides by speeding up the chemical breakdown (i.e., catabolism) of triglyceride-rich lipoproteins that circulate in the body.
  • chemical breakdown i.e., catabolism
  • Fibrates include, but are not limited to, bezafibrate, ciprofibrate, fenofibrate, gemfibrozil, clofibrate, or combinations thereof. Fibrates suitable for inclusion in the compositions or administration in the methods of the invention are disclosed in U.S. Pat. Nos. 4,895,762; 6,074,670; and 6,277,405.
  • Biguanides for use in the compositions and methods of the invention include, but are not limited to, metformin, phenformin, buformin, or combinations thereof. Biguanides suitable for use in the compositions or methods of the invention are also disclosed in U.S. Pat. No. 6,303,146.
  • the combined use of compounds of the invention and a bigaunide may improve glycemic control by enhancing insulin sensitivity in the liver and in muscle.
  • the combination may reduce or avoid cardiovascular risk factors such as dyslipidemia, elevated plasminogen activator inhibitor 1 levels, other fibrinolytic abnormalities, hyperinsulinemia, insulin resistance, and is an effective and safe therapeutic agent for the treatment of type 2 diabetes.
  • compounds of the invention may be used in combination with glitazones, which may increase glucose uptake in muscle and reduced endogenous glucose production.
  • Glitazones include 5-((4-(2-(methyl-2-pyri-dinyl amino)ethoxy)-phenyl)methyl)-2,4-thiazolidinedione, troglitazone, pioglitazone, ciglitazone, WAY-120,744, englitazone, AD 5075, darglitazone, rosiglitazone, combinations thereof, or a pharmaceutically acceptable salt, solvate, clathrate, polymorph, prodrug, or pharmacologically active metabolite thereof.
  • Glitazones suitable for use in the compositions or methods of the invention are disclosed in U.S. Pat. Nos. 4,687,777; 5,002,953; 5,741,803; 5,965,584; 6,150,383; 6,150,384; 6,166,042; 6,166,043; 6,172,090; 6,211,205; 6,271,243; 6,288,095; 6,303,640; and 6,329,404.
  • compositions comprising compounds of the invention and a sulfonylurea or a derivative thereof may increase insulin release from the pancreas and may further insulin levels by reducing hepatic clearance of the hormone.
  • Sulfonylurea-based drugs for use the compositions and methods of the invention include, but are not limited to, glisoxepid, glyburide, acetohexamide, chlorpropamide, glibomuride, tolbutamide, tolazamide, glipizide, gliclazide, gliquidone, glyhexamide, phenbutamide, tolcyclamide, combinations thereof, or a pharmaceutically acceptable salt, solvate, or clathrate.
  • Combination compositions may also include agents that inhibit CETP.
  • agents that inhibit CETP are, for example, Torcetrapib, and S-(2-[([1-(2-ethylbutyl)cyclohexyl]carbonyl)amino]phenyl)-2-methylpropanethioate.
  • Cardiovascular drugs for use in combination with the compounds of the invention to prevent or treat cardiovascular diseases include peripheral antiadrenergic drugs, centrally acting antihypertensive drugs (e.g., methyldopa, methyldopa HCl), antihypertensive direct vasodilators (e.g., diazoxide, hydralazine HCl), drugs affecting renin-angiotensin system, peripheral vasodilators, phentolamine, antianginal drugs, cardiac glycosides, inodilators (e.g., aminone, milrinone, enoximone, fenoximone, imazodan, sulmazole), antidysrhythmic drugs, calcium entry blockers, ranitine, bosentan, and rezulin.
  • peripheral antiadrenergic drugs e.g., centrally acting antihypertensive drugs (e.g., methyldopa, methyldopa HCl
  • compositions of the invention are used in combination therapy with other therapeutics that achieve a specific biological effect.
  • Various medications can lower blood cholesterol levels. They may be prescribed individually or in combination with other drugs. Some of the common types of cholesterol-lowering drugs include statins, resins and nicotinic acid (niacin), gemfibrozil and clofibrate.
  • combination therapy is contemplated utilizing, for example, clofibrate (Atromid-S, which raises the HDL cholesterol levels and lowers triglyceride levels), gemfibrozil (Lopid, which raises HDL cholesterol levels), nicotinic acid (which works in the liver by affecting the production of blood fats and is used to lower triglycerides and LDL cholesterol, and raise HDL (“good”) cholesterol), resins (which are also called bile acid-binding drugs and work in the intestines by promoting increased disposal of cholesterol), including cholestyramine (Questran, Prevalite, Lo-Cholest), colestipol (Colestid) and colesevelam (WelChol), and statins including atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor), pravastatin (Pravachol), rosuvastatin calcium (Crestor), and simvastatin (Zocor).
  • the drugs of first choice for elevated LDL cholesterol are the HMG CoA reductase inhibitors, e.g., atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin.
  • Statin drugs are effective for lowering LDL cholesterol levels, have few immediate short-term side effects, are easy to administer, have high patient acceptance and have few drug-drug interactions.
  • bile acid sequestrants colesevelam, cholestyramine and colestipol—and nicotinic acid (niacin), which have been shown to reduce the risk for coronary heart disease in controlled clinical trials.
  • bile acid sequestrants colesevelam, cholestyramine and colestipol
  • nicotinic acid niacin
