MXPA06009413A - Oxydecahydronaphthalene modulators of hm74 - Google Patents

Abstract

Hosts cells expressing HM74 were used to obtain oxydecalin-like molecules with agonist activity having the following structure (formula I).

Description

HM74 MODULATORS OF OXIDECAHIDRONAFTALENO.

BACKGROUND OF THE INVENTION.

G-protein coupled receptors (GPCRs) are integral membrane receptors that are involved in the transduction of cellular signals. GPCRs respond to a variety of extracellular signals, including neurotransmitters, hormones, odors and light, and can transduce signals to initiate a second messenger response within the cell. Many therapeutic drugs target GPCRs because these receptors mediate a wide variety of physiological responses, including inflammation, vasodilation, heart rhythm, bronchodilation, endocrine secretion, and peristalsis. In general, diseases such as asthma, chronic obstructive pulmonary disease (COPD), psoriasis and rheumatoid arthritis (RA) are considered to have an inflammatory etiology involving helper T cells, monocyte-macrophages and eosinophils. Current anti-inflammatory therapy with corticosteroids is effective in asthma, but is associated with metabolic and endocrine side effects. The same may be true for inhaled formulations that can be absorbed by the lung or nasal mucosa. Currently, there are no satisfactory oral therapies for RA and COPD. Molecular cloning of HM74 from human monocytes predicted that HM74 was a chemokine receptor (Nomura et al., Int. Immunol. (1993) 5 (10): 1239-1249). HM74 is expressed mainly in the bone marrow, spleen, tonsil and trachea. Human cells contain a related but distinct receptor, HM74A. The amino acid sequences of HM74 and HM74A are approximately 95% identical. However, the ligand of HM74A is known, while that of HM74 is not known. Nicacin or nicotinic acid is the ligand of HM74A, Wise et al., J. Biol. Chem. 278: 9869-9874, 2003. However, niacin is a poor activator of HM74. The mouse genome contains an HM74A gene but not an HM74 gene.

In some circumstances, HM74 and HM74A demonstrate co-regulation.

Using Taqman-PCR, the authors of the invention found that the expression of HM74 and HM74A are induced 50 times by TNFs in granulocytes and -20 times for LPS or TNFcr in monocytes. The expressions of HM74 and HM74A are also induced 4-5 times in normal human bronchial epithelial cells with the TH2, IL-4 or IL-13 cytokines, which are known to be important in the etiology of asthma. The expressions of HM74 and HM74A are positively regulated in human primary eosinophils by IL-5. Finally, the expression of pulmonary HM74A is positively regulated in the experimental asthma model in murines. The restricted tissue distribution of HM74 and HM74A, and their regulation, suggest that HM74 and HM74A have a role in inflammatory processes, such as asthma, COPD and RA. Given the role of GPCRs in disease and the ability to treat diseases by modulating the activity of GPCRs, the identification and characterization of GPCR ligands can provide the development of new compositions and methods to treat pathological conditions involving activity. of a GPCR. The present invention identifies and characterizes molecules that are coupled with HM74, and provides compositions and methods for applying the discovery to the identification and treatment of related diseases. SUMMARY OF THE INVENTION The present invention relates to molecules that activate HM74 but not HM74A. In another aspect of the invention, methods for identifying modulators of HM74 are described. For example, a method of interest comprises the steps of providing a chemical moiety, providing a cell that expresses HM74, and determining whether the chemical moiety modulates the signaling activity of HM74, including whether said modulation occurs in the presence or absence of an agonist of the present invention. In a related aspect, chemical residues may include, but are not limited to, peptides, antibodies, agonists, inverse agonists and antagonists. Alternatively, a known modulator can be used in a competitive assay to identify other modulators. Dihydripyrans with a fused ring (also known as oxidecahydronaphthalene and oxidecalin) activate HM74. These compounds produce the selective mobilization of dose-sensitive calcium, in cells expressing HM74, such as CHO cells, 293 cells and L1.2 cells. The oxidecalinas of interest have the following structure: where X is O, NR2 or S; Ri is a CrC18 alkyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C? -C 8 alkenyl which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a Ci-C-iß alkynyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof a C3-C18 aryl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be substituted, or their combinations; or a C5-C? 8 cycloalkyl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be substituted, or combinations thereof; or its combinations; R2 is a group R1; or R2 may be a (C? -C? o) -cycloalkyl (C3-C? o) alkyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; or X and R2 can form a ring; R3 is H or R-i; and Y is carbonyl, a Schiff base, an oxine, a ketal, an acetal, an oxazolidine, a thiazolidine or an enol-ester. In general, the compounds of interest are agonists. Therefore, a compound of interest could be developed as a drug candidate. A compound of interest could also be used to identify other molecules that modulate HM74, for example, by competitive assays. Another aspect of the invention includes therapeutic compositions, wherein said compositions include nucleic acids, antibodies, polypeptides, agonists, inverse agonists and antagonists. In addition, the methods of the invention also include methods for treating disease states and modulating the signaling activity of HM74, administering said therapeutic compositions to a patient in need thereof. These and other aspects of the invention will become apparent by reference to the following detailed description and accompanying drawings. In addition, several references are presented below, which describe some procedures or compositions in more detail. Each of the references hereby incorporated herein by reference, as if indicating the individual incorporation of each of them.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the discovery of molecules that activate HM74. The sequence encoding HM74, Nomura et al., See above, is known. In this specification methods for obtaining, preparing and using the HM74 are provided.

Changes in the sequence coding for HM74 and amino acids are tolerated provided they do not adversely affect the known functional activities of HM74. The present compounds modulate HM74 and are agonists of HM74 and therefore are candidates for drugs for disorders characterized by inflammation, such as asthma. The present compounds can also be used to screen HM74 antagonists. For example, cells expressing HM74 are exposed to a test compound, and then to an agonist of the present invention. The effect of the test compound can then be determined, for example, in the mobilization of calcium, to determine if the test compound reduces the levels of calcium mobilization induced by the agonist of the present invention. It is contemplated that other assays to identify, e.g., agonists and inverse agonists, are within the scope of the present invention. The oxidecalinas of interest have the following structure: where X is O, NR2 or S; R-i is a C C 8 alkyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C C 8 alkenyl which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C? -C-? 8 alkynyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof a C3-C18 aryl, which may contain a side group, may contain a bridge, may contain a heteroatom or it can be substituted, or its combinations; or a C5-Cys cycloalkyl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be substituted, or combinations thereof; or its combinations; R2 is a group R-t; or R2 may be a (C? -C?) cycloalkyl (C3-C10) alkyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; or X and R2 can form a ring; R3 is H or R-i; and Y is carbonyl, a Schiff base, an oxine, a ketal, an acetal, an oxazolidine, a thiazolidine or an enol-ester. In general, the compounds of interest are agonists. Therefore, a compound of interest could be developed as a drug candidate. A compound of interest could also be used to identify other molecules that modulate HM74, for example, by competitive assays. The term "alkyl" means a straight or branched chain hydrocarbon. Representative examples are methyl, ethyl, propyl, isopropyl, butyl, butyl, tert-butyl, sec-butyl, pentyl and hexyl. The hydrocarbon may contain one or more triple unsaturated bonds. The term "alkoxy" means an alkyl group attached to an oxygen atom. Examples are methoxy, ethoxy, propoxy, butoxy and pentoxy. "Aryl" is a ring that is an aromatic hydrocarbon. Examples include phenyl and naphthyl. "Heteroatomo" is generally an atom that differs from those that characterize a molecule. Thus, in a hydrocarbon, any atom that is not a carbon or a hydrogen is a heteroatom. Common biologically acceptable heteroatoms include oxygen, sulfur, and nitrogen. The term "heteroaryl" refers to an aryl group in which one or more carbon atoms are replaced by a heteroatom. Examples are pyridyl, imidazolyl, pyrrolyl, thienyl, furyl, pyranyl, pyrimidinyl, pyridazinyl, indolyl, quinolyl, naphthyridinyl and isoxazolyl. "Branched" means that the structure contains one or more branches in one or more sites. A branch can be a group R as defined above or another side group. The term "cycloalkyl" refers to a cyclic hydrocarbon. Some examples are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. It can contain a bridge of variable length.

