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<SOH> SUMMARY OF THE INVENTION <EOH>The present inventors have made elaborate investigation eagerly to solve the aforementioned problem. As a result, it has been found that crosslinking a tetrafluoroethylene-propylene copolymer or tetrafluoroethylene-propylene-vinylidene fluoride terpolymer prepared in such a manner that the amount of metal contained is reduced to be not larger than a specific amount is effective. That is, the invention provides a fluoro rubber molded product and a method for producing the same as follows. (1) A fluoro rubber molded product characterized by comprising either of a crosslinked tetrafluoroethylene-propylene copolymer and a crosslinked tetrafluoroethylene-propylene-vinylidene fluoride terpolymer, the crosslinked copolymer or terpolymer having a metallic component content of not higher than 1.5% by mass in terms of a quantity based on metallic elements. (2) A fluoro rubber molded product according to the (1), characterized in that the crosslinking is performed with peroxide. (3) A fluoro rubber molded product according to the (2), characterized in that the crosslinking is further performed by ionizing radiation exposure. (4) A fluoro rubber molded product according to the (2) or (3), characterized in that 0.1 to 20 parts bymass of triallyl isocyanurate as a crosslinking coagent are mixed with 100 parts by mass of the tetrafluoroethylene-propylene copolymer. (5) A fluoro rubber molded product according to any one of the (2) through (4), characterized in that the metallic element content is not higher than 5000 ppm. (6) A fluoro rubber molded product according to the (1), characterized in that the crosslinking is performed by ionizing radiation exposure. (7) A fluoro rubber molded product according to any one of the (1) through (6), characterized in that the amount of emission gas is not higher than 3 ppm when the fluoro rubber molded product is held at a temperature of 100° C. for 30 minutes. (8) A method of producing a fluoro rubber molded product, characterized by comprising the step of crosslinking either of a tetrafluoroethylene-propylene copolymer and a tetrafluoroethylene-propylene-vinylidene fluoride terpolymer which is prepared as a starting material to have a metallic component content of not higher than 1.5% by mass in terms of a quantity based on metallic elements. (9) A method of producing a fluoro rubber molded product according to the (8), characterized in that a mixture containing a starting material and a peroxide crosslinking agent or a mixture containing a starting material, a peroxide crosslinking agent and a crosslinking coagent is heat-molded into a crosslinked body. (10) A method of producing a fluoro rubber molded product according to the (9), characterized in that the crosslinked body is further irradiated with an ionizing radiation. (11) A method of producing a fluoro rubber molded product according to the (9) or (10), characterized in that either of a tetrafluoroethylene-propylene copolymer and a tetrafluoroethylene-propylene-vinylidene fluoride terpolymer prepared to have a metallic component content of not higher than 5000 ppm is used as the starting material. (12) A method of producing a fluoro rubber molded product according to the (8), characterized in that the starting material is preformed into a predetermined shape and then a preformed body is irradiated with an ionizing radiation. (13) A method of producing a fluoro rubber molded product according to any one of the (8) through (12), characterized in that the molded product obtained by crosslinking is further subjected to a cleaning treatment using pure water, a heat treatment at a temperature of not lower than 150° C. or a combination of the cleaning treatment and the heat treatment. The invention further provides the following rubber materials as preferred applications of the fluoro rubber molded product described in any one of the (1) through (7). (14) A rubber material for use in a semiconductor producing apparatus or a semiconductor conveying apparatus, characterized by comprising a fluoro rubber molded product according to any one of the (1) through (7). (15) A rubber material for use in a food manufacturing apparatus, a food conveyor or a food storage, characterized by comprising a fluoro rubber molded product according to any one of the (1) through (7). (16) A rubber material for use in a medical appliance, characterized by comprising a fluoro rubber molded product according to any one of the (1) through (7). detailed-description description="Detailed Description" end="lead"? |
Extracts from hop, methods for producing the same and their use |
Novel extracts from hop with an increased content of prenylated chalcones and flavones, methods for producing the same, pharmaceutical preparations comprising such extracts of hop and use of these extracts of hop for the prophylaxis and treatment of pathological conditions caused by oestrogen deficiency or by dysregulations to sex-hormone-related metabolism are described. |
1. Method for obtaining an extract from hop, comprising the steps of: (a) one or more extractions of a drug from hop with a C5-C7-alkane or supercritical CO2 and separating the drug residue from the solution; (b) one or more extractions of the drug residue obtained in step (a) with water at a temperature in the range of 60 to 95% and separating the drug residue; (c) one or more extractions of the drug residue obtained in step (b) with 80-96% (w/w) ethanol and filtration of the obtained extraction solution; and (d) removing the solvent from the combined extraction solutions obtained in step (c) and drying of the obtained residue. 2. Method according to claim 1, wherein the extraction in step (a) is carried out once, twice, or three times. 3. Method according to any one of claims 1 or 2, wherein the solvent in step (a) is selected from the group consisting of n-pentane, n-hexane, and n-heptane. 4. Method according to claim 3, wherein the solvent in step (a) is n-heptane. 5. Method according to anyone of claims 1 to 4, wherein the extraction in step (b) is carried out at about 90° C. 6. Method according to anyone of claims 1 to 5, wherein the solvent in step (c) is 92% (w/w) ethanol. 7. Method according to claim 1, wherein the solvent in step (a) is n-heptane and the solvent in step (c) is 92% (w/w) ethanol. 8. Method according to claim 1, wherein the solvent in step (a) is supercritical CO2 and the solvent in step (c) is 92% (w/w) ethanol. 9. Extract from hop obtainable according to anyone of claims 1 to 8, characterized by having a content of a bitter acids of at least 0.5%, of xanthohumol of at least 2%, and of prenylated flavones selected from the group comprising 6-prenylnaringenin, 8-prenylnaringenin and isoxanthohumol of at least 0.5%. 10. Extract from hop according to claim 9, characterized by having a content of a bitter acids of at least 0.8%, of xanthohumol of at least 3%, and of prenylated flavones selected from the group comprising 6-prenylnaringenin, 8-prenylnaringenin, and isoxanthohumol of at least 0.7%. 11. Pharmaceutical preparation, comprising an extract from hop characterized by having a content of a bitter acids of at least 0.5%, of xanthohumol of at least 2%, and of prenylated flavones selected from the group comprising 6-prenylnaringenin, 8-prenylnaringenin and isoxanthohumol of at least 0.5% and conventional pharmaceutically acceptable additives. 12. Pharmaceutical preparation according to claim 11, comprising an extract from hop, characterized by having a content of a bitter acids of at least 0.8%, of xanthohumol of at least 3%, and of prenylated flavones selected from the group comprising 6-prenylnaringenin, 8-prenylnaringenin, and isoxanthohumol of at least 0.7%. 13. Use of an extract from hop as defined in claim 9 or 10 or a pharmaceutical preparation according to claim 11 or 12 for the preparation of a medicament for the prophylaxis and treatment of pathological diseases caused by a deficiency of oestrogens or a dysregulation of sex-hormone-related metabolism, particularly oestrogen metabolism, selected from the group consisting of climacteric complaints, benign prostate hypertrophy, osteoporosis, Alzheimer's disease and diseases of the cardiovascular system. 14. Use of an extract from hop as defined in claim 9 or 10 or a pharmaceutical preparation according to claim 11 or 12 for the preparation of a medicament for the prophylaxis against sex-hormone-dependent cancers. 15. Use according to claim 14, wherein the sex-hormone-dependent cancers are selected from the group consisting of breast cancer, carcinoma of the prostate, and carcinoma of the uterus. |
Cyclic compound and ppar agonist |
The present invention provides a novel compound having an excellent PPAR agonist action. More specifically, it provides a compound represented by the following formula, a salt thereof, an ester thereof or a hydrate of them. Wherein a, b and c are the same as or different from one another and each represents 0 to 4; R1 to R6 are the same as or different from one another and each represents a hydrogen atom, a hydroxyl group, a cyano group, a halogen atom, etc.; A1 and A2 are the same as or different from each other and each represents a single bond, an oxygen atom, etc.; L, M and T each represent a single bond, an alkylene group having one to six carbon atoms, etc.; W represents a carboxyl group; the partial structure represented by the formula: represents a single bond or a double bond; X represents a single bond, an oxygen atom, —NRX1CQ1O—, etc.; Y represents Y1—Y2— (wherein Y1 represents a 5 to 14-membered aromatic ring having one to four substituents, etc.; and Y2 represents a single bond or a 5 to 14-membered aromatic ring); and the ring Z represents a 5 to 14-membered aromatic ring which have one to four substituents selected form the above-mentioned Group A, may have one or more hetero atoms and may be partially saturated. |
1. A compound represented by the following formula, a salt thereof, an ester thereof or a hydrate of them. Wherein a, b and c are the same as or different from one another and each represents 0, 1, 2, 3 or 4; R1, R2, R3, R4, R5 and R6 are the same as or different from one another and each represents 1) a hydrogen atom, 2) a hydroxyl group, 3) a cyano group, 4) a halogen atom, 5)—N(R7)R8 (wherein R7 and R8 are the same as or different from each other and each represents a hydrogen atom, a cyano group, a formyl group, or an alkyl group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aliphatic acyl group having two to seven carbon atoms, an aromatic acyl group having seven to nineteen carbon atoms, an aliphatic alkoxycarbonyl group having two to seven carbon atoms or an aromatic alkoxycarbonyl group having seven to nineteen carbon atoms, each of which may have one or more substituents), or 6) an alkyl group having one to six carbon atoms, an alkoxy group having one to six carbon atoms, an alkylthio group having one to six carbon atoms, a hydroxyalkyl group-having one to six carbon atoms, a hydroxyalkoxy group having one to six carbon atoms, a hydroxyalkylthio group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, an aminoalkoxy group having one to six carbon atoms, an aminoalkylthio group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, a halogeno-alkoxy group having one to six carbon atoms, a halogeno-alkylthio group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, an alkoxyalkoxy group having two to twelve carbon atoms, an alkoxyalkylthio group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, a cycloalkyloxy group having three to seven carbon atoms, a cycloalkylalkyloxy group having four to thirteen carbon atoms, a cycloalkylthio group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkenyloxy group having two to six carbon atoms, an alkenylthio group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an alkynyloxy group having two to six carbon atoms, an alkynylthio group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an aryloxy group having six to twelve carbon atoms, an arylthio group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an alkylaryloxy group having seven to eighteen carbon atoms, an alkylarylthio group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aralkyloxy group having seven to eighteen carbon atoms or an aralkylthio group having seven to eighteen carbon atoms, each of which may have one or more substituents; A1 and A2 are the same as or different from each other and each represents a single bond, an oxygen atom, a sulfur atom, —SO—, —SO2—, —NRA1— (wherein RA1 represents a hydrogen atom, a cyano group, a formyl group, or an alkyl group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aliphatic acyl group having two to seven carbon atoms or an aromatic acyl group having seven to nineteen carbon atoms, each of which may have one or more substituents, or an aliphatic alkoxycarbonyl group having two to seven carbon atoms or an aromatic alkoxycarbonyl group having seven to nineteen carbon atoms), a group represented by the formula: (wherein RA2 and RA3 are the same as or different from each other and each represents a hydrogen atom, a hydroxyl group, a cyano group, a halogen atom, or an alkyl group having one to six carbon atoms, an alkoxy group having one to six carbon atoms, an alkylthio group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, a hydroxyalkoxy group having one to six carbon atoms, a hydroxyalkylthio group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, an aminoalkoxy group having one to six carbon atoms, an aminoalkylthio group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, a halogeno-alkoxy group having one to six carbon atoms, a halogeno-alkylthio group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, an alkoxyalkoxy group having two to twelve carbon atoms, an alkoxyalkylthio group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, a cycloalkyloxy group having three to seven carbon atoms, a cycloalkylalkyloxy group having four to thirteen carbon atoms, a cycloalkylthio group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkenyloxy group having two to six carbon atoms, an alkenylthio group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an alkynyloxy group having two to six carbon atoms, an alkynylthio group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an aryloxy group having six to twelve carbon atoms, an arylthio group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an alkylaryloxy group having seven to eighteen carbon atoms, an alkylarylthio group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aralkyloxy group having seven to eighteen carbon atoms or an aralkylthio group having seven to eighteen carbon atoms, each of which may have one or more substituents), or —N(RA4)RA5 (wherein RA4 and RA5 are the same as or different from each other and each represents a hydrogen atom, a cyano group, a formyl group, or an alkyl group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aliphatic acyl group having two to seven carbon atoms or an aromatic acyl group having seven to nineteen carbon atoms, each of which may have one or more substituents, or an aliphatic alkoxycarbonyl group having two to seven carbon atoms or an aromatic alkoxycarbonyl group having seven to nineteen carbon atoms); L represents a single bond, or an alkylene group having one to six carbon atoms, an alkenylene group having two to six carbon atoms or an alkynylene group having two to six carbon atoms, each of which may have one or more substituents; M represents a single bond, or an alkylene group having one to six carbon atoms, an alkenylene group having two to six carbon atoms or an alkynylene group having two to six carbon atoms, each of which may have one or more substituents; T represents a single bond, or an alkylene group having one to three carbon atoms, an alkenylene group having two or three carbon atoms or an alkynylene group having two or three carbon atoms, each of which may have one or more substituents; W represents a carboxyl group; the partial structure represented by the formula: represents a single bond or a double bond; X represents a single bond, an oxygen atom, —NRX1CQ1O— (wherein Q1 represents an oxygen atom or a sulfur atom; RX1 represents a hydrogen atom, a cyano group, a formyl group, or an alkyl group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aliphatic acyl group having two to seven carbon atoms or an aromatic acyl group having seven to nineteen carbon atoms, each of which may have one or more substituents, or an aliphatic alkoxycarbonyl group having two to seven carbon atoms or an aromatic alkoxycarbonyl group having seven to nineteen carbon atoms), —OCQ1NRX1— (wherein Q1 and RX1 are as defined above), CQ1NRX1O— (wherein Q1 and RX1 are as defined above), —ONRX1CQ1- (wherein Q1 and RX1 are as defined above), —NRX1CQ1- (wherein Q1 and RX1 are as defined above), —CQ1NRX1— (wherein Q1 and RX1 are as defined above), —NRX1aCQ1NRX1b— (wherein Q1 is as defined above; RX1a and RX1b are the same as or different from each other and each represents a hydrogen atom, a cyano group, a formyl group, or an alkyl group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aliphatic acyl group having two to seven carbon atoms or an aromatic acyl group having seven to nineteen carbon atoms, each of which may have one or more substituents, or an aliphatic alkoxycarbonyl group having two to seven carbon atoms or an aromatic alkoxycarbonyl group having seven to nineteen carbon atoms), -Q2SO2— (wherein Q2 represents an oxygen atom or —NRX10— (wherein RX10 represents a hydrogen atom, a cyano group, a formyl group, or an alkyl group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aliphatic acyl group having two to seven carbon atoms or an aromatic acyl group having seven to nineteen carbon atoms, each of which may have one or more substituents, or an aliphatic alkoxycarbonyl group having two to seven carbon atoms or an aromatic alkoxycarbonyl group having seven to nineteen carbon atoms)), —SO2Q2- (wherein Q2 is as defined above), or a group represented by the formula: (wherein Q1, Q2 and RX1 are as defined above; k represents from 0 to 5; m represents from 1 to 5; n and p are the same as or different from each other and each represents from 1 to 5; RX2, RX3, RX4, RX5, RX6, RX7, RX8 and RX9 are the same as or different from each other and each represents a hydrogen atom, a hydroxyl group, a cyano group, a halogen atom, or an alkyl group having one to six carbon atoms, an alkoxy group having one to six carbon atoms, an alkylthio group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, a hydroxyalkoxy group having one to six carbon atoms, a hydroxyalkylthio group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, an aminoalkoxy group having one to six carbon atoms, an aminoalkylthio group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, a halogeno-alkoxy group having one to six carbon atoms, a halogeno-alkylthio group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, an alkoxyalkoxy group having two to twelve carbon atoms, an alkoxyalkylthio group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, a cycloalkyloxy group having three to seven carbon atoms, a cycloalkylalkyloxy group having four to thirteen carbon atoms, a cycloalkylthio group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkenyloxy group having two to six carbon atoms, an alkenylthio group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an alkynyloxy group having two to six carbon atoms, an alkynylthio group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an aryloxy group having six to twelve carbon atoms, an arylthio group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an alkylaryloxy group having seven to eighteen carbon atoms, an alkylarylthio group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aralkyloxy group having seven to eighteen carbon atoms or an aralkylthio group having seven to eighteen carbon atoms, each of which may have one or more substituents, or —N(RX11)RX12— (wherein RX11 and RX12 are the same as or different from each other and each represents a hydrogen atom, a cyano group, a formyl group, or an alkyl group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aliphatic acyl group having two to seven carbon atoms or an aromatic acyl group having seven to nineteen carbon atoms, each of which may have one or more substituents, or an aliphatic alkoxycarbonyl group having two to seven carbon atoms or an aromatic alkoxycarbonyl group having seven to nineteen carbon atoms), provided that RX2 and RX3, and RX4 and RX5 may together form a ring; and Q3 and Q4 are the same as or different from each other and each represents a single bond, an oxygen atom, (O)S(O) or NRX10 (wherein NRX10 is as defined above)); Y represents Y1—Y2— (wherein Y1 represents a 5 to 14-membered aromatic ring which has one to four substituents selected from the following Group A, may have one or more hetero atoms and may be partially saturated; and Y2 represents a single bond or a 5 to 14-membered aromatic ring which has a substituent selected from the following Group A, may have one or more hetero atoms and may be partially saturated; Group A: a hydrogen atom, a halogen atom, a hydroxyl group, a sulfamoyl group, or an alkyl group having one to six carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkoxy group having one to six carbon atoms, a cycloalkyloxy group having three to seven carbon atoms, a hydroxyalkyl group having one to six carbon atoms, a hydroxyalkoxy group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an acylamino group having two to seven carbon atoms or a 5 to 14-membered heterocyclic group, each of which may have a substituent, provided that when two or more substituents selected from Group A are present, they may together form a ring); and the ring Z represents a 5 to 14-membered aromatic ring which has one to four substituents selected from the above-mentioned Group A, may have one or more hetero atoms and may be partially saturated. 2. The compound according to claim 1, a salt thereof, an ester thereof or a hydrate of them, wherein in the formula (I), c is 0, and A2 is an oxygen atom. 3. The compound according to claim 1 or 2, a salt thereof, an ester thereof or a hydrate of them, wherein in the formula (I), a is 0, b is 1, and A1 is represented by the formula: wherein RA2 and RA3 each represent the same groups as defined above. 4. The compound according to claim 1 or 2, a salt thereof, an ester thereof or a hydrate of them, wherein in the formula (I), a is 2, b is 1, and A1 is a single bond. 5. The compound according to any one of claims 1 to 4, a salt thereof, an ester thereof or a hydrate of them, wherein in the formula (I), X is —CQ1NRX1— (wherein Q1 and RX1 each represent the same groups as defined above). 6. The compound according to any one of claims 1 to 5, a salt thereof, an ester thereof or a hydrate of them, wherein in the formula (I), the ring Z is a 5 to 14-membered aromatic ring which has at least an alkoxy group having one to six carbon atoms, which may have one or more hetero atoms and may be partially saturated. 7. The compound according to any one of claims 1 to 6, a salt thereof, an ester thereof or a hydrate of them, wherein in the formula (I), Y is Y1—Y2— (wherein Y1 represents the same group as defined above, and Y2 is a single bond). 8. The compound according to claim 7, a salt thereof, an ester thereof or a hydrate of them, wherein Y1 is a 5 to 14-membered aromatic ring which has at least an alkoxy group having one to six carbon atoms and may have one or more hetero atoms on the ring. 9. The compound according to any one of claims 1 to 8, a salt thereof, an ester thereof or a hydrate of them, wherein in the formula (I), L is a single bond. 10. The compound according to any one of claims 1 to 9, a salt thereof, an ester thereof or a hydrate of them, wherein in the formula (I), T is an alkylene group having one to six carbon atoms. 11. The compound according to any one of claims 1 to 10, a salt thereof, an ester thereof or a hydrate of them, wherein in the formula (I), the ring Z is represented by the following formula: (which may have a hetero atom on the ring), which may have one to four substituents selected from Group A defined in claim 1. 