  • Both classes of drugs appear to be free of serious side effects. But both can have troublesome side effects and require considerable patient education to achieve adherence. Nicotinic acid is preferred in patients with triglyceride levels that exceed 250 mg/dL because bile acid sequestrants tend to raise triglyceride levels.
  • Angiotensin II causes blood vessels to contract and thereby narrows the blood vessels. The narrowing of the vessels increases the pressure within the vessels and can cause high blood pressure (hypertension).
  • Angiotensin II is formed from angiotensin I in the blood by the enzyme, angiotensin converting enzyme (ACE). ACE inhibitors decrease the production of angiotensin II. As a result, the blood vessels enlarge or dilate, and the blood pressure is reduced.
  • ACE inhibitors that available in the United States include captopril (Capoten), benazepril (Lotensin), enalapril (Vasotec), lisinopril (Prinivil, Zestril) fosinopril (Monopril), ramipril (Altace), perindopril (Aceon), quinapril (Accupril), moexipril (Univasc), and trandolapril (Mavik).
  • combination therapy is contemplated with, for example, acetylsalicylic acid (Aspirin, Ecotrin), choline magnesium salicylate (Trilisate), diclofenac (Voltaren, Cataflam, Voltaren-XR), diflunisal (Dolobid), etodolac (Lodine), fenoprofen (Nalfon), flurbiprofen (Ansaid), ibuprofen (Advil, Motrin, Medipren, Nuprin), indomethacin (Indocin, Indocin-SR), ketoprofen (Orudis, Oruvail), meclofenamate (Meclomen), nabumetone (Relafen), naproxen (Naprosyn, Naprelan, Anaprox, Aleve), oxaprozin (Daypro), phenylbutazone (Butazolidine), piroxicam (Feldene), salsalate (Dispirin, Ecotrin
  • Prescription-only NSAIDs include ibuprofen (B Caudium), aceclofenac (Preservex), acemetacin (Emflex), azapropazone (Rheumox), celecoxib (Celebrex), dexketoprofen (Keral), diclofenac (Voltarol, Diclomax, Arthrotec), diflusinal (Dolobid), etodolac (Lodine), fenbufen (Lederfen), fenoprofen (Fenopron), flurbiprofen (Froben), indometacin, ketoprofen (Orudis, Oruvail), mefenamic acid, meloxicam (Mobic), nabumetone (Relifex), naproxen (Naprosyn, Synflex), phenylbutazone (Butacote), piroxicam (Feldene), sulindac (Clinoril), tenoxicam (
  • anti-thrombosis drugs such as anticoagulant drugs, which inhibit the ability of blood to clot, or coagulate and include dalteparin (Fragmin), danaparoid (Orgaran), enoxaparin (Lovenox), heparin (various), tinzaparin (Innohep), warfarin (Coumadin), and lepirudin (Refludan), and antiplatelet drugs such as aspirin, ticlopidine (Ticlid), clopidogrel (Plavix), tirofiban (Aggrastat) and eptifibatide (Integrilin).
  • anticoagulant drugs such as anticoagulant drugs, which inhibit the ability of blood to clot, or coagulate and include dalteparin (Fragmin), danaparoid (Orgaran), enoxaparin (Lovenox), heparin (various), tinzaparin (Innohep), warfarin (Coumadin), and lepirudin (
  • Still other methods include the use of bivalirudin (selective and reversible thrombin inhibitor), argatroban (reversible inhibitor of thrombin), and low molecular weight heparins (LMWHs), including enoxaparin (Lovenox), dalteparin (Fragmin), ardeparin (Normiflo) fondaparinux and idraparinux.
  • bivalirudin selective and reversible thrombin inhibitor
  • argatroban reversible inhibitor of thrombin
  • LMWHs low molecular weight heparins
  • Still other anti-thrombosis drugs contemplated for use in methods of the invention include fragmin (dalteparin sodium injection) lovenox (enoxaparin sodium), Normiflo (ardeparin sodium), Orgaran (danaparoid sodium), indirect (Antithrombin-Dependent) FXa inhibitors such as fondaparinux (Arixtra®) and idraparinux, direct (Antithrombin-Independent) FXa inhibitors such as BAY 59-7939 [Bayer], DPC-423 [Bristol-Myers Squibb], DX-9065a [Daiichi], LY517717, razaxaban (DPC906), lepirudin (Refludan®), desirudin (Revasc®), bivalirudin (Hirulog®, Angiomax®), argatroban (Novastan®), melagatran, and ximelagatran (Exanta®).
  • the disorder that may be treated by the compositions of the present invention are limited only by the fact that the disorder needs a therapeutic intervention which inhibits platelet aggregation.
  • the doses of the agent may be modified for each individual subject.
  • routes of administration and uses those of skill in the art are referred to the Physician's Desk Reference for generalized descriptions of formulations, routes of administration and patient monitoring used for agents such as AggrastatTM (see e.g., entry at pages 1933-1937, PDR, 57th Edn., 2003), AggrenoxTM (see e.g., entry at pages 1023-1026, PDR, 57th Edn., 2003), AgrylinTM (see e.g., entry at pages 3142-3143, PDR, 57th Edn., 2003), FlolanTM (see e.g., entry at pages 1516-1521, PDR, 57th Edn., 2003), IntegrilinTM (see e.g., entry at pages 2138-2142, PDR, 57
  • the title compound was prepared according to the procedure described in Example 1 by using 5-(ethylthio)-1,3,4-thiadiazole-2-thiol (592 mg, 3.33 mmol), NaH (60% dispersion in mineral oil, 146 mg, 3.66 mmol), and 3-chloropyrazine-2-carbonitrile (422 mg, 3.02 mmol) in DMF and benzene (8 ml, 1/1) by stirring at 90° C. under nitrogen atmosphere overnight.
  • the title compound was prepared according to the procedure described in Example 1 by using 5-(allylthio)-1,3,4-thiadiazole-2-thiol (460 mg, 2.42 mmol), NaH (60% dispersion in mineral oil, 107 mg, 2.66 mmol), and 3-chloropyrazine-2-carbonitrile (338 mg, 2.42 mmol) in DMF and benzene (8 ml, 1/1) by stirring at 85° C. under nitrogen atmosphere overnight.
