US20060189655A1 - Methylphenidate derivatives and uses of them - Google Patents

Methylphenidate derivatives and uses of them Download PDF

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US20060189655A1
US20060189655A1 US11/336,029 US33602906A US2006189655A1 US 20060189655 A1 US20060189655 A1 US 20060189655A1 US 33602906 A US33602906 A US 33602906A US 2006189655 A1 US2006189655 A1 US 2006189655A1
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David Bar-Or
Nagaraja Rao
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Ampio Pharmaceuticals Inc
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Assigned to INSTITUTE FOR MOLECULAR MEDICINE, INC. reassignment INSTITUTE FOR MOLECULAR MEDICINE, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: RAO, NAGARAJA K.R., BAR-OR, DAVID
Publication of US20060189655A1 publication Critical patent/US20060189655A1/en
Priority to US12/260,964 priority patent/US8076485B2/en
Priority to US13/288,179 priority patent/US9463187B2/en
Assigned to AMPIO PHARMACEUTICALS, INC. reassignment AMPIO PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: INSTITUTE FOR MOLECULAR MEDICINE, INC.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D295/145—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P17/00—Drugs for dermatological disorders
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • 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
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • 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

Definitions

  • the invention relates to uses of methylphenidate derivatives.
  • the uses include inhibiting angiogenesis and treating angiogenic diseases and conditions.
  • Methylphenidate is the treatment of choice for children and adults diagnosed with attention deficit/hyperactivity disorder (ADHD), including its inattentive subtype (formerly known as attention deficit disorder or ADD). Certain derivatives of methylphenidate have also been proposed for the treatment of ADD (see U.S. Pat. No. 6,025,502) and for the treatment of other neurological disorders and conditions (see U.S. Pat. Nos. 5,859,249, 6,025,502 and 6,486,177 and PCT application WO 99/36403).
  • ADHD attention deficit/hyperactivity disorder
  • ADD attention deficit/hyperactivity disorder
  • Certain derivatives of methylphenidate have also been proposed for the treatment of ADD (see U.S. Pat. No. 6,025,502) and for the treatment of other neurological disorders and conditions (see U.S. Pat. Nos. 5,859,249, 6,025,502 and 6,486,177 and PCT application WO 99/36403).
  • Methylphenidate is a mild central nervous stimulant and is also taught for treating apathy, fatigue, cognitive decline, and depression in cancer patients, AIDS patients and other seriously ill patients. See U.S. Pat. Nos. 5,908,850, 6,127,385, 6,395,752 and 6,486,177, Challman and Lipsky, Mayo Clin. Proc., 75:711-721 (2000) and Leonard et al., Hum. Psychopharmacol. Clin. Exp., 19:151-180 (2004).
  • the invention provides methods of using a compound of formula I wherein n is an integer from 1 to 5, and each R 1 is independently aryl, heteroaryl, alkyl, cycloalkyl, alkoxy, aryloxy, acyl, carboxyl, hydroxyl, halogen, amino, nitro, sulfo or sulfhydryl. Each alkyl can optionally be substituted with hydroxyl, amino or sulfhydryl.
  • R 2 is hydrogen or lower alkyl.
  • the invention provides a method of inhibiting angiogenesis in an animal.
  • the method comprises administering an effective amount of a compound of formula I, or a pharmaceutically-acceptable salt or a prodrug thereof, to the animal.
  • the invention provides a method of treating an angiogenic disease or condition in an animal.
  • the method comprises administering a therapeutically effective amount of a compound of formula I, or a pharmaceutically-acceptable salt or a prodrug thereof, to the animal.
  • the invention provides a method of treating a proliferative disorder in an animal.
  • the method comprises administering a therapeutically effective amount of a compound of formula I, or a pharmaceutically-acceptable salt or a prodrug thereof, to the animal.
  • the invention also provides a compound of formula IA: where
  • n is an integer from 1 to 5;
  • each R 1 is independently a moiety of the formula —C(O)—R 8 , —OR 7 or —C(O)—O—R 3 ;
  • R 2 is hydrogen or lower alkyl
  • R 3 is hydrogen, alkyl, cycloalkyl or aryl
  • R 7 is aryl
  • R 8 is cycloalkyl or aryl.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and a compound of formula IA: or a salt or prodrug thereof, where
  • n is an integer from 1 to 5;
  • each R 1 is independently a moiety of the formula —C(O)—R 8 , —OR 7 or —C(O)—O—R 3 ;
  • R 2 is hydrogen or lower alkyl
  • R 3 is hydrogen, alkyl, cycloalkyl or aryl
  • R 7 is aryl
  • R 8 is cycloalkyl or aryl.
  • FIGS. 1 A-C are graphs of OD at 530 nm for various additives to peripheral blood lymphocyte (PBL) cultures stimulated with 2 ⁇ g/ml, 5 ⁇ g/ml and 20 ⁇ g/ml phytohemagglutinin (PHA), respectively.
  • PBL peripheral blood lymphocyte
  • FIG. 2 is a graph of OD at 530 nm for various additives to PBL cultures stimulated with 2 ⁇ g/ml PHA.
  • FIG. 3 is a graph of concentration of IL-13 for various additives to PBL cultures stimulated with 2 ⁇ g/ml PHA.
  • FIG. 4 is a graph of concentration of IFN ⁇ for various additives to PBL cultures stimulated with 2 ⁇ g/ml PHA.
  • FIGS. 5 A-B are graphs of OD at 530 nm for various additives to PBL cultures stimulated with 2 ⁇ g/ml and 5 ⁇ g/ml PHA, respectively.
  • FIG. 6 is a graph of concentration of IL-13 for various additives to PBL cultures stimulated with 5 ⁇ g/ml PHA.
  • FIG. 7 is a graph of concentration of TNF ⁇ for various additives to PBL cultures stimulated with 2 ⁇ g/ml PHA.
  • FIG. 8 is a graph of concentration of IL-8 for various additives to PBL cultures stimulated with 2 ⁇ g/ml PHA.
  • FIGS. 9 A-B are graphs of OD for various additives to THP-1 monocyte cultures stimulated with lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • n is an integer from 1 to 5. Preferably n is 1 or 2.
  • Each R 1 which may be the same or different, is aryl, heteroaryl, alkyl, cycloalkyl, alkoxy, aryloxy, acyl, carboxyl, hydroxyl, halogen, amino, nitro, sulfo or sulfhydryl.
  • Each alkyl can optionally be substituted with hydroxyl, amino or sulfhydryl.
  • R 1 is preferably aryl, alkyl, cycloalkyl, alkoxy, aryloxy or acyl. More preferably R 1 is aryl, alkyl or cycloalkyl, even more preferably aryl, most preferably phenyl.
  • R 2 is hydrogen or lower alkyl.
  • R 2 is —CH 3 .
  • the compound of formula II is particularly useful in the present invention:
  • “Acyl” means a moiety of the formula —C(O)—R 3 , wherein R 3 is H, alkyl, cycloalkyl or aryl.
  • Amino means a moiety of the formula —NR 4 R 5 , wherein each of R 4 and R 5 is independently H or lower alkyl, preferably lower alkyl.
  • Alkoxy means a moiety of the formula —OR 6 , wherein R 6 is alkyl.
  • An example of an alkoxy group is methoxy (—O—CH 3 ).