"Heterocycle" is a cycloalkyl in which one or more carbon atoms are replaced by a heteroatom. Examples are pyrrolindinyl, piperidinyl and piperazinyl. The term "heteroalkyl" is an alkyl in which one or more carbon atoms are replaced by a heteroatom. An ether is a heteroalkyl. By "substituted" is meant that the organic base radical has one or more substituent groups. Therefore, an atom or group replaces another atom or group in a molecule. Representative substituents include a halogen, C 1 -C 8 alkyl, -CN, alkoxy, hydroxyl, sulfide, sulfate, sulfonamide, amine, amide, an alcohol, a keto group, C 6 -C 8 aryl, a CC alkyl ? 8 halogenated, a nitrite group or a nitrate group. A "halogen" is, for example, chlorine, fluorine or bromine. An "alkenyl" is a hydrocarbon that contains one or more carbon-carbon double bonds. The hydrocarbon may be branched. The term "ring" means one, one of a plurality of annular structures, or a plurality of annular structures, where two or more of the plurality of rings may be fused, wherein one or more of the plurality of rings may be aromatic , contain a heteroatom, may be substituted, or one of their combinations. The ring can be bicyclic or polycyclic. The ring may contain a bridge of variable length. The term "side group" means an atom or molecule attached to another structure. Therefore, a side group can be a group R as defined above, an alkyl, an aryl, a cycloalkyl, etc. The term "bridge" refers to a connector between two structures. For example, a non-cyclic hydrocarbon, such as an alkyl, alkenyl, and the like, which may contain a heteroatom, may be substituted, may be branched or combinations thereof, may connect two cyclic hydrocarbons, such as aryl or cycloalkyl groups. The bridge can also be contained in a cyclic structure that joins at least two atoms of the cyclic structure. The intramolecular bridge can contain 0, 1, 2, 3, 4 or more atoms. The non-molecular bridge may be linear, branched and may contain substitutions. The compounds of interest contain functional groups that can form derivatives to form prodrugs to enhance bioavailability. Therefore, the present invention contemplates variants of the active compounds of interest, which after administration, are metabolized to a bioactive form. Such precursors of bioactive drugs are also known as bioreversible vehicles, latent drugs, drug delivery systems or prodrugs. ("Bioreversible Carriers in Drug Design" EB Roche, ed., Pergamon, NY, 1987, "Prodrugs as Novel Drug Delivery Systems," Higuchi &Stella, eds., American Chemical Society, DC, 1975) Chemical modification of drugs is aimed at addressing particular aspects of pharmacodynamics, such as how to enhance the availability of a polar compound that must cross a lipid barrier, how to stabilize a compound that is normally susceptible to in vivo degradation, etc. Other reasons for preparing prodrugs include the toxicity of the bioactive drug, lack of specificity, instability, being metabolized at the site of absorption, being absorbed too quickly, patient compliance, such as bad taste, or pain at the injection site, little acceptance by the doctor or also formulation problems. A common modification is esterification, which is not limited to the modification of a carboxylic acid. Chemical processes exist for preparing said derivatives, for example, for amines, mines, sulfur-containing substituents, and also amides. In the case of esters, different substituents may be added, including unbranched, cyclic or branched hydrocarbons, which may be substituted, may contain one or more double or triple bonds, may contain ring structures, the main chain of the hydrocarbon may contain one or more heteroatoms, such as nitrogen, sulfur or oxygen, etc. When considering the R group to construct the ester, another factor to consider is the susceptibility to enzymatic cleavage. Thus, steric loading and conformational factors may be determinants for bioavailability. For example, a branched alkyl group can provide steric hindrance for the accessibility of the esterase to the active site, thereby slowing down the rate of hydrolysis. This may be less convenient, bioavailability is delayed, or convenient, bioavailability is prolonged. In other circumstances, it is convenient to enhance the aqueous solubility of a drug. Examples of substituents that achieve this goal include succinates, sulfates, hemisuccinates, phosphates, amino acids, acetates, amines and the like. Nitrogens of amides, imides, carbonates, hydantoins and the like can be derivatized. Suitable groups for the reaction with nitrogen include hydroxymethyl groups, or hydroxyalkyl groups in general, acyloxyalkyl groups and acyl groups. Carbonyl groups are also sites for forming derivatives. Examples of derivatives are Schiff bases, oxines, ketals, acetals, oxazolidines, thiazolidines and enol-esters. Although the derivatives discussed above comprise covalent linkages of the substituent to the drug, a substituent can be bound to the drug in other ways, for example, by hydrogen bonds, Van der Waals forces, electrostatic forces, hydrophobic interactions and the like. Yet another means to obtain derivatives is to use substituents that are removed from a prodrug by a non-enzymatic mechanism. Examples include prodrugs that contain (2-oxo-1,3-dioxol-4-yl) methyl esters, Mannich bases, oxazolidines, esters with a basic side chain that catalyzes intramolecular hydrolysis, and esters or amides that suffer an intramolecular nucleophilic cyclization-elimination reaction. The mechanism of cyclization can be achieved for drugs containing phenols, alcohols and amines. "Prodrug Design" Testa & Mayer in "Encyclopedia of Pharmaceutical Technology," 2nd ed. V. 3, Swarbrick & Boilan, eds., Marcel Dekker, 2002. Therefore, the present invention contemplates any further modification of the compounds of interest by the practice of known synthesis methods to obtain compounds that once administered react or react in vivo to give a compound that modulates the activity of HM74. The term "equivalent amino acid residues" as used herein means that amino acids occupy substantially the same position within a sequence of a protein, when two or more sequences are aligned for analysis. The preferred HM74 polypeptides of the present invention have an amino acid sequence sufficiently identical to that of wild-type HM74. By "wild" is meant the most widespread form or allele present in a defined population, whether local or with greater scope. The term "sufficiently identical" is used herein to refer to a first amino acid or nucleotide sequence that contains a minimum or sufficient number of amino acid residues or nucleotides identical or equivalent (eg, with a similar side chain) to a second amino acid or nucleotide sequence, such that the first and second amino acid or nucleotide sequences have a common structural domain and / or common functional activity. For example, amino acid or nucleotide sequences containing a common structural domain, which are at least 96% identical to an activity of HM74, are herein defined as sufficiently identical. Used interchangeably herein, an "activity of HM74", "biological activity of HM74" or "functional activity of HM74" refers to an activity exerted by a protein, polypeptide or nucleic acid molecule of HM74, in a cell expressing HM74, determined in vivo or in vitro, according to standard techniques. An activity of HM74 may be a direct activity, such as an association with or an enzymatic activity in a second protein, or an indirect activity, such as a cell signaling activity mediated by the interaction of HM74 with a second protein. In a preferred embodiment, an activity of HM74 includes at least one or more of the following activities: (i) the ability to interact with proteins in the signaling path of HM74; (ii) the ability to interact with a ligand of HM74; (iii) the ability to alter the host cell when activated; (iv) activation by the binding of a molecule of the invention; and (v) the ability to interact with an objective intracellular protein. One aspect of the invention relates to the expression of HM74 in cells that demonstrate a response when HM74 is activated. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA), and DNA or RNA analogues. generated using nucleotide analogs. The nucleic acid molecule can be single-stranded 0 double-stranded, but preferably it is double-stranded DNA. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid does not have sequences that naturally flank the nucleic acid encoding HM74 (i.e., sequences located at the 5 'and 3' ends of the nucleic acid) in the genomic DNA of the organism of which gets the nucleic acid. The nucleic acid molecule of isolated HM74 may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences that naturally flank the open reading frame of the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is obtained. In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, may not have substantially other cellular material, or culture medium, when produced by recombinant techniques, or have substantially no chemical precursors or other chemicals when synthesizes chemically. A nucleic acid molecule of the present invention, for example, a nucleic acid molecule encoding HM74, can be isolated using standard molecular biology techniques and then sequenced (Nomura et al., See above). Using all or a part of the sequence of HM74, nucleic acid molecules of HM74 can be isolated using standard hybridization and cloning techniques (e.g. as described by Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). A nucleic acid molecule of the invention can be amplified using cDNA, mRNA or genomic DNA as a template, and suitable oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid amplified in this way can be cloned into a suitable vector and characterized by analysis of the DNA sequence. In addition, oligonucleotides corresponding to nucleotide sequences of HM74 can be prepared by standard synthetic techniques, for example, using an automatic DNA synthesizer. In addition, the nucleic acid molecule of the invention may comprise only parts of a nucleic acid sequence encoding HM74, eg, a fragment encoding the extracellular domains and / or intracellular domains that produce a detectable intracellular event, when joined 'to him a ligand. Preferably, the detectable intracellular event is one that is observed when the HM74 is activated in a normal host cell. A nucleic acid fragment encoding a "biologically active part of HM74" can be prepared by isolating a part of HM74 that encodes a polypeptide having a biological activity of HM74, by expressing the encoded part of the HM74 protein (eg, by recombinant expression). in vitro), and evaluating the activity of the coded part of HM74. The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of the HM74 described, due to the degeneracy of the genetic code, and therefore they encode substantially the same HM74 protein, as previously described. Those skilled in the art will understand that within a population (e.g., the human population) there may be polymorphisms of the sequence of DNA leading to changes in the amino acid sequences of HM74. Such genetic polymorphism in the sequence encoding HM74 may exist between individuals within a population, due to natural allelic variation. An allele is one of a group of genes that are found alternatively at a given genetic locus. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules that comprise an open reading frame that encodes a HM74 protein, preferably a mammalian HM74 protein. As used herein, the phrase "allelic variant" refers to a nucleotide sequence that is located at a locus of HM74, or to a polypeptide encoded by the nucleotide sequence. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be easily accomplished using hybridization probes to identify the same genetic locus in a variety of individuals. It is intended that any and all such variations of nucleotides and resulting amino acid polymorphisms or variations of HM74 that are the result of natural allelic variation, and that do not alter the functional activity of HM74, are within the scope of the invention. In addition, it is intended that the nucleic acid molecules encoding the HM74 proteins of other species (homologues of HM74) with a nucleotide sequence that differs from that of human HM74, but have substantially the same activity, are within the scope of the invention. The nucleic acid molecules corresponding to the natural and homologous allelic variants of the HM74 cDNA of the invention can be isolated, based on the identity with the human HM74 nucleic acids described herein, using the human cDNAs, or one of their parts, such as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. As used herein, the term "hybrid under stringent conditions" is intended to describe conditions for hybridization and washing, in which the nucleotide sequences typically remain hybridized. Such restrictive conditions are known to those skilled in the art, and can be found in "Current Protocols in Molecular Biology," John Wiley & amp;; Sons, N.Y. (1989), 6.3.16.3.6. A preferred and non-limiting example of stringent hybridization conditions is hybridization in 6 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by one or more washes in 0.2x SSC, 0.1% SDS , at 50-65 ° C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions with the sequence of HM74 or its complement, corresponds to a natural nucleic acid molecule. As used herein, a "natural" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that is found in nature (eg, encodes a natural protein). In addition, of the natural allelic variants of the HM74 sequence that may exist in the population, one skilled in the art will further understand that changes can be introduced by mutation in the nucleotide sequence, thus leading to changes in the amino acid sequence