  • the title compound was prepared according to the procedure described in Example 4 by using 1,3,4-thiadiazole-2,5-dithiol (300 mg, 2.00 mmol), NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol), 1-iodobutane (0.263 ml, 2.30 mmol), NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol), and 3-chloropyrazine-2-carbonitrile (280 mg, 2.00 mmol) in DMF and benzene (8 ml, 1/1) by stirring at room temperature under nitrogen atmosphere overnight.
  • the title compound was prepared according to the procedure described in Example 4 by using 1,3,4-thiadiazole-2,5-dithiol (300 mg, 2.00 mmol), NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol), 1-bromo-2-methylpropane (0.25 ml, 2.30 mmol), NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol), and 3-chloropyrazine-2-carbonitrile (280 mg, 2.00 mmol) in DMF and benzene (8 ml, 1/1) by stirring at room temperature under nitrogen atmosphere overnight.
  • the title compound was prepared according to Example 4 by using 1,3,4-thiadiazole-2,5-dithiol (300 mg, 2.00 mmol), NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol), 1-iodopentane (0.30 ml, 2.30 mmol), NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol), and 3-chloropyrazine-2-carbonitrile (280 mg, 2.00 mmol) in DMF and benzene (8 ml, 1/1) by stirring at room temperature under nitrogen atmosphere overnight.
  • the title compound was prepared according to Example 1 by using 5-(dodecylthio)-1,3,4-thiadiazole-2-thiol (319 mg, 1.00 mmol), NaH (60% dispersion in mineral oil, 44 mg, 1.10 mmol), and 3-chloropyrazine-2-carbonitrile (140 mg, 1.00 mmol) in DMF and benzene (6 ml, 1/1) by stirring at room temperature under nitrogen atmosphere overnight.
  • the title compound was prepared according to the procedure described in Example 1 by using 1,3,4-thiadiazole-2,5-dithiol (945 mg, 6.05 mmol), NaH (60% dispersion in mineral oil, 264 mg, 6.60 mmol), and 3-chloropyrazine-2-carbonitrile (840 mg, 6.00 mmol) in DMF and benzene (10 ml, 1/1) by stirring at 50° C. under nitrogen atmosphere for 4 hr.
  • the title compound was prepared according to Example 1 by using 5-(isopropylthio)-4-methyl-4H-1,2,4-triazole-3-thiol (379 mg, 2.00 mmol), NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol), and 3-chloropyrazine-2-carbonitrile (280 mg, 2.00 mmol) in DMF and benzene (6 ml, 1/1) by stirring at room temperature under nitrogen atmosphere for 2 hr.
  • the title compound was prepared according to Example 1 by using 5-(methylthio)-1,2,4-thiadiazole-3-thiol (328 mg, 2.00 mmol), NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol), and 3-chloropyrazine-2-carbonitrile (280 mg, 2.00 mmol) in DMF and benzene (6 ml, 1/1) by stirring at room temperature under nitrogen atmosphere for 2 hr.
  • the title compound was prepared according to Example 1 by using 5-methyl-1,3,4-thiadiazole-2-thiol (264 mg, 2.00 mmol), NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol), and 3-chloropyrazine-2-carbonitrile (280 mg, 2.00 mmol) in DMF and benzene (6 ml, 1/1) by stirring at room temperature under nitrogen atmosphere for 6 hr.
  • the title compound was prepared according to Example 1 by using 5-butyl-1,3,4-thiadiazole-2-thiol (260 mg, 1.49 mmol), NaH (60% dispersion in mineral oil, 66 mg, 1.65 mmol), and 3-chloropyrazine-2-carbonitrile (210 mg, 1.50 mmol) in DMF and benzene (4 ml, 1/1) by stirring at room temperature under nitrogen atmosphere for 3 hr.
  • the title compound was prepared according to Example 1 by using 4-methyl-4H-1,2,4-triazole-3-thiol (230.5 mg, 2.00 mmol), NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol), and 3-chloropyrazine-2-carbonitrile (280 mg, 2.00 mmol) in DMF and benzene (6 ml, 1/1) by stirring at room temperature under nitrogen atmosphere for 4 hr.
  • the title compound was prepared according to Example 1 by using 1-methyl-1H-imidazole-2-thiol (228 mg, 2.00 mmol), NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol), and 3-chloropyrazine-2-carbonitrile (280 mg, 2.00 mmol) in DMF and benzene (6 ml, 1/1) by stirring at room temperature under nitrogen atmosphere for 4 hr.
  • the title compound was prepared according the procedure described in Example 1 by using 5-(methylthio)-1,3,4-thiadiazole-2-thiol (1.64 g, 10.00 mmol), NaH (60% dispersion in mineral oil, 445 mg, 11.00 mmol), and 2,3-dichloropyrazine (1.04 ml, 10.00 mmol) in DMF and benzene (12 ml, 1/1) by stirring at 50° C. for 20 hr and then at 110° C. for 4 hr under nitrogen atmosphere.
  • LCAT was isolated from culture media of CHO cells that were stably transfected with human LCAT cDNA. LCAT coding sequence was tagged with FLAG and HIS tages at the C-terminus. Purification of tagged recombinant human LCAT protein was performed according to the standard protocol using Ni-NTA agarose beads. Briefly, CHO cultured media were incubated with Ni-NTA column at pH 8.0. The unbound proteins were washed from resin complex with 20 mM imidazole. The His-tagged protein was eluted with 250 mM imidazole and dialysed against 1 ⁇ PBS containing 50 ⁇ M EDTA.
  • Activity of the modified LCAT of the invention and activities of compounds of the invention were determined by measuring change of the rate of conversion of 3 H-labeled cholesterol to cholesteryl ester (CE).
  • CE cholesteryl ester
  • human plasma samples were equilibrated with a trace amount of radiolabeled cholesterol at 4° C. and the rate of cholesterol esterification was measured by TLC analysis after incubation at 37° C. (Dobiasova, supra).
