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    CHOLESTEROL METABOLITE, 5-COLESTEN, 3BETA-25-DIOL, DISSULFATE (25HCDS) FOR METABOLIC DISORDERS, HYPERLIPIDEMIA, DIABETES, FAT HEPATIC DISEASES AND AEROSCLEROSIS. The present invention relates to 5-colesten, 3delta, 25-diol, disulfate (25HCDS) which has been found to be an authentic PPARy agonist and LXR antagonist, and is used for therapy of lipid disorders and inflammatory diseases, including, without limitation, fatty liver, inflammatory diseases, and atherosclerotic diseases.
    公开号:BR112014025081B1
    申请号:R112014025081-2
    申请日:2013-03-15
    公开日:2020-12-08
    发明作者:Shunlin Ren
    申请人:Virginia Commonwealth University；
    IPC主号:
    专利说明:

    CROSS REFERENCE TO RELATED ORDERS
    [001] This application claims the benefit of United States Provisional Patent Applications 61 / 623,203 and 61 / 623,414, both filed on April 12, 2012. The contents of both Provisional Applications are hereby incorporated by reference. DESCRIPTION FIELD OF THE INVENTION
    [002] The invention generally relates to a new cholesterol metabolite, 5-cholesten-3p, 25-diol, disulfate (25HCDS) and uses of it. In particular, the invention provides 25HCDS for the prevention and treatment of diseases such as metabolic lipid disorders and inflammatory disorders, for example, hyperlipidemia, diabetes, fatty liver diseases and atherosclerosis. BACKGROUND OF THE INVENTION
    [003] The liver plays an essential role in maintaining lipid homeostasis. The accumulation of lipids in liver tissues induces non-alcoholic fatty liver disease (NAFLD). NAFLD affects almost a quarter of the general population in the United States and can progress to significant cirrhosis and hematocellular carcinoma. The spectrum of NAFLD ranges from simple non-progressive steatosis to progressive non-alcoholic steatohepatitis (NASH) that results in liver cirrhosis and hepatocellular carcinoma. The pathogenesis of NAFLD is reviewed with a two-step process. The first step is an accumulation of triglycerides and associated lipids in hepatocytes. The second stage is the occurrence of inflammation of the liver. The NAFLD indication feature is characterized by increased intrahepatic triglyceride build-up. Decreasing lipid levels is an important element of successful NAFLD therapy. In mammals, regulatory element binding protein-1c (SREBP-1c) preferably controls lipogenic gene expression; and regulates triglyceride and fatty acid homeostasis. Its role in fatty acid biosynthesis and the development of fatty liver disease is well documented, there is currently no approved treatment for NAFLD.
    [004] Oxisterols can act at multiple points in cholesterol homeostasis and lipid metabolism. The oxy-esterol receptor, LXR, is the sterol-regulated transcription factor of lipid metabolism. Activation of LXR stimulates the expression of cholesterol efflux and clearance through ABCA1 and ABCG5 / 8, but it also down-regulates the expression of SREBP-1c, which in turn regulates at least 32 genes involved in lipid biosynthesis and transport. Therefore, while activation of LXR by synthetic ligands can reduce the serum cholesterol level for protection against atherosclerosis, activation induces liver steatosis and hypertriglyceridemia due to the induction of fatty acid and triglyceride synthesis through the activation of SREBP -1c. Hepatocytes have a limited capacity to store fatty acids in the form of triglycerides. Since the capacity is subdued, cell damage occurs. Excess amounts of free intracellular fatty acids activate the production of reactive oxygen species (ROS), which cause lipotoxicity and activation of inflammatory signaling reaction series, which ultimately induce apoptosis.
    [005] 5-Colesten-3p, 25-diol 3-sulfate (25HC3S) is an oxisterol that has recently been identified in primary rat liver nuclei. 25HC3S is described in WO 2006/047022. This oxisterol can be synthesized by sterol sulfotransferase SULT2B1b of 25-hydroxycholesterol (25HC) by oyster sulfation. Exogenous administration of a similar cholesterol metabolite, 5-cholesten-3p, 25-diol 3p-sulfate (25HCpS), decreases both the expression of SREBP-1 and SREBP-2; blocks processing of SREBP-1c; and suppresses the expression of key enzymes in lipid metabolism including acetyl-CoA carboxylase-1 (ACC-1), fatty acid synthase (FAS) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), subsequently decreasing levels of neutral lipid and cholesterol.
    [006] The results indicate that 25HC3S acts as an LXR antagonist and as a sign of cholesterol satiety; suppresses the fatty acid and triglyceride reaction series by inhibiting LXR / SREBP signaling. In addition, 25HC3S increases IKBP expression; blocks TNFa-induced IKBP degeneration; and decreases NFKB levels. In contrast, 25HC acts in the opposite way, inducing the degradation of IKBP and accumulation of nuclear NFKB. These results indicate that 25HC3S is also involved in inflammatory responses and may represent a link between series of inflammatory reactions and the regulation of lipid homeostasis. SUMMARY OF THE INVENTION
    [007] Another regulatory cholesterol metabolite, 5-cholesten-3p, 25-diol, disulfate (25HCDS) has currently been identified. Studies of 25HCDS indicate that the decreased expression of this naturally occurring metabolite plays an important role both in the accumulation of lipid and cellular damage in hepatocytes and macrophages, thus contributing to the pathogenesis of metabolic disorders. Addition of 25HCDS to hepatocyte and macrophage culture media decreases the sterol regulatory element binding protein mRNA (SREBPs), decreases the processing of SREBPs, and subsequently sub-regulates the key enzymes involved in lipid biosynthesis, inducing levels of intracellular lipid decreased in hepatocytes and macrophages. 25HCDS also increased expression of peroxisome proliferating activator receptor (PPAR), IKB, and peroxisome proliferating activator receptor alpha 1 (PGC-1a) mRNA, decreased nuclear NFkB levels, and pro-cytokine secretion and expression -inflammatory. Significantly, in vivo studies have shown that administration of 25HCDS decreased liver lipids by ~ 20-35% without showing toxicity.
    [008] Thus, the recently discovered cholesterol metabolite, 25HCDS, functions as a PPARY agonist and authentic LXR antagonist that inhibits cholesterol and lipid biosynthesis in hepatocytes and macrophages in vitro and in vivo, in addition to suppressing inflammatory responses by through the PPARY / IKB / NFKB signaling reaction series. 25HCDS, which has been chemically synthesized as described in the example section here, can thus be used as a medicine for the treatment and prevention of metabolic lipid and inflammatory disorders, including hyperlipidemia, atherosclerosis, diabetes, fatty liver diseases, etc.
    [009] Other features and advantages of the present invention will be mentioned in the description of the invention that follows, and will be evident from the description or can be learned in the practice of the invention. The invention will be realized and achieved by the compositions and methods particularly punctuated in the described description and claims thereof.
    [0010] In one aspect, the invention provides the use of a compound which is: (i) 5-cholesten-3,25-diol, disulfate (25HCDS) of the formula
    and / or pharmaceutically acceptable salts thereof, as a medicine.
    [0011] In some ways, the compound is

    [0012] In some aspects, the invention provides the use of the compound in the methods of: reducing lipids in an individual in need of it; reduction of cholesterol and lipid biosynthesis in an individual in need of it; reduction of inflammation in an individual in need of it; treating diabetes in an individual in need of it; treatment of hyperlipidemia in an individual in need of it; treatment of atherosclerosis in an individual in need of it; treatment of fatty liver disease in an individual in need of it; and / or treating inflammatory disease in an individual in need of it. In other respects, the invention provides the use of a compost
    for the manufacture of a medicine for: reduction of lipids in an individual in need of it; reduction of cholesterol and lipid biosynthesis in an individual in need of it; reduction of inflammation in an individual in need of it; treating diabetes in an individual in need of it; treatment of hyperlipidemia in an individual in need of it; treatment of atherosclerosis in an individual in need of it; treatment of fatty liver disease in an individual in need of it; or treating inflammatory disease in an individual in need of it.