of the HM74 protein encoded, without substantially altering the biological activity of the HM74 protein. Thus, nucleotide substitutions can be made which lead to amino acid substitutions in "non-essential" amino acid residues. A "non-essential" amino acid residue is a moiety that can be altered in the natural sequence of HM74 without substantially altering the biological activity. An "essential" amino acid is an amino acid residue necessary for substantial biological activity. For example, amino acid residues that are not conserved or are only semi-preserved between HM74 of different species, may not be essential for the activity, and therefore would be likely targets to alter. Alternatively, amino acid residues that are conserved among the HM74 proteins of different species may be essential for activity, and therefore would not be likely targets to alter. Accordingly, another aspect of the invention relates to nucleic acid molecules that encode HM74 proteins that contain changes in amino acid residues that are not essential for activity. Said proteins HM74 differ from the known amino acid sequence, but retain the biological activity. In one embodiment, the isolated nucleic acid molecule includes a nucleotide sequence that encodes a protein that includes an amino acid sequence that is at least 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of HM74 known. An isolated nucleic acid molecule encoding an HM74 protein having a sequence that differs from that of known HM74 can be created by introducing one or more substitutions, additions or deletions of nucleotides in the nucleotide sequence of the known HM74, so that introduce one or more substitutions, additions or deletions of amino acids in the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made in one or more amino acid residues that are predicted to be nonessential. A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue having a similar side chain. The families of amino acid residues having similar side chains are defined in the art. Families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acid side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine) , threonine, tyrosine and cysteine), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), branched side chains in beta (eg, threonine, valine and isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan and histidine). Therefore, an amino acid residue that is predicted to be non-essential in HM74 is preferably replaced by another amino acid residue from the same side chain family. Alternatively, mutations can be randomly introduced throughout all or a part of a sequence encoding HM74, such as by saturation mutagenesis, and the resulting mutants can be screened according to their biological activity of HM74 to identify mutants that retain the activity . After mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined. Examples of modified nucleotides that can be used to generate nucleic acids of interest include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, -carboxymethylaminomethyluracil, dihydrouracil, D-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, D-D-mannosylkeosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, wibutoxose, pseudoouracil, kerosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, -methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracyl, 3- (3-amino-3-N-2-carboxypropyl) uracil and 2, 6-diaminopurine. One way to affect the function of HM74 is to affect the expression of HM74. Therefore, transcription levels can be manipulated for a lower or higher level of HM74 mRNA, which in turn would lead to a lower or higher level of HM74 expression on the cell surface. One way to achieve such manipulation is to use adjustable promoters that can be introduced at the appropriate site in the genome, near the coding sequence of HM74, for example, by homologous recombination. Accordingly, another aspect of the invention relates to anti-HM74 antibodies. The term "antibody" as used herein, refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, ie, molecules that contain an antigen binding site, that specifically bind to an antigen, such as HM74. A molecule that binds specifically to HM74 is a molecule that binds to HM74 but does not bind substantially to other molecules in the sample, for example, a biological sample that contains natural HM74. Examples of immunologically active parts of immunoglobulin molecules include F (ab) and F (ab ') 2 fragments that can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind to HM74. The term "monoclonal antibody" or "monoclonal antibody composition" as used herein, refers to a population of antibody molecules that contain only one species of an idiotype or a clone of an antigen binding site capable of reaction. immunological with a particular epitope of HM74. Therefore, a monoclonal antibody composition typically exhibits a single binding affinity for a particular epitope of HM74 protein. As is known in the art, other forms of antigen binding to antibodies can be prepared, including fragments, chimeric antibodies, recombinant antibodies, humanized antibodies and the like. Another aspect of the invention relates to vectors, preferably expression vectors, which contain a nucleic acid encoding HM74 (or one of its parts). As used herein, the term "vector" refers to a nucleic acid molecule that can carry another nucleic acid to which it has been linked. One type of vector is a "plasmid" that refers to a circular double-stranded DNA loop, in which additional DNA segments can be ligated. Another type of vector is a viral vector, in which segments of Additional DNA in a viral genome. Some vectors are capable of autonomous replication in a host cell (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell when introduced into the host cell, and therefore replicated together with the host genome. In addition, some vectors, expression vectors, can direct the expression of the genes to which they are operatively linked. In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids (vectors). However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g. , retroviruses with defective replication, adenoviruses and adeno-associated viruses) that have equivalent functions. The recombinant expression vectors of the invention comprise nucleic acid of the invention in a form suitable for the expression of the nucleic acid in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which are operably linked to the nucleic acid to be expressed. In a recombinant expression vector, "operably linked" is meant to mean that the nucleotide sequence of interest is linked to the regulatory sequence (s) in a manner that allows the expression of the sequence of nucleotides (for example, in an in vitro transcription / translation system, or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Said regulatory sequences are described, for example by Goeddel, Gene Expression Technology: Methods in Enzymology Vol. 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of the nucleotide sequence in many types of host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression of the desired protein, etc. The expression vectors of the invention can be introduced into host cells to produce proteins or peptides encoded by nucleic acids as described herein (eg, HM74, mutant forms of HM74, fusion proteins, etc.). The recombinant expression vectors of the invention can be designed for the expression of HM74 in prokaryotic or eukaryotic cells, for example, bacterial cells, such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or cells of mammals. Suitable host cells are discussed in more detail by Goeddel, see above. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, using, for example, regulatory sequences of the T7 promoter and T7 polymerase. In another embodiment, the expression vector of HM74 is a yeast expression vector. Examples of expression vectors in yeast such as S. cerevisiae include pYepSed (Baldari et al., EMBO d. (1987) 6: 229-234), pMFa (Kurjan et al., Cell (1982) 30: 933-943) , pJRY88 (Schuitz et al., Gene (1987) 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, CA) and pPicZ (Invitrogen Corp, San Diego, CA). Alternatively, HM74 can be expressed in insect cells using baculovirus expression vectors. Among the baculovirus vectors available for the expression of proteins in cultured insect cells (e.g., Sf cells 9) include the pAc series (Smith et al., Mol Cell (Biol. (1983) 3: 2156-2165) and the pVL series (Lucklow et al., Virology (1989) 170: 31-39 ). In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDMd (Seed, Nature (1987) 329: 840) and pMT2PC (Kaufman et al., EMBO J. (1987) 6: 187195). When used in mammalian cells, control functions of the expression vector are often provided by viral regulatory elements. For example, the promoters normally used are obtained from polyoma virus, adenovirus 2, cytomegalovirus and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells, see chapters 16 and 17 of Sambrook et al., See above. For the stable transformation of mammalian cells, it is known that depending on the expression vector and the transformation technique used, only a small fraction of the cells can integrate the foreign DNA into the genome. To identify and select the integrants, a gene encoding a selectable marker (e.g., for antibiotic resistance) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. The nucleic acid encoding a selectable marker can be introduced into a host cell in the same vector as that encoding HM74, or it can be introduced into a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (for example, cells that have incorporated the selectable marker gene will survive, while the other cells will die). Compounds of interest that couple and activate HM74 are dihydropyrans with fused ring, which include oxidecalin-type compounds. Oxycalcalin is another name for oxidecahydronaphthalene. Said dihydropyrans have the general structure: The dihydropyrans and oxidecalins of interest can be prepared as taught in U.S. Pat. n °. 6,399,653, WO 97/48691, and von Roedern, Mol. Div. (1998) 3: 253-256. The oxidecalinas of interest have the following structure: where X is O, NR2 or S; Ri is a C? -C? 8 alkyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C C 8 alkenyl which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a CC? 8 alkynyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof a C3-C18 aryl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be substituted, or its combinations; or a C5-C? 8 cycloalkyl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be substituted, or combinations thereof; or its combinations; R 2 is a group R 2 or R 2 may be an alkylC-i-C-ioy-cycloalkyloy-C-io), which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; or X and R2 can form a ring; R3 is H or R- and Y is carbonyl, a Schiff base, an oxine, a ketal, an acetal, an oxazolidine, a thiazolidine or an enol-ester. In general, the compounds of interest are agonists. Therefore, a compound of interest could be developed as a drug candidate. A compound of interest could also be used to identify other molecules that modulate HM74, for example, by competitive assays. Preferred compounds are those in which when R 2 is methyl, ethyl or cyclohexyl, R is not an n-alkyl or a branched C 1 -C 4 alkyl; or when Ri is a C1-C4 alcohol or a branched C4 alkyl which may be substituted with an acetyl group, Ri is not an (C-? - C8) n-alkyl, a branched (C1-C4) alkyl or a phenyl replaced with uncle. The term "alkyl" means a straight or branched chain hydrocarbon. Representative examples are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tere-butyl, sec-butyl, pentyl and hexyl. The hydrocarbon may contain one or more triple unsaturated bonds. The term "alkoxy" means an alkyl group attached to an oxygen atom. Examples are methoxy, ethoxy, propoxy, butoxy and pentoxy. "Aryl" is an aromatic hydrocarbon. Examples include phenyl and naphthyl. "Heteroatomo" is generally an atom that differs from those that characterize a molecule. Thus, in a hydrocarbon, any atom that is not a carbon or a hydrogen is a heteroatom. Common biologically acceptable heteroatoms include oxygen, sulfur, and nitrogen. The term "heteroaryl" refers to an aryl group in which one or more carbon atoms are replaced by a heteroatom. Examples are pyridyl, imidazolyl, pyrrolyl, thienyl, furyl, pyranyl, pyrimidinyl, pyridazinyl, indolyl, quinolyl, naphthyridinyl and isoxazolyl. The term "cycloalkyl" refers to a cyclic hydrocarbon. Some examples are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. "Heterocycle" is a cycloalkyl in which one or more carbon atoms are replaced by a heteroatom. Examples are pyrrolindinyl, piperidinyl and piperazinyl. The term "heteroalkyl" is an alkyl in which one or more carbon atoms are replaced by a heteroatom. An ether is a heteroalkyl. By "substituted" is meant that the organic base radical has one or more substituent groups. Therefore, an atom or group replaces another atom or group in a molecule. Representative substituents include a halogen, C 1 -C 8 alkyl, -CN, alkoxy, hydroxyl, sulfide, sulfate, sulfonamide, amine, amide, an alcohol, a keto group, C 6 -C 8 aryl, a CC alkyl ? 8 halogenated, a nitrite group or a nitrate group. A "halogen" is, for example, chlorine, fluorine or bromine. An "alkenyl" is a hydrocarbon that contains one or more carbon-carbon double bonds. The hydrocarbon may be branched. The term "ring" means one or one of a plurality of annular structures, where two or more of the plurality of rings may be fused, wherein one or more of the plurality of rings may be aromatic, contain a heteroatom, may be replaced, or one of their combinations. The ring may be bicyclic or polycyclic, and may contain a bridge. The compounds of interest bind to HM74 and activate HM74, but do not bind to HM74A.