  • EC 50 represents a compound concentration achieving 50% of maximal activation of LCAT-mediated cholesterol esterification.
  • full-length human LCAT cDNA was isolated from normal human liver cDNA library (BioChain, Hayward, Calif.) with standard protocol and cloned into a pCMV-Flag vector (See Example 18). Recombinant LCAT was expressed in CHO cells and the enzyme secreted from stably transfected cells was harvested in serum-free culture medium. Recombinant LCAT identity was confirmed with anti-human LCAT and anti-Flag antibodies. The activity of recombinant LCAT enzyme was determined using apoAI-liposome substrates prepared by the standard cholate-dialysis procedure (Chen et al. (1982) J. Lipid Res. 23: 680-691.
  • the initial mixture contained egg PC/ 3 H-unesterfied cholesterol/human apoAI (molar ratio of 250:12.5:0.8). After dialysis the proteoliposomes were incorporated with recombinant LCAT protein. LCAT activity was determined by measuring the conversion of radiolabeled cholesterol to cholesteryl ester and expressed in nmol CE/mL per hour.
  • LCAT enzyme stability was measured using the standard ELISA protocol. Briefly, LCAT protein molecules of the plasma samples were captured onto the ELISA plate with an anti-LCAT antibody which was pre-coated to the plate. After a careful wash to remove the unbound molecules, the LCAT protein was detected by using a second anti-LCAT antibody. The LCAT-antibody immunocomplexes were detected and quantified by using HRP detection system. Purified recombinant LCAT protein were used as standard and measured under the same experiment conditions (Kobori et al. (2002) J Lipid Res 43: 325-334).
  • Rodents used for assessing the in vivo effects of the compounds of the invention include BALB/c mice, CD 1 mice, and Syrian hamsters of wild type fed with normal chew. These animals were treated with either vehicle or Compound A (3-(5-ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile) by IP injection. At time points (see Figures) after compound administration, blood samples were collected, plasma was immediately separated, and plasma levels of lipids, lipoprotein and LCAT activity were determined.
  • Results presented in FIG. 2 (A) confirm activity and specificity of the compounds of the invention on LCAT enzyme.
  • This experiment studied the effect of Compound A in LCAT-deficient plasma samples obtained from lcat mutant mice. Ng et al.(1997) J. Biol. Chem. 272:1 5777-81. No LCAT activity was detected in LCAT-deficient (lcat ⁇ / ⁇ ) plasma samples in the presence or absence of Compound A, indicating that the observed interaction of Compound A with LCAT is highly specific.
  • FIG. 2 (B) Results summarized in FIG. 2 (B) illustrate the mechanism of action of compounds of the invention on LCAT enzyme.
  • Two loss-of-function LCAT mutants, H377A and S181A were generated, in which the critical catalytic triad of the enzyme was destroyed as described in Francone et al. (1991) Biochemistry 30: 10074-77; Peelman et al. (1998) Protein Sci. 7: 587-599; and Peelman et al. (2000) Curr. Opin. Lipidol. 11: 155-160. No activity was observed on either nonfunctional enzyme with or without Compound A ( FIG. 2B ).
  • FIG. 3 summarizes data showing that compounds of the invention increase LCAT enzyme activity in a dose dependent manner in BALB/c mice.
  • BALB/c mice male, 7 weeks old
  • IP intra-peritoneal
  • Animals were fed normal chow diet.
  • Compounds were solubilized in DEPG vehicle (containing 20% dimethyl acetamide, 10% ethanol, 50% polylene glycol) and administered to the animals.
  • blood samples were taken from animals and plasma was separated immediately. An aliquot of plasma sample was labeled with 3 H-cholesterol for the LCAT activity assay. Each data point represents the mean of samples from two individual animals. The remaining samples were used for plasma lipid and lipoprotein analyses.
  • Results presented in FIG. 4 demonstrate that treatment with the compounds of the invention increases HDL cholesterol levels in CD1 mice.
  • Plasma samples were collected and HDL cholesterol concentrations were determined standard reagents and assay protocol using clinical analyzer (Infinity).
  • FIG. 5 illustrates the time course of LCAT activation and the levels of HDL in mice following a single doze of Compound A.
  • Male three month old CD1 mice were given Compound A (20 mg/kg) by an IP injection.
  • Each data point represents the mean of measurement from four individual animals per treatment group.
  • Results presented in FIG. 6 demonstrate that treatment with the compound of the invention increases HDL levels and decreases apoB-containing lipoprotein in vivo.
  • Plasma samples were collected and concentrations of total cholesterol (TC), panel B, and HDL cholesterol (panel A) were measured.
  • LpB cholesterol contents were obtained by subtracting HDL from TC (panel C).
  • FIG. 7 provides elution profiles indicating that treatment with the compounds of the invention increases HDL-Ch levels, increases HDL particle size, and decrease TG levels in the VLDL fraction in vivo.
  • TABLE 1 HDL particle size profiles HDL subclass Control T865 2a 12 39 2b 58 56 3a 28 3 3b 1 1 3c 1 1
  • Table 1 summarizes HDL particle size profiles as determined by Gradient Gel Electrophoresis (GGE) indicating an increase of HDL particle size in vivo after treating with the compounds of the invention.
  • GGE Gradient Gel Electrophoresis