  • Alkyl means a monovalent saturated straight-chain or branched hydrocarbon containing 1-8 carbon atoms. Each alkyl may, optionally, be substituted with one or more amino, hydroxyl or sulfhydryl groups.
  • Aryl means a monovalent mono-, bi- or tricyclic aromatic hydrocarbon moiety of 6 to 14 ring carbon atoms. Preferred is phenyl.
  • Aryloxy means a moiety of the formula —OR 7 , wherein R 7 is aryl.
  • An example of an alkoxy group is phenoxy.
  • Carboxyl means a moiety of the formula —C(O)—OR 3 , wherein R 3 is H, alkyl, cycloalkyl or aryl.
  • Cycloalkyl means a saturated, monovalent mono- or bicyclic hydrocarbon moiety of three to ten ring carbon atoms. Preferably the cycloalkyl contains 4-8 ring carbon atoms. The most preferred cycloalkyl is cyclohexyl.
  • Halogen means chlorine, fluorine, bromine or iodine. Preferred is chlorine or bromine.
  • Heteroaryl means a monovalent monocyclic or bicyclic aromatic moiety of 5 to 12 ring atoms containing one, two, or three ring heteroatoms each of which is independently selected from N, O, and S, the remaining ring atoms being C.
  • Haldroxyl means —OH.
  • “Lower alkyl” means a saturated straight-chain or branched hydrocarbon containing 1-4 carbon atoms.
  • Niro means —NO 2 .
  • “Sulfhydryl” means —SH.
  • Prodrug means any compound which releases an active parent drug according to formula I in vivo when such prodrug is administered to a mammalian subject.
  • Prodrugs of a compound of formula I are prepared by modifying one or more functional group(s) present in the compound of formula I in such a way that the modification(s) may be cleaved in vivo to release the parent compound.
  • Prodrugs include compounds of formula I wherein a hydroxy, amino, or sulfhydryl group in a compound of formula I is bonded to any group that may be cleaved in vivo to generate the free hydroxyl, amino, or sulfhydryl group, respectively.
  • prodrugs include, but are not limited to, esters (e.g., acetate, formate, and benzoate derivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) of hydroxy functional groups in compounds of formula I, and the like.
  • esters e.g., acetate, formate, and benzoate derivatives
  • carbamates e.g., N,N-dimethylaminocarbonyl
  • Treating” or “treatment” of a disease or condition includes: (1) preventing the disease or condition, i.e., causing the clinical symptoms of the disease or condition not to develop in a mammal that may be exposed to or predisposed to the disease or condition, but does not yet experience or display symptoms of the disease or condition; (2) inhibiting the disease or condition, i.e., arresting or reducing the development of the disease or condition or its clinical symptoms; or (3) relieving the disease or condition, i.e., causing regression of the disease or condition or its clinical symptoms, including curing the disease or condition.
  • an “effective amount” means the amount of a compound that, when administered to an animal for treating a disease or condition or for causing an effect is sufficient to do so.
  • the “effective amount” can and will most likely vary depending on the compound, the disease or condition and its severity, or the effect sought to be caused, and the age, weight, etc., of the animal to be treated.
  • the compound of the present invention contains one or more chiral centers
  • the compound can be synthesized enantioselectively or a mixture of enantiomers and/or diastereomers can be prepared and separated.
  • the resolution of the compounds of the present invention, their starting materials and/or the intermediates may be carried out by known procedures, e.g., as described in the four volume compendium Optical Resolution Procedures for Chemical Compounds : Optical Resolution Information Center, Manhattan College, Riverdale, N.Y., and in Enantiomers, Racemates and Resolutions , Jean Jacques, Andre Collet and Samuel H. Wilen; John Wiley & Sons, Inc., New York, 1981, which are incorporated herein in their entirety.
  • the resolution of the compounds is based on the differences in the physical properties of diastereomers by attachment, either chemically or enzymatically, of an enantiomerically pure moiety, resulting in forms that are separable by fractional crystallization, distillation or chromatography.
  • the pharmaceutically-acceptable salts of the compounds of formula I may also be used in the practice of the invention.
  • Pharmaceutically-acceptable salts include conventional non-toxic salts, such as salts derived from inorganic acids (such as hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, and the like), organic acids (such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, glutamic, aspartic, benzoic, salicylic, oxalic, ascorbic acid, and the like) or bases (such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation or organic cations derived from N,N-dibenzylethylenediamine, D-glucosamine, or ethylenediamine).
  • the salts are prepared in a conventional manner, e.g., by reacting the free base form of the compound with an acid.
  • Angiogenesis is the process of new blood vessel formation in the body.
  • Angiogenesis is also used herein to mean the same as, or to include, neovascularization, vascularization, arterialization and vasculogenesis.
  • angiogenic diseases and conditions can also be used to treat angiogenic diseases and conditions.
  • An angiogenic disease or condition is a disease or condition involving, caused by, exacerbated by, or dependent on, angiogenesis.
  • Specific angiogenic diseases and conditions treatable according to the invention include neoplastic diseases, hypertrophy (e.g., cardiac hypertrophy induced by thyroid hormone), connective tissue disorders (e.g., rheumatoid arthritis and atherosclerosis), psoriasis, ocular angiogenic diseases, cardiovascular diseases, cerebral vascular diseases, endometriosis, polyposis, obesity, diabetes-associated diseases and hemophiliac joints.
  • the compounds of formula I, pharmaceutically-acceptable salts thereof or prodrugs thereof can also be used to inhibit the vascularization required for embryo implantation, thereby providing a method of birth control.
  • the compounds of formula I, pharmaceutically-acceptable salts thereof or prodrugs thereof will be particularly useful for the treatment of ocular angiogenic diseases.
  • Ocular angiogenic diseases include diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasias, and rubeosis.
  • the compounds of formula I, pharmaceutically-acceptable salts thereof or prodrugs thereof, will be especially useful for the treatment of diabetic retinopathy and macular degeneration.
  • Neoplastic diseases treatable with the compounds of formula I, pharmaceutically-acceptable salts thereof or prodrugs thereof include malignant tumors (e.g., tumors of the bladder, brain, breast, cervix, colon, rectum, kidney, liver, lung, ovary, pancreas, prostate, stomach and uterus), tumor metastasis, and benign tumors (e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas and pyrogenic granulomas)).
  • malignant tumors e.g., tumors of the bladder, brain, breast, cervix, colon, rectum, kidney, liver, lung, ovary, pancreas, prostate, stomach and uterus
  • benign tumors e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas and pyrogenic granulomas
  • the compounds of formula I, pharmaceutically-acceptable salts thereof or prodrugs thereof, will be especially useful for the treatment of tumors of the brain, breast, colon, liver and pancreas, most especially tumors of the brain (e.g., glioblastomas).
  • the compounds of formula I, pharmaceutically-acceptable salts thereof or prodrugs thereof have been found to be able to inhibit the proliferation of cells, reduce the growth of cancer cells, inhibit the production of cytokines, inhibit Ras and RAP-1, and inhibit the production of NF ⁇ B and AP-1.
  • the compounds of formula I, pharmaceutically-acceptable salts thereof or prodrugs thereof will also be particularly useful for the treatment of a variety of proliferative disorders, including angiogenic diseases and conditions, especially neoplastic diseases (see above), and other cancers and other proliferative disorders.
  • Cancers treatable with the compounds of formula I, pharmaceutically-acceptable salts thereof or prodrugs thereof include carcinomas, sarcomas, lymphomas, leukemias, solid tumors and hematologic malignancies.