    [0013] In still other aspects, the invention provides methods of treating an individual, the method of which comprises administering to said individual an effective amount of a compound
    where the method is selected from: a method for reducing lipids in an individual in need of it; a method of reducing cholesterol and lipid biosynthesis in an individual in need of it; a method of reducing inflammation in an individual in need of it; a method of treating diabetes in an individual in need of it; a method of treating hyperlipidemia in an individual in need of it; a method of treating atherosclerosis in an individual in need of it; a method of treating fatty liver disease in an individual in need of it; and a method of treating inflammatory disease in an individual in need of it. In some respects, the compound is administered in an amount ranging from 0.1 mg / kg to 100 mg / kg based on the body weight of that individual, or the compound is administered in an amount ranging from 1 mg / kg to 10 mg / kg, based on the individual's body mass; and / or administration comprises at least one between oral administration, enteral administration, sublingual administration, transdermal administration, intravenous administration, peritoneal administration, parenteral administration, administration by injection, subcutaneous injection and intramuscular injection.
    [0014] In one aspect, the invention provides a compound which is: (i) 5-cholesten-3,25-diol, disulfate (25HCDS) of the formula
    and / or pharmaceutically acceptable salts thereof. In one aspect, the compound itself and pharmaceutically acceptable salts thereof are provided. In another aspect, what is provided is the use of the compound and pharmaceutically acceptable salts thereof as a medicine.
    [0015] In some ways, the compost

    [0016] In some ways, the compound is an isolated compound. In other respects, the compound is substantially pure. In still other aspects, the compound is in solid form. The solid form can be in powder form; and / or in lyophilized form.
    [0017] The invention also provides pharmaceutical compositions comprising a compound which is: (i) 5-cholesten-3,25-diol, disulfate (25HCDS) of the formula
    and (ii) physiologically acceptable excipient, diluent or vehicle.
    [0018] In some ways, the compound is

    [0019] In some respects, the pharmaceutical composition is formulated in unit dosage form. In other respects, the pharmaceutical composition is in solid form. Solid forms of the composition include those in which: the pharmaceutical composition is in the form of a powder, a tablet, a capsule or a lozenge; or the composition comprises the compound in lyophilized form together with a bulking agent, the composition optionally being in a sealed vial, ampoule, syringe or pouch. In some aspects, the pharmaceutical composition comprises a carrier that is a liquid. In this aspect, the compound can be solubilized in the liquid or dispersed in the liquid; and / or the liquid is aqueous; and / or the liquid is sterile water for injection or phosphate buffered saline; and / or the composition is in a sealed vial, ampoule, syringe or bag.
    [0020] The invention also provides processes for producing a
    the process of which comprises reacting 25-hydroxycholesterol with a source of sulfur trioxide and optionally forming a resulting pharmaceutically acceptable salt of the resulting 5-cholesten-3,25-diol (25HCDS). In some respects, the sulfur trioxide source is a sulfur trioxide amine complex. In other respects, the process comprises combining the compound with a physiologically acceptable excipient, diluent or vehicle.
    [0021] As indicated above, the present invention, among other things, provides the compounds specified for use in a method of: reducing lipids in an individual in need thereof; reduction of cholesterol and lipid biosynthesis in an individual in need of it; reduction of inflammation in an individual in need of it; treating diabetes in an individual in need of it; treatment of hyperlipidemia in an individual in need of it; treatment of atherosclerosis in an individual in need of it; treatment of fatty liver disease in an individual in need of it; or treating inflammatory disease in an individual in need of it. For the avoidance of doubt, in this respect the present invention can provide the specified compound for use as a drug in the specified method. In addition, the present invention can provide the specified compound as an active therapeutic ingredient in the specified method. In addition, the present invention can provide the specified compound for use in a method of treating the human or animal body by therapy, the method comprising the specified method. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Characterization of nuclear oxisterol as 5-cholesten-3p, 25-diol disulfate by triple negative quadrupole mass spectrometry. HPLC / MS negative total scan spectrum, HPLC-MS elution profile classified with mass ion 80 of product scan spectrum of m / z 583 and m / z 561 is shown. Figure 2. Analysis of chemically synthesized 25HCDS. MS spectrum of the product. Figure 3. Spectrum of 1H of 25HCDS. The arrows indicate the proton in position 3 of the compound and its chemical resonance deviation in the starting material and in the product. Figure 4. Spectrum of 13C NMR of 25HCDS. The arrows indicate, the carbon 3 and 25 positions of the compound and its chemical resonance deviation in the starting material and in the product. Figure 5A to D. 25HCDS regulates lipid biosynthetic gene expression. A, real-time RT-PCR analysis of SREBP-1c, ACC mRNA levels, and FAS in THP-1 cells treated with 25HCDS at the indicated concentration is shown; B, SREBP-2, HMG-CoA reductase, and LDLR; levels of PPARg and IkB mRNA in THP-1 cells treated with 25HCDS at the indicated times, (C) and under the indicated conditions (D). Expression levels were normalized to GAPDH. Each value represents the average of three separate evaluations + standard deviation. Figure 6.Administration of 25HCDS decreases the accumulation of lipid in liver tissue in liver tissue. The animals were peritoneally injected with 25HCDS once every 3 days for 6 weeks. Hepatic triglyceride, free fatty acid, total cholesterol, free cholesterol, and cholesterol ester, free fatty acid, and triglyceride were determined as described in the example. Each individual level was normalized by protein concentration. All values are expressed as mean ± SD; Symbol * represents p <0.05 versus liver of mice treated by vehicle powered by HFD. DETAILED DESCRIPTION
    [0022] A new cholesterol metabolite 5-cholesten-3p, 25-diol, disulfate (25HCDS), has recently been identified. Administration of 25HCDS substantially decreased the expression of PPARY alpha coactivator 1, PPARY alpha coactivator (PGC-1a), and IKB, and decreased hepatic triglyceride and cholesterol levels by means of LXR-SREBP- signaling reaction series. 1c in vivo in mouse NAFLD models. These findings demonstrate that 25HCDS is a potent regulator involved in lipid metabolism and inflammatory responses.
    [0023] The invention thus provides methods of using 25HCDS for the treatment and prevention of metabolic lipid and inflammatory disorders. In some respects, the method involves administering a therapeutically effective dose of 25HCDS to individuals in need of such treatment, in order to raise the level of 25HCDS in the individual and / or effect beneficial changes in lipid metabolism. Implementation of the methods generally involves identifying patients who are suffering from or at risk of developing lipid metabolic disorders and conditions associated with it, and / or identifying patients who are suffering from or at risk of developing abnormal inflammation, and administering 25HCDS in an acceptable form by an appropriate routine. The identification of suitable individuals can be carried out, for example, using various blood tests, results of liver biopsy, the presence of evident disease symptoms, etc., as is known in the art. Individuals suitable for treatment include those who are identified as suffering from or likely to suffer from a lipid metabolic disorder and / or inflammation. 25HCDS and related pharmaceutical compositions are also provided in accordance with the present invention. These can be used in treatment methods.
    [0024] The 25HCDS can be in the form of a pharmaceutically acceptable salt. The pharmaceutically acceptable salt can be a di-addition salt or a mono-addition salt. A di-addition salt is made up by the loss of hydrogen atoms in each of the two sulfate groups of the 25HCDS molecule. A mono-addition salt is formed by the loss of the hydrogen atom of the 25HCDS molecule (at position 3 or 25 of the molecule).
    [0025] The pharmaceutically acceptable salt may, for example, be an alkali metal salt (for example, a lithium, sodium or potassium salt), an alkaline earth metal salt (for example, a calcium salt) or a ammonium salt. The pharmaceutically acceptable salt can, for example, be a sodium, potassium, calcium, lithium or ammonium salt.
    [0026] An example of such a salt is a 25HCDS sodium salt, for example, a 25HCDS mono-addition sodium salt, such as the mono-addition salt formed by the loss of the hydrogen atom in the sulfate group at the 25 of 25HCDS, that is, the compound having the formula

    [0027] For the avoidance of doubt, it is emphasized that references throughout this specification to "25HCDS" include pharmaceutically acceptable salts of 25HCDS unless explicitly indicated otherwise.
    [0028] Cholesterol contains eight chiral centers, thereby giving rise to a large number of distinguishable isomers. These eight chiral centers are also present at 25HCDS. In general, the 25HCDS used in the present invention can be in any stereoisomeric form or it can be a mixture of any two or more stereoisomeric forms. However, at least 50% by weight, preferably at least 90% by weight and more preferably at least 95% by weight of the 25HCDS can be formulated

    [0029] It will be appreciated that the chirality in each of the eight chiral centers in this formula is analogous to that in native cholesterol. Thus, this stereoisomer corresponds to the stereoisomeric form of the 25HCDS metabolite in vivo.
    [0030] The 25HCDS or a pharmaceutically acceptable salt thereof may be 25HCDS alone or a pharmaceutically acceptable salt thereof. "Isolated" means not included in the tissue material contained within, or extracted from, a human or animal individual. For example, 25HCDS alone or a pharmaceutically acceptable salt thereof is not comprised within a cell. Thus, 25HCDS alone or a pharmaceutically acceptable salt thereof is clearly distinguishable from native 25HCDS which is comprised of tissue material (e.g., a cell) that is contained alone within, or has been extracted from, a human or animal individual.
    [0031] The 25HCDS or a pharmaceutically acceptable salt thereof can be substantially pure. For example, 25HCDS or a pharmaceutically acceptable salt thereof can be supplied in a substantially purified form for the treatment methods.
    [0032] When it is "substantially pure" or "substantially purified" the disulfated oyster (the 25HCDS or a pharmaceutically acceptable salt thereof) can be in a form that is at least about 75%, preferably at least about 80%, more preferably at least about 90%, and more preferably at least about 95% or more free of other chemical species. The substantially pure 25HCDS or a pharmaceutically acceptable salt thereof can, in particular, comprise at least about 90% by weight or at least about 95%, and more preferably at least about 98% by weight, at least about 99% by weight or, even more preferably, at least about 99.5% by weight or at least about 99.8% by weight of 25HCDS or a pharmaceutically acceptable salt thereof.
    [0033] The 25HCDS or a pharmaceutically acceptable salt of the same can be solid. For example, the 25HCDS or a pharmaceutically acceptable salt thereof can be in the form of a powder.
    [0034] The 25HCDS or a pharmaceutically acceptable salt of the same can be in lyophilized form. As is well known, freeze drying is a dehydration process typically used to conserve perishable material or make it more convenient for transportation. There are three main stages to this technique, namely freezing, primary drying and secondary drying. Freezing is typically performed using a freeze drying machine. During primary drying, pressure is controlled by applying appropriate levels of vacuum while sufficient heat is provided to allow any water to sublimate. In the secondary drying process, hydration water is also removed by the application of heat. Typically, the pressure is lowered to encourage further drying. Upon completion of the drying process, the vacuum can be stopped with an inert gas such as nitrogen before sealing or the material can be sealed under vacuum.
    [0035] While it is possible to isolate and purify 25HCDS from living cells, those skilled in the art will recognize that in order to generate sufficient amount of the disulfated oyster, the compound will generally be synthesized, by synthetic chemical methods, or by methods involving the use of recombinant DNA technology (for example, using cloned enzymes to make appropriate cholesterol modifications). An exemplary synthetic scheme is provided in the examples section below.
    [0036] More generally, 25HCDS or a pharmaceutically acceptable salt thereof can be produced synthetically by reacting 25-hydroxycholesterol with a sulfur trioxide source, and optionally forming a pharmaceutically acceptable salt of the resulting product.
    [0037] Any suitable source of sulfur trioxide can be used to convert the two hydroxyl groups (-OH) present in 25-hydroxycholesterol into sulfate groups (-OSO3H). Sulfur-amine trioxide complexes are an exemplary group of sources of sulfur trioxide. Examples of such complexes include sulfur trioxide-trimethylamine complexes (TMAS), sulfur triethylamine complex trioxide (TEAS), sulfur trioxide-dimethylaniline complex (DMAS), sulfur trioxide-dimethylformamide complex (DMFS), complex sulfur trioxide-pyridine (PSS) and sulfur trioxide-polyvinylpyridine complex (PVPS). Typically one to two moles, for example, two to ten moles, of the chosen sulfur trioxide source (such as the sulfur trioxide-amine complex) are used per mole of 25-hydroxycholesterol.
    [0038] The reaction is typically carried out in an inert solvent. The solvent can, for example, be an anhydrous solvent. An exemplary solvent is anhydrous pyridine.
    [0039] A base can also be added, for example, in order to generate the desired pharmaceutically acceptable salt of the disulfate product. One such base is NaOH, which can be used to generate a sodium salt of 25HCDS. It will be readily appreciated that alternative reagents (having different basicities and / or different cations) can be used to generate other pharmaceutically acceptable salts.
    [0040] The reaction temperature can typically be 10 to 100 ° C, for example, 20 to 80 ° C. The reaction time can typically be from 0.1 to 24 hours, for example, from 0.25 to 5 hours.
    [0041] If desired, the product can be purified from the reaction mixture after the reaction has taken place. If desired, the product can be isolated from the reaction mixture after the reaction has taken place.
    [0042] The 25-hydroxycholesterol starting material is a commercially available product. Alternatively, it can be prepared by hydroxylating cholesterol (see, for example, Ogawa et al. Steroids 74: 81-87). The process can therefore also comprise an initial cholesterol hydroxylation step to produce 25-hydroxycholesterol.
    [0043] 25HCDS can be administered in pure form or in a pharmaceutically acceptable formulation. Such formulations (compositions) typically include 25HCDS or a pharmaceutically acceptable salt thereof and a physiologically acceptable (compatible) excipient, diluent or carrier / vehicle. The 25HCDS can be, for example, in the form of a pharmaceutically acceptable salt (for example, an alkali metal salt such as sodium, potassium, calcium, lithium, ammonium, etc.), or another complex.
    [0044] The pharmaceutical composition is sterile. Sterile means substantially free of viable microbes, for example, as determined using USP sterility test (see, "The United States Pharmacopeia", 30th Revision, The United States Pharmacopeial Convention: 2008.). In order to maintain sterility, the pharmaceutical composition can be presented in a sealed package that is able to prevent the entry of viable microbes. For example, in the case of a liquid pharmaceutical composition, the composition can be sealed in a vial or ampoule.
    [0045] It should be understood that pharmaceutically acceptable formulations (compositions) include liquid and solid materials conventionally used to prepare both injectable dosage forms and solid dosage forms such as tablets, lozenges, capsules, as well as aerosol dosage forms . The compound can be formulated with water-based or aqueous vehicles. Water can be used as the vehicle for preparing compositions (for example, injectable compositions), which can also include conventional agents and buffers to make the composition isotonic and maintain a physiologically acceptable pH. Other potential additives (preferably those that are generally considered to be safe [GRAS]) include: dyes; flavorings; surfactants (TWEEN, oleic acid, etc.); solvents, stabilizers, elixirs, and binders or encapsulants (lactose, liposomes, etc.). Solid diluents and excipients include lactose, starch, conventional disintegrating agents, coatings and the like. Preservatives such as methyl paraben or benzalkyl chloride can also be used.
    [0046] In other details, when the composition is in solid form it can be in the form of a powder, a tablet, a capsule or a lozenge. When the composition is in solid form the composition may comprise 25HCDS in lyophilized form together with a bulking agent. A bulking agent is a pharmaceutically inactive and typical and chemically inert substance that can be added to increase its volume. Common bulking agents for use in the preparation of freeze-dried pharmaceutical compositions, which are suitable here, include mannitol and glycine. When the composition is in solid form it can optionally be in a sealed bottle, ampoule, syringe or bag.
    [0047] When the pharmaceutical composition comprises a liquid carrier, the 25HCDS can be solubilized in said liquid or dispersed in said liquid; and / or the liquid can be aqueous; and / or the liquid can be sterile water for injection or phosphate buffered saline. When the pharmaceutical composition comprises a liquid carrier, the composition can be in a sealed bottle, ampoule, syringe or pouch.
    [0048] Depending on the formulation, the active agent 25HCDS is expected to consist of 1% to about 99% by weight of the composition and the carrier "carrier" to constitute about 1% to about 99% by weight of the composition. The pharmaceutical compositions of the present invention can include any pharmaceutically acceptable additives or adjuncts to the extent that they do not prevent or interfere with the therapeutic effect of sulfated oysterol.
    [0049] Administration can be at least one of: oral administration, enteral administration, sublingual administration, transdermal administration, intravenous administration, peritoneal administration, parenteral administration, administration by injection, subcutaneous injection, and intramuscular injection. For example, administration can be oral or parenteral, including intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, etc., or by other routines (for example, transdermal, sublingual, oral, rectal and buccal release, aerosol inhalation, etc.). In a preferred embodiment, administration is oral. In addition, administration of the compound can be performed as a simple mode of therapy, or in conjunction with other therapies, for example, drugs that lower lipid or cholesterol, diet and exercise regimens, etc., as described above for treatment regimens that can be assigned by an individual to detect a metabolic lipid disorder. The administration of 25HCDS to a patient can be intermittent, or at a gradual or continuous, constant or controlled frequency. In addition, the time of day and the number of times per day that the pharmaceutical formulation is administered can vary and are best determined by a skilled clinician such as a physician.
    [0050] The exact dosage of 25HCDS to be administered can vary depending on the age, sex, weight, general state of health of the individual patient, etc., as well as the precise etiology of the disease. However, in general, for administration to mammals (e.g., humans), therapeutically effective dosages are in the range of about 0.1 to about 100 mg or more of compound per kg of body weight for 24 hours, and generally about 0, 5 to about 50 mg of compound per kg of body weight per 24 hours, and often about 1 to about 10 mg of compound per kg of body weight per 24 hours, are effective.
    [0051] A pharmaceutical composition of the invention can be formulated in unit dosage form, that is, the pharmaceutical composition can be in the form of discrete portions, each containing a unit dose of 25HCDS. In this context, a unit dose can comprise, for example, from about 0.1 mg to about 100 mg, or from about 0.5 mg to about 50 mg, or from about 1 mg to about 10 mg 25HCDS.
    [0052] The pharmaceutical composition can be prepared by combining the 25HCDS with the chosen pharmaceutically acceptable excipients, diluents and / or vehicles.
    [0053] While individuals are generally human, veterinary applications of the technology are also contemplated.
    [0054] In other respects, the level of 25HCDS is elevated in an individual in need while increasing endogenous expression / production of 25HCDS. Exemplary methods for doing this include administering to the individual with one or more enzymes responsible for the synthesis of 25HCDS. In some embodiments, the enzymes alone are provided; in other embodiments, nucleic acids encoding the enzymes are provided. The enzymes that are involved in the synthesis of 25HCDS are SULT2Bab and SULT2B1a, and one or both of these can be administered in order to raise endogenous levels of 25HCDS. For example, vectors that contain and express one or both of these enzymes can be provided. Exemplary vectors include, but are not limited to, adenoviral vectors, retroviral vectors, competent replication vectors, herpes viral vectors, etc.
    [0055] Metabolic lipid disorders that can be prevented or treated by raising the levels of 25HCDS in an individual as described here include, but are not limited to: hepatitis (inflammation of the liver) caused mainly by various viruses, but also by some bacterial infections, drugs or chemicals (for example, poisons, alcohol), as well as associated complications such as liver fibrosis; autoimmunity (autoimmune hepatitis) or hereditary conditions; non-alcoholic fatty liver disease (NAFLD) a spectrum disease associated with obesity and characterized by an abundance of fat in the liver, and several syndromes associated with NAFLD (eg, hepatitis, non-alcoholic steatohepatitis (NASH), cirrhosis, liver disease terminal stage, etc.); cirrhosis, that is, the formation of fibrous scar tissue in the liver due to the replacement of dead liver cells (the death of liver cells can be caused, for example, by viral hepatitis, alcoholism or contact with other products toxic to the liver); hemochromatosis, an inherited disease that causes iron to accumulate in the body, eventually inducing liver damage; liver cancer (for example, primary hepatocellular carcinoma or cholangiocarcinoma and mestatic cancers, usually from other parts of the gastrointestinal tract); Wilson's disease, an inherited disease that causes the body to retain copper; primary sclerosing cholangitis, an inflammatory disease of the bile duct, probably immune in nature; primary biliary cirrhosis, an autoimmune disease of small bile ducts; Budd-Chiari syndrome (hepatic vein obstruction); Gilbert's syndrome, a genetic disorder of bilirubin metabolism, discovered in about 5% of the population; glycogen storage disease type II; as well as various pediatric liver diseases, for example, including biliary atresia, alpha-1 antitrypsin deficiency, alagil syndrome, and progressive familial intrahepatic cholestasis, etc. In addition, trauma liver injury can also be treated, for example, injury caused by accidents, gunshot wounds, etc. In addition, liver damage caused by certain drugs can be prevented or treated, for example, drugs such as the anti-arrhythmic agent amiodarone, various antiviral drugs (for example, nucleoside analogs), aspirin (rarely as part of Reye's syndrome in children ), corticosteroids, methotrexate, tamoxifen, tetracycline, etc. are known to cause liver damage. In some modalities, diagnostic and treatment methods are performed in association with (for example, before, during or after) liver surgery in an individual. For example, liver surgery can be liver transplant surgery and the individual being treated can be a donor or a recipient; or liver surgery can be surgery that removes sick or damaged liver tissue, or that removes cancerous tumors, etc.
    [0056] In some modalities, the disease or condition that is prevented or treated is or is caused by hyperlipidemia. By "hyperlipidemia" we mean a condition of abnormally high levels of any or all of the lipids and / or lipoproteins in the blood. Hyperlipidemia includes both primary and secondary subtypes, with primary hyperlipidemia generally being due to genetic causes (such as a mutation in a receptor protein), and secondary hyperlipidemia resulting from other underlying causes such as diabetes. Lipids and lipid composites that can be elevated in an individual and decreased by the treatments described here include, but are not limited to, chylomicrons, very low density lipoproteins, intermediate density lipoproteins, low density lipoproteins (LDLs) and high density lipoproteins (HDLs). In particular, cholesterol (hypercholesteremia) and elevated triglycerides (hypertriglycerideomy) are known to be risk factors for cardiovascular and blood vessel disease due to their influence on atherosclerosis. Elevated lipid can also predispose an individual to other conditions such as acute pancreatitis. The methods of the invention thus can also be used in the treatment or prophylaxis (for example, prophylactic treatment) of conditions that are or are associated with elevated lipids. Such conditions include, for example, but are not limited to: hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, fatty liver (hepatic steatosis), metabolic syndrome, cardiovascular diseases, coronary heart disease, atherosclerosis (i.e., arteriosclerotic vascular disease or ASVD) and associated diseases, pancreatitis acute, various metabolic disorders, such as insulin resistance syndrome, diabetes, polycystic ovary syndrome, fatty liver disease, cachexia, obesity, arteriosclerosis, cerebrovascular disease, gallbladder stones, inflammatory bowel disease, hereditary metabolic disorders such as disorders lipid storage, and the like. In addition, several conditions associated with hyperlipidemia include those described in United States Patents 8,003,795 (Liu, et al) and 8,044,243 (Sharma, et al), the complete contents of both of which are incorporated herein by reference in their wholeness.
    [0057] In some embodiments, diseases and conditions that are prevented or treated include inflammation, and / or diseases and conditions associated with, characterized by or caused by inflammation. These include a large group of disorders that underlie most human diseases. In some modalities, the inflammation is acute, resulting from, for example, an infection, an injury, etc. In other modalities, the inflammation is chronic. In some modalities, the immune system is involved with the inflammatory disorder as seen in both allergic reactions and some myopathies. However, several nonimmune diseases with etiological origins in an inflammatory process can also be treated, including cancer, atherosclerosis, and ischemic heart disease, as well as others listed below.
    [0058] Examples of disorders associated with abnormal inflammation that can be prevented or treated using 25HCDS include, but are not limited to: acne vulgaris, asthma, various autoimmune diseases, celiac disease, chronic prostatitis, glomerulonephritis, various hypersensitivity, inflammatory bowel diseases, pelvic inflammatory diseases, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis, and interstitial cystitis. Also included are inflammatory disorders that occur as a result of the use of both legally prescribed and illicit drugs, as well as inflammation activated by negative cognitions or the consequences of them, for example, caused by stress, violence, or deprivation.
    [0059] In one aspect, the inflammatory disorder that is prevented or treated is an allergic reaction (type 1 hypersensitivity), the result of an inappropriate immune response that activates inflammation. A common example is hay fever, which is caused by a hypersensitive response by skin mast cells to allergens. Several inflammatory responses can mature into a systemic response known as anaphylaxis. Other hypersensitivity reactions (type 2 and type 3) are mediated by antibody reactions and induce inflammation by attracting leukocytes that damage surrounding tissue, and can also be treated as described here.
    [0060] In other respects, inflammatory myopathies are prevented or treated. Such myopathies are caused by the immune system to improperly attack muscle components, inducing signs of muscle inflammation. They can occur in conjunction with other immune disorders, such as systemic sclerosis, and include dermatomyositis, polymyositis, and inclusion body myositis.
    [0061] In one aspect, the methods and compositions of the invention are used to prevent and treat systemic inflammation such as that which is associated with obesity. In such inflammation, the processes involved are identical to tissue inflammation, but systemic inflammation is not confined to a particular tissue, but involves the endothelium and other organ systems. Systemic inflammation can be chronic, and is widely seen in obesity, where many elevated markers of inflammation are seen, including: IL-6 (interleukin-6), IL-8 (interleukin-8), IL-18 (interleukin-18) , TNF-a (tumor necrosis factor alpha), CRP (C-reactive protein), insulin, blood glucose, and leptin. Conditions or diseases associated with high levels of these markers can be prevented or treated as described here. In some embodiments, inflammation can be classified as "chronic low-grade inflammation" in which a two to three-fold increase in systemic cytokine concentrations such as TNF-a, IL-6, and CRP is observed. Waist circumference also correlates significantly with systemic inflammatory responses; a predominant factor in this correlation is due to the autoimmune response activated by adiposity, whereby immune cells "miss" fat deposits for infectious agents such as bacteria and fungi. Systemic inflammation can also be agitated by overeating. Foods high in saturated fat, as well as foods high in calories have been associated with increases in inflammatory markers and the response may become chronic if chronic excess pain.
    [0062] Various facets of the invention are described in the Examples below. However, the information provided in the examples should not be considered as limiting the scope of the invention in any way. EXAMPLE A new cholesterol metabolite, 5-cholesten-3p, 25-diol, disulfate (25HCDS), decreases lipid biosynthesis and suppresses inflammatory responses in vitro and in vivo INTRODUCTION
    [0063] It has been shown that there is widespread deregulation of lipid metabolism in non-alcoholic fatty liver diseases (NAFLD) and, specifically, there are major disturbances in cholesterol metabolism. The potential mechanisms by which such disturbances can induce NAFLD through nuclear receptor signaling remain unclear. In the present study, a new cholesterol metabolite, 5-cholesten-3p, 25-diol, disulfate (25HCDS) was identified in primary rat hepatocytes. As described here, 25HCDS has recently been chemically synthesized and its biological function has been studied. Administration of 25HCDS (25 pM) to human THP-1 macrophages and HepG2 cells, and in vivo to mouse NAFLD animal models, increased expression of PPARY and PPARY (PGC-1a) alpha coactivator and decreased protein expression key factors involved in lipid biosynthesis and pro-inflammatory responses. Administration markedly decreased hepatic lipid levels and suppressed inflammatory responses. Quantitative RT-PCR and Western blot analysis showed that 25HCDS strongly decreased levels of SREBP-1/2 mRNA and suppressed the expression of its response genes including ACC, FAS, and HMG-CoA reductase, and increased levels of IKB and decreased mRNA levels of TNFa and ILp. The results suggest that the inhibition of lipid biosynthesis occurred by blocking SREBP signaling, and suppressing inflammatory responses by increasing the expression of PPARY, PGC-1 a, and IKB expression. Analysis of lipid profiles in liver tissues showed that administration of 25HCDS once every three days for 6 weeks significantly decreased total cholesterol, free fatty acids, and triglycerides by 30, 25, and 20%, respectively. 25HCDS is thus a potent regulator of lipid metabolism and inflammatory responses. MATERIALS AND METHODS Materials:
    [0064] Cell culture reagents and supplies were purchased from GIBCO BRL (Grand Island, NY); 25-hydroxycholesterol from New England Nuclear (Boston, MA). THP-1 and HepG2 cells were obtained from the American Type Culture Collection (Rockville, MD). Reagents for real-time RT-PCR were from AB Applied Biosystems (Warrington WA1 4 SR, UK). The chemicals used in this research were obtained from Sigma Chemical Co. (St. Louis, MO) or Bio-Rad Laboratories (Hercules, CA). Polyclonal rabbit antibodies against SREBP1, SREBP-2 and HMG-CoA reductase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All solvents were obtained from Fisher (Fair Lawn, NJ) unless otherwise indicated. enhanced chemiluminescence (ECL) were purchased from Amersham Biosciences (Piscataway, NJ). Testosterone and 27-hydroxycholesterol were obtained from Research Plus Inc. (Bayonne, NJ). 20 x 20 cm LK6 thin layer chromatography plates (TLC) were purchased from Whatman Inc. (Clifton, NJ). Methods: Chemical synthesis of 5-colesten-3p, 25-diol, disulfate
    [0065] General procedure: 25-Hydroxycholesterol was prepared from cholesterol by the method previously described (Ogawa et al. Steroids 74: 81-87). IR spectra were obtained on KBr disks in a JASCO FT-IR 460 plus spectrometer (Tokyo, Japan). The 1H and 13C NMR spectra were obtained on a Varian 500 Inova (AS500) instrument at 499.62 MHz and 125.64 MHz, respectively. Flow resolution low resolution mass spectra (LR-MS) were recorded by a Thermo Scientific TSQ Quantum Ultra MS equipped with electrospray ionization probe (ESI) in negative ion mode. High resolution mass spectra (HR-MS) were measured using Thermo Scientific LTQ Qrbitrap Discovery MS with ESI probe under negative ion mode. Reverse phase TLC was performed on pre-coated RP-18F254S plates using methanol-water-acetic acid mixtures (90: 10: 1, volume / volume / volume) as the development solvent. The stains were visualized by 50% H2SO4 with heating at 110 oC. A Bond Elute C18 cartridge (10 g; Varian,) was used for sample purification. Oxone® (potassium peroximonosulfate) and acetone were purchased from Sigma-Aldrich Co (St. Louis, MO, USA), and all other reagents were the highest grade, except for organic solvents that were HPLC grade.
    [0066] Synthesis of 5-cholesten-3p, 25-diol, disulfate (25HCDS): To a solution of 25-hydroxycholesterol (30 mg, 0.07 mmol) in anhydrous piperidine (300 pL), sulfur-trioxide complex trimethylamine (45 mg) was added, and the suspension was stirred at 50 ° C for 1 hour. To the reaction mixture, 0.1N of methanolic NaOH (100pL) was added and the mixture was applied to a Sep Pak C18 cartridge, which was prepared with methanol (10 ml) and water (10 ml). The cartridge was successively washed with PBS (25 ml) and water (25 ml), and then the retained 25HCDS was eluted with 60% methanol (10 ml). After 10X dilution with acetonitrile, the solvents were evaporated to dryness under a N2 stream below 40 ° C, and the 25HCDS was obtained in powder form. Yield 25 mg (60%). Cell culture
    [0067] Human THP-1 monocytes and HepG2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and maintained according to the supplier's protocols. THP-1 monocytes were differentiated by macrophages by adding 100 nM phorbol 12-myristate 13-acetate (PMA). When the cells reached ~ 90% confluence, 25HCDS in ethanol (the final concentration of ethanol on average was 0.1%) was added. The cells were harvested at the indicated times for protein, mRNA, and lipid analysis.
    [0068] For the study of expression regulation of HMG CoA, HepG2 or PHH were grown in the media as described above in the presence or absence of mevinoline (50 pM) and mevalonate (0.5 pM). After culturing for 48 hours, oxisterols were added and cultured for an additional 6 hours, and then the cells were harvested for determination of mRNA and protein levels. Determination of Cholesterol Biosynthesis by TLC and HPLC
    [0069] After incubation of THP-1 macrophages or HepG2 cells in media containing different concentrations of 25HCDS as indicated for 6 hours, cells in 60 mm dishes were supplied 3 ml of the same fresh medium containing 5 pCi of [1-14C] acetate. After 2 hours incubation at 37 ° C, the media was removed and the cells were washed twice with phosphate buffered saline (PBS), harvested with rubber policeman as described, and collected in microcentrifuge tubes. The cells were pelleted by centrifugation and the pellets were washed three times by resuspension and pelleting. Subcellular fractions (microsomal, cytosol, and nuclear) were isolated as previously described (2). Cellular or subcellular pellets were resuspended in 0.3 ml of PBS. For each sample, 1.5 pg of testosterone was added as an internal standard. The total lipids were extracted and separated by adding 3 volumes of chloroform: methanol (1: 1). [14C] cholesterol and hydroxycholesterols were isolated in the chloroform phase and separated in TLC (toluene: ethyl acetate, 2/3, volume / volume). Derivatives of [1-14C] acetate were visualized by Fujifilm BAS-1800 II image reader as previously described (1).
    [0070] For analysis of unlabelled sterol products, the extracted lipids were incubated with 2 units of cholesterol oxidase at 37 ° C for 20 minutes. The oxidation reaction was terminated by adding 1.5 ml of methanol followed by 0.5 ml of saturated KCl. The sterols were extracted twice using 3 ml of hexane. The hexane phase was collected and evaporated under a stream of nitrogen. The residues were dissolved in mobile phase solvents for HPLC analysis as previously described (3).
    [0071] [1-14C] acetate derivatives in the chloroform phase were analyzed by HPLC on a silica column (5p x 4.6 mm x 25 cm; Beckman, USA) using HP Series 1100 solvent release system (Hewlett Packard) at a flow rate of 1.3 ml / minute. The column was equilibrated and carried out in a hexane: isopropanol: aceticglacial acid system (965: 25: 10, volume / volume / volume), as the mobile phase. The effluents were collected every 0.5 minutes (0.65 ml per fraction) except as indicated. The counts of [14C] acetate derivatives were determined by scintillation counting. The column was calibrated with [14C] cholesterol, [3H] 25-hydroxycholesterol, and [14C] 27-hydroxycholesterol. Determination of mRNA levels by real-time RT-PCR
    [0072] Total RNA was isolated with SV Total RNA Isolation Kit (Promega, Madison, WI), which included DNase treatment. Total RNA, 2 pg, was used for first strand cDNA synthesis as recommended by the manufacturer (Invitrogen, Carlsbad, CA). Real-time RT-PCR was performed using a suitable dye as an indicator on the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). All primer / probe groups for real-time PCR were TaqMan gene expression assays (Applied Biosystems, Foster City, CA). Amplifications of p-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as internal controls. Expression of relative messenger RNA (mRNA) was quantified with the comparative cycle threshold method (Ct) and was expressed as 2-AACt. Primer sequences suitable for amplification are described, for example, in Ren et al., 2007 (1). Western blot analysis
    [0073] Microsomal fractions were isolated as previously described (4). Microsomal proteins or total extracted from treated cells were separated on a 7.5% SDS-polyacrylamide denaturing gel. Following the SDS-PAGE, the proteins were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were then blocked at 25 ° C for 60 minutes in blocking buffer [PBS, pH 7.4, 0.1% TWEEN® 20 (non-ionic membrane protein solubilizing surfactant, C58H114O26), 5% milk in skim powder). The proteins were then incubated at 4 ° C overnight with a polyclonal rabbit IgG against human SREBP1, SREBP-2, or HMG-CoA reductase. After washing with PBS, pH 7.4, containing 0.05% TWEEN® 20, 1: 2500 spicy goat rabbits peroxidase IgG conjugate in washing solution was added and incubated for 60 minutes. Protein bands were detected using the Amersham ECL plus kit. Positive bands were quantified by the Advanced Image Data Analyzer (Aida Inc., Straubenhardt, Alemamha). Animal Studies
    [0074] Animal studies were approved by the Institutional Animal Care and Use Committee of McGuire Veterans Affairs Medical Center and were conducted in accordance with Declaration of Helsinki, the Guide for the Care and Use of Laboratory Animals, and all applicable regulations. To examine the effect of 25HCDS on diet-induced lipid accumulation in sera and liver, 8-week-old female C57BL / 6J mice (Charles River, Wilmington, MA) were fed a high-fat (HFD) diet (Harlan Teklad, Madison, WI) containing 42% kcal of fat, 43% kcal of carbohydrate, 15% kcal of protein and 0.2% cholesterol for 10 weeks. All mice were housed under identical conditions in an aseptic facility and provided free access to water and food. At the end of each period, the mice were injected intraperitoneally with the vehicle solution (ethanol / PBS; Vehicle), or 25HCDS (25 mg / kg) once every three days for 6 weeks and fasted overnight; and blood samples were collected. Serum triglyceride, total cholesterol, high density lipoprotein cholesterol, glucose, alkaline phosphatase (ALK), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured using standard enzyme techniques in the clinical laboratory at McGuire Veterans Affairs Medical Center. Profiles of lipoproteins in sera were analyzed by HPLC as described below. Quantification of hepatic lipids
    [0075] Liver tissues were homogenized, and the lipids were extracted with a mixture of chloroform and methanol (2: 1), and filtered. The extracts, 0.2 ml, were evaporated to dryness and dissolved in 100 pl of isopropanol containing 10% TRITON ™ X100 (C14H22O (C2H4O) n), a nonionic surfactant) for cholesterol testing (Wako Chemicals USA, Richmond , VA), the NEFA solution (0.5 g EDTA-Na2, 2 g TRITON ™ X-100, 0.76 ml 1N NaOH, and 0.5 g sodium azide / l, pH 6 , 5) for free fatty acid assay (Wako Chemicals USA, Richmond, VA), or isopropanol for triglyceride assay only (Fisher Scientific, Pittsburgh, PA). All tests were performed according to the manufacturer's instructions, respectively. Each lipid concentration was normalized for liver weight. Statistics
    [0076] The data are reported as the mean ± standard deviation. Where indicated, data were subjected to t-test analysis and determined to be significantly different if p <0.05. RESULTS Detection of new cholesterol metabolite in nuclei of primary rat hepatocytes
    [0077] To determine the presence of new cholesterol metabolites in liver nuclei, nuclear fractions were isolated from primary rat hepatocytes. The oxisterols in the methanol / water phases of each fraction were analyzed by LC-MS. The results showed that two of the main molecular ions, m / z 561 in / z 583 (561 + Na) are well-adjusted to the molecule, 5-colesten-3p, 25-diol disulfate (Figure 1). The molecule is more likely synthesized by SULT2B1b and SULT2B1a. Chemical synthesis of oyster, 5-cholesten-3p, 25-diol, nuclear disulfate
    [0078] To confirm its structure and study its role in lipid cell homeostasis and inflammatory responses, 25HCDS was chemically synthesized as described above and purified.
    [0079] The MS analysis of the synthesized compound shows the same molecular weight ion, m / z 561 in / z 583 (+ Na) as the authentic nuclear oxisterol, and the purified product was not contaminated by the starting material, 25- hydroxycholesterol, m / z 401. LR-MS (ESI-negative), m / z: 583.4 (M + Na-2H, 88%), 561.3 (MH, 46%), 481.4 (M- SO3- H, 11%), 463.4 (M-H2SO4-H, 34%), 431.82 (14%), 381.27 (100%) (Figure 2), 1H NMR (CD3OD) δ: 0.72 (3H, s, I8-CH3), 0.97 (3H, d, J 5.0Hz, 21-CH3), 1.03 (3H, s, 19-CH3), 1.14 (6H, s, 26 - and 27-CH3), 4.14 (1H, br, m, 3a-H), 5.39 (1H, br, s, 6-H) (Figure 3), 13C NMR (CD3OD) δ: 12, 45, 19.37, 19.90, 21.82, 22.29, 25.45, 25.51, 27.05, 27.12, 29.39, 29.44, 30.13, 33.16, 33.37, 37.26, 37.32, 37.50, 38.60, 40.52, 41.27, 43.65, 51.78, 57.71, 58.37, 79.98, 85, 93, 123.44, 141.71 (Figure 4). The results indicate that the synthesized molecule is 5-cholesten-3p, 25-diol, disulfate (25HCDS), and that it "fits" the molecule indicated in the nuclear fraction of hepatocyte. 25HCDS inhibits lipid biosynthesis by decreasing ACC, FAS, and HMG-CoA mRNA reductase levels through SREBP signaling
    [0080] To investigate how 25HCDS inhibits lipid biosynthesis, total RNA was isolated from treated THP-1 macrophages. The levels of ACC and FAS mRNA for triglyceride synthesis, and HMG-CoA reductase for cholesterol synthesis in macrophages and hepG2 cells were determined by real-time RT-PCR. As shown in figure 5, the decrease in ACC and FAS levels (Figure 5A), and HMG-CoA reductase mRNA (Figure 5B) following the addition of 25HCDS to cells in the culture were concentration-dependent as shown and time-dependent ( data not shown). These decreases were consistent with the decrease in SREBP1 / 2 expression shown in Figures 5A and 5B. These results indicate that 25HCDS decreases SREBP signaling and subsequently decreases lipid biosynthesis. Interestingly, 25HCDS linearly increased PPARY mRNA levels and coincidentally increased iKBa expression at an early stage and in low concentrations. The results suggest that 25HCDS suppresses inflammatory responses through the PPARY / lKBa signaling reaction series as does 25HC3S. Effects of administration of 25HCDS on lipid homeostasis in mice fed with HFD
    [0081] To study the long-term treatment effects of 25HCDS on lipid homeostasis, 8 week old female C57BL / 6J mice were fed an HFD for 10 weeks, and then divided into two groups. One group was treated with 25HCDS and the other with peritoneal vehicle once every three days for six weeks. During treatment, the mice were fed HFD, and body mass and caloric intake were monitored. No significant difference in these two parameters was observed (data not shown). After 6 weeks of injections, the mice were fasted overnight, and sacrificed. The liver weights of the mice did not show significant differences regardless of diet (data not shown).
    [0082] To study the effect of 25HCDS on hepatic lipid metabolism, hepatic lipid levels and related levels of gene expression were determined. As previously reported, HFD-fed mice exhibited increased levels of triglyceride, total cholesterol, free fatty acid, and triglyceride in the liver when compared to mice fed with feed (data not shown). These increases were significantly reduced by administration of 25HCDS, for example, by 30%, 20% and 18% (p <0.05), respectively, as shown in Figure 6. In addition, gene expression analysis showed that administration 25HCDS significantly decreased the expression of key enzymes and receptors involved in the synthesis of free fatty acid, triglyceride, and cholesterol, as shown in Table 1.
    [0083] Deregulation of lipid metabolism is often associated with inflammatory conditions. Treatment with 25HCDS significantly suppressed the expression of TNFa, and IL1p, in 50%, 36%, respectively (Table 2). These results are consistent with liver function assays that showed that 25HC3S suppresses liver inflammatory responses, decreasing liver damage and alkaline phosphatase activity in sera (data not shown). Interestingly, 25HCDS increased PGC-1a expression 2-fold in the liver. Thus, 25HCDS seems to regulate lipid metabolism and inflammatory responses through LXR, PPARY and PGC-1a signaling. Table 1. Expression of Relative Hepatic mRNA involved in lipid metabolism in mice fed with HFD with or without 25HCDS

    [0084] The animals were treated as described above. All values are expressed as mean ± SD; n = 6-7. * p <0.05 compared to HFD mouse. Abbreviations: HFD, high-fat diet. Table 2. Expression of Relative Hepatic mRNA involved in Inflammatory responses in mice fed with HFD

    [0085] The animals were treated as described above. All values are expressed as mean ± SD; n = 6-7. * p <0.05 compared to HFD mouse. Abbreviations: HFD, high-fat diet. DISCUSSION
    [0086] Cholesterol and triglyceride metabolism are closely associated. Orphan nuclear receptors are ligand-activated transcription factors that regulate the expression of key target genes that are important regulators of many biological events. Receptors for fatty acids (PPARs), oxisterols (LXRs), retinoic acids (RXR), and SREBPs function as sensors for cellular lipid levels, eliciting changes in gene expression in order to maintain lipid homeostasis and protect cells of damage by accumulation of lipid. However, interference between the receiver's activities remains obscure. As shown here, the cholesterol metabolite, 25HCDS, inhibits the expression, processing and activity of SREBP-1c, in vitro and in vivo and increases the expression of PPARY and PGC-1a. It is well documented that SREBPs control lipid biosynthesis, PPARY regulates inflammatory responses, and PGC-1a controls energy homeostasis. Thus, the results show that 25HCDS is a potent regulator of these processes, and plays an important role in maintaining hepatic lipid homeostasis and inflammatory responses. The administration of 25HC3S increases the levels of nuclear PPARY protein and suppresses inflammatory responses, but only slightly increases PPARy mRNA. In contrast, 25HCDS significantly increases PPARY mRNA and PGC-1a mRNA expression, in a time and concentration dependent manner, indicating that 25HCDS is more potent than 25HC3S in regulating lipid metabolism and inflammatory responses.
    [0087] The reactions of 25HCDS biosynthesis and oyster sulfation represent a new series of regulatory feed, which mediates nuclear receptor activity in hepatocytes. The key components of this reaction series are summarized as follows: 1) when intracellular cholesterol levels are increased, the mitochondrial cholesterol-releasing protein, StAR, releases cholesterol in the mitochondria, where regulatory oxisterols, such as 25HC, are synthesized by CYP27A1 . These oxisterols in turn activate LXR, and subsequently down-regulate the expression of target genes involved in fatty acid and triglyceride biosynthesis. In addition, 25HC activates LXR, again down-regulates synthesized cholesterol synthesis by inhibiting HMGR expression and increases ABCA1-mediated cholesterol secretion from cells (formation of HDL). 2) 25HC3S and 25HCDS inactivate LXRs and suppress the processing of SREBP-1c, indicating that these sulfated oxisterols decrease the levels of intracellular lipids inhibiting synthesis; 3) the effects of 25HC on lipid metabolism are opposite to those of 25HC3S and 25HCDS. Thus, intracellular oyster sulfation represents a new regulatory mechanism involved in lipid metabolism, and in the development of NAFLD.
    [0088] Treatment of mouse NAFLD models with 25HCDS decreased hepatic lipid levels. A large number of treatments for NAFLD have been studied. While most seem to improve biochemical markers such as alanine transaminase levels, most have not been shown to reverse histological abnormalities or reduce clinical purposes. 25HCDS suppresses the key gene expressions involved in lipid biosynthesis at the transcriptional level by blocking nuclear receptor activation of LXRs and SREBPs, suppressing the pro-inflammatory cytokines induced by HFD and controlling energy homeostasis through PGC1a. Thus, 25HCDS functions as a potent regulator to effectively reduce liver lipid levels and consequently represents a new agent for the therapy of NALFD and other diseases associated with metabolic lipid. REFERENCES 1.Ren, S., Li, X., Rodriguez-Agudo, D., Gil, G., Hylemon, P., And Pandak, W.M. 2007. Sulfated oxysterol, 25HC3S, is a potent regulator of lipid metabolism in human hepatocytes. Biochem.Biophys. Commun. 360: 802-808. 2.Ren, S., Hylemon, P., Zhang, Z.P., Rodriguez-Agudo, D., Marques, D., Li, X., Zhou, H., Gil, G., And Pandak, W.M. 2006. Identification of a novel sulfonated oxysterol, 5-cholesten-3beta, 25-diol 3-sulfonate, in hepatocyte nuclei and mitochondria. J. Lipid Res. 47: 1081-1090. 3. Pandak, WM, Ren, S., Marques, D., Hall, E., Redford, K., Mallonee, D., Bohdan, P., Heuman, D., Gil, G., And Hylemon, P . 2002. Transport of cholesterol into mitochondria is rate-limiting for bile acid synthesis via the alternative pathway in primary rat hepatocytes. J. Biol. Chem. 277: 48158-48164. 4.Ren, S., Hylemon, P., Marques, D., Hall, E., Redford, K., Gil, G., And Pandak, W.M. 2004. Effect of increasing the expression of cholesterol transporters (StAR, MLN64, and SCP-2) on bile acid synthesis. J. Lipid Res. 45: 2123-2131.
    [0089] Even though it had been described in terms of its preferred modalities, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the attached claims. Consequently, the present invention should not be limited to the modalities as described above, but must also include all modifications and equivalents thereof within the spirit and scope of the description provided here.
    权利要求:
    Claims (16)
    [0001]
    1.Use of a compound which is: (i) 5-cholesten-3,25-diol, disulfate (25HCDS) of the formula:
    [0002]
    2.Use, according to claim 1, characterized by the fact that the compound is:
    [0003]
    3.Use, according to claim 1 or 2, characterized by the fact that: -the drug is for administration in an amount ranging from 0.1 mg / kg to 100 mg / kg of the compound based on the body mass of said individual; and -the administration comprises at least one of oral administration, enteral administration, sublingual administration, transdermal administration, intravenous administration, peritoneal administration, parenteral administration, administration by injection, subcutaneous injection and intramuscular injection.
    [0004]
    4. Use according to any one of claims 1 to 3, characterized by the fact that: - the drug is for administration in an amount ranging from 1 mg / kg to 10 mg / kg, based on the body mass of said individual .
    [0005]
    5. Compound, characterized by the fact that it is a pharmaceutically acceptable salt of (i) 5-cholesten-3,25-diol, disulfate (25HCDS) of the formula:
    [0006]
    6. Compound, characterized by the fact that it is (i) 5-colesten-3,25-diol, disulfate (25HCDS) of the formula:
    [0007]
    7. Compound, according to claim 6, characterized by the fact that it is for: reduction of lipids in an individual in need of it; reduction of cholesterol and lipid biosynthesis in an individual in need of it; reduction of inflammation in an individual in need of it; treating diabetes in an individual in need of it; treatment of hyperlipidemia in an individual in need of it; treatment of atherosclerosis in an individual in need of it; treatment of fatty liver disease in an individual in need of it; treatment of inflammatory disease in an individual in need of it; or treating metabolic lipid disorders in an individual in need of it.
    [0008]
    8. Compound, according to claim 6, characterized by the fact that it is: -in powder form; and / or - in lyophilized form.
    [0009]
    9. Pharmaceutical composition for: reduction of lipids in an individual in need of it; reduction of cholesterol and lipid biosynthesis in an individual in need of it; reduction of inflammation in an individual in need of it; treating diabetes in an individual in need of it; treatment of hyperlipidemia in an individual in need of it; treatment of atherosclerosis in an individual in need of it; treatment of fatty liver disease in an individual in need of it; treatment of inflammatory disease in an individual in need of it; or treating metabolic lipid disorders in an individual in need thereof, characterized by the fact that it comprises: (i) a compound as defined in claim 1 or 2; and (ii) physiologically acceptable excipient, diluent or vehicle.
    [0010]
    10. Composition according to claim 9, characterized by the fact that the composition is in solid form.
    [0011]
    11. Composition according to claim 10, characterized by the fact that: -the composition is in the form of a powder, a tablet, a capsule or a tablet; or - the composition comprises the compound in lyophilized form together with a bulking agent, the composition optionally being in a sealed vial, ampoule, syringe or pouch.
    [0012]
    12. Composition, according to claim 9, characterized by the fact that it comprises a vehicle that is a liquid.
    [0013]
    13. Composition, according to claim 12, characterized by the fact that: -the compound is solubilized in said liquid or dispersed in said liquid; and - said liquid is sterile water for injection or phosphate buffered saline; and - said composition is in a sealed vial, ampoule, syringe or pouch.
    [0014]
    14. Process of producing a compound that is (i) 5-colesten-3,25-diol, disulfate (25HCDS) of the formula:
    [0015]
    15. Process according to claim 14, characterized by the fact that the compound is:
    [0016]
    16. Process for producing a pharmaceutical composition, as defined in any of claims 9 to 13, characterized in that it comprises combining said compound with said physiologically acceptable excipient, diluent or vehicle.
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    同族专利:
    公开号 | 公开日
    HUE035073T2|2018-05-02|
    ES2641841T3|2017-11-14|
    WO2013154752A1|2013-10-17|
    CA2867694A1|2013-10-17|
    PT2836502T|2017-10-27|
    CN104220450A|2014-12-17|
    JP2015512937A|2015-04-30|
    PL2836502T3|2018-01-31|
    CA2867694C|2021-08-31|
    US20180127457A1|2018-05-10|
    JP2019089777A|2019-06-13|
    US20160355544A1|2016-12-08|
    EP2836502B1|2017-08-02|
    CN104220450B|2016-07-06|
    KR102180485B1|2020-11-18|
    EA026683B1|2017-05-31|
    IL234892A|2019-02-28|
    MX2014012324A|2015-05-11|
    MX357046B|2018-06-22|
    US20200157140A1|2020-05-21|
    US20210238219A1|2021-08-05|
    SI2836502T1|2017-11-30|
    EP2836502A1|2015-02-18|
    AU2017268646A1|2017-12-21|
    EA201491859A1|2015-02-27|
    US20150072962A1|2015-03-12|
    CN106083976B|2018-02-09|
    KR20150013520A|2015-02-05|
    AU2013246435B2|2017-10-05|
    DK2836502T3|2017-10-23|
    AU2017268646B2|2019-07-11|
    IN2014KN02366A|2015-05-01|
    JP6125610B2|2017-05-10|
    EP2836502A4|2015-08-19|
    CN106083976A|2016-11-09|
    BR112014025081A2|2018-05-08|
    ZA201406981B|2016-05-25|
    HK1202294A1|2015-09-25|
    AU2013246435A1|2014-10-16|
    JP2021138713A|2021-09-16|
    EP3239163A1|2017-11-01|
    JP2017160213A|2017-09-14|
    US20190135856A1|2019-05-09|
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    法律状态:
    2018-05-22| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2018-10-30| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |
    2019-11-19| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|
    2020-04-07| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-09-01| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-12-08| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/03/2013, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US201261623203P| true| 2012-04-12|2012-04-12|
    US201261623414P| true| 2012-04-12|2012-04-12|
    US61/623,203|2012-04-12|
    US61/623,414|2012-04-12|
    PCT/US2013/031861|WO2013154752A1|2012-04-12|2013-03-15|A NOVEL CHOLESTEROL METABOLITE, 5-CHOLESTEN, 3β-25-DIOL, DISULFATEFOR THERAPY OF METABOLIC DISORDERS, HYPERLIPIDEMIA, DIABETES, FATTY LIVER DISEASES AND ATHEROSCLEROSIS|
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