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Cardiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Neurology (AREA)
  • Obesity (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Neurosurgery (AREA)
  • Vascular Medicine (AREA)
  • Psychiatry (AREA)
  • Ophthalmology & Optometry (AREA)
  • Hospice & Palliative Care (AREA)
  • Urology & Nephrology (AREA)
US11/823,251 2006-06-26 2007-06-26 Methods for treating atherosclerosis Abandoned US20080096900A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US11/823,251 US20080096900A1 (en) 2006-06-26 2007-06-26 Methods for treating atherosclerosis
US13/087,261 US8426358B2 (en) 2006-06-26 2011-04-14 Methods for treating atherosclerosis
US13/851,019 US20130273024A1 (en) 2006-06-26 2013-03-26 Methods for treating atherosclerosis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US81641506P 2006-06-26 2006-06-26
US11/823,251 US20080096900A1 (en) 2006-06-26 2007-06-26 Methods for treating atherosclerosis

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/087,261 Division US8426358B2 (en) 2006-06-26 2011-04-14 Methods for treating atherosclerosis

Publications (1)

Publication Number Publication Date
US20080096900A1 true US20080096900A1 (en) 2008-04-24

Family

ID=38786909

Family Applications (3)

Application Number Title Priority Date Filing Date
US11/823,251 Abandoned US20080096900A1 (en) 2006-06-26 2007-06-26 Methods for treating atherosclerosis
US13/087,261 Active US8426358B2 (en) 2006-06-26 2011-04-14 Methods for treating atherosclerosis
US13/851,019 Abandoned US20130273024A1 (en) 2006-06-26 2013-03-26 Methods for treating atherosclerosis

Family Applications After (2)

Application Number Title Priority Date Filing Date
US13/087,261 Active US8426358B2 (en) 2006-06-26 2011-04-14 Methods for treating atherosclerosis
US13/851,019 Abandoned US20130273024A1 (en) 2006-06-26 2013-03-26 Methods for treating atherosclerosis

Country Status (7)