12. A medicament comprising a compound represented by the following formula, a salt thereof, an ester thereof or a hydrate of them. Wherein a, b and c are the same as or different from one another and each represents 0, 1, 2, 3 or 4; R1, R2, R3, R4, R5 and R6 are the same as or different from one another and each represents 1) a hydrogen atom, 2) a hydroxyl group, 3) a cyano group, 4) a halogen atom, 5)—N(R7)R8 (wherein R7 and R8 are the same as or different from each other and each represents a hydrogen atom, a cyano group, a formyl group, or an alkyl group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aliphatic acyl group having two to seven carbon atoms, an aromatic acyl group having seven to nineteen carbon atoms, an aliphatic alkoxycarbonyl group having two to seven carbon atoms or an aromatic alkoxycarbonyl group having seven to nineteen carbon atoms, each of which may have one or more substituents), or 6) an alkyl group having one to six carbon atoms, an alkoxy group having one to six carbon atoms, an alkylthio group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, a hydroxyalkoxy group having one to six carbon atoms, a hydroxyalkylthio group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, an aminoalkoxy group having one to six carbon atoms, an aminoalkylthio group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, a halogeno-alkoxy group having one to six carbon atoms, a halogeno-alkylthio group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, an alkoxyalkoxy group having two to twelve carbon atoms, an alkoxyalkylthio group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, a cycloalkyloxy group having three to seven carbon atoms, a cycloalkylalkyloxy group having four to thirteen carbon atoms, a cycloalkylthio group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkenyloxy group having two to six carbon atoms, an alkenylthio group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an alkynyloxy group having two to six carbon atoms, an alkynylthio group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an aryloxy group having six to twelve carbon atoms, an arylthio group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an alkylaryloxy group having seven to eighteen carbon atoms, an alkylarylthio group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aralkyloxy group having seven to eighteen carbon atoms or an aralkylthio group having seven to eighteen carbon atoms, each of which may have one or more substituents; A1 and A2 are the same as or different from each other and each represents a single bond, an oxygen atom, a sulfur atom, —SO—, —SO2—, —NRA1— (wherein RA1 represents a hydrogen atom, a cyano group, a formyl group, or an alkyl group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aliphatic acyl group having two to seven carbon atoms or an aromatic acyl group having seven to nineteen carbon atoms, each of which may have one or more substituents, or an aliphatic alkoxycarbonyl group having two to seven carbon atoms or an aromatic alkoxycarbonyl group having seven to nineteen carbon atoms), a group represented by the formula: (wherein RA2 and RA3 are the same as or different from each other and each represents a hydrogen atom, a hydroxyl group, a cyano group, a halogen atom, or an alkyl group having one to six carbon atoms, an alkoxy group having one to six carbon atoms, an alkylthio group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, a hydroxyalkoxy group having one to six carbon atoms, a hydroxyalkylthio group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, an aminoalkoxy group having one to six carbon atoms, an aminoalkylthio group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, a halogeno-alkoxy group having one to six carbon atoms, a halogeno-alkylthio group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, an alkoxyalkoxy group having two to twelve carbon atoms, an alkoxyalkylthio group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, a cycloalkyloxy group having three to seven carbon atoms, a cycloalkylalkyloxy group having four to thirteen carbon atoms, a cycloalkylthio group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkenyloxy group having two to six carbon atoms, an alkenylthio group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an alkynyloxy group having two to six carbon atoms, an alkynylthio group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an aryloxy group having six to twelve carbon atoms, an arylthio group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an alkylaryloxy group having seven to eighteen carbon atoms, an alkylarylthio group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aralkyloxy group having seven to eighteen carbon atoms or an aralkylthio group having seven to eighteen carbon atoms, each of which may have one or more substituents), or —N(RA4)RA5 (wherein RA4 and RA5 are the same as or different from each other and each represents a hydrogen atom, a cyano group, a formyl group, or an alkyl group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aliphatic acyl group having two to seven carbon atoms or an aromatic acyl group having seven to nineteen carbon atoms, each of which may have one or more substituents, or an aliphatic alkoxycarbonyl group having two to seven carbon atoms or an aromatic alkoxycarbonyl group having seven to nineteen carbon atoms); L represents a single bond, or an alkylene group having one to six carbon atoms, an alkenylene group having two to six carbon atoms or an alkynylene group having two to six carbon atoms, each of which may have one or more substituents; M represents a single bond, or an alkylene group having one to six carbon atoms, an alkenylene group having two to six carbon atoms or an alkynylene group having two to six carbon atoms, each of which may have one or more substituents; T represents a single bond, or an alkylene group having one to three carbon atoms, an alkenylene group having two or three carbon atoms or an alkynylene group having two or three carbon atoms, each of which may have one or more substituents; W represents a carboxyl group; the partial structure represented by the formula: represents a single bond or a double bond; X represents a single bond, an oxygen atom, —NRX1CQ1O— (wherein Q1 represents an oxygen atom or a sulfur atom; RX1 represents a hydrogen atom, a cyano group, a formyl group, or an alkyl group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aliphatic acyl group having two to seven carbon atoms or an aromatic acyl group having seven to nineteen carbon atoms, each of which may have one or more substituents, or an aliphatic alkoxycarbonyl group having two to seven carbon atoms or an aromatic alkoxycarbonyl group having seven to nineteen carbon atoms), —OCQ1NRX1— (wherein Q1 and RX1 are as defined above), —CQ1NRX1O— (wherein Q1 and RX1 are as defined above), —ONRX1CQ1- (wherein Q1 and RX1 are as defined above), —NRX1CQ1- (wherein Q1 and RX1 are as defined above), —CQ1NRX1— (wherein Q1 and RX1 are as defined above), —NRX1aCQ1NRX1b— (wherein Q1 is as defined above; RX1a and RX1b are the same as or different from each other and each represents a hydrogen atom, a cyano group, a formyl group, or an alkyl group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aliphatic acyl group having two to seven carbon atoms or an aromatic acyl group having seven to nineteen carbon atoms, each of which may have one or more substituents, or an aliphatic alkoxycarbonyl group having two to seven carbon atoms or an aromatic alkoxycarbonyl group having seven to nineteen carbon atoms), -Q2SO2— (wherein Q2 represents an oxygen atom or —NRX10— (wherein RX10 represents a hydrogen atom, a cyano group, a formyl group, or an alkyl group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aliphatic acyl group having two to seven carbon atoms or an aromatic acyl group having seven to nineteen carbon atoms, each of which may have one or more substituents, or an aliphatic alkoxycarbonyl group having two to seven carbon atoms or an aromatic alkoxycarbonyl group having seven to nineteen carbon atoms)), —SO2Q2- (wherein Q2 is as defined above), or a group represented by the formula: (wherein Q1, Q2 and RX1 are as defined above; k represents from 0 to 5; m represents from 1 to 5; n and p are the same as or different from each other and each represents from 1 to 5; RX2, RX3, RX4, RX5, RX6, RX7, RX8 and RX9 are the same as or different from each other and each represents a hydrogen atom, a hydroxyl group, a cyano group, a halogen atom, or an alkyl group having one to six carbon atoms, an alkoxy group having one to six carbon atoms, an alkylthio group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, a hydroxyalkoxy group having one to six carbon atoms, a hydroxyalkylthio group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, an aminoalkoxy group having one to six carbon atoms, an aminoalkylthio group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, a halogeno-alkoxy group having one to six carbon atoms, a halogeno-alkylthio group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, an alkoxyalkoxy group having two to twelve carbon atoms, an alkoxyalkylthio group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, a cycloalkyloxy group having three to seven carbon atoms, a cycloalkylalkyloxy group having four to thirteen carbon atoms, a cycloalkylthio group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkenyloxy group having two to six carbon atoms, an alkenylthio group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an alkynyloxy group having two to six carbon atoms, an alkynylthio group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an aryloxy group having six to twelve carbon atoms, an arylthio group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an alkylaryloxy group having seven to eighteen carbon atoms, an alkylarylthio group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aralkyloxy group having seven to eighteen carbon atoms or an aralkylthio group having seven to eighteen carbon atoms, each of which may have one or more substituents, or —N(RX11)RX12— (wherein RX11 and RX12 are the same as or different from each other and each represents a hydrogen atom, a cyano group, a formyl group, or an alkyl group having one to six carbon atoms, a hydroxyalkyl group having one to six carbon atoms, an aminoalkyl group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an alkoxyalkyl group having two to twelve carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkenyl group having two to six carbon atoms, an alkynyl group having two to six carbon atoms, an aryl group having six to twelve carbon atoms, an alkylaryl group having seven to eighteen carbon atoms, an aralkyl group having seven to eighteen carbon atoms, an aliphatic acyl group having two to seven carbon atoms or an aromatic acyl group having seven to nineteen carbon atoms, each of which may have one or more substituents, or an aliphatic alkoxycarbonyl group having two to seven carbon atoms or an aromatic alkoxycarbonyl group having seven to nineteen carbon atoms), provided that RX2 and RX3; and RX4 and RX5 may together form a ring; and Q3 and Q4 are the same as or different from each other and each represents a single bond, an oxygen atom, (O)S(O) or NRX10 (wherein NRX10 is as defined above)); Y represents Y1—Y2— (wherein Y1 represents a 5 to 14-membered aromatic ring which has one to four substituents selected from the following Group A and may have one or more hetero atoms; and Y2 represents a single bond or a 5 to 14-membered aromatic ring which has a substituent selected from the following Group A and may have one or more hetero atoms; Group A: a hydrogen atom, a halogen atom, a hydroxyl group, a sulfamoyl group, or an alkyl group having one to six carbon atoms, a cycloalkyl group having three to seven carbon atoms, an alkoxy group having one to six carbon atoms, a cycloalkyloxy group having three to seven carbon atoms, a hydroxyalkyl group having one to-six carbon atoms, a hydroxyalkoxy group having one to six carbon atoms, a halogeno-alkyl group having one to six carbon atoms, an acylamino group having two to seven carbon atoms or a 5 to 14-membered heterocyclic group, each of which may have a substituent, provided that when two or more substituents selected from Group A are present, they may together form a ring); and the ring Z represents a 5 to 14-membered aromatic ring which has one to four substituents selected from the above-mentioned Group A, may have one or more hetero atoms and may be partially saturated. 13. The medicament according to claim 12, which is a PPAR α and γ dual agonist. 14. The medicament according to claim 12, which is a PPAR α, β(δ) and γ triple agonist. 15. The medicament according to any one of claims 12 to 14, which is an insulin sensitizer. 16. The medicament according to any one of claims 12 to 14, which is an agent for preventing or treating diabetes mellitus. 17. The medicament according to any one of claims 12 to 14, which is an agent for preventing or treating syndrome X. 18. The medicament according to any one of claims 12 to 14, which is an agent for preventing or treating diabetic complications. 19. The medicament according to any one of claims 12 to 14, which is an agent for preventing or treating hyperlipemia. 20. The medicament according to any one of claims 12 to 14, which is a lipid-lowering agent. 21. The medicament according to any one of claims 12 to 14, which is an agent for preventing or treating obesity. 22. The medicament according to any one of claims 12 to 14, which is an agent for treating osteoporosis. 23. The medicament according to any one of claims 12 to 14, which is an anti-inflammatory agent. 24. The medicament according to any one of claims 12 to 14, which is an agent for preventing or treating a disease of the digestive organs. 25. The medicament-according to claim 24, wherein the disease of the digestive organs is a disease selected from the group consisting of 1) inflammatory diseases of the digestive organs; 2) proliferative diseases of the digestive organs; and 3) ulcerative diseases of the digestive organs. 26. The medicament according to claim 25, wherein the inflammatory disease of the digestive organs is a disease selected from the group consisting of 1) ulcerative colitis; 2) Crohn's disease; 3) pancreatitis; and (4) gastritis. 27. The medicament according to claim 25, wherein the inflammatory disease of the digestive organs is ulcerative colitis. 28. An agent for preventing or treating a disease against which an action of improving insulin resistance is efficacious, which comprises the compound according to any one of claims 1 to 11 and a pharmacologically acceptable carrier. 29. The medicament according to claim 25, wherein the proliferative disease of the digestive organs is a disease selected from the group consisting of (1) benign tumor of the digestive organs; (2) digestive polyp; (3) hereditary polyposis syndrome; (4) colon cancer; (5j rectum cancer; and (6) stomach cancer. 30. The medicament according to any one of claims 12 to 14, which is an agent for preventing or treating (1) stenocardia and myocardial infarction, and sequelae thereof; (2) senile dementia; and/or (3) cerebrovascular dementia, and whose action is improving energy metabolism. 31. The medicament according to any one of claims 12 to 14, which is an immunomodulatory agent. 32. The medicament according to any one of claims 12 to 14, which is an agent for treating or preventing cancer. 33. A method of preventing or treating a disease against which an action of improving insulin resistance is efficacious, which comprises administering to a patient a pharmacologically effective amount of the compound according to any one of claims 1 to 11, a salt thereof, an ester thereof or a hydrate of them. 34. Use of the compound according to any one of claims 1 to 11, a salt thereof, an ester thereof or a hydrate of them, for producing an agent for preventing or treating a disease against which an action of improving insulin resistance is efficacious. 35. A method of preventing or treating a disease against which a PPAR α and γ dual agonist or a PPAR α, β(δ) and γ triple agonist is efficacious, which comprises administering to a patient a pharmacologically effective amount of the compound according to claim 1, a salt thereof, an ester thereof or a hydrate of them. 36. Use of the compound according to claim 1, a salt thereof, an ester thereof or a hydrate of them, for producing a PPAR α and γ dual agonist or a PPAR α, ε(δ) and γ triple agonist. |
<SOH> TECHNICAL FIELD <EOH>The present invention relates to a novel carboxylic acid compound, a salt thereof and a hydrate of them. More specifically, it relates to the above-mentioned compound which is useful for prevention or treatment of hyperglycemia, hyperlipemia and inflammatory disease, and to a medicament comprising the compound. |
Robust reception of digital broadcast transmission |
A method and apparatus for improving the reception of digitally modulated signals. A main signal and a supplemental signal are provided in the transmitter. The signals may be substantially identical except that the supplemental signal is advanced in time with respect to the main signal. The main and supplemental signals are sent from the transmitter to the receiver modulated on a signal. At the receiver, the supplemental signal is stored in a buffer. If the main signal is undesirably changed during transmission, corresponding portions of the supplement signal are substituted for the undesired portions of the main signal. |
1. A method for improving the reception of transmitted digital broadcast signals, comprising the steps of: producing a first set of program material from a first source in a transmitter; producing a second set of program material from said first source in said transmitter; time delaying said first set with respect to said second set before transmission; transmitting the first and the second set of program materials on a signal for reception by a receiver; applying said first set of program materials received in said receiver to normal reception channels of said receiver; storing said second set of program materials received in said receiver in a buffer in said receiver; detecting an undesired change in said received first set of program materials; and substituting corresponding portions of said signal stored in said buffer for any undesirably changed portions of said first set of program materials. 2. A method as claimed in claim 1 wherein said first and second sets of program material are identical. 3. A method as claimed in claim 1 wherein said first set of program material is produced with a different quality than said second set of program material. 4. A method as claimed in claim 3 wherein the quality of said first set of program material is higher than the resolution of said second set of program material. 5. In a receiver, a method for improving the reception of signals transmitted in the form of synchronously encoded main and supplemental signals, said signals being staggered in time with said supplemental signal being in advance of said main signal, comprising the steps of: storing said supplemental signal in a buffer in the receiver; processing said main signal in said receiver in a normal manner; detecting an undesired change in the processed main signal; and substituting corresponding portions of said stored supplemental signal for any undesirably changed portions of said main signal. 6. A method as claimed in claim 5 wherein said undesired change is related to a quality of said processed main signal and said change is detected by a quality measure of said processed main signal. 7. A method as claimed in claim 6 wherein said quality measure is one or more of a signal-to-noise ratio, bit error rate or packet error rate measure. 8. A method as claimed in claim 5 wherein said main signal and said supplemental signal have different resolutions. 9. A method as claimed in claim 8 wherein the resolution of said main signal is higher than the resolution of said supplemental signal. 10. A system for improving the reception of a digital signals comprising: means for producing a first set of program material from a source in a transmitter; means for producing a second set of program material from said source in said transmitter; means for delaying said first set in time with respect to said second set; means for transmitting a signal carrying said delayed first set and said second set of program material; a receiver having a first and a second channel for receiving said transmitted signal, said second channel having a buffer circuit for storing said second set of program material, and said first channel including means for processing said first set of program material; a detector in said receiver for detecting any undesired change in said processed first set; and means in said receiver for substituting corresponding portions of said stored second set for any undesirably changed portions of said first set. 11. A system as claimed in claim 9 wherein said first and second sets of program material are identical. 12. A system as claimed in claim 9 wherein the resolution of said first set of program material is different from the resolution of said second set of program material. 13. A system as claimed in claim 11 wherein the resolution of said first set of program material is higher than the resolution of said second set of program material. 14. A receiver for improving the reception of a signal transmitted in the form of synchronously encoded main and supplemental signals, said signals being staggered in time with said supplemental signal being in advance of said main signal, comprising: a buffer in said receiver for storing said supplemental signal; a signal processor in said receiver for processing said main signal in a normal manner; a detector in said receiver for detecting any undesired change in said processed main signal; and means coupled to said detector for substituting corresponding portions of said stored supplemental signal for any undesirably changed portions of said main signal. 15. Apparatus as claimed in claim 13 wherein said undesired change in said main signal is a measure of the amplitude of said main signal and said detector includes one or more of a signal-to-noise ratio, bit error rate and packet error. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates to a system for improving the reception of the signal used in digital television. More particularly, the present invention is useful in mobile digital television receivers. 2. Discussion of Related Art Any terrestrial TV system must overcome a number of problems in transmitting signals to a receiver. For example, the United States has adopted eight-level vestigial side band (8-VSB) modulation, as proposed by the Advanced Television Systems Committee (ATSC), as its terrestrial digital television system modulation standard. The VSB system, being a single carrier modulation system, is susceptible to fading caused by multipath and signal attenuation. Any of the signal fading that is frequency selective may be corrected by equalization techniques. However this can result in degraded performance when fading occurs. If the fade is deep, wide and long enough in duration, however, the signal will be lost and the demodulator system in the TV receiver will lose synchronization. Such fading is particularly severe in mobile reception of the signal used in digital television. The present invention seeks to overcome these problems by utilizing two sets of program material from a source in a transmitter. One of the sets is delayed in time with respect to the other. Thus, if the delayed set is used for reception and fading occurs, the set that is advanced in time can be substituted for the faded or missing portion of the signal. While the detailed description of the current invention below focuses on the details of the 8-VSB system, it must be recognized that the solution of the current invention is equally applicable to any digital broadcast transmission system that is subject to a fading channel environment. |
<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with principles of the present invention a method and apparatus for improving the reception of digitally modulated signals operates as follows. A main signal and a supplemental signal are provided in the transmitter. The signals may be substantially identical except that the supplemental signal is advanced in time with respect to the main signal. The main and supplemental signals are sent from the transmitter to the receiver modulated on a signal. At the receiver, the supplemental signal is stored in a buffer. If the main signal is undesirably changed during transmission, corresponding portions of the supplement signal are substituted for the undesired portions of the main signal. |
Alternative compositions and methods for the culture of stem cells |
Methods and cell culture medium for the generation of human pluripotent embryonic stem cells are disclosed. Human embryonic stem cells are cultured with human granulosa feeder cells, muscle cells, Fallopian ductal epithelial cells, bone marrow stromal cells, and skin fibroblasts and the embryonic stem cells maintain their pluripotent phenotype. The human pluripotent embryonic stem cells can be cultured without feeder cells, and in the presence of supplemental growth factors. The human pluripotent embryonic stem cells can be alternatively cultured with conditioned medium obtained from a cell culture capable of maintaining human embryonic stem cells in a pluripotent state, wherein the cell culture is a human granulosa cell culture. |
1. A human pluripotent stem cell culture, comprising a human pluripotent stem cell and a human feeder cell. 2. The human pluripotent stem cell culture of claim 1, wherein the human pluripotent stem cell is an embryonic stem cell. 3. The human pluripotent embryonic stem cell culture of claim 2, wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a bone marrow stromal cell, a granulosa cell or a fibroblast cell. 4. The human pluripotent embryonic stem cell culture of claim 1, wherein the fibroblast cell is a skin keloid fibroblast cell. 5. The human pluripotent embryonic stem cell culture of claim 4, wherein the skin keloid fibroblast cell has the identifying characteristic of ATCC CRL-1762. 6. The human pluripotent stem cell culture of claim 1, wherein the fibroblast cell is a fetal skin fibroblast cell. 7. The human pluripotent embryonic stem cell culture of claim 1, wherein the human feeder cell is a bone marrow stromal cell 8. The human pluripotent embryonic stem cell culture of claim 7, wherein the bone marrow stromal cell has the identifying characteristic of ATCC CRL-11882. 9. The human pluripotent embryonic stem cell culture of claim 1, wherein the human feeder cell is a granulosa cell. 10. The human pluripotent embryonic stem cell culture of claim 1, wherein the human feeder cell is a Fallopian ductal epithelial cell. 11. The human pluripotent embryonic stem cell culture of claim 1, wherein the human feeder cell is a skeletal muscle cell. 12. The human pluripotent embryonic stem cell culture of claim 11, wherein the skeletal muscle cell is a fetal skeletal muscle cell. 13. The human pluripotent embryonic stem cell culture of claim 1, wherein the human feeder cell maintains embryonic stem cells and expresses IL-6 and signals through the gp130 pathway. 14. The human pluripotent embryonic stem cell culture of any of claims 1, 3, 4 or 7, wherein the human feeder cell is mitotically inactivated. 15. A human pluripotent stem cell culture comprising a human pluripotent stem cell and a human feeder cell conditioned medium. 16. The human pluripotent stem cell culture of claim 15, wherein the human pluripotent stem cell is an embryonic stem cell. 17. The human pluripotent stem cell culture of claim 16, wherein the human feeder cell is a skeletal muscle cell, Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, or a fibroblast cell. 18. The human pluripotent stem cell culture of claim 15, wherein the human feeder cell maintains embryonic stem cells and expresses IL-6 and signals through the gp130 pathway. 19. The human pluripotent stem cell culture of claim 17, wherein the fibroblast cell is a skin keloid fibroblast. 20. The human pluripotent embryonic stem cell culture of claim 19, wherein the skin keloid fibroblast cell has the identifying characteristic of ATCC CRL-1762. 21. The human pluripotent stem cell culture of claim 17, wherein the fibroblast cell is a fetal skin fibroblast cell. 22. The human pluripotent embryonic stem cell culture of claim 15, wherein the human feeder cell is a bone marrow stromal cell. 23. The human pluripotent embryonic stem cell culture of claim 22, wherein the bone marrow stromal cell has the identifying characteristic of ATCC CRL-11882. 24. The human pluripotent embryonic stem cell culture of claim 15, wherein the human feeder cell is a granulosa cell. 25. The human pluripotent embryonic stem cell culture of claim 15, wherein the human feeder cell is a Fallopian ductal epithelial cell. 26. The human pluripotent embryonic stem cell culture of claim 15, wherein the human feeder cell is a skeletal muscle cell. 27. The human pluripotent embryonic stem cell culture of claim 26, wherein the skeletal muscle cell is a fetal skeletal muscle cell. 28. The human pluripotent stem cell culture of claim 1 or 15, wherein the human pluripotent embryonic stem cell culture further comprises one or more supplemental growth factors. 29. The human pluripotent stem cell culture of claim 28, wherein one or more supplemental growth factors are selected from the group consisting of SCF, OSM, CNTF, IL-6, IL-6R, FGF, BMP, TNF, and GM-CSF. 30. (Canceled) 31. (Canceled) 32. A tissue generated from the cell culture of claim 1 or 15. 33. A method of maintaining a human pluripotent stem cell culture, comprising culturing a human stem cell on a human feeder cell layer. 34. The method of claim 33, wherein the human stem cell is an embryonic stem cell. 35. The method of claim 33, wherein the human feeder cell layer is a skeletal muscle cell, a granulosa cell, a bone marrow stromal cell, or a fibroblast cell. 36. The method of claim 35, wherein the human fibroblast cell is a skin keloid fibroblast cell. 37. The method of claim 36, wherein the skin keloid fibroblast cell has the identifying characteristic of ATCC CRL-1762. 38. The method of claim 35, wherein the human fibroblast cell is a fetal skin fibroblast cell. 39. The method of claim 33, wherein the human feeder cell is a bone marrow stromal cell. 40. The method of claim 39, wherein the bone marrow stromal cell has the identifying characteristic of ATCC CRL-11882. 41. The method of claim 33, wherein the human feeder cell is a granulosa cell. 42. The method of claim 33, wherein the human feeder cell is a Fallopian ductal epithelial cell. 43. The method of claim 33, wherein the human feeder cell is a skeletal muscle cell. 44. The method of claim 43, wherein the skeletal muscle cell is a fetal skeletal muscle cell. 45. The method of claim 33, wherein the human feeder cell maintains embryonic stem cells and expresses IL-6 and signals through the gp130 pathway. 46. A method of maintaining a human pluripotent embryonic stem cell culture, comprising the steps of: (a) isolating cells from the inner cell mass of a blastocyst; (b) plating the inner cell mass cells, wherein inner cell mass-derived cell masses are formed; and (c) re-plating and maintaining the human embryonic stem cell colony on a human feeder cell layer to thereby maintain a human pluripotent embryonic stem cell. 47. The method of claim 46, wherein the wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin cell, and a skin keloid fibroblast cell. 48. The method of claim 46, wherein the inner cell mass-derived cells are dissociated, and re-plated on a mouse embryonic fibroblast cell. 49. The method of claim 46, further comprising the step of selecting a colony with the characteristics of a human embryonic stem cell after re-plating the inner cell mass cells and before re-plating the human embryonic stem cell colony. 50. The method of claim 49, wherein the wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell. 51. A method of isolating and maintaining a human pluripotent embryonic stem cell culture, comprising the steps of: (a) isolating cells from the inner cell mass of a blastocyst; (b) plating the inner cell mass cells on a human feeder cell, wherein inner cell mass-derived cell masses are formed; and (c) re-plating and maintaining the human embryonic stem cell colony on a human feeder cell to thereby isolate and maintain a human pluripotent embryonic stem cell. 52. The method of claim 51, wherein the wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell. 53. The method of claim 51, wherein the inner cell mass-derived cells are dissociated, and re-plated on a human feeder cell, and a colony is selected with the characteristics of a human embryonic stem cell prior to re-plating the selected human embryonic stem cell colony on a human feeder cell. 54. The method of claim 53, wherein the wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell. 55. A method of maintaining a human pluripotent embryonic stem cell culture, comprising culturing a selected embryonic stem cell colony in a human feeder cell conditioned medium. 56. The method of claim 55, wherein the wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell. 57. The method of claim 55, comprising the steps of: (a) isolating cells from the inner cell mass of a blastocyst; (b) plating the inner cell mass cells on a mouse embryonic fibroblast cell, wherein inner cell mass-derived cell masses are formed; (c) dissociating the inner cell mass-derived cell masses into dissociated cells; (d) re-plating the dissociated cells on a mouse embryonic fibroblast cell; (e) selecting colonies with the characteristics of human ES cells; (f) re-plating the colonies in the absence of a feeder cell; and (g) adding a human feeder cell conditioned medium to thereby maintain a human pluripotent embryonic stem cell. 58. The method of claim 57, wherein the wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell. 59. A method of isolating and maintaining a human pluripotent embryonic stem cell culture, comprising the steps of: (a) isolating cells from the inner cell mass of a blastocyst; (b) plating the inner cell mass cells on a human feeder cell, wherein inner cell mass-derived cell masses are formed; (c) dissociating the inner cell mass-derived cell masses into dissociated cells; (d) re-plating the dissociated cells on a human feeder cell; (e) selecting colonies with the characteristics of human ES cells; (f) re-plating the colonies in the absence of a feeder cell; and (g) adding a human feeder cell conditioned medium to thereby isolate and maintain a human pluripotent embryonic stem cell. 60. The method of claim 59, wherein the wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell. 61. The method of any of claims 33, 46, 51, 55, and 59, further comprising one or more supplemental growth factors. 62. The method of claim 61, wherein one or more supplemental growth factors are selected from the group consisting of SCF, OSM, CNTF, IL-6, IL-6R, FGF, BMP, TNF, and GM-CSF. 63. A human pluripotent embryonic stem cell developed by the method of any of claims 33, 46, 51, 55, and 59. 64. A tissue generated from the cell culture of any of claims 33, 46, 51, 55, and 59. 65. A method of treating a disease using the cell or cell culture of any of claims 1, 15, 33, 46, 51, 55, and 59, wherein the disease is selected from the group consisting of Parkinson's, Alzheimer's, Multiple Sclerosis, spinal cord injuries, stroke, macular degeneration, burns, liver failure, heart disease, diabetes, Duchenne's muscular dystrophy, osteogenesis imperfecta, osteoarthritis, rheumatoid arthritis, anemia, leukemia, breast cancer, solid tumors, and AIDS. 66. The method of claim 65, wherein the disease is Parkinson's or Alzheimer's. |
<SOH> BACKGROUND <EOH>Embryonic stem cells, referred to as ES cells, are derived from the inner cell mass (ICM) of fertilized eggs in blastocyst phase, and can be cultured and maintained in vitro while being kept in an undifferentiated state. ES cells are pluripotent, possessing the capability of developing into any organ or tissue type or, at least potentially, into a complete embryo. For example, ES cells can differentiate and give rise to a succession of mature functional cells. Differentiation has been shown in tissue culture and in vivo. An important application of human ES cells is their use in cell therapy: the treatment of symptoms, diseases, conditions, and disabilities with ES cell derived replacement cells and tissues. Many diseases and disorders result from disruption of cellular function or destruction of tissues of the body. A wide spectrum of diseases may be treated based upon both the possession of a population of cells having multi-lineage potential and an understanding of the mechanisms that regulate embryonic cell development. Pluripotent stem cells that are stimulated in vitro to develop into specialized cells offer the possibility of a renewable source of replacement cells and tissue to treat numerous diseases, conditions, and disabilities. Some of these diseases, conditions, and disabilities include but are not limited to Parkinson's and Alzheimer's diseases and other neurodegenerative disorders, spinal cord injuries, stroke, macular degeneration, burns, liver failure, heart disease, diabetes, Duchenne's muscular dystrophy, osteogenesis imperfecta, osteoarthritis, rheumatoid arthritis, anemia, leukemia, breast cancer and other solid tumors, and AIDS. ES cells have been derived from mouse (Evans and Kaufman, Nature 292:154-156, 1981; Martin, PNAS USA 78:7634-7639, 1981), hamster (Doetschmann et al., Dev. Biol., 127:224-227, 1999), sheep (Handyside et al., Roux's Arch. Dev. Biol., 198:48-55, 1987; Notarianni et al., J. Reprod. Fertil., 43:255-260, 1991; Piedrahita et al., Theriogenology, 34:879-901, 1990), cow (Evans et al., Theriogenology, 33:125-128, 1990), rabbit (Giles et al., Mol. Reprod. Dev., 36:130-138, 1993), mink (Sukoyan et al., Mol. Reprod. Devl, 36:148-158, 1993) and pig (Piedrahita et al., Theriogenology, 29:286, 1988; Evans et al., supra, 1990; Notarianni et al., J. Reprod. Fertil., Suppl. 41:51-56, 1990). Recently, the derivation of human ES cells has been reported (Thomson et al., Science, 282:1145-1147, 1998; Shamblott, et al, Proc. Natl. Acad. Sci. USA, 95:13726-13731, 1998; Reubinoff et al., Nature Biotechnology 18:399-404, 2000 (published erratum Nature Biotechnology 18:559, 2000)). Human ES cells have been isolated from two different tissue sources, however, the characteristics of the derived ES and embryonic germ (EG) cells are very similar (reviewed in Pera et al., J. Cell Science, 113:5-10, 2000). Thomson et al. isolated ES cells from the ICM of surplus human blastocysts that had been donated from fertility clinics (Thomson et al., supra, 1998), while Shamblott et al. isolated stem cells from the gonadal tissues of terminated pregnancies (Shamblott et al., supra, 1998). In neither case were the blastocysts or embryos created for the purpose of research. The ES cell isolated by Thomson et al., and the embryonic germ (EG) cell derived by Shamblott et al. are reported to share certain characteristics: the cells originate from a pluripotent cell population; they maintain a normal karyotype in vitro; they are immortal and can be propagated indefinitely in the embryonic state; and are capable of spontaneous differentiation into somatic cells representative of all three embryonic germ layers in teratomas or in vitro (reviewed in Pera et al., supra, 2000). The culture conditions for the human ES and EG cells differ from the culture conditions for the mouse ES cell. Mouse ES cells are typically derived using fibroblast feeder layers. The fibroblast feeder layers typically are either STO fibroblasts, a transformed cell line, or more often, the mouse ES cell is co-cultured with a primary culture of mouse embryonic fibroblasts (MEFs). These cultures are typically supplemented with leukemia inhibitory factor (LIF). The mouse ES culture medium may alternatively be supplemented with other growth factors that prevent differentiation. Examples of such growth factors are OSM, CNTF, IL-6 in combination with soluble IL-6 R, or other cytokines that signal through the gp130 pathway. Mouse ES cells remain undifferentiated indefinitely in the presence of an embryonic fibroblast feeder layer. Similarly, it is reported that a feeder layer consisting of mitotically inactivated MEFs or other fibroblasts is required for human ES cells to remain in an undifferentiated state (see e.g., U.S. Pat. No. 6,200,806; Amit et al., Developmental Biology 227:271-78, 2000; Odorico et al., Stem Cells 19:193-204, 2001). However, while mouse ES cells will also remain undifferentiated in the absence of an embryonic fibroblast feeder layer so long as the medium is supplemented with LIF (Smith et al., Nature 336:688-690, 1988; Williams et al., Nature 336:684-687, 1988), human ES cells differentiate or die in the absence of a fibroblast feeder layer, even when the medium is supplemented with LIF (Thomson et al., 1998 supra; Reubinoff et al., 2000 supra). The exact role of the MEFs in establishment and maintenance of a ES cell culture is not known. Possible roles for the MEFs include prevention of differentiation or death, or induction of proliferation, by one or some of a number of mechanisms, including, but not limited to the production of cytokines such as LIF, the provision of extracellular matrix components that provide attachment sites for the ES cells, the provision of receptor-style interactions that provide survival signals for the ES cells, the presentation of cytokines to the ES cells, the adsorption of environmental toxins such as heavy metals, or the secretion of growth factors necessary to support the ES cell. While fibroblast feeder layers are critical to the survival and non-differentiation of the human ES cell, mouse embryonic fibroblast feeder cells are labor-intensive to derive, and can vary between lots (Amit et al., supra, 2000). The development and use of non-fibroblast feeder cell layers that are not labor-intensive to establish, and that offer greater consistency than embryonic fibroblast cells would be an advantage to the field. Moreover, the potential applications for the human ES cell are limited when the ES cell is cultured in the presence of non-human feeder cell layers. Ideally, a human ES cell could be cultured with human feeder cell layers, or could be cultured in the presence medium conditioned by human cells. There is no evidence in the prior art showing the long-term isolation and/or maintenance of human pluripotent ES cells on non-fibroblast feeder cells. Others have attempted to isolate human ES cells on non-fibroblast feeder cells, but have not succeeded in maintaining the human ES cells in a pluripotent state for long or indefinite periods of time. Bongso et al. cultured human blastocysts on oviduct epithelial cells in the presence of human LIF (Bongso et al., Human Reproduction 9:2100-2117, 1994). Bongso et al. then separated the ICMs from the trophoblast and feeder cells, and replated the ICM-derived cells in the absence of a feeder layer. This method supported the growth of ICM-derived cells for two subcultures, or at least 18 days, without differentiation; however, the cells subsequently differentiated into fibroblasts or died. Similarly, there is no evidence in the prior art showing the long-term isolation and or maintenance of human pluripotent ES cells in the presence of conditioned media from human cell types. Although the co-culture of human ES cells with conditioned media from mouse embryonic fibroblasts has been reported (Xu et al., Keystone Symposia Abstract Book, Pluripotent Stem Cells: Biology and Applications, February 2001, A. 133), conditioned medium from human cell cultures has not been reported to maintain human ES cells in a pluripotent state. Granulosa cells are the cells that support and nourish the oocyte in the ovary. Granulosa cells are thought to arise from a population of stem cells (Rodgers et al., Mol Cell Endocrinol 22;171(1-2):41-8, 2001; Lavranos et al., Biology of Reproduction 61, 358-366, 1999; Rodgers et al., J Reprod Fertil Suppl 54:343-52, 1999). Initially, a primordial follicle consists of an oocyte surrounded by a single layer of flattened epithelial pregranulosa cells. As the follicle grows, the granulosa cells proliferate radially, reaching a total of tens of thousands of cells in the preovulatory state. Granulosa cells cease dividing at ovulation, and after ovulation, granulosa cells differentiate into the luteal cells of the developing corpus luteum in the ovary. See also generally, Weiss, et al., Eur J Endocrinol. 144(6):677-85, June 2001; Stevenson, Indian J Exp Biol. 2000 December;38(12):1183-91; Hosokawa et al., Endocrinol; 138(11):4679-4687, 1998; Hosokawa et al., Endocrinology 138(11):4688-4700, 1998; Byong-Lyul et al., Mol and Cell. Endocrinology 120:169-176, 1996. Researchers have attempted to use pig granulosa cells as feeder cell to support the isolation and/or maintenance of pig and cow ES cells (Vasil'eva and Vasil'ev, 1995 Russian J. Dev. Biol., 26:167-72, Translated from Ontogenez, 26:206-12, 1995; Vasil'ev and Vasil'eva, 1995 Russian J. Dev. Biol., 26:163-66, Translated from Ontogenez, 26:201-205, 1995). Pig embryos did not attach to pig granulosa cells, and while pig embryonic cells did attach to pig granulosa cells, the cultured embryonic cells produced trophoblast-like cells and not ES-like cells (Vasil'ev and Vasil'eva, 1995 supra). Cow embryos did attach to pig granulosa cells, and formed ES-like cells that could be maintained in culture on granulosa cell feeder layers for three transfers without differentiating (Vasil'eva and Vasil'ev, 1995 supra). Thus the culture conditions which were successful with one large domestic animal were not successful for another domestic animal. The authors acknowledge that the techniques useful for the isolation of ES cells from large domestic animals will differ from those useful for the isolation of ES cells from mice. It is therefore not predictable that a technique successful for the isolation and short-term maintenance of ES-like cells from cows will be useful for the isolation and/or maintenance of human pluripotent stem cells. For the treatment of many human diseases by cell therapy, it may be necessary to direct the differentiation of human ES cells in culture, prior to transplanting the ES cells into the subject. In vitro differentiation may be directed by the addition of supplemental growth factors to the culture medium. Various soluble factors have been used to induce differentiation of mouse ES cells down specific lineages: IL-3 directs cells to become macrophages, mast cells or neutrophils (Wiles, M. V., and Keller, G., Development 111:259-267, 1991); IL-6 directs cells to the erythroid lineage (Biesecker, L. G. and Emerson, S. G., Exp. Hematol., 21:774-778, 1993); retinoic acid induces neuron formation (Slager et al., Dev. Genet. 14:212-224, 1993; Bain et al., Dev. Biol. 168:342-357, 1995); and transforming growth factor (TGF)-β1 induces myogenesis (Slager et al., supra, 1993; Rohwedel et al., Dev. Biol. 164:87-101, 1994). Most of these studies were performed on ES cells that had been induced to form embryoid bodies in culture (Slager et al., supra, 1993; Bain et al., Supra, 1995; Rohwedel et al., supra, 1994). While the use of the soluble factors induced differentiation of different cell lineages, the factors did not induce differentiation of only one cell type; instead, the factors changed the proportion of the different cell types in the cultures. The most comprehensive analysis of human ES cells examined the effects of eight growth factors on the differentiation of cells grown first as embryoid bodies and then disaggregated (Schuldiner et al., 2000; PNAS USA 97:11307-11312). Schuldiner et al. applied basic fibroblast growth factor (bFGF), TGF-β1, activin-A, bone morphogenetic protein 4 (BMP-4), hepatocyte growth factor (HGF), epidermal growth factor (EGF), β nerve growth factor (βNGF), and retinoic acid to the cells, and determined the effects on cell-specific gene expression and cell morphology. TGF-β1 and activin-A induced differentiation of muscle cells; retinoic acid, bFGF, BMP-4, and EGF induced differentiation of ectodermal and mesodermal cells; while NGF and HGF allowed differentiation of cells from all three germ layer lineages. However, none of the growth factors tested directed the differentiation of a uniform and singular cell type. Finally, Reubinoff et al. were able to isolate human neuronal-lineage cells in a relatively pure form from a human ES culture (Reubinoff et al., 2000 supra). The differentiation of neuronal-lineage cells occurred spontaneously when the human ES cell was cultured on mouse embryonic fibroblasts. Reubinoff et al. isolated the areas of differentiated cells and re-plated the cells in serum free medium. The cells formed spheres, which were again re-plated and allowed to attach to an adhesive substrate. Although this procedure provided a relatively pure population, these cells cannot be used for the treatment of humans since they were cultured on mouse feeder cells. Additionally, the differentiation was not directed towards a specific lineage. There is a need, therefore, to develop methods for the directed differentiation of human ES cells that are not cultured with mouse feeder cells. These methods may involve the addition of a supplemental growth factor to the culture medium. There is a need, therefore, to establish culture conditions, such as human feeder cells, or conditioned medium, that allow for greater reproducibility and consistency among cultures, and that allow for the use of the human ES cells in cell therapies. There is also a need to establish methods for selectively differentiating human ES cells into precursors and into the desired and uniform cell lineages, such as the neuronal cell lineage. Large, purified populations of selectively differentiated ES cells will provide a potentially limitless source of cells for cellular therapy treatments and further drug discovery. Selectively differentiated, and reversibly differentiated, ES cells can be used for cell therapy, and transplanted into subjects to treat a number of different conditions and diseases. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides for the isolation and/or maintenance of human pluripotent ES cells. The present invention provides a human pluripotent ES cell culture that includes a human ES cell and a human feeder cell. In a preferred embodiment, the human feeder cell is selected from the group consisting of a human fibroblast cell, a MRC-5 cell, a human embryonic kidney cell, a mesenchymal cell, an osteosarcoma cell, a keratinocyte, a chondrocyte, a Fallopian ductal epithelial cell, a liver cell, a cardiac cell, a bone marrow stromal cell, a granulosa cell, a skeletal muscle cell, and an aortic endothelial cell. In a more preferred embodiment the human feeder cell is selected from the group consisting of a skin keloid fibroblast cell, a fetal skin fibroblast cell, a bone marrow stromal cell, or a skeletal muscle cell. The invention further provides methods for the isolation and maintenance of a human pluripotent ES cell in the presence of a human feeder cell. The invention provides a human pluripotent ES cell culture that includes the human pluripotent ES cell and a conditioned medium. The invention further provides the conditioned medium maintains the human pluripotent ES cell in a pluripotent state. In a preferred embodiment the conditioned medium is obtained from the human feeder cell. The invention further provides for a human pluripotent ES cell culture comprising the human pluripotent ES cell and a human feeder cell factor conditioned medium. The invention provides for a human pluripotent ES cell cultured in the presence of a supplemental growth factor. The invention provides that the supplemental growth factor is selected from the group consisting of SCF, OSM, CNTF, IL-6 in combination with soluble IL-6R, FGF, BMP, TNF, and GM-CSF. In a preferred embodiment, a supplemental growth factor is added to the conditioned medium obtained from the human feeder cell. The invention provides a method of maintaining a human pluripotent ES cell culture, comprising culturing selected ES colonies on a human feeder cell layer. In a preferred embodiment, the feeder cell is selected from the group consisting of a human fibroblast cell, a MRC-5 cell, a human embryonic kidney cell, a mesenchymal cell, an osteosarcoma cell, a keratinocyte, a chondrocyte, a Fallopian ductal epithelial cell, a liver cell, a cardiac cell, a bone marrow stromal cell, a granulosa cell, a skeletal muscle cell, a muscle cell and an aortic endothelial cell. In a more preferred embodiment, the human feeder cell is selected from the group consisting of a skin keloid fibroblast cell, a fetal skin fibroblast cell, a bone marrow stromal cell, a Fallopian ductal epithelial cell, or a skeletal muscle cell. In a preferred embodiment, the feeder cell expresses leukemia inhibitory factor, steel cell factor, and FGF. The invention further provides a method of maintaining a human pluripotent ES cell culture comprising the steps of: (a) isolating cells from the inner cell mass of a blastocyst; (b) plating the inner cell mass cells, wherein inner cell mass-derived cell masses are formed; and (c) re-plating and maintaining the cell masses on a human feeder cell layer to thereby maintain a human pluripotent ES cell. The invention further provides a method of isolating and maintaining a human pluripotent ES cell culture, comprising the steps of: (a) isolating cells from the inner cell mass of a blastocyst; (b) plating the inner cell mass cells on a human feeder cell layer, wherein inner cell mass-derived cell masses are formed; (c) dissociating the mass into dissociated cells; (d) re-plating the dissociated cells on a human feeder cell layer; (e) selecting colonies with the characteristics of human ES cells; (f) re-plating and maintaining the colonies on a human feeder cell layer to thereby maintain a human pluripotent ES cell. The present invention provides a method for isolating and maintaining a human pluripotent ES cell, further comprising culturing the human pluripotent ES cell in the presence of conditioned medium. The invention provides that the conditioned medium is obtained from a human cell. In a more embodiment, the human cell is selected from the group consisting of a skin keloid fibroblast cell, a fetal skin fibroblast cell, a bone marrow stromal cell, a Fallopian ductal epithelial cell or a skeletal muscle cell. The conditioned medium may further comprise a supplemental growth factor. The invention further provides for the isolation and use of a human feeder cell factor. The cell factor can be isolated from a human feeder cell, or the conditioned medium obtained from a human feeder cell. The present invention provides a method of isolating and maintaining a human pluripotent ES cell comprising the steps of: (a) isolating cells from the inner cell mass of a blastocyst; (b) plating the inner cell mass cells on a human feeder cell layer, wherein inner cell mass-derived cell masses are formed; (c) dissociating the mass into dissociated cells; (d) re-plating the dissociated cells on a human feeder cell layer; (e) selecting colonies with the characteristics of human ES cells; and (f) re-plating and maintaining the colonies in a human conditioned medium to thereby maintain a human pluripotent ES cell. In another embodiment, the invention provides for maintaining the human pluripotent ES cell that was re-plated in a human feeder cell conditioned medium further in the presence of a supplemental growth factor, wherein the supplemental growth factor is selected from one or more of the group consisting of SCF, OSM, CNTF, IL-6 in combination with soluble IL-6R, FGF, BMP, TNF, and GM-CSF. The invention further provides culture additions, such as feeder cells or conditioned medium, for the selective differentiation of human ES cells, and for the selectively reversible differentiation of human ES cells. The invention further provides for a human pluripotent ES cell generated by any of the methods described herein. The invention additionally provides for a tissue generated by any of the human pluripotent ES cells described herein. The invention further provides that the cell and tissues generated using the invention can be used in cell therapy to experimentally, therapeutically or prophylactically treat a disease or condition in a human or animal. Preferably the disease is selected from the group consisting of Parkinson's, Alzheimer's, Multiple Sclerosis, spinal cord injuries, stroke, macular degeneration, burns, liver failure, heart disease, diabetes, Duchenne's muscular dystrophy, osteogenesis imperfecta, osteoarthritis, rheumatoid arthritis, anemia, leukemia, breast cancer, solid tumors, and AIDS. In preferred embodiments, the disease is Parkinson's or Alzheimer's. |
Air mattress |
The invention relates to an air mattress comprised of at least one air chamber made of strips of an air-tight, water vapor permeable or water vapor impermeable material that are welded or glued together. The air chamber is provided with a closable valve for letting air in and out, and the air chamber is filled with a down feather material or a synthetic fiber insulation material. |
1-8. (canceled) 9. An air mattress, comprising at least one air chamber of contiguous, permanently attached lengths of an air-impermeable material, said air chamber including a closable valve for admitting and releasing air; wherein said air chamber is filled with a thermally insulting filler material; and said valve is lined with an open-pore material for preventing the escape of said thermally insulating filler material. 10. The air mattress of claim 9, wherein said air mattress includes bottom and top surfaces; and said air chamber is subdivided by at least one strut, said strut being secured to said bottom and top surfaces of said air mattress. 11. The air mattress of claim 10, wherein said at least one strut is air-permeable. 12. The air mattress of claim 10, wherein said at least one strut is air-impermeable. 13. The air mattress of claim 10, wherein said at least one strut is permanently attached to said bottom and top surfaces of said air mattress. 14. The air mattress of claim 10, wherein said at least one strut includes at least two struts; and wherein said at least two struts are evenly disposed within said air mattress and are disposed transverse to at least one of said bottom and top surfaces of said air mattress. 15. The air mattress of claim 9, wherein said valve is a safety valve for preventing the outflow of air from the interior of said air mattress. 16. The air mattress of claim 9, wherein said valve is adapted to be connected to a pump having a flexible casing provided with an inlet valve, and said casing is filled with an open-pore, self-expanding foam. 17. The air mattress of claim 9, wherein said thermally insulating filler material is down. 18. The air mattress of claim 9, wherein said thermally insulating filler material is a synthetic insulating fiber. 19. The air mattress of claim 9, wherein said air permeable material is water-vapor permeable. 20. The air mattress of claim 9, wherein said air permeable material is water-vapor impermeable. 21. The air mattress of claim 10, wherein said struts are directly attached to said bottom and top surfaces of said air mattress. 22. The air mattress of claim 10, wherein said struts are indirectly attached to said bottom and top surfaces of said air mattress through an attachment means. 23. The air mattress of claim 22, wherein said attachment means includes tape. 24. The air mattress of claim 10, wherein said at least one strut is perpendicular to said at least one of said bottom and top surfaces of said air mattress. 25. The air mattress of claim 10, wherein said at least one strut is obliquely disposed to said at least one of said bottom and top surfaces of said air mattress. |
Dna encoding rat bombesin receptor subtype-3 (brs-3) and uses thereof |
A rat bombesin receptor subtype-3 has been isolated, cloned and sequenced. This receptor is characteristic of the G-protein family of receptors. Isolation of rat bombesin receptor subtype-3 may be used to screen and identify novel bombesin receptor modulators that may contribute to the regulation of endocrine processes, metabolism, or the cell cycle. Such compounds may be used in the treatment of conditions that result from deregulated expression of bombesin receptor subtype-3. |
1. An isolated nucleic acid molecule, comprising a sequence of nucleotides that encodes a rat bombesin receptor subtype-3 (BRS-3) protein as set forth in SEQ ID NO:2. 2. The isolated nucleic acid molecule of claim 1 wherein the nucleic acid is DNA. 3. The isolated nucleic acid molecule of claim 1 wherein the nucleic acid is mRNA. 4. The isolated nucleic acid molecule of claim 1 wherein the nucleic acid is cDNA. 5. The isolated nucleic acid molecule of claim 1 wherein the sequence of nucleotides comprises the sequence of nucleotides set forth in SEQ ID NO:1. 6. A vector comprising the nucleic acid molecule of claim 1. 7. A host cell comprising the vector of claim 6. 8. A process for expressing a rat BRS-3 protein in a recombinant host cell, comprising: (a) introducing a vector comprising the nucleic acid of claim 1 into a suitable host cell; and, (b) culturing the host cell under conditions which allow expression of said rat BRS-3 protein. 9. (canceled) 10. An isolated and purified rat bombesin receptor subtype-3 (BRS-3) polypeptide comprising a sequence of amino acids as set forth in SEQ ID NO:2. 11. A method for identifying compounds that modulate rat bombesin receptor subtype-3 expression, comprising contacting a test compound with the rat BRS-3 polypeptide of claim 10 and determining whether the test compound interacts with rat bombesin receptor subtype-3. 12. The polypeptide of claim 10 wherein the amino acids Y298E299S300 are replaced with the amino acids S298Q299T300. 13. The polypeptide of claim 10 wherein the amino acids D306V307P308 are replaced with the amino acids A306M307H308. 14. A chimeric receptor comprising a sequence of amino acid residues as set forth in SEQ ID NO:2, wherein a region defined by amino acid residues 294-311 is replaced by a region defined by amino acid residues 294-311 of SEQ ID NO:4. 15. A chimeric polypeptide comprising an amino acid sequence of rat BRS-3 as set forth in residues 294-311 of SEQ ID NO:2; linked with a non-rat BRS-3 amino acid sequence. 16. An isolated nucleic acid molecule, comprising a sequence of nucleotides that encodes the chimeric polypeptide of claim 15. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Bombesin, a tetradecapeptide originally isolated from frog skin, represents the first member of a large family of regulatory peptides named bombesin-like peptides. Two bombesin-like peptides, gastrin-releasing peptide (GRP) (McDonald et al., Biochem. Biophys. Res. Commun. 90: 227-33 (1979)) and neuromedin B (NMB) (Minamino et al., Biochem. Biophys. Res. Commun. 114: 541-548 (1983)) have been found in mammals. The orphan receptor bombesin receptor subtype-3 (BRS-3, also named BB3) is one of three subtypes of bombesin receptors, which was identified based on its high degree of homology to mammalian bombesin receptors. BRS-3 is a member of the G protein-coupled receptor superfamily and has been cloned from human, mouse and sheep (Whitley et al., J. Mol. Endocrinol. 23: 107-16 (1999)). A naturally occurring high affinity ligand for BRS-3 has not been identified. However, a synthetic peptide, [D-Tyr6-betaAla11-Phe13-Nle14] bombesin (6-14) (hereinafter dYB, comprising SEQ ID NO:10) was shown to have high affinity for all three human bombesin receptor subtypes (Pradhan et al., Eur. J. Pharmacol. 343: 275-87 (1998)). However, the human receptor pharmacology does not always extend to other species homologs. WO 01/10889 (published Feb. 15, 2001) discloses a molecule described as rat BRS-3. However, this receptor molecule appears not to be a full-length receptor as it is only 382 amino acids long. Bombesin, bombesin-like peptides and related receptors participate in a diverse array of physiological processes. BRS-3 has been implicated in the regulation of neuroendocrine function and energy metabolism (Ohki et al. Nature 390: 165-69 (1997)). Mice lacking functional BRS-3 are hyperphagic and have a reduced metabolic rate, which leads to the development of obesity, hypertension and diabetes as adults. Additionally, bombesin-like peptides may contribute to the pathogenesis of some human carcinomas (For review, see Lebacq-Verheyden et al., in Handbook of Experimental Pharmacology, Sporn, M. N. and Roberts, A. B., eds., Vol. 95, pp. 71-124, Springer-Verlag, Berlin). Despite the identification of the cDNA clones encoding bombesin receptor subtypes mentioned above, it would be advantageous to identify additional mammalian genes encoding bombesin receptor subtypes in order to allow screening to identify novel bombesin receptor modulators that may contribute to the regulation of endocrine processes, metabolism, or the cell cycle. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to an isolated or purified nucleic acid molecule (polynucleotide) which encodes a novel rat bombesin receptor subtype-3 (hereinafter rBRS-3). The DNA molecules disclosed herein may be transfected into a host cell of choice wherein the recombinant host cell provides a source for substantial levels of an expressed functional rBRS-3 protein (SEQ ID NO:2). This receptor protein provides a screening target to identify modulators of bombesin and bombesin-like peptides, which may be involved in the pathogenesis of a variety of human disorders when deregulated. The present invention also relates to isolated nucleic acid molecules comprising a sequence of nucleotides that encode a rat BRS-3 protein as set forth in SEQ ID NO:2. The present invention further relates to an isolated nucleic acid molecule which encodes mRNA which expresses a novel rat BRS-3 protein, this DNA molecule comprising the nucleotide sequence disclosed herein as SEQ ID NO:1. The present invention also relates to biologically active fragments or mutants of SEQ ID NO:1, which encode mRNA expressing a novel rBRS-3 protein. Any such biologically active fragment and/or mutant will encode either a protein or protein fragment which at least substantially mimics the pharmacological properties of the rBRS-3 protein, including but not limited to the rBRS-3 protein as set forth in SEQ ID NO:2. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a functional rBRS-3 protein in a eukaryotic cell, such as Xenopus oocytes, so as to be useful for screening for agonists and/or antagonists of rat BRS-3 activity. The present invention further relates to a process for expressing a rat BRS-3 protein in a recombinant host cell, comprising: (a) introducing a vector comprising the nucleic acid of claim 1 into a suitable host cell; and, (b) culturing the host cell under conditions which allow expression of said rat BRS-3 protein. A preferred aspect of this portion of the present invention is disclosed in FIG. 1 (SEQ ID NO:1), which shows a DNA molecule encoding a novel rBRS-3 protein (SEQ ID NO:2). The isolated nucleic acid molecules of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA). The present invention also relates to recombinant vectors and recombinant host cells, both prokaryotic and eukaryotic, which contain the nucleic acid molecules disclosed throughout this specification. A preferred aspect of this portion of the present invention is a substantially purified form of a rat BRS-3 protein which consists of the amino acid sequence disclosed in FIG. 2 (SEQ ID NO:2). Another preferred aspect of the present invention relates to a substantially purified, fully processed (including proteolytic processing, glycosylation and/or phosphorylation), mature BRS-3 protein obtained from a recombinant host cell containing a DNA expression vector comprising nucleotide sequence as set forth in SEQ ID NO:1, which expresses the rBRS-3 protein. It is especially preferred that the recombinant host cell be a eukaryotic host cell, such as a mammalian cell line, or Xenopus oocytes, as noted above. Another preferred aspect of the present invention relates to a substantially purified membrane preparation, partially purified membrane preparation, or cell lysate which has been obtained from a recombinant host cell transformed or transfected with a DNA expression vector which comprises and appropriately expresses a complete open reading frame as set forth in SEQ ID NO:1, resulting in a functional form of rBRS-3. The subcellular membrane fractions and/or membrane-containing cell lysates from the recombinant host cells (both prokaryotic and eukaryotic as well as both stably and transiently transformed cells) contain the functional and processed proteins encoded by the nucleic acids of the present invention. This recombinant-based membrane preparation may comprise a rat BRS-3 protein and is essentially free from contaminating proteins, including but not limited to other rat source proteins. Therefore, a preferred aspect of the invention is a membrane preparation which contains a rat BRS-3 comprising the functional form of the full length BRS-3 protein as disclosed in FIG. 2 (SEQ ID NO:2). These subcellular membrane fractions will comprise either wild type and/or mutant variations which are biologically functional forms of rat BRS-3 at levels substantially above endogenous levels. Any such protein will be useful in various assays described throughout this specification to select for modulators of the rBRS-3 protein. A preferred eukaryotic host cell of choice to express the rBRS-3 molecules of the present invention is a mammalian cell line, or Xenopus oocytes. The present invention also relates to biologically active fragments and/or mutants of a rat BRS-3 protein, comprising the amino acid sequence as set forth in SEQ ID NO:2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for selective modulators, including but not limited to agonists and/or antagonists for rat bombesin and bombesin-like peptide receptor pharmacology. A preferred aspect of the present invention is disclosed in FIG. 2 (SEQ ID NO:2), which indicates the amino acid sequence of the rat BRS-3 protein of the present invention. Characterization of this protein will allow for screening to identify novel bombesin receptor subtype-3 modulators that may have a role in the regulation of endocrine processes or metabolism. As noted above, heterologous expression of rat BRS-3 disclosed herein is contemplated at levels substantially above endogenous levels and will allow for the pharmacological analysis of compounds which may contribute to the pathogeneis of a variety of human disorders associated with deregulated BRS-3 expression. Heterologous cell lines expressing a functional rat BRS-3 (e.g., functional forms of SEQ ID NO: 2), can be used to establish functional or binding assays to identify novel BRS-3 modulators that may be useful in the development of therapeutics for human diseases associated with deregulated BRS-3 expression. The rat BRS-3 receptor proteins of the present invention may be in the form of the “mature” protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production. The present invention also relates to rat BRS-3 fusion constructs, including but not limited to fusion constructs which express a portion of the rat BRS-3 protein linked to various markers, including but in no way limited to GFP (Green fluorescent protein), the MYC epitope, GST, and Fc. Any such fusion constructs may be expressed in the cell line of interest and used to screen for modulators of the rat BRS-3 protein disclosed herein. The present invention relates to methods of expressing rat BRS-3 proteins and biological equivalents disclosed herein, assays employing these gene products, recombinant host cells which comprise DNA constructs which express these proteins, and compounds identified through these assays which act as agonists or antagonists of BRS-3 activity. As used herein, “substantially free from other nucleic acids” means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other nucleic acids. As used interchangeably, the terms “substantially free from other nucleic acids,” “substantially purified,” “isolated nucleic acid” or “purified nucleic acid” also refer to DNA molecules which comprise a coding region for a rat BRS-3 protein that has been purified away from other cellular components. Thus, a rat BRS-3 DNA preparation that is substantially free from other nucleic acids will contain, as a percent of its total nucleic acid, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-rat BRS-3 nucleic acids. Whether a given rat BRS-3 DNA preparation is substantially free from other nucleic acids can be determined by such conventional techniques of assessing nucleic acid purity as, e.g., agarose gel electrophoresis combined with appropriate staining methods, e.g., ethidium bromide staining, or by sequencing. As used herein, “substantially free from other proteins” or “substantially purified” means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other proteins. Thus, a rat BRS-3 protein preparation that is substantially free from other proteins will contain, as a percent of its total protein, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-rat BRS-3 proteins. Whether a given rat BRS-3 protein preparation is substantially free from other proteins can be determined by such conventional techniques of assessing protein purity as, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with appropriate detection methods, e.g., silver staining or immunoblotting. As used interchangeably, the terms “substantially free from other proteins” or “substantially purified”, or “isolated rat BRS-3 protein” or “purified rat BRS-3 protein” also refer to rat BRS-3 protein that has been isolated from a natural source. Use of the term “isolated” or “purified” indicates that rat BRS-3 protein has been removed from its normal cellular environment. Thus, an isolated rat BRS-3 protein may be in a cell-free solution or placed in a different cellular environment from that in which it occurs naturally. The term isolated does not imply that an isolated rat BRS-3 protein is the only protein present, but instead means that an isolated rat BRS-3 protein is substantially free of other proteins and non-amino acid material (e.g., nucleic acids, lipids, carbohydrates) naturally associated with the rat BRS-3 protein in vivo. Thus, a rat BRS-3 protein that is recombinantly expressed in a prokaryotic or eukaryotic cell and substantially purified from this host cell which does not naturally (i.e., without intervention) express this BRS-3 protein is of course “isolated rat BRS-3 protein” under any circumstances referred to herein. As noted above, a rat BRS-3 protein preparation that is an isolated or purified rat BRS-3 protein will be substantially free from other proteins and will contain, as a percent of its total protein, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-rat BRS-3 proteins. As used herein, “a conservative amino acid substitution” refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue. Examples of such conservative substitutions are: substitution of one hydrophobic residue (isoleucine, leucine, valine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for aspartic acid). As used herein, “rBRS” refers to a—rat bombesin receptor subtype-3— As used herein, the term “mammalian” will refer to any mammal, including a human being. |
Chronic pathogen-expressing cell lines |
This application provides a method to establish and construct cell lines expressing pathogens without destruction of the host cells. The invention allows for the formation of cell lines for the purpose of continuous expression, release, and harvesting of the pathogen and maintain the consistency of the final biological pro duct Although the invention is intended for pathogen antigen expression, the invention allows for the production of any antigen by the described methods. The establishment of a chronically infected celline can be used for reagent, diagnostic, quantification, or vaccine purposes. We have used the procedure to select for a host cell line that naturally adapts to HIV-1 replication without affecting the host cell's ability to survive. This allowed for the establishmentof a chronic HIV-1 expressing cell line that continuously expresses HIV-1 particles. |
1. A biologically constructed cell line(s) that has been modified to produce budding particles (i) where the components required for the formation of the budding particles are stability integrated into the said cell line(s) for chronic production of the budding particles; (ii) where the released budding particles could be harvested as a preparation; and (iii) the preparation could be used as a reagent in diagnostic test and/or as a therapeutic agent. 2. The biologically constructed cell line(s) of claim 1 is a cell line selected to tolerate the continuous expression and assemble of intact budding particles that could be harvested from the cell-free culture supernatant. 3. The biologically constructed cell line of claim 1 is a cell line(s) genetically modified to tolerate the continuous expression and assemble of intact budding particles that could be harvested from the cell-free culture supernatant. 4. The genetic modification in claim 3 introduces exogenous genes that are incorporated specifically within the intact budding particles. 5. The genetic modification in claim 3 introduces exogenous genes that are associated passively with the intact budding particles. 6. The exogenous genes in claim 4 are expressed on the surface of said biologically constructed cell line(s) of claim 1. 7. The exogenous gene(s) in claim 6 is selected from the group consisting of a protein, a polypeptide, an antibody, a glycolipid, a lipid, and/or an antigen. 8. The exogenous gene(s) in claim 7 is selected from a pathogen. 9. The exogenous gene(s) in claim 7 is selected from an infectious agent. 10. The exogenous gene(s) in claim 7 is selected from a tumor. 11. The tumor in claim 8 is a tumor-specific transplantation antigen associated with a cancer disease. 12. The budding particles produced from the biologically constructed cell line(s) of claim 1 are infectious. 13. The budding particles produced from the biologically constructed cell line(s) of claim 1 are infectious, but not replication competent. 14. The budding particles produced from the biologically constructed cell line(s) of claim 1 are non-infectious-a virus-like-particle (VLP). 15. The components required for the formation of the budding particles of claim 2 are introduced by infection into the host cell line(s) of claim 1. 16. The components required for the formation of the budding particles of claim 3 are introduced by an expression vector into the host cell line(s) of claim 1. 17. The components required for the formation of the budding particles of claim 4 and claim 5 are introduced by an expression vector into the host cell line(s) of claim 1. 18. The pathogen selected in claim 8 is the entire pathogen. 19. The infectious disease selected in claim 9 is the entire agent that cause said disease. 20. A method for establishing cell line(s) of claim 1 are capable of producing budding particles that incorporates (i) exogenous antigens and (ii) recombinant materials that are released from the cells for collection within the culture supernatant. 21. The harvested budding particles from the cell line(s) of claim 1 can be harvested from culture supernatants. 22. The harvested budding particles of claim 21 can be used as a pharmaceutical drug carrier for therapeutic purposes. 23. The harvested budding particles of claim 21 can be used as a lysate for diagnostic purposes. 24. The harvested budding particles of claim 21 can be used as a starting material for protein isolation. 25. The harvested budding particles of claim 21 can be used as a source for nucleic acid isolation. 26. The cell line(s) of claim 1 can be used as a starting material for protein and/or nucleic acid isolation. 27. The cell line(s) of claim 1 can specifically express human immunodeficiency virus type 1. 28. The harvested budding particles of claim 21 can be human immunodeficiency virus type 1. 29. Biological material expressed from the cell line(s) established in claim 1 could be further induced to express budding particles claim in 21 by treatment of cell line(s) with biological or non-biological chemicals. 30. A use of the budding particles claimed in 21 could be to treat or prevent a disease in an animal, including humans, by administrating to the animal a prophylactic or therapeutic effective amount of inactivated harvested budding particles. 31. The cell line(s) producing the budding particles in claim 30 have been modified to have at least (i) one exogenous antigen such that said antigen can be presented to initiate an immune response; (ii) also contains at least one exogenous antigen fragment bound to a primary surface molecule; (iii) also expresses at least one co-stimulatory molecule. 32. The cell line(s) producing the budding particles in claim 30 have been modified to express a molecule(s) identified in claim 7 that can inhibit a human pathogen. 33. The human pathogen in claim 32 is biological or chemical. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Most, if not all pathogens, destroy their host cell during pathogen replication. Death of living cells can follow more than one possible scenario. It may result from an external injury, from cell killing during acute infection with cytopathic pathogens, or it may be the outcome of activating an internal pathway for cell suicide—programmed cell death. Programmed cell death or apoptosis is a controlled process by which unwanted cells are selectively eliminated. Apoptosis is a normal physiological process of eliminating unwanted cells from living organisms during embryonic and adult development, but can also be induced in cells following exposure to a pathogen. The mechanism by which pathogens cause cell death—either direct killing or indirect—varies with the pathogen and the host cell in question. Controversy surrounds the cause of pathogen-induced cell death in even in the most extensively studied pathogens. For example, in human immunodeficiency virus type 1 (HIV-1)-initiated killing of CD4+ cells T cell death has been reported to be caused by syncytium formation-interaction of the envelope glycoprotein (gp120) with CD4 and subsequent fusion of the cells; influenced by type 1/type 2 cytokine modulation; mediated by specific cell death proteases (caspases) that function in the distal portions of the proteolytic cascades involved in apoptosis; membrane tumor necrosis factor induced cooperative signaling of tumor necrosis factor membrane receptors p55 and p75; Fas-induced apoptosis; and direct interaction of HIV gp120 envelop with the T cell CD4 molecule. Although agreement in the mechanism of cell death is disputed, it is clear that pathogen replication results in host cell destruction. Pathogens replication can only occur inside host cells, commandeering the cell's machinery to reproduce. Infection typically begins when a pathogen encounters a cell with a specific cellular surface receptor molecule that matches the proteins found on the virus. The membranes of the virus and the cell will fuse, followed by release of viral nucleic acids, proteins and enzymes into the cell. Cell-to-cell spread of the pathogen also can occur through the fusion of an infected cell with an uninfected cell. The pathogen nucleic acid moves to the cell nucleus, where in most cases is spliced into the host DNA (for RNA-based pathogens, pathogen encoded reverse transcriptase converts RNA into DNA). Once incorporated into the cellular genome, RNA copies are made that are read by the host cell protein-malcing machinery. After the MRNA is processed in the cell nucleus, it is transported to the cytoplasm. The pathogen co-opts the cellular protein-making machinery to make long chains of viral proteins and enzymes, using the pathogen MRNA as a template. Newly made pathogen proteins, enzymes and nucleic acids gather inside the cell, while the pathogen envelope proteins aggregate within cellular membranes. An immature viral particle forms and pinches off from cellular membranes, acquiring an envelope. Depending on the pathogen, the mature virus particle is either released into the cytoplasm of the cell or released external to the cell. In the case of HIV-1, the outer coat of the virus, known as the viral envelope, is composed of 72 copies (on average) of a complex HIV protein that protrudes from the envelope surface. This protein, known as Env, consists of a cap made of three or four molecules called glycoprotein (gp) 120, and a stem consisting of three or four gp41 molecules that anchor the structure in the viral envelope. Within the envelope of a mature HIV particle is a bullet-shaped core or capsid, made of 2,000 copies of another viral protein, p24. The capsid surrounds two single strands of HIV RNA, each of which has a copy of the virus genes—nine genes in total. Three of these, gag, pol and env, contain information needed to make structural proteins for new virus particles. The env gene, for example, codes for a protein called gp160 that is broken down by a viral enzyme to form gpl20 and gp41, the components of Env. Three regulatory genes, tat, rev and nef, and three auxiliary genes, vif, vpr and vpu, contain information necessary for the production of proteins that control the ability of HIV to infect a cell, produce new copies of virus, or cause disease. The core of HIV also includes a protein called p7, the HIV nucleocapsid protein; and three enzymes that carry out later steps in the virus life cycle: reverse transcriptase, integrase and protease. Another HIV protein called p17, or the HIV matrix protein, lies between the viral core and the viral envelope. The ability to either molecularly clone and subsequently express a gene by recombinant technology, isolate whole pathogens, or purify specific pathogen gene(s), has led to the development of sensitive assay systems for detecting pathogens and for measuring immune responses to their infection. Because early pathogen infection often causes no symptoms, a doctor or other health care worker relies on testing a person's blood for the presence of antibodies (disease-fighting proteins) to the pathogen in question for diagnosis. By early testing, treatment at a time when the individuals' immune systems are most able to combat the pathogen and thus prevent the spread the virus to others could occur. Medical diagnose of pathogen's infection is normally performed by using two different types of antibody tests, ELISA and Western Blot. Diagnostic studies with a number of pathogens show that pathogen burden predicts disease progression. That is, people with high levels of pathogen in their bloodstream are more likely to develop pathogen-related symptoms or to die than individuals with lower levels of pathogen. Methods are available to detect specific antigens or nucleic acid sequences. These techniques can detect pathogen exposures that occur before antibody responses are established. Diagnostic detection for most pathogens exist in first-, second-, and third-line screening procedures and the use of Western Blot analysis is routinely established as a recognized confirmatory method. |
<SOH> SUMMARY OF THE INVENTION <EOH>This invention provides for the formation and establishment of stable chronically expressing pathogen cell lines for the preparation of pathogen antigens and nucleic acids. The established cell lines continually express the pathogen and contain the pathogens DNA stably integrated into the host cells DNA without detrimental effects on cellular viability. Once the line is established, reproducible preparations of pathogen antigens and nucleic acids can be prepared for reagent and diagnostic purposes. The invention is intended for in vitro use for the purpose of pathogen detection and quantification, although purification of native antigens and/or amplification of specific pathogen nucleic acid sequences, therapeutic vaccines, and monitoring or elucidating immune responses in vitro or in vivo can also be envisioned. Establishing continually expressing pathogen antigen(s) cell lines has several advantages over procedures utilizing direct infection of host cell lines for obtaining enriched preparations of pathogen antigens and nucleic acids. Established lines allow reproducibility between preparations by controlling the rate, amount, and level of pathogen antigen transcription and translation. By fixing the number of pathogen genome integration sites in an established cell line (a process that is essentially uncontrollable when cells are directly infected) the level of antigen expression is controlled and synchronize. Synchrony can be achieved by enhancing expression coordinately by treatment with know inducers that up-regulate transcriptional and/or translational/post-translational events. However, even without induction, continuous expression and assemble of virus particles released from cells that cumulate in the culture media during expansion can significantly increase pathogen antigen yields. The chronic expressing pathogen containing cell lines can be established by standard infection procedures, transfection of pathogen containing nucleic acid sequences, transduction of pathogen containing nucleic acid sequences, or by genetically engineering the pathogen antigen of interest by recombinant techniques and assembling the pathogen antigen within a pathogen structures different from the natural native pathogen structure for that antigen of interest. Swapping of pathogen antigens or hybrid chimeric pathogen constructions could be extremely useful for preparations of pathogen antigens that are either not able to be grown, poorly expressed, or poorly released from cellular membranes in culture [such as, but not limited to hepatitis A, B, C, human herpesvirus (HHV)-6, -7, -8 viruses]. In addition, swapped or hybrid pathogen constructions could be a means to express pathogens whose structures are unstable during production and/or purification of the native pathogen that naturally express that antigen (such as, but not limited to Rubella). The present invention simplifies the process of pathogen antigen production over direct infection of host cells by decreasing: (i) labor, (ii) manipulation time, and (iii) expense associated with pathogen antigen production by establishing a process that is generic in design. In one aspect, the invention provides a pathogen antigen preparation containing and expressing all possible antigens by either the introduction of the pathogen by direct infection with the pathogen itself, or transfection by chemical and/or mechanical means by the introduction of a molecular clone of the pathogen into a pre-screened cell line shown to be resistant to or capable of adapting to pathogen antigen expression that are normally detrimental to cell survival. In one embodiment of this aspect, the molecular clone or a nucleic acid preparation containing the pathogen genetic sequence is introduced into a cell line by transfection, which bypasses the cellular membrane to gain access to the cellular chromatin structure. Integration occurs and the cell adapts to the pathogen cytopathic deleterious effect on the cell by decreasing (down-regulating) or eliminating the pathogen cell surface membrane receptors. By this process or by other processes (presently identified or not) that similarly result in the ability of the pathogen-harboring-cell to survive and propagate in culture, a chronically expressing pathogen containing cell line is established (see EXAMPLE 1). In another embodiment, the established pathogen containing line is induced by chemical or mechanical means to enhance the production (on the DNA, RNA, protein, or release level) of pathogen output and hence, the ultimate yield of the pathogen antigens. Yield enhancement could occur by one or more inducers (chemical, mechanical or biological) added simultaneously or sequentially in order to obtain the desired results (see EXAMPLE 2). In another aspect, the invention provides a method to virally transduce [including but not limited to murine leukemia virus (MuLV), adenovirus, adeno-associated virus (AAV), lentivirus, and canarypox vectors] specific pathogen antigen(s) into a cell line for the purpose of over-expressing one or more of the pathogen antigens. In one embodiment of this aspect, the transduced specific pathogen antigen is membrane-bound in the transduced cell in such a fashion that the pathogen antigen will be specifically incorporated or acquired (by either a passive or active process). In this embodiment, the transduced over-expressed pathogen antigen(s) associate with expressed competent intact virus or virus-like particle (VLP) core structures. Association of the specific pathogen antigen(s) with the released competent intact virus-replication competent, or not—or VLP could be enhanced by the inclusion of known sequences in a hybrid recombinant construction with the pathogen antigen, but these hybrid constructions are not necessarily required. These specific sequences could be intracellular/transmembrane sequences or sequences identified to enhance the association of the pathogen antigen to the competent intact virus or VLP. In addition, the competent intact virus or VLP that provides the “carrier function” for the pathogen antigen need not be related to the pathogen antigen in question, but could be a heterologous competent intact virus and/or VLP. However, it could be the pathogen and/or VLP itself and in that case (where the “carry function” is being performed by a VLP of the pathogen), the hybrid recombinant construction with the pathogen antigen could be required for VLP release from the cell. Preferably, the transduced cell line is an established cell line expressing either competent intact viruses or VLP, although it could be a cell line where establishment of the chronic competent intact virus or VLP is introduced after pathogen antigen transduction (see EXAMPLE 3). The invention allows for the “swapping” of antigens with similar functions between pathogen and hybrid constructions, where fusion molecules are created for the purpose of expressing specific pathogen antigens within the backbone structure of another pathogen. The second pathogen provides only a carrier function and is there solely for the expression of the specific pathogen antigen of interest. The swapping or inclusion of antigens could be variant-forms of the same pathogen antigen (see EXAMPLE 4). This concept allows pathogen antigen cassettes to be created for specific antigens of interest into a particular carrier platform (see EXAMPLE 5). In another aspect the invention provides a reproducible source of pathogen specific nucleic acid preparations of DNA and RNA for detection and quantification purposes. This nucleic acid material could be used in nucleic acid based detection and/or quantification test systems as a positive control reagent, but need not be limited to this role. The number of copies of pathogen-specific sequences could be quantified by comparing signal intensities (ELISA-based probe-dependent readings) with cloned fragments after oligonucleotide-dependent polymerase chain reactions (PCR) assays. For RNA, a reverse transcriptase step would be performed prior to PCR analysis. In another embodiment of this aspect, instead of using the purified nucleic acids with known copy number, intact virus particles obtained from harvested supernatants of chronic pathogen containing cell lines could be quantified for pathogen nucleic acid copy number and used in known amounts “spiked” into duplicate samples to determine percent recovery of pathogen specific material from human specimens and/or tissues. In this way the present invention can serve as reagent material for both antigen-based and nucleic acid-based detection kits for the research and diagnostic industry. In another aspect the invention relates to any antigen that could be expressed on, in, or within a virus or virus-like-particle. In one embodiment of this aspect, the antigen is a tumor antigen for a particular form of cancer and used as a diagnostic indicator for progression of the disease. In another embodiment of this aspect, the tumor antigen is a therapeutic product and used to alert the immune system to mount a response against the tumor (see EXAMPLE 6). The invention allows the expression of any protein used for reagent, diagnostic, research, or therapeutic purposes assembled into a virus or virus-like-particle that is released from an established chronically expressing cell line for the purpose of harvesting the antigen by collecting and/or concentrating the virus or virus-like-particle released into the culture supernatant. The major advantage of associating the antigen with a virus or virus-like-particle is the ease of recovering said antigen at a lower cost using generic technology to harvest the antigen when associate with a particle released from an established expressing cell line rather than the antigen released in a soluble-form. Once harvested from the culture supernatant, the antigen-associated particle could be used directly, the particle could be disrupted to form a lysate, or the antigen could be partially and/or fully purified from particle associated material by standard methods used in the art of protein purification. Harvesting could be by ultracentrifugation or by low-speed centrifugation either by differential sedimentation or in combination with techniques to remove or precipitate particulate material from culture fluids. The invention improves current methods of antigen production by providing a method that increase yield, stability, and concentrates antigens of interest. Once concentrated, downstream processing and/or purification of the antigen(s) are simplified. This aspect expands the concept of antigen production from pathogen antigens to the production of any antigen/protein of choice. In summary, the formation and establishment of stable chronically expressing pathogen containing cell lines has a wide range of applications, including but not limited to, in vitro preparation of pathogen antigens, DNA, and RNA for pathogen detection and quantification for use as reagents in diagnostic test for research and industry. The present invention provides a method to form pathogen expressing cell lines. These lines are formed by either direct infection by standard methods or by bypassing cell surface receptors to introduce the pathogen nucleic acid material into a cell of choice. The cell of choice could be prescreen to tolerate the continuous expression and assemble of pathogen particles that are released from the cell, or can be introduced into a cell that does not naturally express the receptor needed for entry, or can be genetically modified to harbor and expression the host pathogen by specific addition or deletion of signals that maintains cellular viability. Specific pathogen antigens can be expressed: (i) as the entire pathogen integrated into the cellular chromatin structure; (ii) as a specific pathogen antigen whose expression is required for the assemble of the pathogen particle; or (iii) as a specific pathogen antigen that associates with another pathogen strain (related or distal to the pathogen of interest) that provides a carrier fimction to said pathogen antigen. In the latter two cases, the pathogen antigen can be unmodified containing its innate sequence or could be genetically modified by standard procedures to enhance its incorporation into either homologous or heterologous pathogen particles. Thus, disclosed are methods for the formation and establishment of chronic expressing pathogen containing cell lines. The present invention particularly concerns: A method for establishing a cell line that expresses a pathogen to levels 10-to 1,000-fold greater than that attained by standard procedures of direct infection and expansion. This level of pathogen production is attained by establishing conditions to generate multiple integration of the pathogen genome into the host cell line, maintaining the cell line through the critical period of adaptation to tolerate this level of pathogen production, elucidating through experimentation the sequence and timed additions of reagents to the culture to further increase pathogen production, and elucidating a method for efficient intact pathogen isolation. |
Method for gene expression |
The invention relates to a method for gene expression in a cell-free transcription/translation system, the reaction solution containing all the components necessary for the transcription/translation mechanism, amino acids, nucleotides, metabolic components which provide energy and which are necessary for synthesis, and the proteins arising during translation being immobilized on a matrix. |
1. A method for gene expression in a cell-free transcription/translation system, the reaction solution containing all components necessary for transcription/translation mechanism, amino acids, nucleotides, metabolic components which provide energy and which are necessary for the synthesis, wherein the proteins arising during transcription/translation are immobilized on a matrix such that the reaction solution is conducted over the matrix disposed outside the reaction vessel. 2. A method according to claim 1, wherein the matrix is disposed in a separate reaction vessel passed by the reaction solution. 3. A method according to claim 1 or 2, wherein the concentration of protein arising during transcription/translation is determined before and behind the matrix by measuring the absorption of light. 4. A method according to claim 1, wherein the matrix specifically binds proteins by hydrophobic, hydrophilic, antigen/antibody, or ionic interactions. 5. A method according to claim 1, wherein the matrix contains anion or cation exchange material or hydroxyapatide. 6. A method according to claim 1, wherein the proteins are expressed as fusion proteins containing a fusion partner. 7. A method according to claim 6, wherein the fusion partners are located at the N terminal, C terminal or within the expressed protein. 8. A method according to claim 6, wherein a matrix is used which specifically binds to the fusion partner. 9. A method according to claim 7, wherein the protein contains at the N terminal or C terminal one or more successive histidines. 10. A method according to claim 1, wherein the matrix carries a metal chelate compound comprising bivalent metal ions. 11. A method according to claim 7, wherein the protein contains at the N terminal or C terminal glutathione S-transferase as a fusion partner. 12. A method according to claim 11, wherein glutathione is coupled to the matrix. 13. A method according to claim 6, wherein the protein contains an amino acid sequence permitting it to bind to streptavidin. 14. A method according to claim 13, wherein streptavidin is coupled to the matrix. 15. A method according to one of claim 1, wherein generated low-molecular metabolic products are extracted during the expression, and consumed necessary metabolic components of the reaction are added in an amount such that the concentrations of the low-molecular metabolic products and the necessary metabolic components during the duration of the reaction differ by less than 80% of their a respective initial concentrations. 16. A method according to claim 1, wherein the necessary metabolic components contain a substance of the group comprising “ATP, UTP, GTP and CTP, pyrophosphate, amino acids, and mixtures of these substances”. 17. A method according to claim 15, wherein the necessary metabolic components are added via a semi-permeable membrane, and the low-molecular metabolic products are extracted via a semi-permeable membrane comprising the same or different semi-permeable membrane. 18. A device for carrying-out a method according to claims 1, comprising a reaction chamber for receiving the reaction solution containing all the components necessary for the transcription/translation mechanism, amino acids, nucleotides, metabolic components which provide energy and which are necessary for the synthesis, and a matrix specifically binding expressed proteins, wherein the matrix is located in a separate reaction vessel, comprising a column outside the reaction vessel, which is connected by an inlet, and an outlet to the reaction vessel. 19. A device according to claim 18, wherein there are provided means before and behind the matrix for determining the protein concentration, such means comprising light absorption, comprising light in the u.v. range. 20. A device according to claims 18, wherein the reaction chamber comprises a section separated by a semi-permeable membrane, to which the reaction solution can be removed or added. 21. The method of claim 2, wherein the separate reaction vessel comprises a column. 22. The method of claim 3, wherein the absorption is measured of light in the u.v. range. 23. The method of claim 9, wherein the histidines comprise 3 to 12 successive histidines. 24. The method of claim 9, wherein the histidines comprise 5 to 9 successive histidines. 25. The method of claim 9, where the histidines comprise 6 successive histidines. 26. The method of claim 10, wherein the metal chelate compound comprises bivalent copper or nickel ions. 27. The method of claim 15, wherein the concentrations of the low-molecular metabolic products and the necessary metabolic components during the duration of the reaction differs by less than 50% of their respective initial concentrations. 28. The method of claim 15, wherein the concentrations of the low-molecular metabolic products and the necessary metabolic component during the duration of the reaction differs by less than 20% of their respective initial concentrations. 29. The method of claim 17, wherein the semi-permeable membrane has cut-off limits in the range of 500 to 20,000 Da. 30. The method of claim 17, wherein the semi-permeable membrane has cut-off limits in the range of 3,000 to 5,000 Da. |
<SOH> FIELD OF THE INVENTION <EOH>The invention relates to a method for gene expression in a cell-free transcription/translation system, the reaction solution containing all the components necessary for the transcription/translation mechanism, amino acids, nucleotides, metabolic components which provide energy and which are necessary for the synthesis, and the proteins arising during the translation being immobilized on a matrix. Further, the invention relates to a device for the cell-free gene expression in a transcription/translation system. |
Device and method for aligning a stack of sheets arranged one above the other |
The invention relates to a device and a method for aligning at least the front edge of several sheets arranged in a stack, one above the other, whilst maintaining the order of the sheets. Said device comprises a stacking table, one side of which is provided with a front edge stop for aligning the front edge of the sheets. A supporting platform, upon which a stack of sheets can be placed with non-aligned front edges, is arranged upstream of the stacking table. The device is provided with a sheet feeder, which removes the sheets from the supporting platform by placing them in an overlapping stream with the front edges placed under the preceding sheets. A turning device is provided downstream of the sheet feeder, said device turning the overlapping stream in such a way that the front edge area of each sheet is freely accessible. Downstream of the turning device, the invention is provided with a conveyer device, by means of which the sheets are conveyed, forming a new stack, to the front edge stop of the stacking table, where their front edges can be aligned. |
1. An apparatus for aligning at least the leading edges of a plurality of sheets arranged one above the other in a group, while maintaining the sequence of the sheets, having a supporting table with a leading-edge stop for aligning the leading edges of the sheets provided on one side, it being the case that arranged upstream of the supporting table is a carrying plate, on which the sheets are set down in a group with non-aligned leading edges, that the apparatus contains a sheet feeder, by means of which the sheets are removed from the carrying plate to form an imbricated stream with underlap imbrication, that arranged downstream of the sheet feeder is a turning arrangement, by means of which the imbricated stream is turned such that each sheet is freely accessible in the region of its leading edge, and that arranged downstream of the turning arrangement is a conveying arrangement, by means of which the sheets are conveyed to the leading-edge stop of the supporting table, with a new group being formed in the process, and are aligned there by way of their leading edges, the apparatus containing a side pull-type lay, against which a side edge of the sheet in the imbricated stream can be aligned laterally, characterized in that the side pull-type lay is arranged between the sheet feeder and turning arrangement. 2. An apparatus for aligning at least the leading edges of a plurality of sheets arranged one above the other in a group, while maintaining the sequence of the sheets, having a supporting table with a leading-edge stop for aligning the leading edges of the sheets provided on one side, it being the case that arranged upstream of the supporting table is a carrying plate, on which the sheets are set down in a group with non-aligned leading edges, that the apparatus contains a sheet feeder, by means of which the sheets are removed from the carrying plate to form an imbricated stream with underlap imbrication, that arranged downstream of the sheet feeder is a turning arrangement, by means of which the imbricated stream is turned such that each sheet is freely accessible in the region of its leading edge, and that arranged downstream of the turning arrangement is a conveying arrangement, by means of which the sheets are conveyed to the leading-edge stop of the supporting table, with a new group being formed in the process, and are aligned there by way of their leading edges, the apparatus containing a side pull-type lay, against which a side edge of the sheets in the imbricated stream can be aligned laterally, characterized in that the conveying speed of the sheets following alignment of the side edges against the side pull-type lay is reduced in order to increase the overlapping of the sheets in the imbricated stream. 3-4. (cancelled) 5. The apparatus as claimed in claim 1, characterized in that the turning arrangement contains a roller, against the circumference of which the sheets of the imbricated stream can be brought into abutment and turned by virtue of the roller being driven in rotation. 6. The apparatus as claimed in claim 5, characterized in that the turning arrangement contains guide belts, by means of which the sheets can be pressed, at least in certain sections, onto the circumference of the roller. 7. The apparatus as claimed in claim 6, characterized in that the guide belts are designed, at least at the outlet of the turning arrangement, as suction belts. 8-9. (cancelled) 10. An apparatus for transporting sheets by means of at least one conveying arrangement having a suction body, it being the case that the suction body is arranged above a stack comprising a plurality of sheets, and imbricated sheets can be fed to the stack between the suction body and stack, the suction-body being mounted in a vertically displaceable manner, characterized in that the suction bodies are designed in the manner of suction rollers which can be driven in rotation. 11. The apparatus as claimed in claim 1, characterized in that the conveying speed of the conveying arrangement can be changed, in particular regulated or controlled. 12. The apparatus as claimed in claim 1, characterized in that the conveying arrangement is designed in the manner of a suction-body unit, of which the suction bodies attach the sheets of the imbricated stream by suction in the region of the freely accessible leading edges and, by way of the corresponding drive action, convey them against the leading-edge stop. 13. The apparatus as claimed in claim 12, characterized in that the suction bodies are designed in the manner of suction rollers which can be driven in rotation. 14. The apparatus as claimed in claim 12, characterized in that the suction-body unit is mounted in a vertically displaceable manner. 15. The apparatus as claimed in claim 14, characterized in that the suction-body unit is mounted resiliently. 16. The apparatus as claimed in claim 14, characterized in that the vertical position of the suction-body unit can be changed in dependence on the height of the newly formed group of sheets. 17. The apparatus as claimed in one of claim 2, characterized in that the turning arrangement contains a roller, against the circumference of which the sheets of the imbricated stream can be brought into abutment and turned by virtue of the roller being driven in rotation. 18. The apparatus as claimed in claim 2, characterized in that the conveying speed of the conveying arrangement can be changed, in particular regulated or controlled. 19. The apparatus as claimed in claim 10, characterized in that the conveying speed of the conveying arrangement can be changed, in particular regulated or controlled. 20. The apparatus as claimed in claim 2, characterized in that the conveying arrangement is designed in the manner of a suction-body unit, of which the suction bodies attach the sheets of the imbricated stream by suction in the region of the freely accessible leading edges and, by way of the corresponding drive action, convey them against the leading-edge stop. 21. The apparatus as claimed in claim 14, characterized in that the suction-body unit is mounted resiliently. 22. The apparatus as claimed in claim 13, characterized in that the vertical position of the suction-body unit can be changed in dependence on the height of the newly formed group of sheets. 23. The apparatus as claimed in claim 15, characterized in that the vertical position of the suction-body unit can be changed in dependence on the height of the newly formed group of sheets. |
Brake control device with a check valve for a motor vehicle |
The invention relates to a brake control device comprising: a liquid reservoir (4), a master cylinder (2), and a valve (14) able to allow the liquid of the cylinder in the reservoir to rise by offering a first minimum flow cross section (31). The valve is able to allow the rise by offering a second minimum flow cross section (33) which is larger than the first cross section when a pressure in the cylinder exceeds a given threshold. |
1. Brake control device comprising: a liquid reservoir (4), a master cylinder (2), and a valve (14; 114; 214; 314; 414; 514; 1014) able to allow the liquid of the cylinder in the reservoir to rise by offering a first minimum flow cross section (31), characterized in that the valve is able to allow the rise by offering a second minimum flow cross section (33) which is larger than the first cross section when a pressure in the cylinder exceeds a given threshold. 2. Device according to claim 1, characterized in that the valve comprises a seat (24; 224; 324; 424; 524; 824; 924; 1024) and a shutter (40; 140; 240; 340; 640; 740; 1040) able to bear against the seat. 3. Device according to claim 2, characterized in that it comprises means (40; 760) of centering the shutter with respect to the seat. 4. Device according to either claim 3, characterized in that the shutter has a density lower than that of the liquid. 5. Device according to claim 4, characterized in that the seat (24; 224) is rigid. 6. Device according to claim 5, characterized in that the seat (24; 224; 424) can be moved with respect to the reservoir when the pressure exceeds the threshold. 7. Device according to claim 6, characterized in that the seat (24; 524; 924) has at least one passage (552; 952) and is designed to allow the liquid to rise through the passage only when the seat moves. 8. Device according to claim 7, characterized in that it comprises means (32; 232; 432) of returning the seat against the rising of the liquid. 9. Device according to claim 8, characterized in that the seat (324; 424; 524; 824; 924; 1024) can be deformed elastically when the pressure exceeds the threshold. 10. Device according to claim 9, characterized in that the seat (324; 424; 824; 1024) is deformable so that the liquid flows around the seat during the rise. 11. Device according to claim 10, characterized in that the seat (524; 924) is deformable so that the liquid flows through the seat during the rise. 12. Device according to claim 11, characterized in that the seat (524; 924) has at least one orifice (552; 952) designed to be open only during deformation. 13. Device according to claim 12, characterized in that the orifice (952) opens to one edge of the seat. 14. Device according to claim 13, characterized in that the orifice (552) extends some distance from the edges of the seat. 15. Device according to claim 14, characterized in that it has at least one relief (1064) designed to cause the shutter (1040) to tip with respect to the seat (1024) when the pressure exceeds the threshold. 16. Device according to claim 15, characterized in that the shutter (340; 640; 740) has at least one passage (228; 628; 728) and is designed to allow the liquid to rise through the pipe when the shutter is in contact with the seat. 17. Device according to claim 16, characterized in that the passage (228) passes right through the shutter (340). 18. Device according to claim 17, characterized in that the passage (328; 728) extends over one face of the shutter able to come into contact with the seat. 19. Device according to claim 18, characterized in that the shutter (40) has one approximately spherical face able to come into contact with the seat. 20. Device according to claim 19, characterized in that the shutter is a ball (40). 21. Device according to claim 19, characterized in that the shutter (140; 240; 340; 640) has the overall shape of a flat cake. 22. Device according to claim 21, characterized in that the valve has an orifice (16) via which liquid can leave toward the master cylinder (2) and which is arranged in such a way that the shutter, in its lowermost position, leaves this orifice open. 23. Device according to claim 22, characterized in that the orifice (16) extends in a vertical wall. 24. Device according to claim 23, characterized in that it comprises means (1070) of retaining the shutter (1040) with respect to the reservoir (4) before the reservoir is assembled with the master cylinder (2). 25. Device according to claim 24, characterized in that the valve is able to allow the liquid to drop from the reservoir into the master cylinder by offering a third minimum flow cross section (35) which is larger than the first cross section (31). 26. Valve for a device for controlling a brake with a liquid reservoir and a master cylinder, the valve being able to allow liquid to rise by offering a first minimum flow cross section (31), characterized in that the valve is able to allow the rise by offering a second minimum flow cross section (33) which is larger than the first cross section when a liquid pressure reaches a given threshold upstream of the valve, with reference to the direction of flow during the rise. |
Method and apparatus for regulating electrical power output of a fuel cell system |
A method and apparatus are provided for regulating electrical power output of a fuel cell system comprising a fuel processing system, a system for supplying an oxidant stream, and a fuel cell to supply power. In a reformer of the fuel processing system a supply fuel is used to produce hydrogen-rich gas to be supplied to the fuel cell. The fuel cell system also contains a controller to set the mass flow of the supply fuel to the fuel processing system and the mass flow of the oxidant stream to the fuel cell, whereby the mass flow of the oxidant stream can be set in dependence on the dynamic response of the fuel processing system and/or the mass flow of the hydrogen-rich gas can be set in dependence on the dynamic response of the system for supplying the oxidant stream. |
1. A method of operating a fuel cell system comprising a fuel cell (3) with an anode stream passage and a cathode stream passage, an anode supply system (1) for supplying a hydrogen-rich gas to the anode stream passage, a cathode supply system (2) for supplying an oxidant stream to the cathode stream passage and a controller (4) for operating the anode and cathode supply systems (1, 2), the method comprising operating one supply system in dependence on the dynamic response of the other supply system, characterized in that the fuel cell system comprises a fuel processing system (1) for converting a supply fuel into a hydrogen-rich gas wherein the supply fuel mass flow ({dot over (m)}Fuel) to the fuel processing system (1) is set in dependence on the dynamic response of the cathode supply system (2) 2. The method of claim 1, wherein the hydrogen-rich gas mass-flow ({dot over (m)}H2) to the anode stream passage is set in dependence on the dynamic response of the cathode supply system (2). 3. The method of claim 1, wherein the oxidant stream mass flow ({dot over (m)}Air) to the cathode stream passage is set in dependence on the dynamic response of the anode supply system (1). 4. The method of claim 1, wherein the oxidant stream mass flow ({dot over (m)}H2) to the cathode stream passage Is set in dependence on the dynamic response of the fuel processing system (1). 5. The method of claim 1, wherein the supply fuel mass flow ({dot over (m)}Fuel) to the fuel processing system is set in dependence on the efficiency of the fuel processing system (1). 6. The method of claim 1, wherein the supply fuel mass flow ({dot over (m)}Fuel) to the fuel processing system (1) is set in dependence on the intrinsic consumption of hydrogen-rich gas in the fuel processing system (1). 7. A fuel cell system comprising: a) a fuel cell (3) comprising an anode stream passage and a cathode stream passage; b) an anode supply system (1) for supplying a hydrogen-rich gas to the anode stream passage; c) a cathode supply system (2) for supplying an oxidant. stream to the cathode stream passage; d) a controller (4) for operating one supply system (1, 2) in dependence on the dynamic response of the other supply system (2, 1) characterized in that the anode supply system. comprises a fuel processing system (1) for converting a supply fuel into a hydrogen-rich gas wherein the controller (4) sets the hydrogen-rich gas mass flow ({dot over (m)}H2) to the anode stream passage in dependence on the dynamic response of the cathode supply system (2). 8. A fuel cell, system as recited in claim 7, wherein the controller (4) sets the supply fuel mass flow ({dot over (m)}Fuel) to the fuel processing system (1) in dependence on the dynamic response of the cathode supply system (2). 9. A fuel cell system as recited in claim 7, wherein the controller (4) sets the oxidant stream mass flow ({dot over (m)}Air) to the cathode stream passage in dependence on the dynamic response of the anode supply system (1). 10. A fuel cell system as recited In claim 7, wherein the controller (4) sets the oxidant stream mass flow ({dot over (m)}Air) to the cathode stream passage in dependence on the dynamic response of the fuel processing system (1). 11. A fuel cell system as recited in claim 7, wherein the controller (4) sets the supply fuel mass flow ({dot over (m)}Fuel) to the fuel processing system (1) in dependence on the effidency (η) of the fuel processing system (1). 12. A fuel cell system as recited in claim 7, wherein the controller (4) sets the supply fuel mass flow ({dot over (m)}Fuel) to the fuel processing system (1) in dependence on the intrinsic consumption of hydrogen-rich gas in the fuel processing system (1). |
<SOH> BACKGROUND <EOH>1. Field of Invention The invention relates to a method and apparatus for regulating electrical power output of a fuel cell system. 2. Description of the Related Art U.S. Pat. No. 5,432,710 A describes a power supply system that contains a fuel cell and a controller. The controller regulates the systems and subsystems of the power supply system by minimizing a cost function in the form of an algebraic equation. This cost function takes into account the power demand of the load, the power demand of the system itself, and the exhaust gases. In dependence on the cost function, the controller sets the mass flow of an oxidant stream and the mass flow of a hydrogen-rich gas for the fuel cell unit, and the mass flow of a fuel for the reformer. JP 59-11270 describes a fuel cell system comprising differential pressure controlling valves for controlling a differential pressure between the pressures of supplied oxygen and hydrogen. The hydrogen is supplied from a hydrogen tank. The differential pressure control valves are connected to each other by a link mechanism. Thus, the operation of one pressure control valve is controlled in dependence on the operation of the respective other differential pressure control valve. JP 5911273 describes a similar fuel cell system with two pressure control valves which are controlled in dependence from the respective other pressure control valve. EP 1 207 578 A2, which is not prepublished, describes a fuel cell system wherein the hydrogen is supplied from a high pressure hydrogen tank to the fuel cell system. In this document it is mentioned that pressure hydrogen supplied from the high pressure hydrogen tank to the fuel cell is based on the air pressure supplied from the air supply side. That means that the pressure of the hydrogen supplied to the fuel cell system is controlled in dependence on the air pressure supply from the air supply side. There is a need for improvement in regulating power supply from a fuel cell to a load. The present invention addresses this need and provides further related advantages. |
<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>The invention provides a method of operating a fuel cell system with the features according to claim 1 . The fuel cell system comprises a fuel cell with an anode stream passage and a cathode stream passage, an anode supply system for supplying a hydrogen-rich gas to the anode stream passage, a cathode supply system for supplying an oxidant stream to the cathode stream passage and a controller for operating the anode and cathode supply systems. The method comprises operating one supply system in dependence on the dynamic response of the other supply system. The hydrogen-rich gas mass flow to the anode stream passage may be set in dependence on the dynamic response of the cathode supply system and/or the oxidant stream mass flow to the cathode stream passage may be set in dependence on the dynamic response of the anode supply system. The invention also provides a method of operating a fuel cell comprising a fuel processing system for converting a supply fuel into a hydrogen-rich gas. In such an embodiment, the supply fuel mass flow to the fuel processing system may be set in dependence on the dynamic response of the cathode supply system and/or the oxidant stream mass flow to the cathode stream passage may be set in dependence on the dynamic response of the fuel processing system. In a further embodiment, the supply fuel mass flow to the fuel processing system may be set in dependence on the efficiency of the fuel processing system. In a still further embodiment, the supply fuel mass flow to the fuel processing system may be set in dependence on the intrinsic consumption of hydrogen-rich gas In the fuel processing system. The Invention also provides a fuel cell system with the features according to claim 7 . The fuel cell system comprises: a) a fuel cell comprising an anode stream passage and a cathode stream passage; b) an anode supply system for supplying a hydrogen-rich gas to the anode stream passage; c) a cathode supply system for supplying an oxidant stream to the cathode stream passage; d) a controller for operating one supply system in dependence on the dynamic response of the other supply system. The anode supply system may comprise a fuel processing system for converting a supply fuel into a hydrogen-rich gas. Pursuant to the invention, the controller may set the hydrogen-rich gas mass flow to the anode stream passage In dependence on the dynamic response of the cathode supply system. Alternatively, the controller may set the supply fuel mass flow to the fuel processing system in dependence on the dynamic response of the cathode supply system. In an alternative embodiment, the controller may set the oxidant stream mass flow to the cathode stream passage in dependence on the dynamic response of the anode supply system or in dependence on the dynamic response of the fuel processing system. In a further alternative embodiment, the controller may set the supply fuel mass flow to the fuel processing system in dependence on the efficiency of the fuel processing system. In a still further alternative embodiment, the controller may set the supply fuel mass flow to the fuel processing system in dependence on the intrinsic consumption of hydrogen-rich gas in the fuel processing system. Many specific details of certain embodiments of the invention are set forth in the detailed description below to provide a thorough understanding of such embodiments. One skilled in the art, however, will understand that the present invention may have additional embodiments, or may be practiced without several of the details described. |
Semiconductor device manufacturing method |
A method of manufacturing a semiconductor device for realizing a semiconductor device which is suitable for enhancing the operating speed thereof and which is high in quality and reliability is provided. The method of manufacturing a semiconductor device is a method of manufacturing a semiconductor device including a barrier film (7) having a copper diffusion preventive function and formed on a copper-containing metallic wire (9), the method including the steps of: conducting electroplating by use of an electroplating liquid containing a catalyst metal (10) added thereto so as thereby to form the metallic wiring (2) containing the catalyst metal (10); and conducting electroless plating by use of the catalyst metal (10) exposed at the surface of the metallic wiring (2) as a catalyst so as thereby to form the barrier film (7) having the copper diffusion preventive function on the metallic wiring (2). |
1. A method of manufacturing a semiconductor device comprising a barrier film having a copper diffusion preventive function and formed on a copper-containing metallic wiring, said method comprising the steps of: conducting electroplating by use of an electroplating liquid containing a catalyst metal added thereto so as thereby to form said metallic wiring containing said catalyst metal; and conducting electroless plating by use of said catalyst exposed at the surface of said metallic wiring as a catalyst so as thereby to form said barrier film having said copper diffusion preventive function on said metallic wiring. 2. The method of manufacturing a semiconductor device according to claim 1, wherein said catalyst metal is added to said electroplating liquid in a complexed form. 3. The method of manufacturing a semiconductor device according to claim 1, wherein said catalyst metal is one selected from the group consisting of Au, Pt, Pd, Ag, Ni, and Co. 4. The method of manufacturing a semiconductor device according to claim 1, wherein said barrier film is comprised of a cobalt alloy or a nickel alloy. |
<SOH> BACKGROUND ART <EOH>Conventionally, aluminum-based alloys are used as a material of a minute wiring of a high-density integrated circuit formed on a semiconductor wafer. For enhancing the operating speed of a semiconductor device, however, it is necessary to use a material lower in resistivity than aluminum-based alloys as the wiring material, and copper, silver and the like are preferable for use as such a low-resistivity material. Particularly, copper is expected as a next-generation material because it has a low resistivity of 1.8 μΩcm, it is therefore advantageous for enhancing the operating speed of a semiconductor device, and it is higher in electro-migration resistance than aluminum-based alloys by about one order. In formation of a wiring by use of copper, generally, the so-called Damascene process is used because dry etching of copper is difficult to carry out. The process includes the steps of preliminarily forming a groove in an inter-layer insulating film formed, for example, of silicon oxide, filling the groove with a wiring material (copper), and removing the excess wiring material by chemical mechanical polishing (hereinafter referred to as CMP), thereby forming a wiring. Further, there has been known the dual Damascene process including the steps of forming connection holes (via holes) and a wiring groove (trench), then collectively filling these with a wiring material, and removing the excess wiring material by CMP. Meanwhile, the copper wiring is generally used in the form of a multi-layer structure. In this case, for the purpose of preventing the diffusion of copper into the inter-layer insulating film, a barrier film consisting of silicon nitride, silicon carbide or the like is formed before the formation of the wiring. However, since the barrier film is absent on the surface of the copper wiring immediately after the CMP, the barrier film for functioning as a copper diffusion preventive layer is formed before the formation of the upper-layer wiring. In this case, since copper is easily oxidized in an oxygen-containing atmosphere even at a low temperature of around 150° C., a silicon nitride film (SiN) a silicon carbide film (SiC) or the like is ordinarily used as the barrier layer. It should be noted here that silicon nitride (SiN) and silicon carbide (SiC) are higher in relative dielectric constant than silicon oxide (SiO 2 ), which leads to the problems that the effective dielectric constant of the semiconductor device including the copper wiring will be high, the semiconductor will be high in RC delay (delay of the wiring due to resistance and capacitance), and the electro-migration resistance at the interface between the SiN or SiC constituting the barrier film and copper will be weak. In view of the above problems, formation of a film of CoWP on the surface of the copper wiring after the CMP as a material which is excellent in prevention of copper diffusion, improvement of RC delay, and electro-migration resistance has been proposed by U.S. Pat. No. 5,695,810 (USE OF COBALT TUNGSTEN PHOSPHITE AS A BARRIER MATERIAL FOR COPPER METALLIZATION). Furthermore, CoWP has the characteristic feature that a film thereof can be selectively formed only on the copper wiring by electroless plating. A conventional semiconductor device using CoWP as the barrier film is shown in FIG. 21 . The semiconductor device includes a copper-containing metallic wiring, on which is formed a barrier film composed of CoWP and having a copper diffusion preventive function. The semiconductor device has a constitution in which lower-layer wirings 102 a and 102 b as the copper-containing metallic wirings (hereinafter referred to as Cu wirings) are used to fill grooves provided in an insulating layer 103 a , on a substrate 101 preliminarily provided with devices (not shown) such as transistors. The insulating layer 103 a is formed, for example, of SiOC, and a barrier metal film 104 a formed, for example, of TaN is formed between the lower-layer wirings 102 a , 102 b and the insulating layer 103 a . In addition, an etch stopper layer 105 formed of SiC, for example, is formed between the substrate 101 and the insulating layer 103 a , for preventing the diffusion of Cu from the lower-layer wirings 102 a and 102 b into the substrate 101 . Besides, an insulating film 103 b is provided on the lower-layer wirings 102 a and 102 b and the insulating layer 103 a , with an SiN film for copper diffusion prevention therebetween. The insulating film 103 b is formed of SiO 2 , for example. Furthermore, an insulating film 103 c is formed on the insulating film 103 b , with an SiN film for copper diffusion prevention therebetween, and upper-layer wirings 106 a and 106 b as copper-containing metallic wirings are formed in grooves provided in the insulating layer 103 b and the insulating layer 103 c , with a barrier metal film 104 b consisting, for example, of TaN therebetween. A barrier film 108 consisting of CoWP and having a copper diffusion preventing function is formed on the upper-layer wirings 106 a and 106 b , i.e., on the surfaces not covered with the barrier metal film 104 b , i.e., the top surfaces in FIG. 21 , of the upper-layer wirings 106 a and 106 b , with a palladium (Pd) replacement layer 107 therebetween. To manufacture the above-mentioned semiconductor device, electroless plating with CoWP is applied onto the copper wiring, to form the barrier layer. Now, the method and principle of forming a film of CoWP on the copper wiring by electroless plating will be described in brief. In order to selectively forming the film of CoWP on the copper wiring by the electroless plating method, a catalyst layer for starting electroless plating is needed. Copper is low in catalytic activity, and, therefore, it does not function as a sufficient catalyst for deposition of CoWP. In general, therefore, a method in which a catalytic metal layer of palladium (Pd) or the like is preliminarily formed on the copper surface by replacement plating. The replacement plating utilizes the differences in ionization tendency between different metals. Since Cu is electrochemically baser than Pd, when Cu is immersed in an HCl solution of PdCl 2 , for example, electrons librated attendant on the dissolution of Cu are transferred onto the ions of Pd which is a noble metal in the solution, resulting in the formation of a film of Pd on the surface of Cu which is the baser metal. Since the Pd replacement does not occur on the surfaces of insulating films which necessarily are not metallic, the catalytically active layer is formed only on Cu. Subsequently, an electroless plating reaction starts only on the Cu wiring, with the Pd layer as a catalyst, resulting in the formation of a barrier metal layer formed of CoWP. The above-mentioned method, however, has the problem that the Cu wiring is damaged by etching when the catalytically active layer is formed on the Cu surface by the Pd replacement plating. Particularly, holes are locally formed in Cu along Cu grains, and, where etching is vigorous, the Cu wiring may be damaged to such an extent as to cause line breakage. As a result, the resistance of the Cu wiring is raised by as much as 30%, for example, where the Cu wiring is severely damaged. Furthermore, it is difficult to fill the holes, generated between the Cu grains, by the formation of the CoWP film. As a result, even after the formation of the CoWP, voids would be left in the Cu wiring, and the electro-migration resistance would be rapidly worsened starting from the voids. The present invention has been devised in consideration of the above-mentioned circumstances of the prior art. Accordingly, it is an object of the present invention to provide a method of manufacturing a semiconductor device for realizing a semiconductor device which is suitable for enhancing the operating speed thereof and is high in quality and reliability. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 is a vertical sectional view showing one example of the configuration of a semiconductor device produced by applying the present invention. FIG. 2 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device according to the present invention. FIG. 3 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device according to the present invention. FIG. 4 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device according to the present invention. FIG. 5 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device according to the present invention. FIG. 6 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device according to the present invention. FIG. 7 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device according to the present invention. FIG. 8 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device according to the present invention. FIG. 9 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device according to the present invention. FIG. 10 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device according to the present invention. FIG. 11 is a vertical sectional view showing the condition where a lower-layer wiring has been formed by applying the present invention. FIG. 12 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device in the case where the present invention is applied to the dual Damascene process. FIG. 13 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device in the case where the present invention is applied to the dual Damascene process. FIG. 14 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device in the case where the present invention is applied to the dual Damascene process. FIG. 15 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device in the case where the present invention is applied to the dual Damascene process. FIG. 16 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device in the case where the present invention is applied to the dual Damascene process. FIG. 17 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device in the case where the present invention is applied to the dual Damascene process. FIG. 18 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device in the case where the present invention is applied to the dual Damascene process. FIG. 19 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device in the case where the present invention is applied to the dual Damascene process. FIG. 20 is a vertical sectional view for illustrating the method of manufacturing a semiconductor device in the case where the present invention is applied to the dual Damascene process. FIG. 21 is a vertical sectional view showing one example of the configuration of a semiconductor device according to the related art. detailed-description description="Detailed Description" end="lead"? |
Filter system comprising a bulk acoustic wave resonator |
The invention relates to a filter device equipped with at least one bulk acoustic wave resonator, which comprises a resonator unit and a reflection element (2) comprising several layers (6, 7, 8). The layers (6, 7, 8) each comprise a mixture of at least two materials. Within each layer (6, 7, 8), the composition of the mixture varies continuously and periodically relative to the layer thickness. |
1. A filter device equipped with at least one bulk acoustic wave resonator, which comprises a resonator unit and a reflection element (2), wherein the reflection element (2) comprises several layers (6, 7, 8), which each comprise a mixture of at least two materials, wherein, within each layer (6, 7, 8), the composition of the mixture varies continuously and periodically relative to the layer thickness. 2. A filter device as claimed in claim 1, characterized in that the reflection element (2) comprises a mixture of SiO2 and Ta2O5 or SiO2 and Si3N4. 3. A mobile radio device equipped with a filter device with at least one bulk acoustic wave resonator, which comprises a resonator unit and a reflection element (2), wherein the reflection element (2) comprises several layers (6, 7, 8), which each comprise a mixture of at least two materials, wherein, within each layer (6, 7, 8), the composition of the mixture varies continuously and periodically relative to the layer thickness. 4. A transmitter equipped with a filter device with at least one bulk acoustic wave resonator, which comprises a resonator unit and a reflection element (2), wherein the reflector element (2) comprises several layers (6, 7, 8), which each comprise a mixture of at least two materials, wherein, within each layer (6, 7, 8), the composition of the mixture varies continuously and periodically relative to the layer thickness. 5. A receiver equipped with a filter device with at least one bulk acoustic wave resonator, which comprises a resonator unit and a reflection element (2), wherein the reflection element (2) comprises several layers (6, 7, 8), which each comprise a mixture of at least two materials, wherein, within each layer (6, 7, 8), the composition of the mixture varies continuously and periodically relative to the layer thickness. 6. A wireless data transmission system equipped with a filter device with at least one bulk acoustic wave resonator, which comprises a resonator unit and a reflection element (2), wherein the reflector element (2) comprises multiple layers (6, 7, 8), which each comprise a mixture of at least two materials, wherein, within each layer (6, 7, 8), the composition of the mixture varies continuously and periodically relative to the layer thickness. 7. A bulk acoustic wave resonator, which comprises a resonator unit and a reflection element (2), wherein the reflection element (2) comprises several layers (6, 7, 8), which each comprise a mixture of at least two materials, wherein, within each layer (6, 7, 8), the composition of the mixture varies continuously and periodically relative to the layer thickness. |
Expression of modified antibodies in avian cells |
The present invention relates to construct and methods which allow the expression of immunoglobulins or functional fragments thereof which have been altered to that they are humanised. The expression of the immunoglobulins or fragments thereof takes place in avian cells, and the constructs used have been altered such that the expression levels in avian cells are higher than what would have been expected by simply using a humanised construct. The alterations are based on changing codons so that each amino acid of the codon that is used is the one which is most often found in avians. |
1. A DNA construct which when transfected into an avian cell will allow the production of an antibody molecule or functional fragment of said molecule, and which comprises at least one sequence comprising the variable domain of an immunoglobulin heavy chain and at least one sequence comprising the variable domain of an immunoglobulin light chain, and wherein the DNA construct is based on a non-avian sequence and one or more of the codons in the DNA construct have been altered such that for the amino acid being encoded, the codon used is that which most frequently appears in avians. 2. A DNA construct as claimed in claim 1, wherein the construct also contains an avian signal peptide sequence. 3. A construct as claimed in the previous claims, wherein the construct is cloned into a viral vector. 4. A construct as described in claim 3, wherein the viral vector is a lentivirus vector. 5. A DNA construct as claimed in claims 2 to 4, wherein the avian signal peptide sequence is a signal peptide sequence from an egg white protein. 6. A DNA construct as described in claim 5, wherein the egg white protein is chosen from the list lysozyme, ovalbumin, ovatransferrin or ovamucoid. 7. A DNA construct as claimed in any of the previous claims, wherein the construct also includes immunoglobulin constant regions for dimerisation and recruitment of effector functions. 8. A DNA construct as claimed in claim 7, wherein the immunoglobulin constant regions are CH2 and CH3. 9. A DNA construct as claimed in claims 7 or 8, wherein the immunoglobulin constant regions are human constant regions. 10. A DNA construct as described in any of the previous claims, wherein the construct may be transfected into an avian cell using electroporation. 11. A DNA construct as claimed in claims 1 to 9, wherein the construct may be transfected into an avian cell using lipofection. 12. A DNA construct as claimed in claim 1 to 9, wherein the construct may be directly injected into the nucleus of an avian. 13. A DNA construct as claimed in claim 12, wherein the construct may be directly injected into the germinal disc of an oocyte. 14. A DNA construct as described in any of the previous claims, wherein codon usage in the construct is maximised for those codons most frequently appearing in avians. 15. An avian cell containing the construct described in claims 1 to 15, which expresses an immunoglobulin molecule or functional fragment of said molecule. 16. An avian cell as described in claim 16, wherein the expressed immunoglobulin molecule or functional fragment thereof shows an avian glycosylation pattern. 17. An avian cell as described in claims 16 or 17, wherein the immunoglobulin or fragment thereof is expressed at a higher expression level than a standard human construct or humanised construct. 18. A method for producing avian cells capable of expressing an immunoglobulin molecule or functional fragment of said molecule, comprising transfecting an avian cell with the DNA construct as described in claims 1 to 15. 19. Preferably the avian cell is selected from a list of chicken cell, duck cell, turkey cell, quail cell or ostrich cell. 20. An immunoglobulin or functional fragment thereof which is produced using the method described in claims 19 and 20. 21. A transgenic avian which expresses the construct described in claims 1 to 15. 22. A transgenic avian as described in claim 22, wherein the molecule coded for by the construct is expressed in the egg of the transgenic avian. 23. A transgenic avian as claimed in claim 23, wherein the construct is expressed in the egg white. 24. A transgenic avian as described in claim 23, wherein the construct is expressed in the egg yolk. 25. A transgenic avian as described in claims 22 to 25, wherein the expressed immunoglobulin shows an avian glycosylation pattern. |
Liquid crystal polymer technology chemicals and applications |
Liquid phase liquid crystal polymers (LCPs) are disclosed having a composition and structure that can be varied to provide desirable properties. The liquid phase LCPs have polyiminoborane, polyaminoborane, and/or borozine polymer backbone molecules, with silicon and/or phosphorous side chain molecules linked to the backbone that provide a degree of alignment assigned an Order Parameter (S), defined as S=⅓[3 cos2 θ−1], where θ is the angle between the axis of an LCP molecule and the vertical direction. The inventive liquid phase LCPs have an average Order Parameter in the range of about 0.2 to about 0.99 and are applicable to a number of rinse, coolant, lubricant, sterilization and other protectant processes. |
1. A liquid phase liquid crystal polymer (LCP) compound comprising a polyiminoborane [BNH2]x, polyaminoborane [BNH4]x, and/or borozine polymer [B3N3H6]x backbone and at least one silicon and/or phosphorous side chain linked to the backbone, the compound exhibiting an average Order Parameter (S) in the range between about 0.2 to about 0.99 calculated by S=⅓[3 cos2 θ−1], where θ is the angle between the axis of a liquid phase LCP molecule and the vertical direction. 2. A liquid phase liquid crystal polymer (LCP) comprising a compound selected from the group consisting of (—BH—NR—BH—NR′—)x, (—BH2—NHR—BH—HNR′—)x, and combinations thereof, where X in the range of about 10 to about 90, R and R′ are (CH3)3SiO[SiO(CH3)2]nSi(CH3)3 and n is in the range of 1 to 130. 3. A rinse, lubricant, coolant, and/or protectant process for a part comprising treating the part with a liquid phase liquid crystal polymer (LCP) comprising a backbone chain of compounds selected from the group consisting of [BNH2]x, [BNH4]x, [B3N3H6]x, and combinations thereof, and at least one side chain comprising a compound selected from the group consisting of silicon, phosphorous, and combinations thereof linked to the backbone to form PIB, PAB, and/or PBZ, where R and R′ are (CH3)3SiO[SiO(CH3)2]nSi(CH3)3 and n is in the range of 20-100 for rinse processes, n is in the range of 1-10 for lubricant/cooling processes, n is in the range of 100-130 for surface sterilization processes, and n is in the range of 5-100 for protectant processes. 4. A method for rinsing process chemicals from an apparatus comprising contacting the processed apparatus under conditions sufficient to reduce the process chemical from the apparatus with a liquid phase liquid crystal polymer (LCP) having molecules of a polyiminoborane [BNH2]x, polyaminoborane [BNH4]x, and/or borozine polymer [B3N3H6]x, backbone, and at least one silicon and/or phosphorous side chain linked to the backbone to form PIB, PAB, and/or PBZ to result in an average Order Parameter (S) in the range of between about 0.2 to about 0.99 calculated by S=⅓[3 cos2 θ−1], where θ is the angle between the axis of a liquid phase LCP molecule and the vertical direction. 5. The method of claim 4 wherein the average Order Parameter is in the range of about 0.5 to about 0.8. 6. A method for lubricating and/or cooling machined parts comprising contacting the part under conditions sufficient to lubricate and/or cool the part with a liquid phase liquid crystal polymer (LCP) having a polyiminoborane [BNH2]x, polyaminoborane [BNH4]x, and/or borozine polymer [B3N3H6]x, backbone, and at least one silicon and/or phosphorous side chain linked to the backbone to yield an average Order Parameter (S) in the range between about 0.2 to about 0.99 calculated by S=⅓[3 cos2 θ−1], where θ is the angle between the axis of a liquid phase LCP molecule and the vertical direction. 7. The method of claim 5 wherein the part is selected from the group consisting of a metal part, a ceramic part, and combinations thereof. 8. An engine fluid comprising a liquid phase liquid crystal polymer (LCP) having a polyiminoborane [BNH2]x, polyaminoborane [BNH4]x, and/or borozine polymer [B3N3H6]x backbone, and at least one silicon and/or phosphorous side chain linked to the backbone to yield an average Order Parameter in the range between about 0.2 to about 0.99 calculated by S=⅓[3 cos2 θ−1], where θ is the angle between the axis of a liquid phase LCP molecule and the vertical direction. 9. The engine fluid of claim 8 selected from the group consisting of a motor oil, a transmission fluid, a brake fluid, a power steering fluid, a hydraulic fluid, and combinations thereof. 10. A method for sterilizing a surface comprising contacting the surface under conditions sufficient to coat the surface with a liquid phase liquid crystal polymer (LCP) film having a polyiminoborane [BNH2]x, polyaminoborane [BNH4]x, and/or borozine [B3N3H6]x backbone, and at least one silicon and/or phosphorous side chain linked to the backbone to yield an average Order Parameter (S) in the range between about 0.2 to about 0.99 calculated by S=⅓[3 cos2 θ−1], where θ is the angle between the axis of a liquid phase LCP molecule and the vertical direction. 11. A method for protecting a surface comprising contacting the surface under conditions sufficient to coat the surface with a liquid phase liquid crystal polymer (LCP) film having a polyiminoborane [BNH2]x, polyaminoborane [BNH4]x, and/or borozine [B3N3H6]x backbone, and at least one silicon and/or phosphorous side chain linked to the backbone to yield an average Order Parameter (S) in the range between about 0.2 to about 0.99 calculated by S=⅓[3 cos2 θ−1], where θ is the angle between the axis of a liquid phase LCP molecule and the vertical direction. 12. The method of claim 111 wherein the liquid phase LCP protects against a hazard selected from the group consisting of dehydration, moisture, microorganisms, macroorganisms, ultraviolet light, and combinations thereof. 13. A method for selecting a liquid phase liquid crystal polymer (LCP) for an indicated use comprising providing a liquid phase LCP comprising molecules of a polyiminoborane [BNH2]x, polyaminoborane [BNH4]x and/or borozine [B3N3H6]x backbone, and adding to the backbone at least one side chain containing molecules selected from the group consisting of silicon, phosphorous, and combinations thereof, to result in the liquid phase LCP having a desired average Order Parameter less than a solid phase and calculated by S=⅓[3 cos2 θ−1], where θ is the angle between the axis of a liquid phase LCP molecule and the vertical direction. 14. The method of claim 13 further comprising thereafter using the resulting liquid phase LCP for the indicated use selected from the group consisting of a rinsing agent, a lubricant/coolant, a protectant, and combinations thereof. 15. The method of claim 13 wherein the liquid phase LCP is used as a rinsing agent with an average Order Parameter in the range of about 0.2 to about 0.99, more particularly in the range of about 0.5 to about 0.8. 16. The method of claim 13 wherein the liquid phase LCP is used as a lubricant/coolant with an average Order Parameter in the range of 0.2 to about 0.99. 17. The method of claim 13 wherein the liquid phase LCP is used as a protectant to sterilize a surface with an average Order Parameter in the range of about 0.97 to about 0.99. 18. The method of claim 13 wherein the liquid phase LCP is used as a surface protectant with an average Order Parameter in the range of about 0.2 to about 0.99. |
<SOH> BACKGROUND <EOH>Crystal polymers are long chain linear molecules that have a preferred orientation. Most crystal polymers are solids under ambient conditions; however, they are conventionally referred to as liquid crystal polymers because they have characteristics intermediate between solid and liquid phases. Such polymers have found extensive use in the electronic market sector, and are widely used in displays for products such as digital watches, television sets, etc. Further structural and chemical modifications of these polymers would be useful to determine additional properties and applications. |
<SOH> SUMMARY OF THE INVENTION <EOH>One class of crystal polymers are in a liquid phase, rather than a solid phase, and are termed liquid phase liquid crystal polymers (LCPs). The molecules in liquid phase LCPs are aligned to a degree that is less than the alignment of molecules in solid phase liquid crystal polymers, but greater than the randomly oriented molecules in liquids. Compositions and structures of liquid phase liquid crystal polymers (LCPs) are varied to provide desirable properties. The inventive liquid phase LCPs have a polyiminoborane [BNH 2 ] x , and/or polyaminoborane [BNH 4 ] x backbone, with various compositional and structural silicon and/or phosphorous side chain linkages to the backbone. For example, in one embodiment, the ratio of the length of the side chain to the length of the main chain is varied. The linkages provide a degree of alignment to the liquid phase LCP molecules that is quantified by an Order Parameter. The inventive liquid phase LCPs have an average Order Parameter (S) in the range of about 0.2 to about 0.99, such that they exist in a liquid, rather than a solid, phase. Thus, although isolated compounds may have an Order Parameter greater than 0.99, the average of the Order Parameters will be less than that of a solid phase composition, that is, less than 1.0. The inventive liquid phase LCPs are applicable to a number of uses including, but not limited to, compositions and methods for rinsing, lubricating, cooling, and protecting from agents such as moisture, light, pests, bacteria, etc. In one embodiment, liquid phase LCPs having relatively lower Order Parameters within the 0.2 to 0.99 range are used for rinsing processes, and liquid phase LCPs having relatively higher Order Parameters within this range are used for surface protectant processes, e.g., surface sterilization. Liquid phase LCPs having any Order Parameter within this range may also be used for lubricant, coolant, and other protectant processes. Additionally, as will be appreciated by those skilled in the art, the Order Parameter values may overlap, and the compounds may be used for more than one application. One embodiment of the invention is a composition and method using the inventive liquid phase LCPs for rinsing process chemicals, for example, rinsing acids or alkaline solutions from processed metal products, to approach a zero discharge for the rinse agent. In this embodiment, liquid phase LCPs are prepared that have a density/specific gravity that is sufficiently different from the process chemical to be removed, such that two distinct immiscible layers form, analogous to the separation of oil and water. The process chemical(s) being rinsed from the product can therefore be reclaimed in full strength and can be returned in an uncontaminated state to the original process. The LCPs can similarly be recovered from the rinsing process. This method reduces the volume of rinse water that is required, and can approach or achieve zero discharge of water from typical chemical processes. Another embodiment of the invention is a composition and method using the inventive liquid phase LCPs as a lubricant and/or a coolant, including cutting fluids for machining, grinding, drilling, milling, stamping, etc. processes. The liquid phase LCPs may be used as a hydraulic fluid and/or an engine fluid. Other embodiments of the invention are compositions and methods of the inventive liquid phase LCPs as environmentally safe protectants. They can, for example, be applied to inert surfaces to protect against bacteria (e.g., hospital surfaces), applied to vegetation to retain moisture and to retard fires (e.g., forest fires), applied to lumber and other construction material to protect against termites, mold, mildew, etc., applied to biological and non-biological surfaces to protect against damage by ultraviolet light, as well as numerous other uses. These and other embodiments of the invention will be apparent in view of the following figures and detailed description. |
Amino-phthalazinone derivatives active as kinase inhibitors, process for their preparation and pharmaceutical compositions containing them |
Compounds which are amino-phthalazinone derivatives according to formula 1 and pharmaceutically acceptable salts thereof, together with pharmaceutical compositions comprising them are disclosed; these compounds or compostions are useful in the treatment of diseases caused by and/or associated with an altered protein kinase activity such as cancer, cell proliferative disorders, Alzheimer's disease, viral infections, autoimmune diseases and neurodegenerative disorders. |
1. A method for treating diseases caused by and/or associated with an altered protein kinase activity which comprises administering to a mammal in need thereof an effective amount of an amino-phthalazinone derivative represented by formula (I) wherein Ra and Rb are, each independently, a hydrogen atom or a group, optionally further substituted, selected from straight or branched C1-C6 alkyl, C3-C6 cycloalkyl or cycloalkyl C1-C6 alkyl, aryl, aryl C1-C6 alkyl, 5 to 7 membered heterocyclyl or heterocyclyl C1-C6 alkyl with from 1 to 3 heteroatoms selected among nitrogen, oxygen or sulfur; or one of Ra or Rb is hydrogen or an optionally substituted straight or branched Cl-C6 alkyl group, and the other is a group selected from —COR′, —CONHR′, —COOR′ or —SO2R′, wherein R′ is hydrogen or an optionally substituted group selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl or heterocyclylalkyl, as set forth above; R1 is a group of formula —CHR4R5 wherein R4 and R5 are, each independently, hydrogen or an optionally substituted group selected from straight or branched C1-C6 alkyl, C3-C6 cycloalkyl or cycloalkyl C1-C6 alkyl, aryl, aryl C1-C6 alkyl, 5 to 7 membered heterocyclyl or heterocyclyl C1-C6 alkyl with from 1 to 3 heteroatoms selected among nitrogen, oxygen or sulfur; or R1 is a group of formula —NHR′, —NR′COR″, —NR′CONHR″ or —NR′SO2R″, wherein R′ has the above reported meanings other than hydrogen, and R″ is hydrogen or has the meanings set forth above for R′; R2 is a hydrogen atom or it is a group, optionally further substituted, selected from straight or branched C1-C6 alkyl, C3-C6 cycloalkyl or cycloalkyl C1-C6 alkyl, aryl, aryl C1-C6 alkyl, 5 to 7 membered heterocyclyl or heterocyclyl C1-C6 alkyl with from 1 to 3 heteroatoms selected among nitrogen, oxygen or sulfur; any R3, being placed in one or more of the free positions 5, 6 and 8 of the phthalazinone ring are, independently from each other, halogen, nitro, carboxy, cyano or a group optionally further substituted selected from straight or branched C1-C6 alkyl, C3-C6 cycloalkyl or cycloalkyl C1-C6 alkyl, aryl, aryl C1-C6 alkyl, 5 to 7 membered heterocyclyl or heterocyclyl Cl-C6 alkyl with from 1 to 3 heteroatoms selected among nitrogen, oxygen or sulfur; or R3 is a group selected from —COR′, —CONHR′, —SO2R′, —NR′R″, —NR′COR″, —NR′CONHR′ or —NR′SO2R″, wherein R′ and R″ are, the same or different, hydrogen or a group as set forth above; m is 0 or an integer from 1 to 3; or a pharmaceutically acceptable salt thereof. 2. The method of claim 1 wherein the disease caused by and/or associated with an altered protein kinase activity is a cell proliferative disorder selected from the group consisting of cancer, Alzheimer's disease, viral infections, auto-immune diseases and neurodegenerative disorders. 3. The method of claim 2 wherein the cancer is selected from carcinoma, squamous cell carcinoma, hematopoietic tumors of lymphoid or myeloid lineage, tumors of mesenchymal origin, tumors of the central and peripheral nervous system, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratocanthoma, thyroid follicular cancer and Kaposi's sarcoma. 4. The method of claim 1 wherein the cell proliferative disorder is selected from benign prostate hyperplasia, familial adenomatosis, polyposis, neuro-fibromatosis, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post-surgical stenosis and restenosis. 5. The method of claim 1 which provides tumor angiogenesis and metastasis inhibition. 6. The method of claim 1 further comprising subjecting the mammal in need thereof to a radiation therapy or chemotherapy regimen in combination with at least one cytostatic or cytotoxic agent. 7. The method of claim 1 wherein the mammal in need thereof is a human. 8. The method of claim 1 wherein, within the compounds of formula (I), one of Ra and Rb is a hydrogen atom and the other is a group —COR′, —CONHR′, —COOR′ or —SO2R′, wherein R′ is as defined in claim 1. 9. The method of claim 1 wherein, within the compounds of formula (I), one of Ra and Rb is a hydrogen atom and the other is a group —COR′, —CONHR′, —COOR′ or —SO2R′, R2 is hydrogen, m is 0 and R1 and R′ are as defined in claim 1. 10. A method for inhibiting protein kinase activity which comprises contacting the said kinase with an effective amount of a compound as defined in claim 1. 11. An amino-phthalazinone derivative represented by formula (I) wherein Ra and Rb are, each independently, a hydrogen atom or a group, optionally further substituted, selected from straight or branched C1-C6 alkyl, C3-C6 cycloalkyl or cycloalkyl C1-C6 alkyl, aryl, aryl C1-C6 alkyl, 5 to 7 membered heterocyclyl or heterocyclyl C1-C6 alkyl with from 1 to 3 heteroatoms selected among nitrogen, oxygen or sulfur; or one of Ra or Rb is hydrogen or an optionally substituted straight or branched C1-C6 alkyl group, and the other is a group selected from —COR′, —CONHR′, —COOR′ or —SO2R′, wherein R′ is hydrogen or an optionally substituted group selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl or heterocyclylalkyl, as set forth above; R1 is a group of formula —CHR4R5 wherein R4 and R5 are, each independently, hydrogen or an optionally substituted group selected from straight or branched C1-C6 alkyl, C3-C6 cycloalkyl or cycloalkyl C1-C6 alkyl, aryl, aryl C1-C6 alkyl, 5 to 7 membered heterocyclyl or heterocyclyl C1-C6 alkyl with from 1 to 3 heteroatoms selected among nitrogen, oxygen or sulfur; or R1 is a group of formula —NHR′, —NR′COR″, —NR′CONHR″ or —NR′SO2R″, wherein R′ has the above reported meanings other than hydrogen, and R″ is hydrogen or has the meanings set forth above for R′; R2 is a hydrogen atom or it is a group, optionally further substituted, selected from straight or branched C1-C6 alkyl, C3-C6 cycloalkyl or cycloalkyl C1-C6 alkyl, aryl, aryl C1-C6 alkyl, 5 to 7 membered heterocyclyl or heterocyclyl C1-C6 alkyl with from 1 to 3 heteroatoms selected among nitrogen, oxygen or sulfur; any R3, being placed in one or more of the free positions 5, 6 and 8 of the phthalazinone ring are, independently from each other, halogen, nitro, carboxy, cyano or a group optionally further substituted selected from straight or branched C1-C6 alkyl, C3-C6 cycloalkyl or cycloalkyl C1-C6 alkyl, aryl, aryl C1-C6 alkyl, 5 to 7 membered heterocyclyl or heterocyclyl C1-C6 alkyl with from 1 to 3 heteroatoms selected among nitrogen, oxygen or sulfur; or R3 is a group selected from —COR′, —CONHR′, —SO2R′, —NR′R″, —NR′COR″, —NR′CONHR′ or —NR′SO2R″, wherein R′ and R″ are, the same or different, hydrogen or a group as set forth above; m is 0 or an integer from 1 to 3; or a pharmaceutically acceptable salt thereof; the compounds N-[3,4-dihydro-4-oxo-1-(4-pyridinylmethyl)-6-phthalazinyl]-acetamide and N-[3,4-dihydro-4-oxo-1-(4-pyridinylmethyl)-6-phthalazinyl]-2,2,2-trifluoro-acetamide, being excluded. 12. A compound of formula (I) according to claim 11 wherein one of Ra or Rb is a hydrogen atom or an optionally substituted straight or branched C1-C6 alkyl group and the other is a group —COR′ wherein R′ is an optionally substituted group selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl or heterocyclylalkyl, as defined in claim 11, and R1, R2, R3 and m are as defined in claim 11. 13. A compound of formula (I) according to claim 12 wherein R1 is a group —CHR4R5 wherein R4 and R5 are as defined in claim 11, R2 is hydrogen and m is 0. 14. A compound of formula (I) according to claim 11 wherein one of Ra or Rb is a hydrogen atom or an optionally substituted straight or branched C1-C6 alkyl group and the other is a group —CONHR′ wherein R′ is a hydrogen atom or an optionally substituted group selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl or heterocyclylalkyl, as defined in claim 11, and R1, R2, R3 and m are as defined in claim 11. 15. A compound of formula (I) according to claim 14 wherein R1 is a group —CHR4R5 wherein R4 and R5 are as defined in claim 11, R2 is hydrogen and m is 0. 16. A compound of formula (I) according to claim 11 wherein one of Ra or Rb is a hydrogen atom or an optionally substituted straight or branched C1-C6 alkyl group and the other is a group —COOR′ wherein R′ is a hydrogen atom or an optionally substituted group selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl or heterocyclylalkyl, as defined in claim 11, and R1, R2, R3 and m are as defined in claim 11. 17. A compound of formula (I) according to claim 16 wherein R1 is a group —CHR4R5 wherein R4 and R5 are as defined in claim 11, R2 is hydrogen and m is 0. 18. A compound of formula (I) according to claim 11 wherein one of Ra or Rb is a hydrogen atom or an optionally substituted straight or branched C1-C6 alkyl group and the other is a group —SO2R′ wherein R′ is an optionally substituted group selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl or heterocyclylalkyl, as defined in claim 11, and R1, R2, R3 and m are as defined in claim 11. 19. A compound of formula (I) according to claim 18 wherein R1 is a group —CHR4R5 wherein R4 and R5 are as defined in claim 11, R2 is hydrogen and m is 0. 20. A compound of formula (I) according to claim 11 wherein Ra and Rb are both hydrogen atoms and R1, R2, R3 and m are as defined in claim 11. 21. A compound of formula (I) according to claim 20 wherein R1 is a group —CHR4R5 wherein R4 and R5 are as defined in claim 11, R2 is hydrogen and m is 0. 22. A compound of formula (I) according to claim 11 wherein one of Ra or Rb is a hydrogen atom or an optionally substituted straight or branched C1-C6 alkyl and the other is a group, optionally further substituted, selected from alkyl, cycloalkylalkyl, arylalkyl or heterocyclylalkyl as defined in claim 11, and R1, R2, R3 and m are as defined in claim 11. 23. A compound of formula (I) according to claim 22 wherein R1 is a group —CHR4R5 wherein R4 and R5 are as above defined, R2 is hydrogen and m is 0. 24. A compound of formula (I) as defined in claim 11, optionally in the form of a pharmaceutically acceptable salt, selected from: 1. 4-(4-Oxo-6-propionylamino-3,4-dihydro-phthalazin-1-ylmethyl)-benzoic acid methyl ester; 2. 4-[4-Oxo-6-(4-trifluoromethyl-benzoylamino)-3,4-dihydro-phthalazin-1-ylmethyl]-benzoic acid methyl ester; 3. 4-{6-[(Furan-2-carbonyl)-amino]-4-oxo-3,4-dihydro-phthalazin-1-ylmethyl}-benzoic acid methyl ester; 4. 4-[6-(3,4-Dimethoxy-benzoylamino)-4-oxo-3,4-dihydro-phthalazin-1-ylmethyl]-benzoic acid methyl ester; 5. 4-[6-(3-Cyclopentyl-propionylamino)-4-oxo-3,4-dihydro-phthalazin-1-ylmethyl]-benzoic acid methyl ester; 6. 4-[4-Oxo-6-(2-propyl-pentanoylamino)-3,4-dihydro-phthalazin-1-ylmethyl]-benzoic acid methyl ester; 7. 4-{4-Oxo-6-[3-(3-trifluoromethyl-phenyl)-ureido]-3,4-dihydro-phthalazin-1-ylmethyl}-benzoic acid methyl ester; 8. 4-{6-[3-(3-Methoxy-phenyl)-ureido]-4-oxo-3,4-dihydro-phthalazin-1-ylmethyl)-benzoic acid methyl ester; 9. 4-[4-Oxo-6-(3-p-tolyl-ureido)-3,4-dihydro-phthalazin-1-ylmethyl]-benzoic acid methyl ester; 10. 4-{6-[3-(2,4-Difluoro-phenyl)-ureido]-4-oxo-3,4-dihydro-phthalazin-1-ylmethyl}-benzoic acid methyl ester; 11. 4-{6-[3-(3,4-Dichloro-phenyl)-ureido]-4-oxo-3,4-dihydro-phthalazin-1-ylmethyl}-benzoic acid methyl ester; 12. 4-[4-Oxo-6-(3-pyridin-3-yl-ureido)-3,4-dihydro-phthalazin-1-ylmethyl]-benzoic acid methyl ester; 13. 4-(6-Amino-4-oxo-3,4-dihydro-phthalazin-1-ylmethyl)-benzoic acid methyl ester; 14. N-[1-(4-Chloro-3-fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-propionamide; 15. N-[1-[4-Chloro-3-fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-4-trifluoromethyl-benzamide; 16. Furan-2-carboxylic acid [1-(4-chloro-3-fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 17. N-[1-(4-Chloro-3-fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3,4-dimethoxy-benzamide; 18. N-[l-(4-Chloro-3-fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-cyclopentyl-propionamide; 19. 2-Propyl-pentanoic acid [1-(4-chloro-3-fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl)-amide; 20. 1-[1-(4-Chloro-3-fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3-trifluoromethyl-phenyl)-urea; 21. 1-[1-(4-Chloro-3-fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3-methoxy-phenyl)-urea; 22. 1-[1-(4-Chloro-3-fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-p-tolyl-urea; 23. 1-[1-(4-Chloro-3-fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(2,4-difluoro-phenyl)-urea; 24. 1-[1-(4-Chloro-3-fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3,4-dichloro-phenyl)-urea; 25. 1-[1-(4-Chloro-3-fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-pyridin-3-yl-urea; 26. 7-Amino-4-(4-chloro-3-fluoro-benzyl)-2H-phthalazin-1-one; 27. N-{1-[(E)-3-(4-Nitro-phenyl)-allyl]-4-oxo-3,4-dihydro-phthalazin-6-yl}-propionamide; 28. N-{1-[(E)-3-(4-Nitro-phenyl)-allyl]-4-oxo-3,4-dihydro-phthalazin-6-yl}-4-trifluoromethyl-benzamide; 29. Furan-2-carboxylic acid {1-[(E)-3-(4-nitro-phenyl)-allyl]-4-oxo-3,4-dihydro-phthalazin-6-yl}-amide; 30. N-{1-[(E)-3-(4-Nitro-phenyl)-allyl]-4-oxo-3,4-dihydro-phthalazin-6-yl}-3,4-dimethoxy-benzamide; 31. N-{1-[(E)-3-(4-Nitro-phenyl)-allyl]-4-oxo-3,4-dihydro-phthalazin-6-yl}-3-cyclopentyl-propionamide; 32. 2-Propyl-pentanoic acid {1-[(E)-3-(4-nitro-phenyl)-allyl]-4-oxo-3,4-dihydro-phthalazin-6-yl}-amide; 33. 1-{1-[(E)-3-(4-Nitro-phenyl)-allyl]-4-oxo-3,4-dihydro-phthalazin-6-yl}-3-(3-trifluoromethyl-phenyl)-urea; 34. 1-{1-[(E)-3-(4-Nitro-phenyl)-allyl]-4-oxo-3,4-dihydro-phthalazin-6-yl}-(3-methoxy-phenyl)-urea; 35. 1-1-((E)-3-(4-Nitro-phenyl)-allyl]-4-oxo-3,4-dihydro-phthalazin-6-yl}-3-p-tolyl-urea; 36. 1-{1-[(E)-3-(4-Nitro-phenyl)-allyl]-4-oxo-3,4-dihydro-phthalazin-6-yl}-3-(2,4-difluoro-phenyl)-urea; 37. 1-{1-[(E)-3-(4-Nitro-phenyl)-allyl]-4-oxo-3,4-dihydro-phthalazin-6-yl}-3-(3,4-dichloro-phenyl)-urea; 38. 1-{1-[(E)-3-(4-Nitro-phenyl)-allyl]-4-oxo-3,4-dihydro-phthalazin-6-yl}-3-pyridin-3-yl-urea; 39. 7-Amino-4-[(E)-3-(4-nitro-phenyl)-allyl]-2H-phthalazin-1-one; 40. N-(4-Oxo-1-thiophen-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-propionamide; 41. N-(4-Oxo-1-thiophen-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-4-trifluoromethyl-benzamide; 42. Furan-2-carboxylic acid (4-oxo-1-thiophen-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-amide; 43. N-(4-Oxo-1-thiophen-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3,4-dimethoxy-benzamide; 44. N-(4-Oxo-1-thiophen-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-cyclopentyl-propionamide; 45. 2-Propyl-pentanoic acid (4-oxo-1-thiophen-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-amide; 46. 1-(4-Oxo-1-thiophen-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-(3-trifluoromethyl-phenyl)-urea; 47. 1-(4-Oxo-1-thiophen-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-(3-methoxy-phenyl)-urea; 48. 1-(4-Oxo-1-thiophen-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-p-tolyl-urea; 49. 1-(4-Oxo-1-thiophen-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-(2,4-difluoro-phenyl)-urea; 50. 1-(4-Oxo-1-thiophen-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-(3,4-dichloro-phenyl)-urea; 51. 1-(4-Oxo-1-thiophen-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-pyridin-3-yl-urea; 52. 7-Amino-4-thiophen-3-ylmethyl-2H-phthalazin-1-one; 53. N-[1-(3-Methoxy-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-propionamide; 54. N-[1-(3-Methoxy-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-4-trifluoromethyl-benzamide; 55. Furan-2-carboxylic acid [1-(3-methoxy-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 56. N-[1-(3-Methoxy-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3,4-dimethoxy-benzamide; 57. N-[1-(3-Methoxy-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-cyclopentyl-propionamide; 58. 2-Propyl-pentanoic acid [1-(3-methoxy-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 59. 1-[1-(3-Methoxy-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3-trifluoromethyl-phenyl)-urea; 60. 1-[l-(3-Methoxy-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-(3-methoxy-phenyl)-urea; 61. 1-[1-(3-Methoxy-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-p-tolyl-urea; 62. 1-[1-(3-Methoxy-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(2,4-difluoro-phenyl)-urea; 63. 1-[1-(3-Methoxy-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3,4-dichloro-phenyl)-urea; 64. 1-[1- (3-Methoxy-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-pyridin-3-yl-urea; 65. 7-Amino-4-(3-methoxy-benzyl)-2H-phthalazin-1-one; 66. N-(4-Oxo-1-propyl-3,4-dihydro-phthalazin-6-yl)-propionamide; 67. N-(4-Oxo-1-propyl-3,4-dihydro-phthalazin-6-yl)-4-trifluoromethyl-benzamide; 68. Furan-2-carboxylic acid (4-oxo-1-propyl-3,4-dihydro-phthalazin-6-yl)-amide; 69. N-(4-Oxo-1-propyl-3,4-dihydro-phthalazin-6-yl)-3,4-dimethoxy-benzamide; 70. N-(4-Oxo-1-propyl-3,4-dihydro-phthalazin-6-yl)-3-cyclopentyl-propionamide; 71. 2-Propyl-pentanoic acid (4-oxo-1-propyl-3,4-dihydro-phthalazin-6-yl)-amide; 72. 1-(4-Oxo-1-propyl-3,4-dihydro-phthalazin-6-yl)-3-(3-trifluoromethyl-phenyl)-urea; 73. 1-(4-Oxo-1-propyl-3,4-dihydro-phthalazin-6-yl)-(3-methoxy-phenyl)-urea; 74. 1-(4-Oxo-1-propyl-3,4-dihydro-phthalazin-6-yl)-3-p-tolyl-urea; 75. 1-(4-Oxo-1-propyl-3,4-dihydro-phthalazin-6-yl)-3-(2,4-difluoro-phenyl)-urea; 76. 1-(4-Oxo-1-propyl-3,4-dihydro-phthalazin-6-yl)-3-(3,4-dichloro-phenyl)-urea; 77. 1-(4-Oxo-1-propyl-3,4-dihydro-phthalazin-6-yl)-3-pyridin-3-yl-urea; 78. 7-Amino-4-propyl-2H-phthalazin-1-one; 79. N-[1-(3,3-Dimethyl-butyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-propionamide; 80. N-[1-(3,3-Dimethyl-butyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-4-trifluoromethyl-benzamide; 81. Furan-2-carboxylic acid [1-(3,3-dimethyl-butyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 82. N-[1-(3,3-Dimethyl-butyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3,4-dimethoxy-benzamide; 83. N-[1-(3,3-Dimethyl-butyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-cyclopentyl-propionamide; 84. 2-Propyl-pentanoic acid [1-(3,3-dimethyl-butyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 85. 1-[1-(3,3-Dimethyl-butyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3-trifluoromethyl-phenyl)-urea; 86. 1-[1-(3,3-Dimethyl-butyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-(3-methoxy-phenyl)-urea; 87. 1-[1-(3,3-Dimethyl-butyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-p-tolyl-urea; 88. 1-[1-(3,3-Dimethyl-butyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(2,4-difluoro-phenyl)-urea; 89. 1-[1-(3,3-Dimethyl-butyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3,4-dichloro-phenyl)-urea; 90. 1-[1-(3,3-Dimethyl-butyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-pyridin-3-yl-urea; 91. 7-Amino-4-(3,3-dimethyl-butyl)-2H-phthalazin-1-one; 92. N-[4-Oxo-1-(3-phenyl-propyl)-3,4-dihydro-phthalazin-6-yl]-propionamide; 93. N-[4-Oxo-1-(3-phenyl-propyl)-3,4-dihydro-phthalazin-6-yl]-4-trifluoromethyl-benzamide; 94. Furan-2-carboxylic acid [4-oxo-1-(3-phenyl-propyl)-3,4-dihydro-phthalazin-6-yl]-amide; 95. N-[4-Oxo-1-(3-phenyl-propyl)-3,4-dihydro-phthalazin-6-yl]-3,4-dimethoxy-benzamide; 96. N-[4-Oxo-1-(3-phenyl-propyl)-3,4-dihydro-phthalazin-6-yl]-3-cyclopentyl-propionamide; 97. 2-Propyl-pentanoic acid [4-oxo-1-(3-phenyl-propyl)-3,4-dihydro-phthalazin-6-yl]-amide; 98. 1-[4-Oxo-1-(3-phenyl-propyl)-3,4-dihydro-phthalazin-6-yl]-3-(3-trifluoromethyl-phenyl)-urea; 99. 1-[4-Oxo-1-(3-phenyl-propyl)-3,4-dihydro-phthalazin-6-yl]-(3-methoxy-phenyl)-urea; 100. 1-[4-Oxo-1-(3-phenyl-propyl)-3,4-dihydro-phthalazin-6-yl]-3-p-tolyl-urea; 101. 1-[4-Oxo-1-(3-phenyl-propyl)-3,4-dihydro-phthalazin-6-yl]-3-(2,4-difluoro-phenyl)-urea; 102. 1-[4-Oxo-1-(3-phenyl-propyl)-3,4-dihydro-phthalazin-6-yl]-3-(3,4-dichloro-phenyl)-urea; 103. 1-[4-Oxo-1-(3-phenyl-propyl)-3,4-dihydro-phthalazin-6-yl]-3-pyridin-3-yl-urea; 104. 7-Amino-4-(3-phenyl-propyl)-2H-phthalazin-1-one; 105. N-(4-Oxo-1-pyridin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-propionamide; 106. N-(4-Oxo-1-pyridin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-4-trifluoromethyl-benzamide; 107. Furan-2-carboxylic acid (4-oxo-1-pyridin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-amide; 108. N-(4-Oxo-1-pyridin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-succinamic acid ethyl ester; 109. N-(4-Oxo-1-pyridin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-cyclopentyl-propionamide; 110. 2-Propyl-pentanoic acid (4-oxo-1-pyridin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-amide; 111. 1-(4-Oxo-1-pyridin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-(3-trifluoromethyl-phenyl)-urea; 112. 1-(3-Methoxy-phenyl)-3-(4-oxo-1-pyridin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-urea; 113. 1-(4-Oxo-1-pyridin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-p-tolyl-urea; 114. 1-(2,4-Difluoro-phenyl)-3-(4-oxo-1-pyridin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-urea; 115. 1-(3,4-Dichloro-phenyl)-3-(4-oxo-1-pyridin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-urea; 116. 1-(4-Oxo-1-pyridin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-pyridin-3-yl-urea; 117. 7-Amino-4-pyridin-3-ylmethyl-2H-phthalazin-1-one 118. N-(4-Oxo-1-pyridin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-benzamide; 119. N-[1-(4-Chloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-propionamide; 120. N-[1-(4-Chloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-4-trifluoromethyl-benzamide; 121. Furan-2-carboxylic acid [1-(4-chloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 122. N-[1-(4-Chloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-succinamic acid ethyl ester; 123. N-[1-(4-Chloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-cyclopentyl-propionamide; 124. 2-Propyl-pentanoic acid [1-(4-chloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 125. 1-[1-(4-Chloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3-trifluoromethyl-phenyl)-urea; 126. 1-[1-(4-Chloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3-methoxy-phenyl)-urea; 127. 1-[1-(4-Chloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-p-tolyl-urea; 128. 1-[1-(4-Chloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(2,4-difluoro-phenyl)-urea; 129. 1-[1-(4-Chloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3,4-dichloro-phenyl)-urea; 130. 1-[1-(4-Chloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-pyridin-3-yl-urea; 131. 7-Amino-4-(4-Chloro-benzyl)-2H-phthalazin-1-one; 132. N-[1-(4-Cyano-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-propionamide; 133. N-[1-(4-Cyano-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-4-trifluoromethyl-benzamide; 134. Furan-2-carboxylic acid [1-(4-cyano-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 135. N-[1-(4-Cyano-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-succinamic acid ethyl ester; 136. N-[1-(4-Cyano-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-cyclopentyl-propionamide; 137. 2-Propyl-pentanoic acid [1-(4-cyano-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 138. 1-[1-(4-Cyano-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3-trifluoromethyl-phenyl)-urea; 139. 1-[1-(4-Cyano-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3-methoxy-phenyl)-urea; 140. 1-[1-(4-Cyano-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-p-tolyl-urea; 141. 1-[1-(4-Cyano-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(2,4-difluoro-phenyl)-urea; 142. 1-[1-(4-Cyano-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3,4-dichloro-phenyl)-urea; 143. 1-[1-(4-Cyano-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-pyridin-3-yl-urea; 144. 7-Amino-4-(4-Cyano-benzyl)-2H-phthalazin-1-one; 145. N-[1-(3-Fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-propionamide; 146. N-[1-(3-Fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-4-trifluoromethyl-benzamide; 147. Furan-2-carboxylic acid [1-(3-fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 148. N-[1-(3-Fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-succinamic acid ethyl ester; 149. N-[1-(3-Fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-cyclopentyl-propionamide; 150. 2-Propyl-pentanoic acid [1-(3-fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 151. 1-[1-(3-Fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3-trifluoromethyl-phenyl)-urea; 152. 1-[1-(3-Fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3-methoxy-phenyl)-urea; 153. 1-[1-(3-Fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-p-tolyl-urea; 154. 1-[1-(3-Fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(2,4-difluoro-phenyl)-urea; 155. 1-[1-(3-Fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3,4-dichloro-phenyl)-urea; 156. 1-[1-(3-Fluoro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-pyridin-3-yl-urea; 157. 7-Amino-4-(3-Fluoro-benzyl)-2H-phthalazin-1-one; 158. N-[1-(3-Methyl-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-propionamide; 159. N-[1-(3-Methyl-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-4-trifluoromethyl-benzamide; 160. Furan-2-carboxylic acid [1-(3-methyl-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 161. N-[1-(3-Methyl-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-succinamic acid ethyl ester; 162. N-[1-(3-Methyl-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-cyclopentyl-propionamide; 163. 2-Propyl-pentanoic acid [1-(3-methyl-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 164. 1-[1-(3-Methyl-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3-trifluoromethyl-phenyl)-urea; 165. 1-[1-(3-Methyl-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3-methoxy-phenyl)-urea; 166. 1-[1-(3-Methyl-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-p-tolyl-urea; 167. 1-[1-(3-Methyl-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(2,4-difluoro-phenyl)-urea; 168. 1-[1-(3-Methyl-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3,4-dichloro-phenyl)-urea; 169. 1-[1-(3-Methyl-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-pyridin-3-yl-urea; 170. 7-Amino-4-(3-Methyl-benzyl)-2H-phthalazin-1-one; 171. N-[1-(2,4-Dichloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-propionamide; 172. N-[1-(2,4-Dichloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-4-trifluoromethyl-benzamide; 173. Furan-2-carboxylic acid [1-(2,4-dichloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 174. N-[1-(2,4-Dichloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-succinamic acid ethyl ester; 175. N-[1-(2,4-Dichloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-cyclopentyl-propionamide; 176. 2-Propyl-pentanoic acid [1-(2,4-dichloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-amide; 177. 1-[1-(2,4-Dichloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3-trifluoromethyl-phenyl)-urea; 178. 1-[1-(2,4-Dichloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3-methoxy-phenyl)-urea; 179. 1-[1-(2,4-Dichloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-p-tolyl-urea; 180. 1-[1-(2,4-Dichloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(2,4-difluoro-phenyl)-urea; 181. 1-[1-(2,4-Dichloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-(3,4-dichloro-phenyl)-urea; 182. 1-[1-(2,4-Dichloro-benzyl)-4-oxo-3,4-dihydro-phthalazin-6-yl]-3-pyridin-3-yl-urea; 183. 7-Amino-4-(2,4-dichloro-benzyl)-2H-phthalazin-1-one; 184. N-(4-Oxo-1-quinolin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-propionamide; 185. N-(4-Oxo-1-quinolin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-4-trifluoromethyl-benzamide; 186. Furan-2-carboxylic acid (4-oxo-1-quinolin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-amide; 187. N-(4-Oxo-1-quinolin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-succinamic acid ethyl ester; 188. N-(4-Oxo-1-quinolin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-cyclopentyl-propionamide; 189. 2-Propyl-pentanoic acid (4-oxo-1-quinolin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-amide; 190. 1-(4-Oxo-1-quinolin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-(3-trifluoromethyl-phenyl)-urea; 191. 1-(4-Oxo-1-quinolin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-(3-methoxy-phenyl)-urea; 192. 1-(4-Oxo-1-quinolin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-p-tolyl-urea; 193. 1-(4-Oxo-1-quinolin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-(2,4-difluoro-phenyl)-urea; 194. 1-(4-Oxo-1-quinolin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-(3,4-dichloro-phenyl)-urea; 195. 1-(4-Oxo-1-quinolin-3-ylmethyl-3,4-dihydro-phthalazin-6-yl)-3-pyridin-3-yl-urea; 196. 7-Amino-4-quinolin-3-ylmethyl-2H-phthalazin-1-one; 197. N-[4-Oxo-1-(2-trifluoromethyl-benzyl)-3,4-dihydro-phthalazin-6-yl]-propionamide; 198. N-[4-Oxo-1-(2-trifluoromethyl-benzyl)-3,4-dihydro-phthalazin-6-yl]-4-trifluoromethyl-benzamide; 199. Furan-2-carboxylic acid [4-oxo-1-(2-trifluoromethyl-benzyl)-3,4-dihydro-phthalazin-6-yl]-amide; 200. N-[4-Oxo-1-(2-trifluoromethyl-benzyl)-3,4-dihydro-phthalazin-6-yl]-succinamic acid ethyl ester; 201. N-[4-Oxo-1-(2-trifluoromethyl-benzyl)-3,4-dihydro-phthalazin-6-yl]-3-cyclopentyl-propionamide; 202. 2-Propyl-pentanoic acid [4-oxo-1-(2-trifluoromethyl-benzyl)-3,4-dihydro-phthalazin-6-yl]-amide; 203. 1-[4-Oxo-1-(2-trifluoromethyl-benzyl)-3,4-dihydro-phthalazin-6-yl]-3-(3-trifluoromethyl-phenyl)-urea; 204. 1-[4-Oxo-1-(2-trifluoromethyl-benzyl)-3,4-dihydro-phthalazin-6-yl]-3-(3-methoxy-phenyl)-urea; 205. 1-[4-Oxo-1-(2-trifluoromethyl-benzyl)-3,4-dihydro-phthalazin-6-yl]-3-p-tolyl-urea; 206. 1-[4-Oxo-1-(2-trifluoromethyl-benzyl)-3,4-dihydro-phthalazin-6-yl]-3-(2,4-difluoro-phenyl)-urea; 207. 1-[4-Oxo-1-(2-trifluoromethyl-benzyl)-3,4-dihydro-phthalazin-6-yl]-3-(3,4-dichloro-phenyl)-urea; 208. 1-[4-Oxo-1-(2-trifluoromethyl-benzyl)-3,4-dihydro-phthalazin-6-yl]-3-pyridin-3-yl-urea; 209. 7-Amino-4-(2-trifluoromethyl-benzyl)-2H-phthalazin-1-one. 25. A process for preparing the compounds of formula (I) as defined in claim 11, and the pharmaceutically acceptable salts thereof, which process comprises: a) reacting a compound of formula (II) wherein R3 and m are as defined in claim 11 and Hal represents a halogen atom, with a suitable phosphine derivative (PL3), under optional reductive conditions, so as to obtain a compound of formula (III) wherein P is a phosphorous atom and L are the phosphine ligands; b) reacting the compound of formula (III) with an aldehydic Resin-CHO, in the presence of a suitable reducing agent, so as to obtain a resin supported compound of formula (IV) wherein R3, m, P, L, Hal and the Resin are as above defined; c) reacting the compound of formula (IV) with a carbonyl derivative of formula (V) or a nitroso derivative of formula (VI) R4—CO—R5 (V) R′—NO (VI) wherein R4, R5 and R′ are as defined in claim 11; so as to obtain the compound of formula (VII) wherein A is a group ═CR4R5 or ═NR′, respectively; and optionally reacting the compound of formula (VII) according to any one of the alternative steps d.1) or d.2) below d.1) with one of the derivatives of formula (VIII), (IX), (X) or (XI), under optional basic conditions, R′COZ (VIII), R′NCO (IX), R′OCOZ (X), R′SO2Z (XI) wherein Z is a halogen atom or a suitable leaving group and R′ is as defined in claim 11, so as to obtain the compound of formula (XII) wherein Rb is —COR′, —CONHR′, —COOR′ or —SO2R′, respectively; or d.2) with a compound of formula (XIII) Rb—Z (XIII) wherein Z is a halogen atom and Rb is alkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl or heterocyclylalkyl as defined in claim 11, so as to obtain the corresponding compound of the above formula (XII); e) reacting the thus obtained compounds of formula (VII) or (XII) with a hydrazine derivative of formula (XIV) R2—NH—NH2 (XIV) wherein R2 is as defined in claim 11, so as to obtain the compounds of formula (XV) or (XVI), respectively wherein Rb, R2, R3, m and the Resin are as above defined and R1 is a group of formula —CHR4R5 or —NHR′ wherein R4, R5 and R′ are as above defined; f) reacting the compounds of formula (XV) or (XVI) under acidic conditions so as to obtain the compound of formula (I) and, whenever desired, converting it into another compound of formula (I) and/or into a pharmaceutically acceptable salt thereof. 26. The process of claim 25 wherein, in step a), the phosphine derivative is triphenylphosphine PPh3. 27. The process of claim 25 wherein, in step b), the reducing agent is selected from pyridine-borane complex, sodium cyanoboron hydride, sodium triacetate boron hydride or dimethylsufide borane. 28. The process of claim 25 wherein, in step d.1), it is used a compound of formula (VIII), (X) or (XI), wherein Z is a chlorine atom. 29. The process of claim 25 wherein, in step f), acidic conditions are reached by using trifluoroacetic acid. 30. Any specific compound of formula (I), as defined in claim 11, which is obtainable, for instance through a combinatorial chemistry technique according to the process as set forth in claim 25, by first reacting the compound of formula (IV) with each one of the aldehyde derivatives of formula (V), as set forth in table I; by reacting any of the resultant compounds of formula (VII) with each one of the acyl chloride derivatives of formula (VIII), as set forth in table II; by reacting any of the resultant compounds of formula (XII) with hydrazine; and by working according to step f) of the process of claim 25. 31. Any specific compound of formula (I), as defined in claim 11, which is obtainable, for instance through a combinatorial chemistry technique according to the process as set forth in claim 25, by first reacting the compound of formula (IV) with each one of the aldehyde derivatives of formula (V), as set forth in table I; by reacting any of the resultant compounds of formula (VII) with each one of the isocyanate derivatives of formula (IX), as set forth in table III; by reacting any of the resultant compounds of formula (XII) with hydrazine; and by working according to step f) of the process of claim 25. 32. A library of two or more amino-phthalazinone derivatives of formula (I) wherein Ra and Rb are, each independently, a hydrogen atom or a group, optionally further substituted, selected from straight or branched C1-C6 alkyl, C3-C6 cycloalkyl or cycloalkyl C1-C6 alkyl, aryl, aryl C1-C6 alkyl, 5 to 7 membered heterocyclyl or heterocyclyl C1-C6 alkyl with from 1 to 3 heteroatoms selected among nitrogen, oxygen or sulfur; or one of Ra or Rb is hydrogen or an optionally substituted straight or branched C1-C6 alkyl group, and the other is a group selected from —COR′, —CONHR′, —COOR′ or —SO2R′, wherein R′ is hydrogen or an optionally substituted group selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl or heterocyclylalkyl, as set forth above; R1 is a group of formula —CHR4R5 wherein R4 and R5 are, each independently, hydrogen or an optionally substituted group selected from straight or branched C1-C6 alkyl, C3-C6 cycloalkyl or cycloalkyl C1-C6 alkyl, aryl, aryl C1-C6 alkyl, 5 to 7 membered heterocyclyl or heterocyclyl C1-C6 alkyl with from 1 to 3 heteroatoms selected among nitrogen, oxygen or sulfur; or R1 is a group of formula —NHR′, —NR′ COR″, —NR′CONHR″ or —NR′SO2R″, wherein R′ has the above reported meanings other than hydrogen, and R″ is hydrogen or has the meanings set forth above for R′; R2 is a hydrogen atom or it is a group, optionally further substituted, selected from straight or branched C1-C6 alkyl, C3-C6 cycloalkyl or cycloalkyl C1-C6 alkyl, aryl, aryl C1-C6 alkyl, 5 to 7 membered heterocyclyl or heterocyclyl C1-C6 alkyl with from 1 to 3 heteroatoms selected among nitrogen, oxygen or sulfur; any R3, being placed in one or more of the free positions 5, 6 and 8 of the phthalazinone ring are, independently from each other, halogen, nitro, carboxy, cyano or a group optionally further substituted selected from straight or branched C1-C6 alkyl, C3-C6 cycloalkyl or cycloalkyl C1-C6 alkyl, aryl, aryl C1-C6 alkyl, 5 to 7 membered heterocyclyl or heterocyclyl C1-C6 alkyl with from 1 to 3 heteroatoms selected among nitrogen, oxygen or sulfur; or R3 is a group selected from —COR′, —CONHR′, —SO2R′, —NR′R″, —NR′ COR″, —NR′CONHR′ or —NR′SO2R″, wherein R′ and R″ are, the same or different, hydrogen or a group as set forth above; m is 0 or an integer from 1 to 3; or a pharmaceutically acceptable salt thereof. 33. A pharmaceutical composition comprising an effective amount of a compound of formula (I) as defined in claim 11 and, at least, one pharmaceutically acceptable excipient, carrier or diluent. 34. A pharmaceutical composition according to claim 33 further comprising one or more chemotherapeutic agents, as a combined preparation for simultaneous, separate or sequential use in anticancer therapy. 35. A product or kit comprising a compound of claim 11 or a pharmaceutical composition thereof as defined in claim 33, and one or more chemotherapeutic agents, as a combined preparation for simultaneous, separate or sequential use in anticancer therapy. 36. A compound of formula (I) or a pharmaceutically acceptable salt thereof, as defined in claim 11, for use as a medicament. 37. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, as defined in claim 11, in the manufacture of a medicament for treating diseases caused by and/or associated with an altered protein kinase activity. 38. Use according to claim 37 for treating tumors. |
Method for transmitting data from an emitter to a plurality of receivers |
A method is provided for transmitting data from a sender to a number of receivers. Also provided are an emitting and/or receiving unit and a communication system. The present invention seeks to provide a method, an emitting and/or receiving unit and a communication system for the efficient, resource saving and energy saving transmission of data to a group of receivers of a point-to-multipoint service. To this end, a point-to-multipoint service is carried out as an extension of a broadcast service CBS in a multi-layer protocol system, providing a multimedia transmission and/or a multicast service, preferably in the form of a multimedia broadcast/multicast service MBMS, during the distribution and/or planning of the use of system resources and the use of a discontinuous reception DRX. |
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