  • Specific cancers treatable with the compounds of formula I, pharmaceutically-acceptable salts thereof or prodrugs thereof include brain cancers, head and neck cancers, breast cancers, ovarian cancers, prostate cancers, gastric cancers, colon cancers, pancreatic cancers, bladder cancers, thyroid cancers, hepatic cancers, lung cancers, bone cancers and skin cancers.
  • the compounds of formula I, pharmaceutically-acceptable salts thereof or prodrugs thereof will be especially useful for the treatment of brain cancers, breast cancers, colon cancers, liver cancers, pancreatic cancers, skin cancers, lymphomas and leukemias.
  • proliferative disorders include mesangial cell proliferation disorders, fibrotic disorders and hyperproliferative skin disorders.
  • Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells.
  • Mesangial cell proliferative disorders include renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes and glomerulopathies.
  • Fibrotic disorders refer to the abnormal formation of extracellular matrices. Examples of fibrotic disorders include hepatic cirrhosis, pulmonary fibrosis and atherosclerosis.
  • Hyperproliferative skin disorders include psoriasis, skin cancer and epidermal hyperproliferation.
  • a compound of formula I, pharmaceutically-acceptable salt thereof or prodrug thereof is administered to the animal.
  • the animal is a mammal, such as a rabbit, goat, dog, cat, horse or human. Most preferably, the animal is a human.
  • Effective dosage forms, modes of administration and dosage amounts for the compounds of the invention may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the particular compound employed, the disease or condition to be treated, the severity of the disease or condition, the route(s) of administration, the rate of excretion of the compound, the duration of the treatment, the identify of any other active ingredient(s)s being administered to the animal, the age, size and species of the animal, and like factors known in the medical and veterinary arts. In general, a suitable daily dose of a compound of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect.
  • the daily dosage will be determined by an attending physician or veterinarian within the scope of sound medical judgment. If desired, the effective daily dose may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day. Administration of the compound should be continued until an acceptable response is achieved.
  • the compounds useful in the present invention may be administered to an animal patient for therapy by any suitable route of administration, including orally, nasally, rectally, vaginally, parenterally (e.g., intravenously, intraspinally, intraperitoneally, subcutaneously, or intramuscularly), intracisternally, transdermally, intracranially, intracerebrally, and topically (including buccally and sublingually).
  • suitable routes of administration including orally and topically.
  • compositions useful in the invention comprise one or more compounds of formula I, or pharmaceutically-acceptable salts or prodrugs thereof, as active ingredient(s) in admixture with one or more pharmaceutically-acceptable carriers and, optionally, with one or more other compounds, active ingredient(s) or other materials.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the animal.
  • Pharmaceutically-acceptable carriers are well known in the art. Regardless of the route of administration selected, the compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington's Pharmaceutical Sciences.
  • Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules or as a solution or a suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and the like, each containing a predetermined amount of a compound or compounds useful in the present invention as an active ingredient.
  • a compound or compounds useful in the present invention may also be administered as bolus, electuary or paste.
  • the active ingredient(s) is (are) mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter.
  • compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • opacifying agents include polymeric substances and waxes.
  • the active ingredient can also be in microencapsulated form.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compound(s), may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • compositions for intraocular injection of a compound or compounds of the invention into the eyeball include solutions, emulsions, suspensions, particles, capsules, microspheres, liposomes, matrices, etc. See, e.g., U.S. Pat. No. 6,060,463, U.S. Patent Application Publication No. 2005/0101582, and PCT application WO 2004/043480, the complete disclosures of which are incorporated herein by reference.
  • a pharmaceutical formulation for intraocular injection may comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, suspensions or emulsions, which may contain antioxidants, buffers, suspending agents, thickening agents or viscosity-enhancing agents (such as a hyaluronic acid polymer).
  • suitable aqueous and nonaqueous carriers include water, saline (preferably 0.9%), dextrose in water (preferably 5%), buffers, dimethylsulfoxide, alcohols and polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like).
  • compositions may also contain adjuvants such as wetting agents and emulsifying agents and dispersing agents.
  • prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as polymers and gelatin.
  • injectable depot forms can be made by incorporating the drug into microcapsules or microspheres made of biodegradable polymers such as polylactide-polyglycolide. Examples of other biodegradable polymers include poly(orthoesters), poly(glycolic) acid, poly(lactic) acid, polycaprolactone and poly(anhydrides).
  • Depot injectable formulations are also prepared by entrapping the drug in liposomes (composed of the usual ingredients, such as dipalmitoyl phosphatidylcholine) or microemulsions which are compatible with eye tissue.
  • liposomes Composed of the usual ingredients, such as dipalmitoyl phosphatidylcholine
  • microemulsions which are compatible with eye tissue.
  • the rate of drug release from microcapsules, microspheres and liposomes can be controlled.
  • the compounds of the invention can also be administered surgically as an ocular implant.
  • a reservoir container having a diffusible wall of polyvinyl alcohol or polyvinyl acetate and containing a compound or compounds of the invention can be implanted in or on the sclera.
  • a compound or compounds of the invention can be incorporated into a polymeric matrix made of a polymer, such as polycaprolactone, poly(glycolic) acid, poly(lactic) acid, poly(anhydride), or a lipid, such as sebacic acid, and may be implanted on the sclera or in the eye. This is usually accomplished with the animal receiving a topical or local anesthetic and using a small incision made behind the cornea. The matrix is then inserted through the incision and sutured to the sclera.
  • a preferred embodiment of the invention is local topical administration of the compounds of the invention to the eye, and a particularly preferred embodiment of the invention is a topical pharmaceutical composition suitable for application to the eye.
  • Topical pharmaceutical compositions suitable for application to the eye include solutions, suspensions, dispersions, drops, gels, hydrogels and ointments. See, e.g., U.S. Pat. No. 5,407,926 and PCT applications WO 2004/058289, WO 01/30337 and WO 01/68053, the complete disclosures of all of which are incorporated herein by reference.
  • Topical formulations suitable for application to the eye for treatment of an angiogenic disease or condition comprise one or more compounds of the invention in an aqueous or nonaqueous base.
  • the topical formulations can also include absorption enhancers, permeation enhancers, thickening agents, viscosity enhancers, agents for adjusting and/or maintaining the pH, agents to adjust the osmotic pressure, preservatives, surfactants, buffers, salts (preferably sodium chloride), suspending agents, dispersing agents, solubilizing agents, stabilizers and/or tonicity agents.
  • Topical formulations suitable for application to the eye for treatment of an angiogenic disease or condition will preferably comprise an absorption or permeation enhancer to promote absorption or permeation of the compound or compounds of the invention into the eye and/or a thickening agent or viscosity enhancer that is capable of increasing the residence time of a compound or compounds of the invention in the eye.
  • an absorption or permeation enhancer to promote absorption or permeation of the compound or compounds of the invention into the eye and/or a thickening agent or viscosity enhancer that is capable of increasing the residence time of a compound or compounds of the invention in the eye.
  • exemplary absorption/permeation enhancers include methysulfonylmethane, alone or in combination with dimethylsulfoxide, carboxylic acids and surfactants.
  • thickening agents and viscosity enhancers include dextrans, polyethylene glycols, polyvinylpyrrolidone, polysaccharide gels, Gelrite®, cellulosic polymers (such as hydroxypropyl methylcellulose), carboxyl-containing polymers (such as polymers or copolymers of acrylic acid), polyvinyl alcohol and hyaluronic acid or a salt thereof.
  • Liquid dosage forms can be prepared, for example, by dissolving, dispersing, suspending, etc. a compound or compounds of the invention in a vehicle, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like, to form a solution, dispersion or suspension.
  • a vehicle such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like
  • the pharmaceutical formulation may also contain minor amounts of non-toxic auxillary substances, such as wetting or emulsifying agents, pH buffering agents and the like, for example sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
  • Aqueous solutions and suspensions can include, in addition to a compound or compounds of the invention, preservatives, surfactants, buffers, salts (preferably sodium chloride), tonicity agents and water. If suspensions are used, the particle sizes should be less than 10 ⁇ m to minimize eye irritation. If solutions or suspensions are used, the amount delivered to the eye should not exceed 50 ⁇ l to avoid excessive spillage from the eye.
  • Colloidal suspensions are generally formed from microparticles (i.e., microspheres, nanospheres, microcapsules or nanocapsules, where microspheres and nanospheres are generally monolithic particles of a polymer matrix in which the formulation is trapped, adsorbed, or otherwise contained, while with microcapsules and nanocapsules the formulation is actually encapsulated).
  • the upper limit for the size of these microparticles is about 5 ⁇ to about 10 ⁇ .
  • Ophthalmic ointments include a compound or compounds of the invention in an appropriate base, such as mineral oil, liquid lanolin, white petrolatum, a combination of two or all three of the foregoing, or polyethylene-mineral oil gel.
  • An appropriate base such as mineral oil, liquid lanolin, white petrolatum, a combination of two or all three of the foregoing, or polyethylene-mineral oil gel.
  • a preservative may optionally be included.
  • Ophthalmic gels include a compound or compounds of the invention suspended in a hydrophilic base, such as Carpobol-940 or a combination of ethanol, water and propylene glycol (e.g., in a ratio of 40:40:20).
  • a gelling agent such as hydroxylethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose or ammoniated glycyrrhizinate, is used.
  • a preservative and/or a tonicity agent may optionally be included.
  • Hydrogels are formed by incorporation of a swellable, gel-forming polymer, such as those listed above as thickening agents or viscosity enhancers, except that a formulation referred to in the art as a “hydrogel” typically has a higher viscosity than a formulation referred to as a “thickened” solution or suspension.
  • a formulation may also be prepared so to form a hydrogel in situ following application to the eye.
  • Such gels are liquid at room temperature but gel at higher temperatures (and thus are termed “thermoreversible” hydrogels), such as when placed in contact with body fluids.
  • Biocompatible polymers that impart this property include acrylic acid polymers and copolymers, N-isopropylacrylamide derivatives and ABA block copolymers of ethylene oxide and propylene oxide (conventionally referred to as “poloxamers” and available under the Pluronic® tradename from BASF-Wayndotte).
  • Preferred dispersions are liposomal, in which case the formulation is enclosed within liposomes (microscopic vesicles composed of alternating aqueous compartments and lipid bilayers).
  • Eye drops can be formulated with an aqueous or nonaqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Drops can be delivered by means of a simple eye dropper-capped bottle or by means of a plastic bottle adapted to deliver liquid contents dropwise by means of a specially shaped closure.
  • the compounds of the invention can also be applied topically by means of drug-impregnated solid carrier that is inserted into the eye.
  • Drug release is generally effected by dissolution or bioerosion of the polymer, osmosis, or combinations thereof.
  • matrix-type delivery systems can be used. Such systems include hydrophilic soft contact lenses impregnated or soaked with the desired compound of the invention, as well as biodegradable or soluble devices that need not be removed after placement in the eye.
  • These soluble ocular inserts can be composed of any degradable substance that can be tolerated by the eye and that is compatible with the compound of the invention that is to be administered.
  • Such substances include, but are not limited to, poly(vinyl alcohol), polymers and copolymers of polyacrylamide, ethylacrylate and vinylpyrrolidone, as well as cross-linked polypeptides or polysaccharides, such as chitin.
  • Dosage forms for the other types of topical administration (i.e., not to the eye) or for transdermal administration of compounds of the invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants.
  • the active ingredient may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to the active ingredient, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to the active ingredient, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances.
  • Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of compounds of the invention to the body.
  • dosage forms can be made by dissolving, dispersing or otherwise incorporating one or more compounds of the invention in a proper medium, such as an elastomeric matrix material.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.
  • Formulations of the pharmaceutical compositions for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • compositions include those suitable for administration by inhalation or insufflation or for nasal or intraocular administration.
  • the compounds of the invention are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the composition may take the form of a dry powder, for example, a powder mix of one or more compounds of the invention and a suitable powder base, such as lactose or starch.
  • a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator, insufflator or a metered-dose inhaler.
  • compounds useful in the invention may be administered by means of nose drops or a liquid spray, such as by means of a plastic bottle atomizer or metered-dose inhaler.
  • Liquid sprays are conveniently delivered from pressurized packs. Typical of atomizers are the Mistometer (Wintrop) and Medihaler (Riker).
  • Drops such as eye drops or nose drops, may be formulated with an aqueous or nonaqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Drops can be delivered by means of a simple eye dropper-capped bottle or by means of a plastic bottle adapted to deliver liquid contents dropwise by means of a specially shaped closure.
  • compositions suitable for parenteral administrations comprise one or more compounds useful in the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions.
  • isotonic agents such as sugars, sodium chloride, and the like in the compositions.
  • prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monosterate and gelatin.
  • the absorption of the active ingredient(s) in order to prolong the effect of the active ingredient(s), it is desirable to slow the absorption of the active ingredient(s) from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the active ingredient(s) then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered active ingredient(s) is accomplished by dissolving or suspending the active ingredient(s) in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the active ingredient(s) in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of active ingredient(s) to polymer, and the nature of the particular polymer employed, the rate of release of the active ingredient(s) can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the active ingredient(s) in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.
  • sterile liquid carrier for example water for injection
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
  • Whole blood was drawn from GR283, a human volunteer with known allergies, into a glass vacutainer tube containing no anticoagulant. This blood was allowed to clot, and the serum was removed by centrifugation and then heat inactivated by placing it in a water bath at 56° C. for 30 minutes.
  • Whole blood from GR283 was also drawn into a glass vacutainer tube containing heparin and used for peripheral blood lymphocytes (PBL) isolation as follows.
  • Whole blood was layered over room temperature Histopaque 1077 solution and centrifuged at 2000 rpm for 15 minutes at room temperature. Cells at the plasma-Histopaque interface were then removed and washed with culture medium (IMDM medium with 10% heat-inactivated GR283 serum plus 1% penicillin/streptomycin) at 37° C.
  • culture medium IMDM medium with 10% heat-inactivated GR283 serum plus 1% penicillin/streptomycin
  • the compound of formula II (see above) and methylphenidate (both obtained from Dr. Jeffrey D. Winkler, University of Pennsylvania, Philadelphia, Pa.) in culture medium were added to wells of a 96-well plate to give final concentrations of 5 ⁇ g/ml, 15 ⁇ g/ml and 50 ⁇ g/ml of the compound of formula II and of methylphenidate.
  • Sterile 18 M ⁇ water, the solvent for the compound of formula II, and dexamethasone (obtained from Sigma) final concentration of 10 ⁇ g/ml in water
  • GR283's PBL in culture medium were added to the wells to give a final concentration of 150,000 cells per well, and the plates were incubated at 37° C., 5% CO 2 for 24 hours. After this incubation, phytohemagglutinin (PHA) in culture medium was added to give final concentrations of 2 ⁇ g/ml, 5 ⁇ g/ml or 20 ⁇ g/ml, final total volume of 200 ⁇ l/well, and the cells were incubated for an additional 72 hours at 37° C., 5% CO 2 . All cultures were performed in triplicate.
  • PHA phytohemagglutinin
  • cell clumping was examined by photographing representative wells with a digital camera mounted to an inverted microscope.
  • the compound of formula II reduced the amount of cell clumping induced by 5 ⁇ g/ml PHA in a dose-dependent manner.
  • the compound of formula II attenuated cell clumping, presumably, as a result of decreased expression of cellular adhesion molecules on the surfaces of the cells.
  • FIGS. 1 A-C The results of the proliferation assays are presented in FIGS. 1 A-C. As can be seen from FIGS. 1 A-C, the compound of formula II (Cpd.
  • GR467's PBL in culture medium were added to the wells to give a final concentration of 150,000 cells per well, and the plates were incubated at 37° C., 5% CO 2 for 24 hours. After this incubation, PHA was added to give a final concentration of 2 ⁇ g/ml, final total volume of 200 ⁇ l/well, and the cells were incubated for an additional 72 hours at 37° C., 5% CO 2 . All cultures were performed in triplicate.
  • the release of cytokines by the PBL was also measured by culturing the PBL in 1 ml tubes, at 1.3 ⁇ 10 6 cells per ml, with 15 ⁇ g/ml of the compound of formula II, 15 ⁇ g/ml methylphenidate or 10 ⁇ M dexamethasone at 37° C., 5% CO 2 for 24 hours. After this incubation, PHA was added to give a final concentration of 2 ⁇ g/ml, and the cells were incubated for an additional 96 hours at 37° C., 5% CO 2 . All cultures were performed in triplicate. Cells were then removed by centrifugation at 1000 rpm for 10 minutes, and the culture medium collected.
  • IL-13 is made by activated T H 2 cells, and IL-13's primary targets are B-cells and monocytes. IL-13 stimulates humoral immune responses, and it has been implicated in the pathogenesis of asthma. IL-13 is secreted by lymphoma cell lines and may be an autocrine growth factor. IL-13 is also expressed in pancreatic cancer. However, IL-13 has also been reported to inhibit the growth of other types of tumors, such as gliomas and renal cell carcinomas
  • IFN ⁇ is a proinflammatory cytokine made by activated T-cells and other cells. IFN ⁇ can activate neutrophils, endothelial cells and macrophages, as well as cause an increase in MHC molecule expression. IFN ⁇ drives the cell-mediated immune response. IFN ⁇ plays an important role in the immune-mediated rejection of established tumors. IFN ⁇ has antiproliferative effects on some tumors, can have apoptotic effects on others, can induce the production of angiostatic chemokines and enhances the immunogenicity of tumor cells.
  • IL-13 and interferon gamma (IFN ⁇ ) into the culture medium was measured by ELISA.
  • ELISA strip well plates were coated with 10 ⁇ g/ml of antibody (in phosphate-buffered saline (PBS)) to IL-13 and 4 ⁇ g/ml of antibody to IFN ⁇ (in PBS) overnight at room temperature. The plates were then blocked using a 4% BSA solution in PBS for one hour, followed by the addition of 50 ⁇ l of experimental culture medium per well in duplicate.
  • PBS phosphate-buffered saline
  • IL-13 The results for IL-13 are shown in FIG. 3 .
  • the compound of formula II (Cpd. II) and dexamethasone (Dex) significantly inhibited IL-13 release induced by PHA.
  • Methylphenidate (MP) did not inhibit the release of IL-13.
  • methylphenidate increased the release of IL-13 by the PHA-stimulated cells.
  • the results for IFN ⁇ are shown in FIG. 4 .
  • the compound of formula II (Cpd. II) and dexamethasone (Dex) significantly inhibited IFN ⁇ release in both unstimulated cells and in cells stimulated with PHA.
  • Methylphenidate (MP) had some effect on the release of IFN ⁇ by unstimulated cells, but did not significantly suppress the release of IFN ⁇ from cells stimulated with PHA. Indeed, methylphenidate increased the release of IFN ⁇ by the PHA-stimulated cells.
  • GR191's PBL in culture medium were added to the wells to give a final concentration of 150,000 cells per well, and the plates were incubated at 37° C., 5% CO 2 for 24 hours. After this incubation, PHA was added to give final concentrations of 2 ⁇ g/ml and 5 ⁇ g/ml, final total volume of 200 ⁇ l/well, and the cells were incubated for an additional 72 hours at 37° C., 5% CO 2 . All cultures were performed in triplicate.
  • the release of cytokines by the PBL was also measured by culturing the PBL in 1 ml tubes, at 1 ⁇ 10 6 cells per ml, with 15 ⁇ g/ml and 50 ⁇ g/ml of the compound of formula II or 10 ⁇ M dexamethasone at 37° C., 5% CO 2 for 24 hours. After this incubation, PHA was added to give a final concentration of 5 ⁇ g/ml, and the cells were incubated for an additional 72 hours at 37° C., 5% CO 2 . All cultures were performed in triplicate. Cells were then removed by centrifugation at 1000 rpm for 10 minutes.
  • the supernatants were collected, and the concentrations of IL-13 and tumor necrosis factor alpha (TNF ⁇ ) in the supernatants were measured by ELISA.
  • the IL-13 ELISA was performed as described in Example 2. The results are presented in FIG. 6 . As can be seen in FIG. 6 , the compound of formula II (Cpd. II) and dexamethasone (Dex) significantly inhibited the release of IL-13 from the PHA-stimulated PBL.
  • TNF ⁇ is a proinflammatory cytokine made by activated T-cells and other cells. TNF ⁇ causes endothelial cells to express adhesion molecules and may play a role in the recruitment of immune cells to the sites of inflammation. TNF ⁇ has been detected in multiple solid and hemotologic malignancies. A number of different intracellular signals are induced by TNF ⁇ , including signals for both cells survival through NF ⁇ B and AP-1 and cell death through caspase activation. NF ⁇ B is a key regulator of cell survival and promoter of carcinogenesis in multiple tumor types.
  • the TNF ⁇ ELISA was performed as described in Example 2 using matched pair antibodies from Pierce Endogen (2 ⁇ g/ml for the coating antibody and 250 ng/ml for the second antibody). The results are presented in FIG. 7 . As can be seen in FIG. 7 , the compound of formula II (Cpd. II) and dexamethasone (Dex) significantly inhibited the release of TNF ⁇ from PHA-stimulated PBL.
  • the cells were further analyzed by flow cytometry. Annexin was used to determine populations of dead or dying cells. Anti-CD69 antibody was used to establish the level of cellular activation. Antibody to T-cell receptor ⁇ (TCR) was also used. Recombinant Annexin 5 (PE and FITC conjugates) and the antibodies were all purchase from Caltag (Burlingham, Calif.) and used following the manufacturer's recommendations. The following results were observed.
  • Annexin staining of TCR-positive cells increased from 7.3% (background) to 45% and 23% with 50 ⁇ g/ml and 15 ⁇ g/ml of the compound of formula II, respectively, signifying an increase in cell death in the T-cell population.
  • Stimulation with PHA at 5 ⁇ g/ml increased the annexin staining of TCR-positive cells to 67%. This indicates that PHA can also induce cell death in the T-cell population.
  • Cell death decreased slightly as a result of treatment with PHA plus 15 ⁇ g/ml of the compound of formula II (62% of the TCR-positive cells stained for annexin with PHA and IMM 0001 versus 67% with PHA alone).
  • CD69+TCR staining (activated T cells) was not detected in any of the controls (nil, compound of formula II alone and dexamethasone alone).
  • PHA increased CD69+TCR staining to 84%. Only PHA caused T-cell activation as detectable by increased CD 69 staining.
  • CD69+TCR staining of PHA-stimulated cells dropped from 84% to 54% with 50 ⁇ g/ml of the compound of formula II and to 64% with 15 ⁇ g/ml of the compound of formula II.
  • Dexamethasone was less effective than the compound of formula II at reducing the CD69+TCR staining of PHA-stimulated cells.
  • the compound of formula II is more effective at decreasing T-cell activation than dexamethasone, a potent anti-inflammatory.
  • GR-192's PBL were cultured in 1 ml tubes, at 1.3 ⁇ 10 6 cells per ml, with 15 ⁇ g/ml of the compound of formula II (in culture medium made using 10% heat-inactivated GR-192 serum) or 10 ⁇ M dexamethasone, at 37° C., 5% CO 2 for 24 hours. After this incubation, PHA was added to give a final concentration of 2 ⁇ g/ml, and the cells were incubated for an additional 96 hours at 37° C., 5% CO 2 . All cultures were performed in triplicate. Cells were then removed by centrifugation at 1000 rpm for 10 minutes, and the culture medium collected.
  • IL-8 is a pro-inflammatory cytokine and a potent chemoattractant and activator of neutrophils. It has also been reported to be a chemoattractant and activator of T-lymphocytes and eosinophils. IL-8 is produced by immune cells (including lymphocytes, neutrophils, monocytes and macrophages), fibroblasts and epithelial cells. IL-8 has potent angiogenic activity.
  • ELISA strip well plates were coated with 2 ⁇ g/ml of antibody to IL-8 (in phosphate-buffered saline (PBS)) overnight at room temperature. The plates were then blocked using a 4% BSA solution in PBS for one hour, followed by the addition of 50 ⁇ l of experimental culture medium per well in duplicate. The plates were incubated at room temperature for one hour and then washed using 50 mM Tris pH 8.0 with 0.1% Tween 20. Then, solutions of 100 ng/ml biotinylated second antibody to IL-8 were made in blocking buffer, and 100 ⁇ l were added per well.
  • PBS phosphate-buffered saline
  • TRiPS human T-lymphocyte cell line
  • Cells were incubated with antibody for 30 minutes on ice, then washed with cold medium without FBS and combined with approximately 2 ⁇ 10 6 4000R-irradiated normal human donor peripheral blood leukocytes (PBL), as feeder cells, in medium plus 50 U/ml human IL-2 (Xenometrix). Cultures were expanded by the addition of fresh IMDM medium with FBS plus IL-2 on day 3. Day of culture is measured from the day of stimulation with OKT3. Cells can be used for experiments starting on day 7 (at maximum proliferation), typically on day 14 (most sensitive to re-stimulation) and up until day 21 (resting cells approaching senescence).
  • PBL peripheral blood leukocytes
  • Activation experiments were performed by withdrawing an aliquot of cells and washing twice with warmed (37° C.) IMDM.
  • 2 ⁇ 10 5 viable cells were pre-incubated in a total volume of 0.9 ml warmed IMDM medium containing 15 ⁇ g/ml of the compound of formula II or 10 ⁇ M dexamethasone for 15 minutes at 37° C.
  • An aliquot of 2 ⁇ 10 5 CD3/CD28 Dynabeads (Dynal), as activating stimulus, in 0.1 ml warmed IMDM was then added, and the cultures incubated 24 hours at 37° C.
  • Supernatants of the cell cultures were harvested after pelleting the cells by centrifugation.
  • Cytokine content was assayed by specific IL-8 ELISA as described above. It was found that the compound of formula II had no effect on IL-8 production by the TRiPS cell line.
  • THP-1 is a monocyte cell line obtained from American Type Culture Collection (ATCC) (catalog no. TIB-202). THP-1 cells were placed in medium (RPMI containing 10% FCS and 8 ng/ml monothioglycerol (obtained from Sigma)) at a concentration of 250,000 cells per ml and incubated with 15 ⁇ g/ml of compound of formula II or 10 ⁇ M dexamethasone for one hour at 37° C. and 5% CO 2 . After 1 hour, lipopolysaccharide (LPS) (obtained from Sigma) was added to the cultures to give a final concentration of 200 ng/ml, and the cells were then incubated for an additional 4 hours or for an additional 24 hours. After the incubation, the cells were centrifuged, and the supernatants were collected. The concentrations of IL-8 and TNF ⁇ in the supernatants were determined by ELISA.
  • medium RPMI containing 10% FCS and 8 ng/ml monothio
  • the concentrations of IL-8 in the supernatants were determined by ELISA performed as described in Example 4. The results are presented in Table 1 below. As can be seen in Table 1, the compound of formula II (Cpd. II) and dexamethasone (Dex) significantly inhibited the release of IL-8 from the LPS-stimulated monocytes.
  • TNF ⁇ ELISA was performed as described in Example 2. The results are presented in Table 2 below. As can be seen in Table 2, the compound of formula II (Cpd. II) and dexamethasone (Dex) significantly inhibited the release of TNF ⁇ from the LPS-stimulated monocytes.
  • the Jurkat T-lymphocyte leukemia cell line was obtained from American Type Culture Collection (ATCC), Rockville, Md. (catalog no. TIB-152).
  • Jurkat cells at 1 ⁇ 10 5 cells/ml, were cultured at 37° C. and 5% CO 2 in IMDM medium (ATCC) with 10% FCS for 72 hours with 7.5 ⁇ g/ml or 15 ⁇ g/ml of the compound of formula II (Cpd II).
  • Passage 4 i.e., four cell population doublings
  • human umbilical vein endothelial cells (HUVECs), human source lot number 9713 (obtained from ATCC) in 1 ml of endothelial growth medium-2 (EGM-2) (obtained from Cambrex) were mixed with 30 ⁇ g of the compound of formula II (Cpd II) in endothelial basal medium-2 (EBM-2) (Cambrex) or 30 ⁇ g methylphenidate (MP) in EBM-2.
  • Water (vehicle for the two test compounds) was used as a control, and the PI3 kinase inhibitor, LY 294002 (Sigma), at 50 ⁇ M, was included as a positive control.
  • the cells were seeded at 10,000 cells/well into the wells of a plate contained in a tube formation assay kit obtained from BD Biosciences, Rockville, Md.
  • the wells of the plate contained an extracellular matrix protein gel.
  • Fetal calf serum (FCS) ATCC was added to a final concentration of 5% to initiate tube formation.
  • FCS Fetal calf serum
  • the plates were incubated for 18 hours at 37° C. and 5% CO 2 . Following the incubation, the plates were photographed with a digital camera attached to an inverted microscope (Olympus IMT-2 set at a phase contrast (PC) of 10).
  • endothelial cells When endothelial cells are cultured on extracellular matrix protein gels in the presence of angiogenic signals, they arrange themselves into structures loosely resembling capillary blood vessels. To establish the basal tube formation for this assay, cells were treated with the same amount of water as present in the solutions of Cpd II and MP. This treatment produced a lattice of endothelial cell structures with multiple branch points. Treatment with Cpd II and LY 294002 reduced the amount of branching and cellular interaction in the wells, leaving the cells in isolated clusters. MP had no observable effect on the ability of the endothelial cells to organize into structures resembling capillary blood vessels. These data indicate that Cpd II, but not MP, interferes with this step of angiogenesis.
  • HUVECs Passage 4 HUVECs, lot number 9713, in either EGM-2 plus 50 ng/ml vascular endothelial growth factor (VEGF) (obtained from Sigma) or in EGM-2 complete medium (containing 2% FCS, hydrocortisone, human fibroblast growth factor B, VEGF, recombinant insulin-like growth factor-1, ascorbate, human epithelial growth factor, gentamycin and heparin) (obtained from Cambrex) were put into the wells of a 96-well tissue culture plate at 5,000 cells/well.
  • VEGF vascular endothelial growth factor
  • EGM-2 complete medium containing 2% FCS, hydrocortisone, human fibroblast growth factor B, VEGF, recombinant insulin-like growth factor-1, ascorbate, human epithelial growth factor, gentamycin and heparin
  • the following additives were added to the cells: water (vehicle control); 5 ⁇ g/ml of the compound of formula II (Cpd II); 15 ⁇ g/ml Cpd II; or 30 ⁇ g/ml of Cpd II.
  • cell proliferation was evaluated by the Promega cell titer assay as described in Example 1, except that the plates were incubated for only 2 hours after addition of the Promega cell titer reagent.
  • HepG2 is a human hepatic cancer cell line, which was obtained from ATCC. HepG2 cells were grown to confluence in 25 cm 2 flasks in IMDM medium containing 10% FCS. Then, the cells were trypsinized as follows. The medium in each flask was aspirated and replaced with 5 ml of 0.025% trypsin/EDTA (Cambrex). The cells were monitored on a microscope until they no longer adhered to the flasks. Then, 5 ml of trypsin neutralizing solution (TNS) (Cambrex) were added to each flask to stop the reaction. The cell suspension was centrifuged at 1000 rpm for 10 minutes, and the supernatants were aspirated.
  • TMS trypsin neutralizing solution
  • the cells were reconstituted in fresh medium and counted. Then, 4 ml of the cell suspension in medium containing at 1.22 ⁇ 10 6 cells/ml were mixed with an additional 1 ml of medium. Next, 0.5 ml/well of the resulting cell suspension was added to wells in a 24-well culture plate (about 500,000 cells/well). The cells were treated as indicated in Table 5 below and incubated for 24 hours at 37° C., with or without 5% CO 2. The supernatants were removed from the wells and centrifuged to remove debris. Next, the supernatants were analyzed for erythropoietin (EPO) production. EPO was measured by ELISA using a kit obtained from R & D Systems, Minneapolis, Minn. (catalog no. DE900) following the manufacturer's instructions.
  • EPO erythropoietin
  • Passage 4 HUVECs were put into the wells of a 48-well tissue culture plate at 20,000 cells/well in 500 ⁇ l of EGM-2 complete medium (but without serum or ascorbate) supplemented with ITSS (insulin, transferrin and sodium selenite) (obtained from Sigma). Also, passage 4 HUVECs, human source lot number 7016 (obtained from ATCC), were put into the wells of a 48-well tissue culture plate at 20,000 cells/well in 500 ⁇ l of EGM-2 complete medium (but without serum or ascorbate) supplemented with ITSS.
  • ITSS insulin, transferrin and sodium selenite
  • the following additives were added to the cells: water (vehicle control) and 15 ⁇ g/ml of the compound of formula II (Cpd II). After incubation for 1 hour at 37° C. and 5% CO 2 , LPS was added to give a final concentration of 200 ng/ml, and the cells were incubated overnight at 37° C. and 5% CO 2 . After this incubation, the supernatants were collected, and the amount of IL-8 in the supernatants determined by ELISA as described in Example 4.
  • HUVECs human source lot number 8710 (obtained from ATCC), were put into the wells of a 24-well tissue culture plate at 5,000 cells/well in EGM-2 medium and cultured for 72 hours at 37° C. and 5% CO 2 . Then, the medium was replaced with fresh medium, and the following additives were added to the cells: water (vehicle control); 1 ⁇ g/ml, 5 ⁇ g/ml, 10 ⁇ g/ml, 15 ⁇ g/ml or 30 ⁇ g/ml of the compound of formula II (Cpd II); 15 ⁇ g/ml methylphenidate (MP); 10 ⁇ M LY 294002; or 10 ⁇ M dexamethasone (Dex).
  • water vehicle control
  • TNF ⁇ (Pierce) was added to give a final concentration of 10 ng/ml, and the cells were incubated for an additional 18 hours at 37° C. and 5% CO 2 . After this incubation, the supernatants were collected, and the amount of IL-8 in the supernatants determined by ELISA as described in Example 4.
  • NF ⁇ B transcription factor ⁇ B
  • NF ⁇ B is implicated in the regulation of the expression of a wide variety of genes that code for mediators of the immune, acute phase and inflammatory responses.
  • NF ⁇ B is a key regulator of cell survival and promoter of carcinogenesis.
  • the p50/p65 heterodimers and the p50 homodimers are the most common dimers found the NF ⁇ B signaling pathway.
  • NF ⁇ B can be activated by a number of stimuli, including components of bacterial cell walls, such as lipopolysaccharide, or inflammatory cytokines, such as TNF ⁇ or IL-1 ⁇ .
  • Activator protein-1 is a transcription factor that is activated during the cell cycle to promote cell survival, differentiation and adaptive responses.
  • AP-1 proteins play a role in the expression of many genes involved in proliferation and cell cycle progression. For instance, cell transformation by oncogenes that function in the growth factor signal transduction pathway, such as ras, rasF and mek, results in a high increase in AP-1 component protein expression. Therefore, AP-1 regulated genes support the invasive process observed during malignancy and metastasis.
  • AP-1 belongs to a large family of structurally related transcription factors that includes ATFI-4, c-Fos, c-Jun, c-Myc and C/EBP.
  • AP-1 is composed of a mixture of heterodimeric complexes of proteins derived from the Fos and Jun families, including c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB and JunD.
  • AP-1 dimers bind to DNA on a TPA-response element (TRE).
  • TRE TPA-response element
  • AP-1 expression is induced by multiple stimuli such as serum, growth factors, phorbol esters, oncogenes, cytokines of the TGF-13, TNF and interferon families, neuronal depolarization and cellular stress.
  • the amount of NF ⁇ B was determined using a TransAMTM NF ⁇ B p65/NF ⁇ B p50 Transcription Factor Assay Kit and a Nuclear Extract Kit from Active Motif North America, Carlsbad, Calif., according to the manufacturer's instructions. Briefly, a nuclear extract of the cells was prepared using the Nuclear Extract Kit. Then, the nuclear extract was added to the wells of the 96-well plate of the TransAMTM kit. Oligonucleotide containing an NF ⁇ B consensus binding site was immobilized in the wells, and the activated NF ⁇ B contained in the nuclear extract was bound to the oligonucleotide.
  • HRP horseradish peroxidase
  • the amount of c-Jun was determined using a TransAMTM AP-1 Family Transcription Factor Assay Kit and a Nuclear Extract Kit from Active Motif North America, Carlsbad, Calif., according to manufacturer's instructions. Briefly, a nuclear extract of the cells was prepared using the Nuclear Extract Kit. Then, the nuclear extract was added to the wells of a 96-well plate in which oligonucleotide containing a TPA-responsive element (TRE) was immobilized. Activator protein-1 (AP-1) dimers contained in the nuclear extract were bound to this oligonucleotide and were detected using an antibody specific for c-Jun. A secondary antibody conjugated to horseradish peroxidase (HRP) was next added to provide a calorimetric readout that was quantified by spectrophotometry (measurement at 450 nm).
  • HRP horseradish peroxidase
  • VEGF treatment of HUVECs caused almost a doubling of activated NF ⁇ B as detected by the TransAM assay.
  • Cpd II at 15 ⁇ g/ml and 5 ⁇ g/ml reduced the amount of activated NF ⁇ B back to basal levels.
  • VEGF treatment of HUVECs caused an increase of c-Jun.
  • Cpd II at 15 ⁇ g/ml and 5 ⁇ g/ml completely eliminated the increase in the amount of c-Jun.
  • Passage 8 human iliac artery endothelial cells (HIAECs) (obtained from ATCC; catalog no. CC-2545) were grown to confluence in 25 cm 2 flasks in EGM-2 medium. Eighteen hours prior to the experiment, the medium was replaced with EGM-2 medium containing 0.1% FCS plus heparin, GA 1000 (gentamycin) and bovine pituitary extract (all from Cambrex) to place the cells in a resting state.
  • HAAECs human iliac artery endothelial cells
  • the medium was aspirated from the flasks, and the following additives were added to the flasks in fresh medium (total volume of 5 ml/flask): 15 ⁇ g/ml of the compound of formula II (Cpd II) or 10 ⁇ M LY 294002.
  • the flasks were incubated 2 hours at 37° C., 5% CO 2 After this incubation, VEGF or TNF ⁇ was added to give a final concentration 10 ng/ml, and the flasks were incubated for an additional 30 minutes.
  • NF ⁇ B was determined using a TransAMTM NF ⁇ B p65/NF ⁇ B p50 Transcription Factor Assay Kit and a Nuclear Extract Kit from Active Motif North America, Carlsbad, Calif., as described in Example 12.
  • TNF ⁇ treatment of HUVECs caused an extremely large increase in the amount of activated NF ⁇ B as detected by the TransAM assay.
  • Cpd II at 15 ⁇ g/ml reduced the amount of activated NF ⁇ B about 82%.
  • the treatment with VEGF did not result in as large an increase in activated NF ⁇ B as achieved with TNF ⁇ , but the increased amount was reduced 70% by Cpd. II.
  • Day 18 TRiPS cells 1 ⁇ 10 6 were incubated for 30 minutes at 37° C., either with nothing added (“Nil”), with 1 ⁇ l CD3/CD28 Dynabeads (Dynal, Oslo, Norway) (“CD3/CD28 beads”) per 100,000 cells, or with CD3/CD28 beads and 15 ⁇ g/ml of the compound of formula II (Cpd II). After the incubation, the cells were lysed in Cell-Lytic Mammalian Cell Extraction Reagent (Sigma). After centrifugation to pellet cellular debris, the supernatants (cell extracts) were obtained.
  • Cell-Lytic Mammalian Cell Extraction Reagent Sigma
  • the cell extracts were then analyzed using a Custom AntibodyArrayTM manufactured by Hypromatrix Inc., Worcester, Mass., following the manufacturer's instructions.
  • the Custom AntibodyArrayTM is a nylon membrane blotted with antibodies to the proteins listed below. Briefly, the cell extracts were incubated with duplicate Custom AntibodyArrayTM's for 2 hours at room temperature with slow shaking, followed by three washes with Tris buffer (150 mM NaCl, 25 mM Tris, 0.05% Tween-20, pH 7.5). HRP-labeled antibodies specific for phosphorylated-tyrosine, phosphorylated-serine and phosphorylated-threonine in Tris buffer were added, and the arrays incubated for 2 hours.
  • MC/9 murine fibroblast cell line obtained from ATCC, catalog no. CRL-8305
  • the culture medium was Delbecco's Modified Eagle's Medium (DMEM) (obtained from Cambrex) containing 10% FCS.
  • DMEM Delbecco's Modified Eagle's Medium
  • NGF murine nerve growth factor
  • TSTIM a culture supplement prepared from rats and containing concanavalin A which was obtained from BD Biosciences
  • THP-1 cells were placed in medium (RPMI containing 10% FCS and 8 ng/ml monothioglycerol) at a concentration of 250,000 cells per ml and incubated with 5 ⁇ g/ml of compound of formula II (Cpd II) or 15 ⁇ g/ml of Cpd II for one hour at 37° C. and 5% CO 2 . After 1 hour, lipopolysaccharide (LPS) was added to the cultures to give a final concentration of 200 ng/ml, and the cells were then incubated for an additional 24 hours. After the incubation, the amount of NF ⁇ B and c-Jun were determined as described in Example 12.
  • medium RPMI containing 10% FCS and 8 ng/ml monothioglycerol
  • the amount of c-Fos was determined using a TransAMTM AP-1 Family Transcription Factor Assay Kit and a Nuclear Extract Kit from Active Motif North America, Carlsbad, Calif., according to manufacturer's instructions. Briefly, a nuclear extract of the cells was prepared using the Nuclear Extract Kit. Then, the nuclear extract was added to the wells of a 96-well plate in which oligonucleotide containing a TPA-responsive element (TRE) was immobilized. Activator protein-1 (AP-1) dimers contained in the nuclear extract were bound to this oligonucleotide and were detected using an antibody specific for c-Fos.
  • TRE TPA-responsive element
  • HRP horseradish peroxidase
  • Pulldown assays were performed using Pierce EZ-Detect activation kits according to the manufacturer's instructions utilizing GST-RAF-1-RBD and GST-RalGDS-RBD for Ras and RAP-1 respectively. Briefly, 400 ⁇ g total protein from each extract was combined with recombinant protein and glutathione resin and incubated at 4° C. for one hour with gentle shaking. The resin was then washed to remove unbound protein and the activated Ras and RAP-1 proteins were removed by boiling in the presence of SDS-PAGE loading dye containing reducing agent. Ras and RAP-1 western blots were performed to visualize the proteins using antibodies supplied with the kit. Densitometry of the X-ray films was done by scanning and computer analysis.

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US20090275614A1 (en) * 2005-01-20 2009-11-05 David Bar-Or Methylphenidate Derivatives and Uses of Them
US20100105698A1 (en) * 2008-05-27 2010-04-29 Dmi Life Sciences, Inc. Therapeutic Methods and Compounds

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JP5867182B2 (ja) * 2012-03-08 2016-02-24 株式会社島津製作所 ガスクロマトグラフ
EP3296292A1 (fr) 2016-09-19 2018-03-21 Université de Liège Procede pour la preparation d'alkyl phenidates

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