Country Link
US (3) US20080096900A1 (fr)
EP (2) EP2037928B1 (fr)
AT (1) ATE540681T1 (fr)
AU (1) AU2007265475B2 (fr)
CA (2) CA2787343C (fr)
ES (1) ES2376396T3 (fr)
WO (1) WO2008002591A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010029552A1 (fr) * 2008-09-10 2010-03-18 Transpharma Medical Ltd. Libération transdermique d’oligosaccharides
WO2011081667A1 (fr) * 2009-12-31 2011-07-07 Immunitor USA, Inc. Composition pour le traitement ou la prévention de l'athérosclérose, de l'obésité et de troubles liés à l'obésité
WO2011140429A1 (fr) 2010-05-06 2011-11-10 Alphacore Pharma Llc Administration d'ester de cholestéryle à des tissus stéroïdogènes
US20120107298A1 (en) * 2009-06-12 2012-05-03 Auerbach Bruce J Use of LCAT for Treating Anemia and Red Blood Cell Dysfunction
WO2018019911A1 (fr) 2016-07-27 2018-02-01 Hartis-Pharma Sarl Combinaisons thérapeutiques pour traiter les troubles des globules rouges

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5968547A (en) 1997-02-24 1999-10-19 Euro-Celtique, S.A. Method of providing sustained analgesia with buprenorphine
KR20100040937A (ko) 2007-07-26 2010-04-21 암젠 인크 변형된 레시틴-콜레스테롤 아실트랜스퍼라제 효소
AP2013006678A0 (en) * 2010-08-16 2013-01-31 Boehringer Ingelheim Int Oxadiazole inhibitors of leukotriene production
WO2012027322A1 (fr) 2010-08-26 2012-03-01 Boehringer Ingelheim International Gmbh Composés oxadiazole, inhibiteurs de la production de leucotriènes
AU2011305667A1 (en) 2010-09-23 2013-03-21 Boehringer Ingelheim International Gmbh Oxadiazole inhibitors of leukotriene production
EP2619196B1 (fr) 2010-09-23 2015-09-16 Boehringer Ingelheim International GmbH Inhibiteurs oxadiazole de production de leucotriènes
WO2013086443A1 (fr) 2011-12-08 2013-06-13 Amgen Inc. Protéines agonistes de liaison à l'antigène lcat humain et leur utilisation thérapeutique
AR089853A1 (es) 2012-02-01 2014-09-24 Boehringer Ingelheim Int Inhibidores de oxadiazol de la produccion de leucotrienos para terapia de combinacion, composicion farmaceutica, uso
EP2862861B1 (fr) 2012-06-14 2016-01-06 Daiichi Sankyo Company, Limited Dérivé de pipéridinylpyrazolopyridine
WO2015050148A1 (fr) * 2013-10-04 2015-04-09 第一三共株式会社 Dérivé de 3-arylpyrazolopyridine
WO2015087996A1 (fr) * 2013-12-13 2015-06-18 第一三共株式会社 Dérivé d'imidazopyridine
CA2932707C (fr) * 2013-12-13 2018-09-04 Daiichi Sankyo Company, Limited Derive de 5-hydroxy-4-(trifluoromethyl)pyrazolopyridine
WO2015111545A1 (fr) * 2014-01-21 2015-07-30 第一三共株式会社 Dérivé condensé de pyrazole
TWI713534B (zh) 2015-06-11 2020-12-21 日商第一三共股份有限公司 5-羥基-4-(三氟甲基)吡唑并吡啶衍生物之結晶及其用途
US20200338118A1 (en) * 2017-11-15 2020-10-29 Juan Carlos Parodi System for optimal hydration
JPWO2019189046A1 (ja) * 2018-03-30 2021-04-08 第一三共株式会社 Ｌｃａｔ欠損症治療薬