The oxidecalins of interest can be synthesized as is known in the art, U.S. Pat. No. 6,399,653. The oxidecalin-type compounds of the invention can be incorporated into pharmaceutical compositions suitable for administration. Said compositions typically comprise the active ingredient and a pharmaceutically acceptable carrier. As used herein, the expression "Pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of said media and agents for pharmaceutically active substances is known in the art. Except to the extent that any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Complementary active compounds can also be incorporated into the compositions. A pharmaceutical composition of the invention is formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates, and tonicity adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as HCl or NaOH. The parenteral preparation can be enclosed in ampules, disposable syringes, or multiple dose vials of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when soluble in water) or sterile dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF; Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile, and must be fluid to the extent that it can be easily injected with a syringe. The composition must be stable under the conditions of manufacture and storage, and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, maintaining the necessary particle size in the case of dispersion, and using surfactants. The prevention of the action of microorganisms can be achieved by different antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include in the composition isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride. Prolonged absorption of the injectable compositions can be carried out by including in the composition an agent that retards absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent, with one or a combination of the ingredients listed above, as necessary, followed by sterilization by filtration. In general, the dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients from those enumerated above. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, which give a powder of the active ingredient plus any additional desired ingredient from its solution previously sterilized by filtration. Oral compositions generally include an inert diluent or an edible carrier. The compositions can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches or capsules. Oral compositions can also be prepared using a fluid carrier to provide a liquid syrup or formulation, or to be used as a buccal elixir, in which the compound in the fluid carrier is applied orally, and is shaken and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, pills and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a slip agent such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate or orange flavor. For administration by inhalation, the compounds are provided in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be transmucosally or transdermally. For transmucosal or transdermal administration, they are used in the penetrating formulations suitable for the barrier to be penetrated.

In general such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be carried out by the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, balms, gels or creams, as is generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases, such as cocoa butter and other glycerides) or retention enemas for rectal delivery. In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene-vinyl acetate, polyanhydrides, poly (glycolic acid), collagen, polyorthoesters and poly (lactic acid). Methods for preparing such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes directed to cells infected with monoclonal antibodies) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in the form of a dosage unit to facilitate administration and for uniformity of dosage. The unit dosage form, as used herein, refers to physically discrete units in the form of unit dosages for the subject to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect, associated with the required pharmaceutical carrier. Depending on the type and severity of the disease, an initial candidate dosage to administer to the patient is from about 1 g / kg to 15 mg / kg (e.g., 0.1 to 20 mg / kg) of active ingredient, either for example, in one or more separate administrations, or by continuous infusion. A typical daily dosage may be in the range of about 1 g / kg to 100 mg / kg or more, depending on the aforementioned factors. For repeated administrations over several days or longer, depending on the condition, the treatment is maintained until the desired elimination of the symptoms of the disease occurs. However, other dosage regimens may be useful. The progress of the therapy is easily controlled by conventional techniques and tests. An exemplary dosing regimen is described in WO 94/04188. The specification of the unit dosage forms of the invention is given and directly depends on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the technique for composing said active compound for the treatment of individuals. The pharmaceutical compositions can be included in a package, pack or dispenser, along with instructions for administration. The HM74 modulators of interest can be used for screening assays and methods of treatment (eg, therapeutic and prophylactic). The HM74 modulators of interest can be used to screen other drugs or compounds that modulate the activity or expression of HM74, as well as to treat disorders characterized by inflammation. Modulators of interest may also be useful in states resulting from insufficient or excessive production of HM74 protein, or production of HM74 protein forms that have lower activity or aberrant activity compared to wild-type HM74 protein. The invention also relates to novel modulators of HM74 identified by screening assays, and to their uses for treatments as described herein. The invention provides a method (also referred to hereinafter as a "screening method") for identifying modulators, i.e., candidate or test compounds or agents (eg, peptides, peptidomimetics, small molecules, antibodies or other drugs) that are bind to HM74 or have a stimulatory or inhibitory effect, for example on the expression of HM74 or the activity of HM74. In one embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate the activity of HM74. Therefore, screening assays can be used to identify other furosemide-type and oxidecalin-type compounds that modulate HM74. Such modulators can also be used in competitive assays to identify other modulators such as HM74 antagonists. In one embodiment, an assay is a cell-based assay, in which a cell expressing a membrane-bound form of HM74 at the cell surface is contacted with a test compound, and the capacity of the trial to activate the HM74. The cell can be, for example, a yeast cell or a cell of mammalian origin. The determination of the ability of the test compound to activate HM74 can be carried out, for example, by coupling the test compound with a radioisotope or enzymatic marker, so that the binding of the test compound to HM74 can be determined by detecting the labeled compound in a complex with HM74, or where HM74 is located. For example, the test compounds can be labeled with 125 I, 35 S, 14 C or 3 H, directly or indirectly, and the radioisotope can be detected by direct counting of radio emission or by scintillation counting. Alternatively, the test compounds can be labeled enzymatically, for example, with horseradish peroxidase, alkaline phosphatase or luciferase, and the enzyme label can be detected by determining the conversion of a suitable substrate to the product. In a preferred embodiment, the assay comprises contacting a cell expressing a membrane-bound form of HM74 at the cell surface, with a known compound that binds to HM74 together with a test compound, to determine the capacity of the compound of test to compete with the known compound to interact with an HM74. In another embodiment, an assay is a cell-based assay that comprises contacting a cell that expresses a membrane-bound HM74 form on the cell surface with a test compound, and determining the ability of the test compound to modulate (p. (eg, stimulate or inhibit) the activity of HM74. The determination of the ability of the test compound to modulate the activity of HM74 can be carried out, for example, by determining the ability of the test compound to activate or inhibit HM74. This can be evidenced when the activated HM74 interacts with an intracellular or membrane target molecule associated with the signaling pathway. As used herein, a "target molecule" is a molecule with which HM74 binds or interacts in nature, for example, a molecule on the surface of a cell that expresses an HM74, a molecule on the surface of a second cell, a molecule in the extracellular medium, a molecule associated with the inner surface of a cell membrane, or a cytoplasmic molecule. A target molecule of HM74 may be a non-molecule of HM74. In one embodiment, a target molecule of HM74 is a component of a signal transduction pathway that facilitates the transduction of an extracellular signal (e.g., a signal generated by binding of a compound to HM74) through the cell membrane and within the cell. The target can be, for example, a second intercellular protein having catalytic activity or a protein that facilitates the association of downstream signaling molecules with HM74. The determination of the ability of HM74 to bind or interact with a target molecule of HM74 can be carried out by one of the methods described above to determine direct binding. In a preferred embodiment, the determination of the ability of HM74 to bind or interact with a target molecule of HM74 can be carried out by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting the induction of a second target cell messenger (eg, intracellular Ca2 +, diacylglycerol, IP3 etc.), by detecting the catalytic / enzymatic activity of the target on a suitable substrate , detecting the induction of a reporter gene (eg, a regulatory element responsive to HM74 operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase) or detecting a cellular response, e.g., differentiation cellular or cell proliferation. In yet another embodiment, an assay of the present invention is a cell-free assay, which comprises contacting the HM74 with a test compound and determining the ability of the test compound to bind to the HM74. The binding of the test compound to HM74 can be determined directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the HM74 with a known compound of the invention together with a test compound, and determining the ability of the test compound to affect the activity of HM74 of the known compound described herein. . The determination of the ability of the test compound to interact with HM74 comprises determining the ability of the test compound to preferentially bind to HM74 compared to the binding of the known compound described herein. In another embodiment, an assay is a cell-free assay, which comprises contacting the HM74 with a test compound and determining the ability of the test compound to modulate (eg, stimulate or inhibit) the activity of HM74. The determination of the ability of the test compound to modulate the activity of HM74 can be carried out, for example, by determining the ability of activated HM74 to bind to a target molecule of HM74 by one of the methods described above to determine direct binding . In an alternative embodiment, the determination of the ability of the test compound to modulate the activity of HM74 can be carried out by determining the ability of HM74 to further modulate a target molecule of HM74. For example, the catalytic / enzymatic activity of the target molecule can be determined on a suitable substrate, as previously described. In yet another embodiment, the cellless assay comprises contacting the HM74 with a known compound that binds to HM74 and a test compound and determining the ability of the test compound to interact with an HM74, wherein the determination of the ability of the test compound to interact with an HM74 comprises determining the ability of HM74 to preferentially bind or modulate the activity of a target molecule of HM74. The receptors can be activated by molecules that are not ligands that do not necessarily inhibit the binding of the ligand, but produce structural changes in the receptor to allow the binding of the G protein or perhaps the aggregation, dimerization or grouping of the receptor, which can produce activation . The cell-free assays of the present invention are capable of being used with both the soluble form and the membrane-bound form of HM74. In the case of assays without cells comprising the membrane-bound form of HM74, it may be convenient to use a solubilizing agent, so that the membrane-bound form of HM74 is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecyl maltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton X-100, Triton X-114, Thesit. ®, isotridecilpoli (ethylene glycol ether) n, 3 - [(3-colamidopropyl) dimethylamino] -1 -propane sulphonate (CHAPS), 3 - [(3-cholamidopropyl) dimethylamino] -2-hydroxy-1 -propane-sulfonate (CHAPSO) or N-dodecyl = N, N-dimethyl-3-ammonium-1 -propane-sulfonate. In another embodiment, HM74 is altered to be in a constant active state when expressed in a host cell. The alteration of HM74 can make the receptor active without having to bind ligand. One way to obtain an activated receptor is to alter HM74 to interact with G proteins without ligand binding. The alteration mimics the conformational changes of the receptor when the ligand binds, which allow the receptor to bind intracellular G proteins. One such method is provided in WO 00/22129.

WO 00/22129 shows particular amino acids in the region of TM6 and IC3 that produce constitutive activity. Methods for incorporating particular amino acids into HM74 are known in the art, such as site-directed mutagenesis, subcloning etc. Then, the altered HM74 molecule is expressed in a host cell to produce a constitutively active HM74. Then, the activated cell is exposed to molecules that are suspected to be agonists, antagonists, inverse agonists, etc., of HM74, molecules that alter the activity of HM74. Molecules that alter the activity of the G protein are directed to the treatment of disorders associated with the altered metabolism of HM74 using known methods in pharmaceutical development. In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either HM74 or one of its target molecules, to facilitate the separation of the complex forms from those which do not form one or both of the proteins complex, as well as to adapt the automation of the trial. The binding of a test compound to HM74, or the interaction of HM74 with a target molecule in the presence and absence of a candidate compound, can be carried out in any suitable vessel to contact the reagents. Examples of such containers include microtiter plates, test tubes and microcentrifuge tubes.

In one embodiment, a fusion protein can be provided that adds a domain that allows one or both proteins to bind to a matrix. For example, glutathione-S-transferase / HM74 fusion proteins or glutathione-S-transferase / target fusion proteins can be adsorbed onto glutathione-Sepharose beads (Sigma Chemical, St. Louis, MO) or derivatized microtiter plates. with glutathione. Then, this complex is combined with the test compound and the target protein or the non-adsorbed HM74 protein and the mixture is incubated under conditions that lead to complex formation (eg, under saline and physiological pH conditions). After incubation, the beads or wells of the microtiter plate are washed to remove any unbound components and the complex formation is measured directly or indirectly, for example, as described above. Alternatively, complexes of the matrix can be dissociated, and the level of activity or binding of HM74 determined using standard techniques. Other techniques for immobilizing proteins in matrices can also be used in the screening assays of the invention. For example, HM74 or one of its target molecules can be immobilized, using the conjugation of biotin and streptavidin. Biotinylated HM74 or target molecules can be prepared from biotin-NHS (N-hydroxysuccinimide) using techniques known in the art (eg, biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilizing into wells of well plates. 96 wells coated with streptavidin (Pierce Chemicals). Alternatively, antibodies reactive with HM74 or target molecules can be attached to the wells of the plate, but do not interfere with the binding of the HM74 protein to a target molecule, and detach the target molecule or HM74 trapped in the wells by conjugation with the antibody. Among the methods to detect such complexes, in addition to those described above for the immobilized complexes in GST, complex immunodetection is included using antibodies reactive with HM74 or target molecule, as well as assays with linked enzymes that are based on detecting the enzymatic activity associated with HM74 or target molecule. In another embodiment, modulators of HM74 expression are identified in a method in which a cell is contacted with a candidate compound, and expression of the HM74 mRNA or protein in the cell is determined. The level of expression of the HM74 mRNA or protein in the presence of the candidate compound is compared to the expression level of the HM74 RNA or protein in the absence of the candidate compound. Then, the candidate compound can be identified as a modulator of HM74 expression based on this comparison. For example, when the expression of the HM74 mRNA or protein is higher (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator or agonist of the HM74 mRNA or protein. Alternatively, when the expression of the HM74 mRNA or protein is lower (statistically significantly lower) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor or antagonist of the HM74 mRNA or protein. If the activity of HM74 decreases in the presence of a ligand or agonist, or in a constitutive HM74, below the initial values, the candidate compound is identified as an inverse agonist. The level of expression of the HM74 mRNA or protein in the cells can be determined by methods described herein to detect the mRNA or protein of HM74. Since large quantities of pure HM74 can be prepared, the physical characterization of the conformation of probable function zones for rational drug design can be determined. For example, the IC3 region of the molecule and the EC domains are regions of particular interest. Once the shape and ionic configuration of a region have been differentiated, candidate drugs interacting with those regions can be configured, and then assayed in intact cells, animals and patients. Among the methods that would allow obtaining such information from the structure, include X-ray crystallography, NMR spectroscopy, molecular modeling, etc. The 3-D structure can also lead to the identification of analogous conformational sites in other known proteins, where there are known drugs that act at a particular site. These drugs or their derivatives may be useful with HM74. The invention also relates to new agents identified by the screening assays described above, and to their uses for treatments, as described herein. The present invention provides both prophylactic and therapeutic methods for treating a subject at risk (or susceptible) to suffering from a disorder, or having a disorder associated with the expression or aberrant activity of HM74. Such disorders include, but are not limited to, inflammatory disorders such as asthma, chronic obstructive pulmonary disease, and rheumatoid arthritis. In one aspect, the invention provides a method for preventing in a subject a disease or condition associated with the expression or aberrant activity of HM74, by administering to the subject an agent that modulates the expression of HM74 or at least one activity of HM74. Subjects who are at risk of suffering from a disease caused or contributed to by the aberrant expression or activity of the HM74, can be identified, for example, by any or a combination of diagnostic or prognostic assays as described herein.

The administration of a prophylactic agent can occur before the characteristic symptoms of the HM74 aberration manifest, so as to prevent, or alternatively, delay the progression of a disease or disorder. Depending on the type of aberration of HM74, for example, an HM74 agonist agent or an HM74 antagonist can be used to treat the subject. The suitable agent can be determined based on screening assays described herein. Another aspect of the invention relates to methods for modulating the expression or activity of HM74 for therapeutic purposes. The method of modulation of the invention involves contacting a cell with an agent that modulates one or more of the activities of HM74 associated with the cell. An agent that modulates the activity of HM74 can be an agent as described herein, such as a furosemide or oxidecalin, nucleic acid or a protein, a natural cognate ligand of a HM74 protein, a peptide, a peptidomimetic of HM74 or another small molecule. In one embodiment, the agent stimulates one or more biological activities of HM74. In another embodiment, the agent inhibits one or more biological activities of HM74. Examples of such inhibitory agents include anti-HM74 antibodies. The modulation methods can be carried out in vitro (for example, by culturing the cell with the agent), or alternatively live (for example, by administering the agent to a subject). As such, the present invention provides methods for treating an individual suffering from a disease or disorder characterized by the expression or aberrant activity of an HM74. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein) or a combination of agents that modulate (e.g., positively or negatively regulate) the expression or activity of HM74. . The stimulation of the activity of HM74 is convenient in situations in which the HM74 is negatively regulated abnormally and / or in which it is probable that the greater activity of the HM74 has a beneficial effect. Conversely, the inhibition of the activity of HM74 is convenient in situations in which HM74 is abnormally regulated in an abnormal manner and / or in which it is probable that the lower activity of HM74 has a beneficial effect. The invention is illustrated in more detail with the following examples which should not be considered limiting. The contents of all references, patents and published patent applications cited throughout the application are hereby incorporated by reference.

Example 1 - Generation of mammalian cells expressing HM74 The cDNA encoding HM74 is cloned into an expression vector and transfected into mammalian cells, such as CHO cells or 293 cells. To generate mammalian cells overexpressing HM74, mammalian cells are plated in a six-well 35-mm tissue culture plate (3 x 10 5 mammal cells per well (ATCC Catalog No. CRL-1573)) in 2 ml of DMEM medium (Gibco / BRL, Catalog No. 11765-054) in the presence of 10% fetal bovine serum (Gibco / BRL Catalog No. 1600-044). The cells are then incubated at 37 ° C in a C02 incubator until the cells are 50-80% confluent. The cloned cDNA nucleic acid sequence of HM74 is inserted into a pcDNA 3.1 cloning vector (Invitrogen, Catalog No. V790-20). 2 μg of the DNA is diluted in 100 μl of Ham F12 medium without serum. Separately, 25 μl of Lipofectamine reagent (Life Technologies, Catalog No. 18324-020) is diluted in 100 μl of Ham F12 medium without serum.

The DNA solution and the Lipofectamine solution are then mixed gently and incubated at room temperature for 45 minutes to allow the formation of DNA-lipid complexes. The cells are rinsed once with 2 ml of Ham F12 medium without serum. For each transfection (six transfections in a six-well plate), 0.8 ml of Ham F12 medium without serum is added to the solution containing the DNA-lipid complexes (total volume 0.2 ml) and mixed gently. Then, with the resulting mixture (hereinafter the "transfection mixture"), the clarified cells are covered (0.8 ml + 0.2 ml). No antibacterial reagents are added. Then, the cells are incubated with the DNA-lipid complexes for 16 hours at 37 ° C in a C02 incubator to allow transfection. After the completion of the incubation period, 1 ml of Ham F12 medium containing 10% fetal bovine serum is added to the cells without first separating the transfection mixture. 18 hours after transfection, the medium covering the cells is aspirated. Then, the cells are washed with PBS pH 2-4 (Gibco / BRL Catalog No. 10010-023) and the PBS is replaced by HAM F12 medium containing 5% serum ("selective medium"). 72 hours after transfection, the cells are diluted ten times in selective medium containing the antibacterial agent Geneticin with 400 g / ml (Life Technologies, Catalog No. 11811).

Example 2 - Agonist Assay To screen agonists of human HM74, HM74 can be artificially coupled to a Gq mechanism. The activation of the Gq mechanism stimulates the release of Ca2 + from the vesicles of the sarcoplasmic reticulum inside the cell. Ca2 + is released into the cytoplasm where it can be detected using Ca2 + chelating dyes. A fluorometric imaging plate reader or FLIPR® device (Molecular Devices) is used to monitor any resulting changes in fluorescence. The activity of an agonist is reflected by any increase in fluorescence. CHO-Kl cells expressing HM74 are previously designed to express an indiscriminate form of Gq protein (Ga16). To prepare said cells, CHO cells coupled with Gcr16 (Molecular Devices LIVEWARETM, Catalog No. RD-HGA16) are obtained commercially and the protocol of Example 1 is followed to facilitate the expression of HM74 in these cells. Cells are maintained in logarithmic growth phase at 37 ° C and 5% C02 in Ham F12 medium (Gibco / BRL, Catalog No. 11765-054) containing 10% fetal bovine serum, 100 IU / ml penicillin (Gibco / BRL, Catalog No. 15140-148), streptomycin 100 // g / ml (Catalog No. 15140-148, Gibco / BRL), geneticin 400 / g / ml (G418) (Gibco / BRL, Catalog No. 10131- 035) and zeocin 200 // g / ml (Invitrogen, Catalog No. R250-05). One day before a test, 12,500 cells / well of CHO-K1 cells are plated in 384-well clear bottom assay plates, with a volume per well of 50 μl (Greiner / Marsh, Catalog No. N58102 ) using a Multidrop 96/384 device (Labsystems, Type 832). The cells are incubated at 37 ° C in a humidified incubator with 5% C02 (water jacket incubator and C02 Forma Scientific Model 3110). The following stock solutions are prepared: a 1M stock solution of Hepes (pH 7.5) (Gibco / BRL, Catalog No. 15630-080); a 250 mM stock solution of probenicide (Sigma, Catalog No. P8761) prepared in 1 N NaOH; and a 1 mM stock solution of Fluo 4-AM dye (Molecular Probes, Catalog No. Fl 4202) prepared in DMSO (Sigma D2650). The reaction buffer is prepared with 1000 ml of Hank's balanced salt solution (Fisher / Mediatech, Catalog No. MT21023), 20 ml of the 1M Hepes stock solution and 10 ml of the 250 mM probenicidal stock solution. To prepare the loading buffer, 1.6 ml of the stock solution of 1 mM Fluo 4-AM dye is mixed with 0.32 ml of pluronic acid (Molecular Probes, Catalog No. P6866) and then mixed with 400 ml of the previous reaction buffer and 4 ml of fetal bovine serum. One hour before the assay, 50 μl of freshly prepared loading buffer is added to each well of the 384-well plate using a device Multidrop 96/384. The cells are incubated at 37 ° C in a humidified incubator to maximize dye uptake. Immediately before the assay, the cells are washed twice with 90 μl of reaction buffer using an EMBLA 384 cell washer (Skatron; Model No. 12386) with the suction head fixed 10 mm above the bottom of the plate, leaving 45 μl of buffer per well. The CCD camera (Princeton Instruments) of the FLIPR® ll instrument (Molecular Devices) is fixed with a diaphragm aperture of 2.0 and an exposure time of 0.4 seconds. The camera is used to control the accuracy of the dye charge on the cell plates. A compound library containing possible oxidizecalin-type compounds at a concentration of about 10 μM per well, each in physiological saline buffer, is tested. The changes in fluorescence are measured for 10 seconds before adding the compound. After adding the compound, the fluorescence is measured every second during the first minute, followed by exposures taken every six seconds for a total experimental analysis time of three minutes. Aliquots of 5 μl of the stock solution of compound 100 // M are added after the tenth scan, giving a final concentration of compound in the cells of 10 μ. The maximum fluorescence changes during the first 80 scans are recorded as a measure of the agonist activity, and compared to the maximum fluorescence change induced by ATP 10 μU ATP (Sigma A9062). It was found that a series of oxidecalin-type compounds activated the HM74.

Example 3 - Antagonist Assay To screen human HM74 antagonists, HM74 can be artificially coupled to a Gq mechanism. As in example 2, a FLIPR® apparatus is used to control any changes that occur in fluorescence. The activity of an antagonist is reflected by any decrease in fluorescence. CHO-K1 cells expressing HM74 are previously designed to express an indiscriminate form of the Gq protein (G16), as described in Example 2. The cells are maintained in logarithmic growth phase at 37 ° C and C02 at 5 ° C. % in Ham F12 medium (Gibco / BRL, Catalog No. 11765-054) containing 10% fetal bovine serum, 100 IU / ml penicillin (Gibco / BRL, Catalog No. 15140-148), streptomycin 100 // g / ml (Catalog No. 15140-148, Gibco / BRL), geneticin 400 / g / ml (G418) (Gibco / BRL, Catalog No. 10131-035) and zeocin 200 // g / ml (Invitrogen, Catalog No. R250 -05). One day before a test, 12,500 cells / well of CHO-K1 cells are plated in 384-well black / clear assay plates with a 50 μl well volume (Greiner / Marsh, Catalog n ° N58102) using a Multidrop 96/384 device (Greiner / Marsh, Catalog No. N58102). The cells are allowed to incubate at 37 ° C in humidified 5% C02. The following stock solutions are prepared: a 1M stock solution of Hepes (pH 7.5) (Gibco / BRL, Catalog No. 15630-080); a 250 mM stock solution of probenicide (Sigma, Catalog No. P8761) prepared in 1 N NaOH; a stock solution of Fluo 4-AM 1 mM dye (Molecular Probes, Catalog No. F14202) prepared in DMSO (Sigma D2650); and a stock solution of ligand or antagonist. The reaction buffer is prepared with 1000 ml of Hank's balanced salt solution (Fisher / Mediatech, Catalog No. MT21023), 20 ml of the 1 M Hepes stock solution and 10 ml of the 250 mM probenicidal stock solution and CaCl2 1 mM. To prepare the loading buffer, 80 μl of the 1 mM Fluo 4-AM dye stock solution is mixed with 16 μl pluronic acid (Molecular Probes, Catalog No. P6866) and then mixed with 20 ml of the buffer. previous reaction and 0.2 ml of fetal bovine serum. Thirty minutes before the assay, 30 μl of freshly prepared loading buffer is added to each well of the 384-well plate using a Multidrop 96/384 device. Cells are incubated at 37 ° C in a humidified C02 incubator to maximize dye uptake. Immediately before the assay, the cells are washed 3 times with 100 μl of reaction buffer using an EMBLA 384 cell washer with the suction head fixed at least 40 mm above the bottom of the plate, leaving 45 μl of buffer by pocilio. μl of the stock solution of 100 μM antagonist compound is added to the cells using a Platemate-384 pipettor (Matrix) device. The concentration of compound during the incubation step is approximately 10 μM. The cells are placed in the FLIPR® ll apparatus and the fluorescence of the plate is measured every second for the first minute followed by exposures taken every six seconds for a total experimental analysis time of three minutes. After the tenth scan, antagonist or ligand (10 μM) is added. After each addition, the 384 tips of the micropipettes are washed with 20 μl 0.01% DMSO in water. Cells with HM74 can be exposed to an identified agonist or not, before testing candidate antagonists.

Example 4 - Receptor Binding Assay To prepare membrane fractions containing HM74, CHO cell lines expressing HM74 are collected by incubation in phosphate buffered saline (10 ml) containing 1 mM EDTA. The cells are washed 3 more times with phosphate buffered saline containing 1 mM EDTA (10 ml) before resuspending in 5 ml of buffer A (50 mM Tris-HCl (pH 7.8) (Sigma T6791), 5 mM MgCl2 (Sigma M8266) and 1 M EGTA (Sigma 0396) .The cells are then disrupted in a tissue homogenizer (Polytron, Kinemetica, Model PT 10/35) for 1 minute. The resulting homogenate is centrifuged in a Sorvall Instruments RC3B refrigerated centrifuge at 49,000 X g at 4 ° C for 20 minutes. The resulting pellet is resuspended in 25 ml of buffer A and the centrifugation step is repeated 3 times. After the final centrifugation, the pellet is resuspended in 5 ml of buffer A, aliquoted, and stored at -70 ° C. A receptor binding assay is carried out using the membrane fraction and a radiolabeled agonist of interest as a tracer. The assay is carried out in a 96-well plate (Beckman Instruments). The binding reaction consists of 18 μg of the CHO cell preparation in the presence of radioactive agonist (0.01 nM-25 nM) in a final volume of 0.2 ml buffer A containing 1% bovine serum albumin ( Sigma, Catalog No. 34287) (see, Im et al., J. Biol. Chem. (2000) 275 (19): 14281-14286). The reaction is incubated for 1 hour at room temperature. The reaction is terminated by filtration through Whatman GF / C filters in a multichannel collector (Brandell) which is pretreated with 0.3% polyethylenimine (Sigma, Catalog No. P3143) and 0.1% bovine serum albumin. % (BSA) for 1 hour. The mixture is applied to the filter and incubated for one hour. The filters are washed 6 times with 1 ml ice-cold 50 mM Tris-HCl, pH 7.6. The specific binding is calculated based on the difference between the total binding and the non-specific binding (background) for each tracer concentration, by measuring the radioactivity. 8 to 16 concentration data are obtained to determine the binding of the agonist to the receptor achieved in a state of equilibrium between the agonist and the receptor (binding parameters in equilibrium). In a competitive assay, a test compound is added to the mixture to compete for the binding of the radioactive agonist to the receptor (competitive binding values). Inhibition curves are prepared to determine the concentration needed to achieve 50% inhibition of binding (IC50).

Example 5 - Small agonist molecules A series of oxidecalin-type molecules were exposed to cells expressing HM74 as described above. The target molecules were labeled to determine if binding to HM74 occurred. The union was detected by determining the degree of marking of the cells after washing. The binding was also determined by isolating HM74 by 2-D gel electrophoresis, and determining the degree of labeling associated with this protein. After determining this binding, or independently of the determination of the binding, the ability of a candidate agonist to activate HM74 was determined. The FLIPR assay was used to evaluate intracellular calcium mobilization at the binding of the target molecule to HM74. Thus, the oxidecalin molecules that produce calcium mobilization were identified. Having now described the invention, those skilled in the art will know that various changes and modifications of the teachings herein can be made, without departing from the spirit and scope of the invention taught herein. All references cited in this specification are incorporated in their entirety in this specification.

Claims (6)

1. A therapeutic method for modulating the signaling activity of HM74 or signal transduction, in a patient in need of treatment, which comprises administering to said patient a molecule having the structure: where X is O, NR2 or S; R-t is a C -? - C? 8 alkyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C 1 -C 18 alkenyl which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C Cis alkynyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof a C3-C18 aryl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be substituted, or your combinations; or a C5-C18 cycloalkyl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be substituted, or combinations thereof; or its combinations; R2 is a group R-i, an alkyl (C? -C? O) -cycloalkyl (C3-C? O), which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; or X and R2 can form a ring; R3 is H or R1; and Y is carbonyl, a Schiff base, an oxine, a ketal, an acetal, an oxazolidine, a thiazolidine or an enol-ester.

2. A method for identifying an HM74 agonist, comprising contacting an agonist potential with a cell expressing HM74, and determining whether in the presence of said agonist potential the signaling activity of HM74 is greater with respect to the activity of HM74 in the absence of said potential agonist, in which said agonist potential has the structure where X is O, NR2 or S; Ri is an alkyl CrC-is, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C C 8 alkenyl which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C -? - C? 8 alkynyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof a C3-C? 8 aryl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be substituted, or combinations thereof; or a C5-C? 8 cycloalkyl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be substituted, or combinations thereof; or its combinations; R2 is a group R-i, a (C? -C10) alkyl-cycloalkyl (C3-C-? O), which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; or X and R2 can form a ring; R3 is H or R-r, and Y is carbonyl, a Schiff base, an oxine, a ketal, an acetal, an oxazolidine, a thiazolidine or an enol-ester.

3. A method for identifying an inverse agonist of HM74, comprising contacting an inverse agonist potential with a cell expressing HM74, and determining whether in the presence of said inverse agonist potential the activity of HM74 is less with respect to the activity of HM74 in the absence of said inverse agonist potential, and it is lower in the presence of an agonist, in which said inverse agonist potential has the structure: where X is O, NR2 or S; Ri is a C 1 -C 8 alkyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C? -C18 alkenyl which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C? -C? 8 alkynyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof a C3-C18 aryl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be replaced, or their combinations; or a C5-C-? 8 cycloalkyl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be substituted, or combinations thereof; or its combinations; R2 is a group R ^ a (C? -C10) alkyl-cycloalkyl (C3-C? O), which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; or X and R2 can form a ring; And it is carbonyl or oxygen bound to a Schiff base, an oxine, a ketal, an acetal, an oxazolidine, a thiazolidine or an enol-ester.

4. A method for identifying an HM74 antagonist, comprising contacting a potential antagonist with a cell expressing HM74, and determining whether in the presence of said potential antagonist the signaling activity of HM74 is less with respect to the activity of HM74 in the presence of a agonist, in which said potential antagonist has the structure: where X is O, NR2 or S; Ri is a C C 8 alkyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C C 8 alkenyl which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C? -C18 alkynyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof a C3-C? 8 aryl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be replaced, or their combinations; or a C5-C? 8 cycloalkyl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be substituted, or combinations thereof; or its combinations; R2 is a Ri group, an alkyiCyCioJ-cycloalkyloiCs-Cio), which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; or X and R2 can form a ring; R3 is H or R-i; and Y is carbonyl, a Schiff base, an oxine, a ketal, an acetal, an oxazolidine, a thiazolidine or an enol-ester.

5. A method for modulating inflammation comprising exposing a patient in need of treatment to an inflammation modulating amount, of a pharmaceutical composition comprising a compound of the formula: where X is O, NR2 or S; Ri is a CrC 8 alkyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C C18 alkenyl which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C? -C, 8 alkynyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof a C3-C? 8 aryl, which may contain a side group, may contain a bridge, may contain a heteroatom or it may be substituted, or its combinations; or a C5-C? 8 cycloalkyl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be substituted, or combinations thereof; or its combinations; R2 is a group R-i, an alkyl (C? -C? O) -cycloalkyl (C3-C? O), which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; or X and R2 can form a ring; And it is carbonyl, a Schiff base, an oxine, a ketal, an acetal, an oxazolidine, a thiazolidine or an enol-ester.

6. A compound of formula: where X is O, NR2 or S; Ri is a C? -C-? 8 alkyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; an alkenyl CrC-is which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; a C? -C? 8 alkynyl, which may be branched, may contain a heteroatom or may be substituted, or combinations thereof a C3-C? 8 aryl, which may contain a side group, may contain a bridge, may contain a heteroatom or it may be substituted, or its combinations; or a C5-C18 cycloalkyl, which may contain a side group, may contain a bridge, may contain a heteroatom or may be substituted, or combinations thereof; or its combinations; R2 is a group Ri, an alkyl (C? -C10) -cycloalkyl (C3-C? O), which may be branched, may contain a heteroatom or may be substituted, or combinations thereof; or X and R2 can form a ring; R3 is H or Ri; and Y is carbonyl, a Schiff base, an oxine, a ketal, an acetal, an oxazolidine, a thiazolidine or an enol-ester. with the proviso that when R2 is methyl, ethyl or cyclohexyl, Ri is not an n-alkyl or a branched C1-C4 alkyl; or when R2 is a C1-C4 alcohol or a branched C1-C4 alkyl which may be substituted with an acetyl group, R1 is not an (C8) n-alkyl, a branched (C? -C) alkyl or a substituted phenyl with uncle.