Family Cites Families (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3691016A (en) 1970-04-17 1972-09-12 Monsanto Co Process for the preparation of insoluble enzymes
CA1023287A (fr) 1972-12-08 1977-12-27 Boehringer Mannheim G.M.B.H. Procede de fixation d'une proteine sur un substrat
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4195128A (en) 1976-05-03 1980-03-25 Bayer Aktiengesellschaft Polymeric carrier bound ligands
US4330440A (en) 1977-02-08 1982-05-18 Development Finance Corporation Of New Zealand Activated matrix and method of activation
CA1093991A (fr) 1977-02-17 1981-01-20 Hideo Hirohara Traduction non-disponible
US4229537A (en) 1978-02-09 1980-10-21 New York University Preparation of trichloro-s-triazine activated supports for coupling ligands
US4289872A (en) 1979-04-06 1981-09-15 Allied Corporation Macromolecular highly branched homogeneous compound based on lysine units
JPS6023084B2 (ja) 1979-07-11 1985-06-05 味の素株式会社 代用血液
PT72878B (en) 1980-04-24 1983-03-29 Merck & Co Inc Process for preparing mannich-base hydroxamic acid pro-drugs for the improved delivery of non-steroidal anti-inflammatory agents
US4640835A (en) 1981-10-30 1987-02-03 Nippon Chemiphar Company, Ltd. Plasminogen activator derivatives
US5354772A (en) 1982-11-22 1994-10-11 Sandoz Pharm. Corp. Indole analogs of mevalonolactone and derivatives thereof
US4496689A (en) 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
AR240698A1 (es) 1985-01-19 1990-09-28 Takeda Chemical Industries Ltd Procedimiento para preparar compuestos de 5-(4-(2-(5-etil-2-piridil)-etoxi)benzil)-2,4-tiazolidindiona y sus sales
DE3675588D1 (de) 1985-06-19 1990-12-20 Ajinomoto Kk Haemoglobin, das an ein poly(alkenylenoxid) gebunden ist.
JPS63167409A (ja) 1986-12-27 1988-07-11 Yamaha Corp 磁気記録体
US4681893A (en) 1986-05-30 1987-07-21 Warner-Lambert Company Trans-6-[2-(3- or 4-carboxamido-substituted pyrrol-1-yl)alkyl]-4-hydroxypyran-2-one inhibitors of cholesterol synthesis
US4791192A (en) 1986-06-26 1988-12-13 Takeda Chemical Industries, Ltd. Chemically modified protein with polyethyleneglycol
EP0318512B1 (fr) 1986-08-18 1998-06-17 Emisphere Technologies, Inc. Systemes de distribution pour agents pharmacologiques
US5229490A (en) 1987-05-06 1993-07-20 The Rockefeller University Multiple antigen peptide system
ES2137915T3 (es) 1987-09-04 2000-01-01 Beecham Group Plc Derivados de tiazolidindiona sustituida.
US6288095B1 (en) 1987-09-04 2001-09-11 Beecham Group P.L.C. Compounds
EP0315456B1 (fr) 1987-11-05 1994-06-01 Hybritech Incorporated Immunoglobulines modifiées au moyen d'un polysaccharide présentant un potentiel immunogène réduit ou des cinétiques pharmacologiques améliorées
FI94339C (fi) 1989-07-21 1995-08-25 Warner Lambert Co Menetelmä farmaseuttisesti käyttökelpoisen /R-(R*,R*)/-2-(4-fluorifenyyli)- , -dihydroksi-5-(1-metyylietyyli)-3-fenyyli-4-/(fenyyliamino)karbonyyli/-1H-pyrroli-1-heptaanihapon ja sen farmaseuttisesti hyväksyttävien suolojen valmistamiseksi
US5013556A (en) 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
AU643141B2 (en) 1991-03-15 1993-11-04 Amgen, Inc. Pulmonary administration of granulocyte colony stimulating factor
HU9203780D0 (en) 1991-12-12 1993-03-29 Sandoz Ag Stabilized pharmaceutical products of hmg-coa reductase inhibitor and method for producing them
ATE156158T1 (de) 1992-04-14 1997-08-15 Cornell Res Foundation Inc Makromoleküle auf basis von dendritischen polymeren und verfahren zur herstellung
US5792451A (en) 1994-03-02 1998-08-11 Emisphere Technologies, Inc. Oral drug delivery compositions and methods
US5741803A (en) 1992-09-05 1998-04-21 Smithkline Beecham Plc Substituted thiazolidinedionle derivatives
EP0680320B1 (fr) 1993-01-19 1999-04-14 Warner-Lambert Company Formulation ci-981, orale, stable et son procede de preparation
US6309853B1 (en) 1994-08-17 2001-10-30 The Rockfeller University Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof
US5824784A (en) 1994-10-12 1998-10-20 Amgen Inc. N-terminally chemically modified protein compositions and methods
FR2731710B1 (fr) 1995-03-14 1997-04-30 Rhone Poulenc Rorer Sa Virus recombinants exprimant la lecithine cholesterol acyltransferase et utilisations en therapie genique
US5739277A (en) 1995-04-14 1998-04-14 Genentech Inc. Altered polypeptides with increased half-life
US6096871A (en) 1995-04-14 2000-08-01 Genentech, Inc. Polypeptides altered to contain an epitope from the Fc region of an IgG molecule for increased half-life
TWI238064B (en) 1995-06-20 2005-08-21 Takeda Chemical Industries Ltd A pharmaceutical composition for prophylaxis and treatment of diabetes
EA000474B1 (ru) 1995-07-17 1999-08-26 Варнер-Ламберт Компани Кристаллическая кислая кальциевая соль [r-(r*, r*)]-2-(4-фторфенил)-бета, дельта-дигидрокси-5-(1-метилэтил)-3-фенил-4-[(фениламино)карбонил]-1н-пиррол-1-гептановой кислоты (аторвастатин)
CA2236982C (fr) * 1995-11-09 2010-07-27 The Government Of The United States Of America Represented By The Secretary Of The Department Of Health And Human Services Utilisation de la lecithine-cholesterol acyltransferase (lcat) dans le traitement de l'atherosclerose
DE69731289D1 (de) 1996-03-18 2004-11-25 Univ Texas Immunglobulinähnliche domäne mit erhöhten halbwertszeiten
FR2758459B1 (fr) 1997-01-17 1999-05-07 Pharma Pass Composition pharmaceutique de fenofibrate presentant une biodisponibilite elevee et son procede de preparation
DE69836626T2 (de) * 1997-04-11 2007-04-05 Takeda Pharmaceutical Co. Ltd. Proteine mit lecithin-cholesterin acetyltransferase-ähnlicher aktivität, deren herstellung und verwendung
US6692909B1 (en) * 1998-04-01 2004-02-17 Whitehead Institute For Biomedical Research Coding sequence polymorphisms in vascular pathology genes
EP0974356B1 (fr) 1998-07-15 2003-09-24 Merck Sante Comprimés contenant une combinaison de glibenclamide et de metformine
US6660843B1 (en) 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
EP1263458B1 (fr) 2000-03-08 2005-11-16 Novo Nordisk A/S Reduction du cholesterol seriques
US6596544B1 (en) * 2000-03-31 2003-07-22 The Regents Of The University Of California Functional assay of high-density lipoprotein
CA2486127C (fr) * 2002-05-17 2014-03-11 Esperion Therapeutics, Inc. Methode de traitement de troubles dyslipidemiques
WO2004004774A2 (fr) * 2002-07-03 2004-01-15 Esperion Therapeutics, Inc. Compositions pharmaceutiques et methodes de traitement, de prevention et de prise en charge des troubles lies au cholesterol, des dyslipidemies et des troubles apparentes
EP1539210A4 (fr) 2002-09-06 2006-06-07 Bayer Pharmaceuticals Corp Agonistes modifies du recepteur glp-1 et leurs procedes pharmacologiques d'utilisation
KR20050114283A (ko) 2003-04-22 2005-12-05 아바니르 파마슈티컬스 고콜레스테롤혈증을 치료하기 위한 역 콜레스테롤 수송의매개제
WO2004103310A2 (fr) * 2003-05-20 2004-12-02 New England Medical Center Hospitals, Inc. Procedes et compositions destinees au traitement de troubles metaboliques

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010029552A1 (fr) * 2008-09-10 2010-03-18 Transpharma Medical Ltd. Libération transdermique d’oligosaccharides
US20110150976A1 (en) * 2008-09-10 2011-06-23 Transpharma Medical Ltd. Transdermal delivery of oligosaccharides
US20120107298A1 (en) * 2009-06-12 2012-05-03 Auerbach Bruce J Use of LCAT for Treating Anemia and Red Blood Cell Dysfunction
US8492108B2 (en) * 2009-06-12 2013-07-23 Bruce J. Auerbach Methods of treating anemia and red blood cell dysfunction with lecithin cholesterol acyltransferase
WO2011081667A1 (fr) * 2009-12-31 2011-07-07 Immunitor USA, Inc. Composition pour le traitement ou la prévention de l'athérosclérose, de l'obésité et de troubles liés à l'obésité
WO2011140429A1 (fr) 2010-05-06 2011-11-10 Alphacore Pharma Llc Administration d'ester de cholestéryle à des tissus stéroïdogènes
WO2018019911A1 (fr) 2016-07-27 2018-02-01 Hartis-Pharma Sarl Combinaisons thérapeutiques pour traiter les troubles des globules rouges

Also Published As

Publication number Publication date
US20110206652A1 (en) 2011-08-25
CA2654661A1 (fr) 2008-01-03
WO2008002591A3 (fr) 2008-05-08
ES2376396T3 (es) 2012-03-13
EP2037928A2 (fr) 2009-03-25
AU2007265475B2 (en) 2010-08-19
EP2452683A2 (fr) 2012-05-16
US20130273024A1 (en) 2013-10-17
EP2037928B1 (fr) 2012-01-11
CA2787343C (fr) 2016-08-02
WO2008002591A2 (fr) 2008-01-03
CA2654661C (fr) 2012-10-23
US8426358B2 (en) 2013-04-23
ATE540681T1 (de) 2012-01-15
EP2452683A3 (fr) 2012-08-22
CA2787343A1 (fr) 2008-01-03
AU2007265475A1 (en) 2008-01-03

Similar Documents

Publication Publication Date Title
US8426358B2 (en) Methods for treating atherosclerosis
US8703926B1 (en) Modified lecithin-cholesterol acyltransferase enzymes
CN1177934C (zh) 腺病毒介导的肿瘤内投送血管生成拮抗剂用于肿瘤的治疗
AU2006214190A1 (en) Phosphoinositide 3-kinase inhibitors for inhibiting leukocyte accumulation
JP2010536759A (ja) 原核生物フェニルアラニンアンモニアリアーゼの組成物、及び、その組成物を用いる癌の治療方法
AU2014201310A1 (en) Modified Lecithin-Cholesterol Acyltransferase Enzymes
US20060153825A1 (en) Use of protein histidine phosphatase

Legal Events

Date Code Title Description
AS Assignment

Owner name: AMGEN INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KAYSER, FRANK;LABELLE, MARC;SHAN, BEI;AND OTHERS;REEL/FRAME:020226/0272;SIGNING DATES FROM 20071105 TO 20071106

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION