US20070275939A1 - Nuclear sulfated oxysterol, potent regulator of cholesterol homeostasis, for therapy of hypercholesterolemia, hyperlipidemia, and atherosclerosis - Google Patents

Nuclear sulfated oxysterol, potent regulator of cholesterol homeostasis, for therapy of hypercholesterolemia, hyperlipidemia, and atherosclerosis Download PDF

Info

Publication number
US20070275939A1
US20070275939A1 US11/739,330 US73933007A US2007275939A1 US 20070275939 A1 US20070275939 A1 US 20070275939A1 US 73933007 A US73933007 A US 73933007A US 2007275939 A1 US2007275939 A1 US 2007275939A1
Authority
US
United States
Prior art keywords
cholesterol
nuclear
oxysterol
cells
25hc3s
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/739,330
Other languages
English (en)
Inventor
Shunlin Ren
William Pandak
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Virginia Commonwealth University
US Department of Veterans Affairs VA
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to US11/739,330 priority Critical patent/US20070275939A1/en
Assigned to VIRGINIA COMMONWEALTH UNIVERSITY reassignment VIRGINIA COMMONWEALTH UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PANDAK, WILLIAM N., REN, SHULIN
Publication of US20070275939A1 publication Critical patent/US20070275939A1/en
Priority to US12/708,803 priority patent/US8399441B2/en
Priority to US13/766,839 priority patent/US9321802B2/en
Priority to US15/042,244 priority patent/US10144759B2/en
Priority to US16/154,007 priority patent/US10844089B2/en
Priority to US17/060,886 priority patent/US11384115B2/en
Assigned to THE UNITED STATES GOVERNMENT AS REPRESENTED BY THE DEPARTMENT OF VETERANS AFFAIRS, VIRGINIA COMMONWEALTH UNIVERSITY reassignment THE UNITED STATES GOVERNMENT AS REPRESENTED BY THE DEPARTMENT OF VETERANS AFFAIRS ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: VIRGINIA COMMONWEALTH UNIVERSITY
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J31/00—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring
    • C07J31/006—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring not covered by C07J31/003
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane

Definitions

  • the invention generally relates to lipid-lowering therapies.
  • the invention provides a nuclear steroid metabolite, 5-cholesten-3 ⁇ , 25-diol 3-sulphate, that increases cholesterol secretion and degradation, and is thus useful for the treatment and prevention of hypercholesterolemia, hyperlipidemia, and atherosclerosis.
  • Cholesterol is used by the body for the manufacture and repair of cell membranes, and the synthesis of steroid hormones and vitamin D, and is transformed to bile acids in the liver.
  • the average American consumes about 450 mg of cholesterol each day and produces an additional 500 to 1,000 mg in the liver and other tissues.
  • Another source is the 500 to 1,000 mg of biliary cholesterol that is secreted into the intestine daily; about 50 percent is reabsorbed (enterohepatic circulation).
  • hypercholesterolemia High serum cholesterol levels (hypercholesterolemia) are associated with the accumulation of cholesterol in arterial walls, and can result in atherosclerosis.
  • the plaques that characterize atherosclerosis inhibit blood flow and promote clot formation, and can ultimately cause death or severe disability via heart attacks and/or stroke.
  • a number of therapeutic agents for the treatment of hypercholesterolemia have been developed and are widely prescribed by physicians. Unfortunately, only about 35% of patients with hypercholesterolemia are responsive to the currently available therapies. Thus, there is an ongoing need to develop agents and methodologies to decrease intracellular and serum cholesterol levels.
  • the present invention provides a novel sulfated oxysterol, 5-cholesten-3 ⁇ , 25-diol 3-sulphate, with potent cholesterol lowering properties.
  • 5-Cholesten-3 ⁇ , 25-diol 3-sulphate is a nuclear sterol metabolite that increases cholesterol secretion and degradation (bile acid synthesis). The increase in cholesterol degradation and decrease in cholesterol synthesis can lead to lower levels of intracellular and serum cholesterols.
  • the sulfated oxysterol is useful for preventing or treating diseases associated with elevated cholesterol, such as hypercholesterolemia, hyperlipidemia, gallstone, cholestatic liver disease, and atherosclerosis.
  • composition comprises 5-cholesten-3 ⁇ , 25-diol 3-sulphate, and a pharmaceutically acceptable carrier.
  • the method comprises the step of administering 5-cholesten-3 ⁇ , 25-diol 3-sulphate to the patient in an amount sufficient to lower serum cholesterol and triglyceride levels in the patient.
  • the invention further provides a method to treat or prevent pathological conditions associated with high serum cholesterol and triglyceride levels in a patient in need thereof.
  • the method comprises the step of administering 5-cholesten-3 ⁇ , 25-diol 3-sulphate to the patient in an amount sufficient to lower serum cholesterol levels in the patient, and to prevent or treat the pathological condition.
  • the pathological condition is, for example, hypercholesterolemia, hyperlipidemia, or atherosclerosis.
  • the invention further provides a method of increasing cholesterol secretion or degradation in cells.
  • the method comprises the step of increasing a level of 5-cholesten-3 ⁇ , 25-diol 3-sulphate in the cells.
  • the method may be carried out by exposing the cells to 5-cholesten-3 ⁇ , 25-diol 3-sulphate.
  • FIG. 1 Subcellular fractionation protocol. For details see “Experimental Procedures”.
  • FIG. 2A -C Effects of overexpression of StarD1 on Cyp7A1 mRNA expression in primary rat hepatocytes.
  • FIG. 3 Thin layer chromatographic analysis of the chloroform extractable cholesterol derivatives. Rat primary hepatocytes were infected with the indicated viruses. Forty-eight hrs later cells were harvested and nuclear lipids extracted and analyzed as explained under “Experimental Procedures”.
  • FIGS. 4A and B Phase distribution of [ 14 C]cholesterol derivatives in nuclei of primary rat hepatocytes following overexpression of StarD1 and CYP7A1. Rat primary hepatocytes were infected with the indicated viruses. Forty-eight hrs later cells were harvested and subcellular fractions prepared. Fractions E and F were processed for lipid analysis as explained under Experimental Procedures”.
  • A Nuclear inner membrane (Fraction E).
  • B Nuclear digests (Fraction F).
  • FIG. 5A -C HPLC analysis of [ 14 C]cholesterol derivatives in the nuclear fraction (Fraction D) and non-nuclear fraction (Fraction A). Twenty-four hrs following the indicated recombinant adenovirus infection, cells were harvested and nuclear and non-nuclear fractions (Fractions A and D) were isolated, extracted by the Folch method and the methanol/water phase analyzed.
  • A nuclear extracts (Fraction D) 195 nm profiles.
  • B nuclear extracts (Fraction D) 14 C profiles.
  • C non-nuclear extracts (Fraction A) 14 C profiles. In each case, nuclear methanol/water extracts of the equivalent of 5 ⁇ 10 6 cells were loaded.
  • FIG. 6A -F HPLC analysis of the cholesterol derivatives extracted from the nuclei, mitochondria, and culture media.
  • Rat primary hepatocytes were infected with the StarD1 adenovirus and two hrs later [ 14 C]cholesterol was added to the media. Twenty-four hrs following, cells and culture media were harvested. Nuclei and mitochondria were isolated as described under “Experimental Procedures”. Total lipids were extracted from the nuclei, mitochondria, and culture media by Folch partitioning into methanol phase, and analyzed by HPLC as described in “Experimental Procedures”.
  • D-F radioactivity profiles.
  • FIG. 7A -D Characterization of the nuclear oxysterol by enzymatic digestion followed by HPLC and TLC.
  • Nuclear [ 14 C]oxysterol derivatives were isolated from StarD1 overexpressing rat primary hepatocytes and digested with 1 mg/ml of sulfatase in acetic acid buffer, pH 5.0, overnight.
  • Total lipids were extracted with chloroform/methanol and separated by Folch partitioning.
  • the products in the chloroform and methanol/water phase were analyzed by HPLC using a mixture of 965 ml hexane, 25 ml isopropanol, and 10 ml acetic acid as mobile phase, 1.3 ml/min flow rate and 14 C quantified.
  • A HPLC elution profile of the sulfatase digestion products.
  • B HPLC elution profile of [ 14 C]27-hydroxycholesterol.
  • C HPLC elution profile of [ 14 C]25-hydroxycholesterol.
  • D products from the chloroform phase were further analyzed by TLC using a mixture of toluene:ethyl acetate (2:3) as developing solvent.
  • 27-C represents 27-hydroxycholesterol;
  • P sulfatase digestion products of the purified nuclear oxysterols; 25-C, 25-hydroxycholesterol.
  • FIG. 8A -C Characterization of nuclear oxysterol by negative ion-triple quadruple mass spectrometry (LC/MS/MS).
  • A A selected ion chromatogram of mass ion at m/z 481;
  • B the Q1 full scan spectrum;
  • C product scan spectrum of a/z 481.
  • the amu represents atomic mass units, and cps, counts per second.
  • FIG. 9A -B Effect of the nuclear oxysterol on cholesterol uptake and bile acid biosynthesis.
  • Rat primary hepatocyes were treated with nuclear extracts (methanol/water phase) (A and B) or purified nuclear oxysterol dissolved in control nuclear extract (C) 24 hrs after plating them. Then [ 14 C]cholesterol was added as described in FIG. 1 .
  • Culture media were then harvested at 0, 6, 12, and 24 hrs and radioactivity quantified (A).
  • Bile acid synthesis rates (B and C) were measured as the conversion of [ 14 C]cholesterol into methanol/water extractable counts as described in (9).
  • FIG. 10 ABCA1 (A1), ABCG1 (G1), LDL receptor (LDLR), ABCG5 (G5), and ABCG8 (G8) gene expression in primary mouse hepatocytes following addition of the purified nuclear oxysterol.
  • A1 A1
  • ABCG1 G1
  • LDL receptor LDL receptor
  • ABCG5 G5
  • ABCG8 G8 gene expression in primary mouse hepatocytes following addition of the purified nuclear oxysterol.
  • ⁇ -Actin mRNA was used as total mRNA internal standard.
  • the gene expression levels in cells with StarD1 overexpression were compared with those in control cells.
  • Ten ⁇ g of total RNA was used for cDNA preparation (RT) and 10 ng of cDNA was used for PCR. The expression levels were normalized to ⁇ -actin.
  • FIG. 11 Biosynthesis pathway of nuclear sulfated oxysterol.
  • cholesterol is delivered into mitochondria where 25-hydroxylase (25-OHLase) and hydroxylcholesterol sulfate transferase 2b (HST2b) locate, and converted to be 25-hydroxycholesterol 3-sulfate.
  • This sulfated oxysterol translocates to nucleus and regulates gene expressions involved in cholesterol metabolism.
  • FIG. 12A -D (A) addition of sulfate group onto 3 ⁇ -position of 25-hydroxycholesterol for the synthesis of the novel nuclear oxysterol by incubation with sulfur trioxide triethyl amine complex; (B) mass spectrophometric analysis of the product after incubation with the sulfur trioxide and purified by HPLC. Mass ion, m/z 481, represents 25-hydroxycholesterol (M.W. 482)+Sulfate group (M.W.80); (C) nuclear magnetic resonance (NMR) analysis of the 25-hydroxycholesterol 3-sulfate as starting material for the synthesis.
  • NMR nuclear magnetic resonance
  • FIG. 13A -C TLC and HPLC analysis of the newly synthesized [ 14 C]cholesterol. After incubation of the 25HC3S-treated cells with [1- 14 C]acetate for 2 hrs, the cells were harvested. The total neutral lipids were extracted with chloroform/methanol and partitioned into chloroform phase. The [ 14 C]-acetate derivatives were analyzed by thin layer chromatography (TLC) and HPLC.
  • TLC analysis of the [ 14 C]-acetate derivatives the chloroform phase extracts of the equivalent of 5 ⁇ 10 6 cells were loaded onto each lane, separated by developing system of tuluene:acetyl acetate, and visualized by Imagine Reader.
  • FIG. 14A -H HPLC analysis of cholesterol levels in microsomal fractions.
  • the total lipids were extracted from 25HC3S-treated (left panels) or 25HC- (right panels) treated HepG2 cells.
  • ⁇ , ⁇ -Unsaturated ketones were generated by incubating the extracted sterols with cholesterol oxidase and were analyzed by normal phase HPLC:
  • Panels A-C the lipids from the cells treated with 0, 3, and 6 ⁇ M of 25HC3S; D.
  • a summary of a series experiments the lipids from the cells treated with 0, 3, 6, 12, and 25 ⁇ M of 25HC3S.
  • Panels E-G the lipids from the cells treated with 0, 3, and 6 ⁇ M of 25HC; D.
  • a summary of a series experiments the lipids from the cells treated with 0, 3, 6, 12, and 25 ⁇ M of 25HC.
  • the data represent a typical result from one of three independent experiments.
  • FIG. 15A -D 25HC3S regulates HMGR mRNA Expression.
  • Real time RT-PCR analysis of HMG CoA R reductase expression in HepG2 cells (Panels A and B). Western blot analysis of protein levels of HMG CoA reductase (Panel C).
  • Total RNAs were purified using SV Total RNA Isolation Kit (Promega) from the cells treated with 25HC3S at concentrations as indicated (A) and incubation at 12 ⁇ M of 25HC3S for the different times (effect on HMG CoA mRNA) (Panel B).
  • Two ⁇ g of total RNA was used for cDNA preparation (RT) and performed as manufacture recommended (Invitrogen), and 10 ng of cDNA was used for real time PCR.
  • the expression levels were normalized to GAPDH.
  • the bands of HMG CoA reductase (HMGR) were qualitated by laser density scanning.
  • the data from three experiments were summarized in Panel D. Each value represents mean of three experiments ⁇ standard derivation.
  • FIG. 16A -C Effects of 25HC and 25HC3S on the levels of HMG CoA reductase mRNA in hepatocytes.
  • Total RNA was extracted from the HepG2 cells cultured either in 10% FBS media (Panel A) or in 10% lipid-depleted sera (Panel B) and treated with either 25HC or 25HC3S as indicated.
  • the real time RT-PCR analysis was performed as described in FIG. 16 .
  • the data represent a typical result from one of three independent experiments.
  • the 25HC or 25HC3S affects the HMG CoA reductase expression in primary human hepatocytes (Panel C).
  • the data represent a typical result from one of three independent experiments.
  • FIG. 17A -E HPLC analysis of 25HC in PHH cells treated with 25HC.
  • the total lipids were extracted from 25HC-treated PHH cells.
  • ⁇ , ⁇ -Unsaturated ketones were generated by incubating the extracted sterols with cholesterol oxidase and were analyzed by normal phase HPLC:
  • Panels A-E the lipids from the cells treated with 0, 3, 6, 12, 25 ⁇ M of 25HC as indicated; The data represent a typical result from two independent experiments.
  • FIG. 18A -D Western blot analysis of SREBPs activation following 25HC3S or 25HC treatment in HepG2 cells.
  • Total proteins were extracted from HepG2 cells treated with 25HC in ethanol (0.1%) (Panel A and C) or 25HC3S in DMSO (0.1%) (Panel B and D), cultured in the media in absence (Panels A and B) or presence (Panels C and D) of mevinolin (50 mM) and mevalonate (0.5 mM).
  • SREBP-1 and SREBP-2 protein levels in the cells were determined by Western blot analysis. The extracted protein (100 ⁇ g) was loaded onto each lane for each condition as indicated. The data represent a typical result from one of three independent experiments. The trends of the protein levels are highly reproducible.
  • FIG. 19 Relative levels of cholesterol in HepG2 microsomal fractions (y axis) vs concentration of 25HC3S (x axis).
  • FIG. 20 Lipids in mouse sera after injection of 25HC3S.
  • the present invention is based on the discovery of 5-cholesten-3 ⁇ , 25-diol 3-sulphate, a novel sulfated oxysterol with potent cholesterol lowering properties.
  • the chemical structure of the sulfated oxysterol is as follows:
  • This sulfated oxysterol is a nuclear sterol metabolite that decreases cholesterol and triglyceride levels in serum.
  • the compound increases cholesterol secretion by increasing expression of cholesterol transporters in hepatocytes. The increase in cholesterol secretion and degradation ultimately leads to lower levels of serum cholesterol.
  • the sulfated oxysterol is made in the mitochondrion and translocates to the nucleus of the cell, where it acts to up-regulate genes involved in cholesterol metabolism.
  • 5-Cholesten-3 ⁇ , 25-diol 3-sulphate is thus useful for preventing or treating diseases associated with elevated cholesterol (hypercholesterolemia), such as hyperlipidemia, atherosclerosis, coronary heart disease, stroke, and cholestatic liver disease.
  • hypercholesterolemia hyperlipidemia
  • atherosclerosis atherosclerosis
  • coronary heart disease coronary heart disease
  • stroke stroke
  • cholestatic liver disease This sulfated oxysterol is especially suitable for in vivo use because it is a biosynthesized authentic compound (hydroxylation and sulfation of cholesterol) in vivo.
  • 5-cholesten-3 ⁇ , 25-diol 3-sulphate should have few or no toxic side effects when administered to patients.
  • the invention provides methods of preparing and administering 5-cholesten-3 ⁇ , 25-diol 3-sulphate.
  • the compound of the invention will be provided in a substantially purified form for use in the methods of the invention.
  • substantially purified we mean that the sulfated oxysterol is provided in a form that is at least about 75%, preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95% or more free from other chemical species, e.g. other macromolecules such as proteins or peptides, nucleic acids, lipids, and other cholesterol-related species (e.g. other cholesterol derivatives such as cholesterol metabolites, chemically modified forms of cholesterol such as various other hydroxylated cholesterol species, etc.).
  • the sulfated oxysterol of the invention may be isolated and purified from living cells.
  • Example 1 One embodiment of this method is described in Example 1 in the Examples section below.
  • the compound may also be synthesized, either by synthetic chemical means, or by methods which involve the use of recombinant DNA technology (e.g. by using cloned enzymes to carry out suitable modifications of cholesterol).
  • An exemplary synthesis scheme for 5-cholesten-3 ⁇ , 25-diol 3-sulphate is as follows: A mixture of 25-hydroxycholesterol (0.1 mmol) and sulfur trioxide triethyl amine complex (0.12 mmol) in dry toluene was heated to 60 degree for 24 hours under nitrogen, then cooled and the solvent was evaporated under reduced pressure. The residue was purified by flash chromatography to afford the product as a white solid.
  • the methods of the invention are useful for the treatment or prevention of conditions associated with high levels of cholesterol and triglyceride (hyperlipidemia). Such conditions may be either caused or exacerbated by high cholesterol and triglyceride, and include but are not limited to hyperlipidemia, atherosclerosis, heart disease, stroke, Alzheimer's, gallstone diseases, cholestatic liver diseases, etc.
  • hyperlipidemia we mean that a disease condition has already developed, and the methods of the invention are used to ameliorate symptoms of the disease condition, either to stop or decrease progression of the disease, or to reverse symptoms of the disease, either partially or fully.
  • prevent we mean that the compounds of the present invention may be administered to patients prophylactically prior to the development of disease symptoms, e.g. to one who has high cholesterol but has not yet developed atherosclerosis, or to one who does not yet have high cholesterol but is at high risk for developing high cholesterol (e.g. as determined by genetic factors, family history, etc.)
  • high cholesterol generally relates to cholesterol levels in serum in the range of about 200 mg/dl or more.
  • a determination of “high cholesterol” is typically made by a health professional such as a physician, and the established meaning of “high cholesterol” may vary somewhat from professional to professional. Further, the precise definition may vary somewhat depending on the state of the art, e.g. on findings from studies which investigate the relationship between cholesterol levels and diseases. Nevertheless, those of skill in the art will be able to identify suitable candidates for administration of the sulfated oxysterol of the present invention.
  • lowering cholesterol levels we mean that the level of free serum cholesterol in a patient is decreased by at least about 10% to 30%, and preferably at least about 30 to 50%, and more preferably at least about 50 to 70%, and most preferably at least about 70 to about 100%, or more, in comparison to the level of cholesterol in the patient prior to administration of the sulfated oxysterol.
  • the extent of the decrease may be determined by comparison to a similar untreated control individual to whom the compound is not administered.
  • Those of skill in the art are familiar with such determinations, e.g. the use of controls, or the measurement of cholesterol levels in the blood before and after administration of an agent that lowers cholesterol.
  • Implementation of the methods of the invention will generally involve identifying patients suffering from or at risk for developing conditions associated with high cholesterol, and administering the compound of the present invention in an acceptable form by an appropriate route.
  • the exact dosage to be administered may vary depending on the age, gender, weight and overall health status of the individual patient, as well as the precise etiology of the disease. However, in general for administration in mammals (e.g. humans), dosages in the range of from about 0.1 to about 100 ⁇ g or more of compound per kg of body weight per 24 hr., and preferably about 0.1 to about 50 ⁇ g of compound per kg of body weight per 24 hr., and more preferably about 0.1 to about 10 ⁇ g of compound per kg of body weight per 24 hr. are effective.
  • Administration may be oral or parenteral, including intravenously, intramuscularly, subcutaneously, intradermal injection, intraperitoneal injection, etc., or by other routes (e.g. transdermal, sublingual, oral, rectal and buccal delivery, inhalation of an aerosol, etc.).
  • administration is oral.
  • administration of the compound may be carried out as a single mode of therapy, or in conjunction with other therapies, e.g. other cholesterol lowering drugs, exercise and diet regimens, etc.
  • the compounds may be administered in the pure form or in a pharmaceutically acceptable formulation including suitable elixirs, binders, and the like (generally referred to a “carriers”) or as pharmaceutically acceptable salts (e.g. alkali metal salts such as sodium, potassium, calcium or lithium salts, ammonium, etc.) or other complexes.
  • suitable elixirs, binders, and the like generally referred to a “carriers”
  • pharmaceutically acceptable salts e.g. alkali metal salts such as sodium, potassium, calcium or lithium salts, ammonium, etc.
  • the pharmaceutically acceptable formulations include liquid and solid materials conventionally utilized to prepare both injectable dosage forms and solid dosage forms such as tablets and capsules and aerosolized dosage forms.
  • the compounds may be formulated with aqueous or oil based vehicles. Water may be used as the carrier for the preparation of compositions (e.g.
  • injectable compositions which may also include conventional buffers and agents to render the composition isotonic.
  • Other potential additives and other materials include: colorants; 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 benzalkium chloride may also be used.
  • the active composition will consist of about 1% to about 99% of the composition and the vehicular “carrier” will constitute about 1% to about 99% of the composition.
  • the pharmaceutical compositions of the present invention may include any suitable pharmaceutically acceptable additives or adjuncts to the extent that they do not hinder or interfere with the therapeutic effect of the sulfated oxysterol.
  • the administration of the compound of the present invention may be intermittent, or at a gradual or continuous, constant or controlled rate to a patient.
  • the time of day and the number of times per day that the pharmaceutical formulation is administered may vary and are best determined by a skilled practitioner such as a physician.
  • the effective dose can vary depending upon factors such as the mode of delivery, gender, age, and other conditions of the patient, as well as the extent or progression of the disease condition being treated.
  • the compounds may be provided alone, or in combination with other medications or treatment modalities.
  • the compound of the invention may also be used for research purposes.
  • Sterol ligands play key roles in maintenance of the cholesterol homeostasis.
  • the present study has identified a novel regulatory nuclear sulfated oxysterol, which is generated in mitochondria, translocates to the nucleus, and upregulates the rate of bile acid synthesis.
  • a mitochondria cholesterol transport protein (StarD1)
  • StarD1 mitochondria cholesterol transport protein
  • bile acid synthesis increased by 5-fold.
  • [ 14 C]oxysterol derivatives with retention time at 11.50 min in HPLC elution profile was dramatically increased both in the mitochondria and in the nucleus, but not in culture media.
  • the oxysterol product could be extracted into the chloroform phase from the methanol/water phase after sulfatase treatment, and had the same physical properties as 25-hydroxycholesterol.
  • LC/MS/MS analysis showed the nuclear oxysterol with a molecular ion, m/z 481, in the Q1 full scan spectrum, and the presence of fragment ions at m/z 59, 80, 97, and 123 in its product scan spectrum.
  • the nuclear oxysterol derivative can be characterized as 5-cholesten-3 ⁇ , 25-diol 3-sulphate.
  • the biotransformation of cholesterol to primary bile acids occurs via two main pathways in hepatocytes (1).
  • the “neutral” pathway is considered to be the major pathway at least in humans and rats (2).
  • the sterol nucleus is modified before the side-chain, beginning with hydroxylation of cholesterol at the 7 ⁇ position.
  • This reaction is catalyzed by cholesterol 7 ⁇ -hydroxylase (CYP7A1), the first and rate-limiting step of this pathway.
  • CYP7A1 cholesterol 7 ⁇ -hydroxylase
  • the ability to lower plasma cholesterol levels via the pharmacological control of CYP7A1 expression represents a therapeutic approach that has been in use for the last 30 years and is still of intense research interest.
  • CYP7A1 Multiple negative and positive modulators of CYP7A1 transcription have been identified both in tissue culture systems and in vivo (3) and many of these modulators are oxysterols, such as hydroxy-cholesterol molecules and bile acids. They function by activating nuclear receptors, such as liver X receptor (LXR) and farnesoid X receptor (FXR), which in turn regulate the expression of regulatory genes involved in bile acid biosynthesis, such as CYP7A1 and sterol 12 ⁇ -hydroxylase (CYP8B1), the enzyme specific for cholic acid synthesis.
  • LXR liver X receptor
  • FXR farnesoid X receptor
  • Oxysterols are also key regulatory molecules for the expression of many other genes involved in the homeostasis of cholesterol, and other lipids, such as 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, low density lipoprotein (LDL) receptor, some ATP-binding cassette transporters, like the ABCA1 and ABCG8, and many others. They function by modulating the activity of either nuclear receptors or other transcriptional factors, such as the sterol regulatory binding proteins (SREBPs) (4-6). Thus, characterizing endogenous synthesized oxysterols and their mechanism of action is critical for a better understanding of lipid homeostasis.
  • HMG-CoA 3-hydroxy-3-methyl-glutaryl coenzyme A reductase
  • LDL low density lipoprotein
  • SREBPs sterol regulatory binding proteins
  • the initial step in the “acidic” pathway is catalyzed by the enzyme mitochondrial sterol 27-hydroxylase (CYP27A1).
  • CYP27A1 mitochondrial sterol 27-hydroxylase
  • the oxysterol intermediates of the “acidic” pathway such as 25-, or 27-hydroxycholesterol have been shown in vitro to be potent regulators in cholesterol homeostasis (7).
  • Increased CYP27A1 activity in peripheral tissues may both down-regulate cholesterol synthesis through the SREBP pathway, and enhance the efflux of cholesterol and its elimination via LXR (8).
  • the physiological and authentic in vivo LXR ligand is unknown (9).
  • StarD1 steroidogenic acute regulatory protein
  • the adenovirus construct used in this study was obtained through the Massey Cancer Center Shared Resource Facility of the Virginia Commonwealth University as previously described (14).
  • rat hepatocyte cultures prepared as previously described (16), were plated on 150 mm tissue culture dishes ( ⁇ 2.5 ⁇ 10 7 cells) in Williams' E medium containing dexamethasone (0.1 ⁇ M). Cells were maintained in the absence of thyroid hormone. Twenty-four hrs after plating, culture medium was removed, and 2.5 ml of fresh medium was added. Cells were then infected with recombinant adenovirus encoding either the StarD1 (Ad-CMV-StarD1) or the CYP7A1 (Ad-CMV-CYP7A1) cDNAs in front of the human cytomegalovirus promoter (CMV) or no cDNA, as a control virus.
  • CMV human cytomegalovirus promoter
  • the viruses were allowed to incubate for at least 2 hrs in minimal culture medium with gentle shaking of the plates every 15 min. After 2 hrs of infection, unbound virus was removed, replaced with 20 ml of fresh medium, and 2.5 ⁇ Ci of [ 14 C]cholesterol was added. After 48 hrs, cells were then harvested and processed for nuclei isolation as described (17) with minor modification ( FIG. 1 ). Briefly, cells were disrupted by Dounce homogenization in buffer A (10 mM HEPES-KOH at pH 7.6, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM dithiothreitol, 1 mM sodium EDTA, 1 mM EGTA) and spun at 1,000 ⁇ g for 10 min.
  • buffer A (10 mM HEPES-KOH at pH 7.6, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM dithiothreitol, 1 mM sodium EDTA, 1 mM
  • the nuclear pellet was further fractionated by resuspension in 2.5 ml of a 1:1 mixture of buffer A and buffer B (2.4 M Sucrose, 15 mM KCl, 2 mM sodium EDTA, 0.15 mM spermine, 0.15 mM spermidine, 0.5 mM dithiothreitol) and centrifuged at 100,000 ⁇ g for 1 hr at 4° C. through a 1 ml cushion of 3:7 mixture of buffer A and B.
  • the washed nuclear pellet was resuspended in buffer A containing 0.5% (v/v) Nonidet P-40 and centrifuged at 1000 ⁇ g for 10 min at 4° C.
  • the supernatant is designated as nuclear attached membrane (fraction C) and the pellets as purified nuclei (fraction D).
  • Fraction D The purified nuclei (Fraction D) were resuspended and digested by 2 mg/ml of DNase I in 50 mM of acetic buffer, pH 5.0, 10 mM MgCl 2 at 37° C. for 2 hrs. After centrifugation at 10,000 ⁇ g for 20 min, the supernatant was designed as inner nuclear membrane (fraction E). The pellets were further digested by 2 mg/ml of proteinase K in phosphate buffered saline solution (PBS) at 50° C. for 16 hrs and the solution was designed as nuclear protease digests (fraction F).
  • PBS phosphate buffered saline solution
  • the [ 14 C]cholesterol/cholesterol derivatives in chloroform and methanol/water phases were examined by thin layer chromatograph (TLC) (E. Merck, Darmstadt, Germany) using different developing solvent systems: toluene:ethyl acetate (2:3, v/v) for the [ 14 C]cholesterol/oxysterols in chloroform phase, and ethyl acetate:cyclohexane:acetic acid (92:28:12; v/v/v) for those in methanol/water phases.
  • TLC thin layer chromatograph
  • the [ 14 C]cholesterol/cholesterol derivatives were visualized in Phosphorimager using Fuji imaging plates (Fujifilm BAS-1800II, Fuji Photo Film Co., LTD, Japan).
  • the purified nuclear [ 14 C]cholesterol derivatives were digested with 2 mg/ml of sulfatase (EC 3.1.6.1) (Sigma, St Louis, Mo.) in 50 mM of acetic buffer, pH 5.0 by incubation at 37° C. for 4 hrs.
  • the products were extracted into chloroform phase from methanol/water phase by adding 3.3 volume of methanol:chloroform (1:1, v/v) to reaction solution.
  • [ 14 C]Cholesterol derivatives in both chloroform and methanol/water phases were then analyzed by TLC and HPLC as stated above.
  • Reverse phase liquid chromatographic separation was performed on an HP series 1100 system (Agilent Technologies, Palo Alto, Calif.) and CTC HTS-PAL autosampler (Leap Technologies, Carrboro, N.C.). Separation was carried on a ThermoKeystone Aquasil C18 column (5 ⁇ m, 2.1 mm ⁇ 100 mm).
  • the mobile phase consisted of (A), 0.1% formic acid in water, and (B), 0.1% formic acid in acetonitrile.
  • the 20 min gradient was as follows: 0-10.0 min, 10%-95% B linear; 10.0-15.0 min, 95% B; 15.0-15.1 min, 95%-10% B linear; 15.1-20.0 min, 10% B.
  • the mass detector was an API 4000 (MDS Sciex, Toronto, Canada)
  • the elution stream (0.3 ml/min) from the HPLC apparatus was introduced into a MDS Sciex API 4000 Triple Quadrapole Mass Spectrometer with a Turbo IonSpray ionization (ESI) source for the analyses.
  • the mass spectrometer was operated in negative ion modes and data were acquired using both full scan mode as well as the product ion mode for MS/MS.
  • the sample was reconstituted into methanol:water (20:80, v/v).
  • the solution containing the fraction of 11.50 min peak was infused into the LC/MS/MS system to optimize ESI-MS-MS parameters.
  • the optimized parameters for Q1 full scan under the negative mode were: CUR: 10; GS1: 40; GS2: 40; TEM:400; IS: ⁇ 4500; DP: ⁇ 150; EP: ⁇ 10.
  • the optimized parameters for the product scan of 481 under the negative mode were: CUR:10; GS1: 40; GS2: 40; TEM: 400; IS: ⁇ 4500; CAD: 5; DP: ⁇ 150; EP: ⁇ 10; CE: 50; CXP: ⁇ 15.
  • Bile acid synthetic rates were determined by the addition of 2.5 ⁇ Ci of [ 14 C]cholesterol to each P150 mm plate of confluent primary rat hepatocyte cultures ( ⁇ 2.5 ⁇ 10 7 cells) 24 hrs after plating. Media and cells were harvested 48 hrs after viral infection. Conversion of [ 14 C]cholesterol into [ 14 C]-methanol-water soluble products was determined by scintillation counting after extraction with chloroform-methanol (2:1, vol/vol) of cells and of culture media. Rates of bile acid biosynthesis following recombinant adenovirus infection were calculated as the ratio of [ 14 C]-methanol-water soluble counts to the sum of chloroform plus methanol-water counts. Individual bile acids were identified by HPLC analysis as described above.
  • Time points for conversion of [ 14 C]-cholesterol to [ 14 C]-bile acids were carried out using P150 mm tissue culture dishes Aliquots (1/100) of media were collected in duplicate in a microfuge tube and kept frozen until analysis. A mini Folch extraction was carried out by adding 3 volume of methanol:chloroform (1:1) to the culture medium. The tubes were vigorously vortexed and centrifuged at 16,000 g for 6 min. The phases were collected separately and counted.
  • a Novel Nuclear Oxysterol is Generated in Mitochondria and Translocated to the Nucleus in Primary Rat Hepatocytes upon StarD1 Overexpression.
  • the 195 nm profiles were relatively similar in the nuclear fraction (Fraction D) from cells infected either with the StarD1, CYP7A 1, or null recombinant viruses, except for an extra peak with retention time of 11.51 min, which was located between glycocholic acid (10.75 min) and taurochenodeoxycholic acid (12.50 min) ( FIG. 5A ).
  • This 11.50 min peak was the only fraction that contained 14 C-labelled cholesterol derivatives and was detectable only in StarD1 overexpressing cells ( FIG. 5B ).
  • the non-nuclear fraction (Fraction A) from the StarD1 overexpressing cells contained two major [ 14 C]cholesterol derivative peaks with retention times at 5 and 11.50 min. The 11.50 min peak was not detected in cells infected with either the control or the CYP7A1 viruses ( FIG. 5C ).
  • the nuclear oxysterol products were extracted into the chloroform phase from the methanol/water phase and show the same retention time as 25-hydroxycholesterol, but not 27-, no 24-hydroxycholesterol in our HPLC system ( FIG. 7A -C), and the same relative mobility as 25-hydroxycholesterol on the TLC plate ( FIG. 7D ), suggesting that the nuclear oxysterol derivative is a sulfated 25-hydroxycholesterol.
  • the purified nuclear sulfated oxysterol was further analyzed by LC/MS/MS Mass Spectrometry.
  • LC/MS Q1 full scan mode, 350 to 550 atomic mass units, amu
  • the Q1 full scan spectrum showed the peak at 10.75 min mainly contained molecule ions, m/z 480.1 and 481.5 ( FIG. 8B ).
  • m/z 480.1 did not contain a sulfate group on hydroxyl group (m/z 97) (data not shown). However, the molecule ion, m/z 481.5, corresponds to sulfate group 97 and cholesterol (MW 386).
  • the nuclear oxysterol derivative can be characterized as 5-cholesten-3 ⁇ , 25-diol 3-sulphate (25-hydroxycholesterol 3-sulfate) as shown in ( FIG. 8C ).
  • mitochondrial cholesterol transport proteins such as StarD1
  • StarD1 proteins deliver cholesterol into mitochondria where it is metabolized to 25-OH cholesterol 3-sulfate (the nuclear sulfated oxysterol).
  • the generated nuclear oxysterol derivative is then translocated into the nucleus probably by binding and activating the nuclear oxysterol receptor(s).
  • the nuclear sulfated oxysterol-nuclear receptor(s) complex may enter into the nuclei and regulate gene expression involved in cholesterol metabolism.
  • FIG. 11 A possible mechanism is proposed in FIG. 11 .
  • 25-Hydroxycholesterol 3-sulfate may be a Potential Authentic Ligand of Nuclear Sterol Receptor(s).
  • 25-hydroxycholesterol 3-sulfate is a water-soluble compound. It is thus reasonable to propose that 25-hydroxycholesterol 3-sulfate serves as a potent nuclear sterol regulators in vivo.
  • 25-Hydroxylases 25-OHLase
  • Hydroxycholesterol Sulfotransferase 2 HS12
  • StarD1 delivers cholesterol into the mitochondria and generates a novel nuclear sulfated oxysterol, 25-hydroxycholesterol 3-sulphate. This data suggests that a new pathway of cholesterol metabolism is responsible for generating this nuclear oxysterol.
  • Our present report shows the presence of 25-hydroxycholesterol 3-sulfate in both the mitochondria and the nucleus but not in culture media, suggesting that 25-hydroxycholesterol 3-sulfate is generated in mitochondria and translocates exclusively to the nucleus. To biosynthesize this potent nuclear sterol regulator, two reactions should be involved: 25-hydroxylation and 3 ⁇ -sulfation of cholesterol.
  • 25-hydroxycholesterol 3-sulfate may be glucuronidated for further catabolism and secretion via the bile as 24-hydroxycholesterol 3-sulfate (26).
  • 24-hydroxycholesterol 3-sulfate 26
  • the [ 14 C]-cholesterol derivative with a retention time of 5 min is the glucuronidation product of 25-hydroxycholesterol 3-sulfate ( FIGS. 5 and 6 ).
  • Steroid sulfate conjugates may play an important role in the maintenance of cholesterol homeostasis.
  • An interesting development in recent years has been the realization that steroid sulfoconjugates play important roles in well-characterized biological effects, such as serving as potent neuroexcitatory agents, which are distinct from the well-known role of unconjugated steroids as ligands for nuclear receptors to regulate gene expression (27).
  • Several sulfated sterols have been reported to be widely distributed in steroidogenesis tissues (26) and to circulate in plasma at concentrations ranging from 328-924 ⁇ g/100 ml, with a blood production rate of 35-163 mg/day (28).
  • 24-hydroxycholesterol 3-sulfate 24-glucuronide (based on MS/MS analysis) was reported to be in the serum and urine of children with severe cholestatic liver diseases (26).
  • the 3-sulfate of 24-hydroxycholesterol is the major hydroxycholesterol sulfate found in meconium and infant feces (29;30), and is most likely excreted via bile. Bile excretion of this oxysterol would be impaired in cholestasis, leading to its increased concentration of 24-hydroxycholesterol 3-sulfate in hepatocytes and conceivably leading to its glucuronidation.
  • Sulfated sterols play an important, but unclear, role in the normal development and physiology of skin, where an epidermal sterol sulfate cycle has been described (24).
  • the present results show that the nuclear extract containing the sulfated oxysterol and the purified nuclear sulfated oxysterol dramatically increased the rates of bile acid synthesis, strongly suggesting that the nuclear sulfated oxysterol may play an important role in cholesterol metabolism.
  • LXR ⁇ -deficient mice LXR ⁇ / ⁇ dysregulate the CYP7A1 gene and several other important lipid-associated genes (3). Studies utilizing these animals confirmed the essential function of LXR ⁇ as a major sensor of dietary cholesterol and an activator of the bile acid synthetic pathway in mice.
  • the present report with evidence showing that a potent regulatory nuclear sulfated oxysterol that is generated in the mitochondria translocates into the nucleus, provides a new clue regarding the role of oxysterol(s) in the regulation of intracellular cholesterol homeostasis. It is reasonable to hypothesize that the regulatory nuclear sulfated oxysterol generated in mitochondria translocates into nucleus, activates nuclear oxysterol receptor(s), and up-regulates bile acid synthesis.
  • the nuclear sulfated oxysterol serves as a ligand of nuclear sterol receptor(s).
  • FIG. 12A shows a schematic illustration of the synthesis of the novel sulfated oxysterol of the invention by addition of a sulfate group to the 3 ⁇ -position of 25-hydroxycholesterol. Synthesis was carried out as follows: A mixture of 25-hydroxycholesterol (0.1 mmol) and sulfur trioxide triethyl amine complex (0.12 mmol) in dry toluene was heated to 60 degree for 24 hours under nitrogen, then cooled and the solvent was evaporated under reduced pressure. The residue was purified by flash chromatography to afford the product as a white solid using the method of described above.
  • FIG. 12B shows the mass spectrophotometric analysis of the HPLC purified product, which suggests that the sulfate group has been successfully added to 25-hydroxycholesterol.
  • FIGS. 12C and 12D show NMR data for the starting material, 25-hydroxycholesterol, and product, respectively. As can be seen, the resonance of C3 in the molecule has been shifted from 3.35 ppm in the original compound to 4.12 ppm in the product, suggesting that the desired product, 3 ⁇ -sulfated 25-hydroxycholesterol, has been formed.
  • a Nuclear Oxysterol, 25HC3S, Decreases Cholesterol Synthesis Via Inhibition of SREBP-1 Activation in Hepatocytes
  • 25HC3S a novel oxysterol, 5-cholesten-3 ⁇ , 25-diol 3-sulfate
  • This oxysterol was also detected in human liver nuclei.
  • 25HC3S was chemically synthesized. Addition of varying concentrations of 25HC3S to HepG2 cells markedly inhibited cholesterol biosynthesis and significantly decreased microsomal cholesterol.
  • Real time RT-PCR and Western blot analysis shows that 25HC3S strongly decreased HMG CoA reductase mRNA levels in HepG2 cells and primary human hepatocytes.
  • 25HC 25-hydroxycholesterol
  • 25HC3S inhibited the activation of steroid response element binding protein (SREBP-1) in absence or presence of mevinolin and mevalonate, indicating that cholesterol biosynthesis inhibition occurred through blocking SREBP-1 activation, and subsequently the expression of HMG CoA reductase in the human hepatocytes.
  • SREBP-1 steroid response element binding protein
  • the “acidic” pathway of bile acid biosynthesis is initiated by the mitochondrial enzyme sterol 27-hydroxylase (CYP27A1).
  • Oxysterol intermediates of the “acidic” pathway such as 27-hydroxycholesterol (27HC) and 25-hydroxycholesterol (25HC) have been shown to be regulators of cholesterol homeostasis (1;2).
  • These oxysterols represent regulatory molecules for the expression of many other genes encoding enzymes involved in cholesterol biosynthesis and transport (3-5).
  • increased CYP27A1 activity in peripheral tissues could both down-regulate cholesterol synthesis through generating regulatory oxysterols and the steroid response element binding proteins (SREBPs) pathway, and enhance the cellular efflux of cholesterol, i.e. its elimination, via liver oxysterol receptor, LXR (6).
  • SREBPs steroid response element binding proteins
  • a novel oxysterol, 5-cholesten-3 ⁇ , 25-diol 3-sulfate 25HC3S was found and characterized in mitochondria and nuclei of primary hepatocytes following overexpression of StarD1.
  • This oxysterol was also present in human liver nuclei (10). The results suggested that the oxysterol is synthesized in the mitochondria and translocated to the nuclei. Oxysteorls in nuclei should be able to play important roles in maintenance of intracellular cholesterol homeostasis. However, the function of this nuclear oxysterol, 25HC3S, remains unknown.
  • the synthesized compound was analyzed by a MDS Sciex ABI 4000 Triple Quadrapole Mass Spectrometer (MDS Sciex, Toronto, Canada) with a Turbo IonSpray ionization (ESI) source and the mass spectrometer was operated in negative ion modes and data were acquired using full scan mode as previously described (10).
  • MDS Sciex ABI 4000 Triple Quadrapole Mass Spectrometer MDS Sciex, Toronto, Canada
  • ESI Turbo IonSpray ionization
  • HepG2 cells were grown in MEM containing non-essential amino acids, 0.03M NaHCO 3 , 10% FBS, 1 mM L-glutamine, 1 mM sodium pyruvate and 1% Pen/Strep and incubated at 37° C. in 5% CO 2 . When cells reached at ⁇ 90% confluency, the oxysterols in DMSO or in ethanol (final concentration, 0.1%) and/or [1- 14 C]acetate for cholesterol synthesis was added or otherwise as indicated. Microsomal and cytosol fractions were isolated from broken cells as described previously (10).
  • hepatocytes Primary human hepatocytes were purchased from an NIH-approved facility (Liver Tissue Procurement Distribution System, Univ. of Minnesota). Cells were obtained from a random sampling of males and females 18-69 yr of age. Experiments were performed as cells became available to corroborate findings in experiments conducted in HepG2 cells as previous described (13).
  • [1- 14 C]Acetate derivatives in the chloroform phase were analyzed by HPLC on an Ultrasphere Silica column (5 ⁇ 4.6 mm ⁇ 25 cm; Backman, USA) using HP Series 1100 solvent delivery system (Hewlett Packard) at 1.3 ml/min flow rate.
  • the column was equilibrated and run in a solvent system of hexane:isopropanol:glacial acetic acid (965:25:10, v/v/v), as the mobile phase.
  • the effluents were collected every 0.5 min (0.65 ml per fraction) except as indicated.
  • the counts in [ 14 C]acetate derivatives were determined by Scintillation Counting.
  • the column was calibrated with [ 14 C]cholesterol, [ 3 H]25HC, and [ 14 C]27-hydroxycholesterol.
  • Real-time PCR was performed using SYBR Green on ABI 7500 Fast Real-Time PCR System (Applied Biosystems). The final reaction mixture contained 5 ng of cDNA, 100 nM of each primer, 10 ⁇ l of 2 ⁇ SYBR® Green PCR Master Mix (Applied Biosystems), and RNase-free water to complete the reaction mixture volume to 20 ⁇ l. All reactions were performed in triplicate. The PCR was carried out for 40 cycles at 95° C. for 15 s and 60° C. for 1 min.
  • the fluorescence was read during the reaction, allowing a continuous monitoring of the amount of PCR product.
  • the data was normalized to internal control- ⁇ -actin or GAPDH mRNA.
  • the sequences of primers for HMG CoA reductase used in real-time PCR are ACCTTTCCAGAGCAAGCACATT (SEQ ID NO: 1) (Forward) and AGGACCTAAAATTGCCATTCCA (SEQ ID NO: 2) (Reverse).
  • Microsomal proteins (80 ⁇ g) were separated on a 7.5% SDS-polyacrylamide denaturing gel according to the method of Laemmli (14). Following SDS-PAGE (Hoefer Vertical Slab Gel Unit; Heofer, San Francisco, Calif.), proteins were electrophoretically transferred overnight (4° C.) to Immobilon-P membranes using a Hoefer Trans Blot Electrophoretic Transfer cell. The membranes were then blocked for 90 minutes (25° C.) in blocking buffer (PBS, pH 7.4, 0.1% Tween, 5% non-fat dry milk).
  • PBS pH 7.4, 0.1% Tween, 5% non-fat dry milk
  • Proteins were then incubated for 90 minutes (25° C.) or overnight (4° C.) with a rabbit polyclonal IgG (1:2500-1:20,000) against human SREBP-1, SREBP-2, or HMG CoA reductase. After washing, a secondary antibody (goat anti-rabbit IgG-Horse-radish peroxidase conjugate, 1:2500) was added to the blocking solution (25° C., 90 minutes). Protein bands were detected using the Amersham ECL plus Kit.
  • FIG. 13 summarizes the effects of 25HC3S on cholesterol biosynthesis.
  • FIG. 15A To investigate how 25HC3S inhibits cholesterol biosynthesis, total mRNA were isolated from HepG2 cells following incubation in 10% FBS fresh media containing different concentrations of 25HC3S ( FIG. 15A ).
  • the mRNA levels of HMG CoA reductase were determined by real time RT-PCR.
  • FIG. 15A there was concentration dependent decreases in HMG CoA reductase mRNA following the addition of 25HC3S to the cells in culture.
  • the addition of 25HC3S to HepG2 cells also lead to a marked decrease in the levels of HMG CoA reductase protein ( FIGS. 15C and D).
  • Western blot analysis shows that 50% of its protein level was decreased in a concentration dependent following the addition of 25HC3S to culture media ( FIGS. 15C and D).
  • mRNA levels of HMG CoA reductase in 25HC or 25HC3S-treated HepG2 cells were analyzed by real time RT-PCR. Both of the compounds can inhibit HMG CoA reductase in a similar fashion as shown in FIG. 16A .
  • 25HC3S inhibits HMG CoA reductase expression via lipids uptake in the culture media
  • HepG2 cells were incubated in media containing lipid-depleted serum for 2 hrs, which increases expression of HMG CoA reductase by four-fold as previously reported (15). Cells were incubated for another 6 hrs following the addition of 25HC3S.
  • 25HC did not significantly affect the levels of HMG CoA reductase mRNA ( ⁇ 15% at 25 mM).
  • 25HC3S inhibited HMG CoA reductase mRNA at a similar level ( ⁇ 75%) as that in HepG2 cells ( FIG. 16C ).
  • HPLC analysis showed that the increasing levels of 25HC in the cells are concentration dependent ( FIG. 17A -E), indicating that 25HC can still enter to the cells under this culture condition as in FBS containing media.
  • HMG CoA reductase gene expression is regulated by SREBP-1 and SREBP-2 (18).
  • SREBPs When SREBPs are activated, the cholesterol biosynthesis will increase (19).
  • SREBP regulatory system is involved in the inhibition of HMG CoA reductase mRNA levels and inhibition of cholesterol biosynthesis by 25HC3S, total cellular protein was extracted from HepG2 cells treated with 25HC3S or 25HC at different concentration ( FIG. 18 ).
  • the precursor and mature forms of SREBPs were determined by Western blot analysis. As expected, the decreases of the mature forms of SREBP-1 and the increases of the precursor form of SREBP-1 following addition of 25HC3S and 25HC were dose dependent ( FIGS. 18A and 7B ).
  • SREBP-2 only slightly decreased ( FIGS. 18A and 7B ). It was observed that the activation of SREBP-1 was much more sensitive to the treatment with 25HC and 25HC3S than that of SREBP-2.
  • the inhibition of SREBP-1 maturation fits the decrease in HMG CoA reductase mRNA, at 3 ⁇ M of 25HC or 12 ⁇ M of 25HC3S, 85% of the mRNA of HMG CoA reductase and SREBP1 was inhibited.
  • HepG2 cells were incubated in media containing 50 ⁇ M of mevinolin and 0.5 ⁇ M of mevalonate. Under this condition, SREBPs and HMG CoA expressions are upregulated.
  • 25HC3S shows more potent inhibition on SREBP-1 activation than 25HC.
  • the nuclear oxysterol, 25HC3S is most likely to inhibit the activation of SREBP-1 and subsequently inhibits the expression HMG CoA reductase.
  • 25HC3S inhibits the cholesterol biosynthesis and the expression of the key enzyme, HMG CoA reductase.
  • 25HC3S inhibits this expression in primary human hepatocytes as well as HepG2.
  • 25HC3S was found in the human liver nuclei and its levels were dramatically increased following overexpression of mitochondrial cholesterol delivery protein, StarD1, in primary rat hepatocytes, indicating that 25HC3S is most likely synthesized in the mitochondria and translocated to the nuclei for the regulation of gene expression involved in cholesterol homeostasis (10).
  • the oxysterol, 25HC3S plays an important role in maintenance of the intracellular cholesterol homeostasis, and suggested that the StarD1 protein may serve as a sensor of intracellular cholesterol levels.
  • StarD1 protein may deliver cholesterol to mitochondria where cholesterol is converted to be potent regulatory oxysterols such as 25HC, 27HC, and 25HC3S. Those oxysterols play important roles in maintenance of cholesterol homeostasis.
  • 25HC can be sulfated by hydroxysteroid sulfotransferase 2B1b (SULT2B1b) to be sulfated 25HC or sulfated 25HC can be degraded by sulfatase to be 25HC.
  • hydroxysteroid sulfotransferase 2B1b SUVLT2B1b
  • sulfated 25HC can be degraded by sulfatase to be 25HC.
  • Our HPLC analysis data shows that no 25HC was generated following addition of 25HC3S up to 50 ⁇ M ( FIG. 17 , left panels) suggesting that 25HC3S was not degraded during the culture time.
  • 25HC3S and 25HC inhibit cholesterol synthesis by two different mechanisms, both involving the proteins that control sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors that activate genes encoding enzymes of lipids biosynthesis.
  • SREBPs sterol regulatory element-binding proteins
  • SCAP SREBP cleavage-activating protein
  • NH 2 -terminal domain released by the process can enter the nucleus where it activates transcription of the gene encoding HMG CoA reductase and more than 30 other genes whose products are necessary for lipid synthesis (18).
  • SCAP When 25HC is delivered to cells in ethanol or when cholesterol is delivered in LDL, SCAP becomes trapped in the ER. The bound-SREBP is no longer carried to the Golgi apparatus, and the NH2-terminal domain can not enter the nucleus (20). As a result, transcription of the lipid biosynthetic genes declines. Retention of the SCAP-SREBP complex in the ER is mediated by the sterol-induced binding of SCAP to Insigs (Insig-1 and Insig-2) in the ER membrane (20;21). When mixtures of cholesterol and 25HC are added to cultured cells, SCAP is induced to bind to Insig 1 and Insig-2, and thus can not transport SREBPs to the Golgi body (3) and to be activated. J.L.
  • the oxysterol, 25HC3S inhibits the cholesterol biosynthesis through inhibiting the activation of SREBP-1 and SREBP-2, and subsequently inhibit the expression HMG CoA reductase in HepG2 cells and primary human hepatocytes.
  • SREBP-1 and SREBP-2 activation stimulate HMG CoA reductase by 30 folds and 38 folds, respectively (18).
  • SREBP-1 also strongly stimulates fatty acid synthase but not SREBP-2 (18).
  • SREBP-1 and SREBP-2 are directly mediated by 25HC3S to regulate the expression regulation of the key enzyme, HMG CoA.
  • nuclear oxysterol did not increase the activities of alkaline phosphotase, serum glutamate pyruvate transaminase (SGPT), and serum glutamic-oxaloacetic transaminase (SGOT) in sera suggesting that the nuclear oxysterol is non-toxic (data not shown).
  • mice were provided with either a normal or high cholesterol diet for 8 days, and treated with the nuclear oxysterol as described above. Liver tissues were collected for pathohistochemistry studies (Sudan IV staining) and the results showed that administration of the oxysterol significantly decreased triglyceride levels in liver tissues of mice that were fed normally and also of mice that were fed a high cholesterol diet (data not shown).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Steroid Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
US11/739,330 2004-10-25 2007-04-24 Nuclear sulfated oxysterol, potent regulator of cholesterol homeostasis, for therapy of hypercholesterolemia, hyperlipidemia, and atherosclerosis Abandoned US20070275939A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US11/739,330 US20070275939A1 (en) 2004-10-25 2007-04-24 Nuclear sulfated oxysterol, potent regulator of cholesterol homeostasis, for therapy of hypercholesterolemia, hyperlipidemia, and atherosclerosis
US12/708,803 US8399441B2 (en) 2004-10-25 2010-02-19 Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US13/766,839 US9321802B2 (en) 2004-10-25 2013-02-14 Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US15/042,244 US10144759B2 (en) 2004-10-25 2016-02-12 Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US16/154,007 US10844089B2 (en) 2004-10-25 2018-10-08 Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US17/060,886 US11384115B2 (en) 2004-10-25 2020-10-01 Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US62153704P 2004-10-25 2004-10-25
PCT/US2005/033874 WO2006047022A1 (fr) 2004-10-25 2005-09-21 Oxysterol sulfate nucleaire, regulateur homeostatique du cholesterol agissant puissamment dans le traitement de l'hypercholesterolemie, de l'hyperlipidemie et de l'atherosclerose
US11/739,330 US20070275939A1 (en) 2004-10-25 2007-04-24 Nuclear sulfated oxysterol, potent regulator of cholesterol homeostasis, for therapy of hypercholesterolemia, hyperlipidemia, and atherosclerosis

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2005/033874 Continuation-In-Part WO2006047022A1 (fr) 2004-10-25 2005-09-21 Oxysterol sulfate nucleaire, regulateur homeostatique du cholesterol agissant puissamment dans le traitement de l'hypercholesterolemie, de l'hyperlipidemie et de l'atherosclerose

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US12/708,803 Continuation-In-Part US8399441B2 (en) 2004-10-25 2010-02-19 Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US13/766,839 Continuation-In-Part US9321802B2 (en) 2004-10-25 2013-02-14 Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis

Publications (1)

Publication Number Publication Date
US20070275939A1 true US20070275939A1 (en) 2007-11-29

Family

ID=36228107

Family Applications (4)

Application Number Title Priority Date Filing Date
US11/739,330 Abandoned US20070275939A1 (en) 2004-10-25 2007-04-24 Nuclear sulfated oxysterol, potent regulator of cholesterol homeostasis, for therapy of hypercholesterolemia, hyperlipidemia, and atherosclerosis
US15/042,244 Active US10144759B2 (en) 2004-10-25 2016-02-12 Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US16/154,007 Active US10844089B2 (en) 2004-10-25 2018-10-08 Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US17/060,886 Active US11384115B2 (en) 2004-10-25 2020-10-01 Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis

Family Applications After (3)

Application Number Title Priority Date Filing Date
US15/042,244 Active US10144759B2 (en) 2004-10-25 2016-02-12 Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US16/154,007 Active US10844089B2 (en) 2004-10-25 2018-10-08 Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US17/060,886 Active US11384115B2 (en) 2004-10-25 2020-10-01 Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis

Country Status (2)

Country Link
US (4) US20070275939A1 (fr)
WO (1) WO2006047022A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013154752A1 (fr) * 2012-04-12 2013-10-17 Virgina Commonwealth University Nouveau métabolite du cholestérol, 5-cholestène, 3β-25-diol, disulfate (25hcds) pour la thérapie de troubles métaboliques, de l'hyperlipidémie, du diabète, des stéatoses hépatiques et de l'athérosclérose
WO2016058000A1 (fr) 2014-10-10 2016-04-14 Virginia Commonwealth University Sulfates de cholestérol oxygénés pour le traitement de troubles causés par une diminution de l'activité de la leptine ou un trouble de stockage des lipides, ou les deux
US9321802B2 (en) 2004-10-25 2016-04-26 Virginia Commonwealth University Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US9480692B2 (en) 2011-04-06 2016-11-01 Virginia Commonwealth University Sulfated-oxysterol and oxysterol sulfation by hydroxysterol sulfotransferase promote lipid homeostasis and liver proliferation
WO2018026781A1 (fr) * 2016-08-02 2018-02-08 Virginia Commonwealth University Compositions comprenant du 5-cholestène-3, 25-diol,3-sulfate (25 h3s) ou un sel pharmaceutiquement acceptable de celui-ci et au moins un oligosaccharide cyclique
US10144759B2 (en) 2004-10-25 2018-12-04 Virginia Commonwealth University Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US10272097B2 (en) 2013-12-24 2019-04-30 Virginia Commonwealth University Uses of oxygenated cholesterol sulfates (OCS)
WO2021067297A1 (fr) * 2019-09-30 2021-04-08 Durect Corporation Traitement de l'hépatite alcoolique

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018026837A1 (fr) * 2016-08-02 2018-02-08 Durect Corporation Compositions comprenant du sulfate de cholestérol oxygéné et au moins du polyalkylène glycol, de la carboxyméthylcellulose ou du polyoxylglycéride
US11612619B2 (en) 2018-11-01 2023-03-28 National Institute Of Health (Nih), U.S. Dept. Of Health And Human Services (Dhhs), U.S. Government Compostions and methods for enabling cholesterol catabolism in human cells
MX2022008065A (es) * 2019-12-27 2022-07-27 Durect Corp 25-hidroxi-colest-5-en-3-sulfato de sodio cristalino y cristalino liquido y metodos para preparar los mismos.
CN111214474A (zh) * 2020-02-03 2020-06-02 中国海洋大学 一种海参甾醇硫酸酯在抗动脉粥样硬化制品中的应用
CA3183119A1 (fr) * 2020-05-22 2021-11-25 Durect Corporation Traitement de la steatohepatite non alcoolique (shna)
CN114487185B (zh) * 2022-01-21 2024-05-28 武汉迈特维尔生物科技有限公司 一种胆固醇通路的分离鉴定方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4427668A (en) * 1982-04-01 1984-01-24 New York University 26-Hydroxycholesterol and derivatives and analogs thereof in regulation of cholesterol accumulation in body tissue

Family Cites Families (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BE794362A (fr) 1972-01-22 1973-07-23 Merck Patent Gmbh Sulfates hydrosolubles de sterine
US3928397A (en) 1973-03-02 1975-12-23 Eisai Co Ltd New 5-cholestene derivatives and preparation thereof
US3822254A (en) 1973-05-21 1974-07-02 Hoffmann La Roche Synthesis of 25-hydroxycholesterol
US4264512A (en) * 1975-09-29 1981-04-28 The Regents Of The University Of California 3-Deoxy-1α-hydroxy- and 3-deoxy-1α,25-dihydroxycholecalciferol and processes for the preparation thereof
US4202891A (en) 1977-05-16 1980-05-13 Kandutsch Andrew A 15-Oxygenated sterol compounds and the use of such compounds to inhibit the biosynthesis of sterols
GB1595020A (en) 1977-06-24 1981-08-05 Chugai Pharmaceutical Co Ltd 3a-hydroxy steroid derivatives of the cholestane series
US4743597A (en) 1986-01-27 1988-05-10 Javitt Norman B Composition comprising an oxygenated cholesterol and use thereof for topical treatment of diseases
US5510340A (en) 1992-06-12 1996-04-23 Sri International Antihypercholesterolemic compounds and related pharmaceutical compositions and methods of use
US5371077A (en) 1992-08-03 1994-12-06 William Marsh Rice University Side chain derivatized 15-oxygenated sterols, methods of using them and a process for preparing them
US5599659A (en) 1993-03-11 1997-02-04 Breonics, Inc. Preservation solution for ex vivo, warm preservation of tissues, explants,organs and vascular endothelial cells comprising retinal-derived fibroblast growth factor, cyclodextrin and chondroitin sulfate
US5587368A (en) 1993-11-30 1996-12-24 New York University Administration of a 27-hydroxycholesterol or related compound or sterol-27-hydroxylase stimulant to prevent restenosis following vascular endothelial injury
US6645953B2 (en) 1995-06-23 2003-11-11 Novo Nordisk A/S Meiosis regulating compounds
AU718246B2 (en) 1995-06-23 2000-04-13 Novo Nordisk A/S Meiosis regulating compounds
US20030153541A1 (en) 1997-10-31 2003-08-14 Robert Dudley Novel anticholesterol compositions and method for using same
IL139241A0 (en) 1998-05-13 2001-11-25 Novo Nordisk As Meiosis regulating compounds
AU3701199A (en) 1998-05-13 1999-11-29 Novo Nordisk A/S Meiosis regulating compounds
US7374779B2 (en) 1999-02-26 2008-05-20 Lipocine, Inc. Pharmaceutical formulations and systems for improved absorption and multistage release of active agents
US6248363B1 (en) 1999-11-23 2001-06-19 Lipocine, Inc. Solid carriers for improved delivery of active ingredients in pharmaceutical compositions
US20060025393A1 (en) 1999-04-30 2006-02-02 Shutsung Liao Steroid derivatives
KR20020028876A (ko) 1999-04-30 2002-04-17 추후제출 스테로이드 유도체
AU1291901A (en) 1999-09-01 2001-03-26 University Of British Columbia, The Compositions and methods for modulating hdl cholesterol and triglyceride levels
JP4018312B2 (ja) 2000-02-21 2007-12-05 株式会社ルネサステクノロジ 無線通信装置
JP2005508281A (ja) 2001-02-08 2005-03-31 ザ ユニバーシティー オブ シカゴ ステロイド誘導体
US20020164310A1 (en) 2001-03-02 2002-11-07 Mgvs Ltd. Nucleic acid constructs, cells transformed therewith and methods utilizing same for inducing liver regeneration and alleviation of portal hypertension
WO2002090375A2 (fr) 2001-05-03 2002-11-14 The University Of Chicago Agonistes du recepteur x hepatique
US20070197484A1 (en) 2001-05-03 2007-08-23 Ching Song Method of treating disorder related to high cholesterol concentration
TW200300078A (en) 2001-11-08 2003-05-16 Univ Chicago Method of treating disorder related to high cholesterol concentration
EA012416B1 (ru) 2004-06-30 2009-10-30 Янссен Фармацевтика Н.В. Производные замещенного 2-алкилхиназолинона как ингибиторы parp
US8399441B2 (en) 2004-10-25 2013-03-19 Virginia Commonwealth University Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
WO2006047022A1 (fr) 2004-10-25 2006-05-04 Virginia Commonwealth University Oxysterol sulfate nucleaire, regulateur homeostatique du cholesterol agissant puissamment dans le traitement de l'hypercholesterolemie, de l'hyperlipidemie et de l'atherosclerose
DE102006021181B3 (de) 2006-05-06 2007-05-03 Laux, Thomas, Dr. Zahnprothesen-Trägerimplantat
GB0625965D0 (en) 2006-12-23 2007-02-07 Renovo Ltd Medicaments for wound healing
US8831674B2 (en) 2007-09-27 2014-09-09 Multi-Tech Systems, Inc. Message server
CN101952281B (zh) 2008-01-04 2014-04-02 株式会社Lg生命科学 具有细胞、组织及器官保存效果的吲哚及吲唑衍生物
WO2011077245A2 (fr) 2009-12-23 2011-06-30 Fondazione Centro San Raffaele Del Monte Tabor Compositions
US9034859B2 (en) 2011-04-06 2015-05-19 Virginia Commonwealth University Sulfated oxysterol and oxysterol sulfation by hydroxysterol sulfotransferase promote lipid homeostasis and liver proliferation
CN107936076B (zh) 2011-09-08 2021-10-15 萨奇治疗股份有限公司 神经活性类固醇、组合物、及其用途
US8665030B2 (en) 2011-12-14 2014-03-04 Taiwan Semiconductor Manufacturing Company, Ltd. Voltage-controlled oscillator
EP3239163A1 (fr) 2012-04-12 2017-11-01 Virginia Commonwealth University Nouveau métabolite du cholestérol, 5-cholestène-3,25-diol, disulfate (25hcds) pour la thérapie de troubles métaboliques, de l'hyperlipidémie, du diabète, des stéatoses hépatiques et de l'athérosclérose
JP6209791B2 (ja) 2012-07-17 2017-10-11 リージェンツ オブ ザ ユニバーシティ オブ ミシガン 白内障を処置するためのα−クリスタリン凝集の阻害剤
EP2842547A1 (fr) 2013-08-27 2015-03-04 Freund Pharmatec Ltd. Compositions de fénofibrate améliorées
US10272097B2 (en) 2013-12-24 2019-04-30 Virginia Commonwealth University Uses of oxygenated cholesterol sulfates (OCS)
EA034524B1 (ru) 2014-10-10 2020-02-17 Вирджиния Коммонвелт Юниверсити Сульфаты окисленного холестерина для лечения пониженной лептиновой активности
JP2018522070A (ja) 2015-07-27 2018-08-09 キャタコア・インコーポレイテッドＣａｔａｃｏｒｅ， Ｉｎｃ． 白内障の処置用組成物
SI3494125T1 (sl) 2016-08-02 2022-10-28 Virginia Commonwealth University Sestavki, ki vsebujejo 5-holesten-3, 25-diol, 3-sulfat(25HC3S) ali njegovo farmacevtsko sprejemljivo sol in vsaj en cikličen oligosaharid
WO2018026837A1 (fr) 2016-08-02 2018-02-08 Durect Corporation Compositions comprenant du sulfate de cholestérol oxygéné et au moins du polyalkylène glycol, de la carboxyméthylcellulose ou du polyoxylglycéride
AU2017306140A1 (en) 2016-08-02 2019-02-21 Durect Corporation Use of oxygenated cholesterol sulfates (OCS) to treat inflammatory skin disease and skin lesions

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4427668A (en) * 1982-04-01 1984-01-24 New York University 26-Hydroxycholesterol and derivatives and analogs thereof in regulation of cholesterol accumulation in body tissue

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11384115B2 (en) 2004-10-25 2022-07-12 Virginia Commonwealth University Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US9321802B2 (en) 2004-10-25 2016-04-26 Virginia Commonwealth University Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US10844089B2 (en) 2004-10-25 2020-11-24 Virginia Commonwealth University Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US10144759B2 (en) 2004-10-25 2018-12-04 Virginia Commonwealth University Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US9480692B2 (en) 2011-04-06 2016-11-01 Virginia Commonwealth University Sulfated-oxysterol and oxysterol sulfation by hydroxysterol sulfotransferase promote lipid homeostasis and liver proliferation
KR102180485B1 (ko) 2012-04-12 2020-11-18 버지니아 커먼웰스 유니버시티 대사 장애, 고지질혈증, 당뇨병, 지방간 질환 및 죽상동맥경화증의 치료를 위한 신규한 콜레스테롤 대사물질, 5-콜레스텐-3β,25-디올, 디설페이트 (２５ＨＣＤＳ)
KR20150013520A (ko) * 2012-04-12 2015-02-05 버지니아 커먼웰스 유니버시티 대사 장애, 고지질혈증, 당뇨병, 지방간 질환 및 죽상동맥경화증의 치료를 위한 신규한 콜레스테롤 대사물질, 5-콜레스텐, 3β-25-디올, 디설페이트 (２５ＨＣＤＳ)
EA026683B1 (ru) * 2012-04-12 2017-05-31 Вирджиния Коммонвелт Юниверсити МЕТАБОЛИТ ХОЛЕСТЕРИНА ДИСУЛЬФАТ 5-ХОЛЕСТЕН-3β,25-ДИОЛА (25HCDS) ДЛЯ ТЕРАПИИ НАРУШЕНИЙ ОБМЕНА ВЕЩЕСТВ, ГИПЕРЛИПИДЕМИИ, ДИАБЕТА, ЖИРОВЫХ БОЛЕЗНЕЙ ПЕЧЕНИ И АТЕРОСКЛЕРОЗА
WO2013154752A1 (fr) * 2012-04-12 2013-10-17 Virgina Commonwealth University Nouveau métabolite du cholestérol, 5-cholestène, 3β-25-diol, disulfate (25hcds) pour la thérapie de troubles métaboliques, de l'hyperlipidémie, du diabète, des stéatoses hépatiques et de l'athérosclérose
US10272097B2 (en) 2013-12-24 2019-04-30 Virginia Commonwealth University Uses of oxygenated cholesterol sulfates (OCS)
US10786517B2 (en) * 2013-12-24 2020-09-29 Durect Corporation Uses of oxygenated cholesterol sulfates (OCS)
JP2020186241A (ja) * 2013-12-24 2020-11-19 ヴァージニア コモンウェルス ユニバーシティ 酸素化コレステロール硫酸塩（ｏｃｓ）の使用
US11612609B2 (en) 2013-12-24 2023-03-28 Durect Corporation Uses of oxygenated cholesterol sulfates (OCS)
JP7289030B2 (ja) 2013-12-24 2023-06-09 ヴァージニア コモンウェルス ユニバーシティ 酸素化コレステロール硫酸塩（ｏｃｓ）の使用
WO2016058000A1 (fr) 2014-10-10 2016-04-14 Virginia Commonwealth University Sulfates de cholestérol oxygénés pour le traitement de troubles causés par une diminution de l'activité de la leptine ou un trouble de stockage des lipides, ou les deux
CN109983025A (zh) * 2016-08-02 2019-07-05 弗吉尼亚联邦大学 包含5-胆甾烯-3,25-二醇,3-硫酸酯(25hc3s)或其药学上可接受的盐和至少一种环状寡糖的组合物
WO2018026781A1 (fr) * 2016-08-02 2018-02-08 Virginia Commonwealth University Compositions comprenant du 5-cholestène-3, 25-diol,3-sulfate (25 h3s) ou un sel pharmaceutiquement acceptable de celui-ci et au moins un oligosaccharide cyclique
US11406646B2 (en) 2016-08-02 2022-08-09 Virginia Commonwealth University Compositions comprising 5-cholesten-3, 25-diol, 3-sulfate (25HC3S) or pharmaceutically acceptable salt thereof and at least one cyclic oligosaccharide
WO2021067297A1 (fr) * 2019-09-30 2021-04-08 Durect Corporation Traitement de l'hépatite alcoolique

Also Published As

Publication number Publication date
US20160264615A1 (en) 2016-09-15
US20190169225A1 (en) 2019-06-06
US20210147469A1 (en) 2021-05-20
US20180346509A9 (en) 2018-12-06
US10144759B2 (en) 2018-12-04
WO2006047022A1 (fr) 2006-05-04
US11384115B2 (en) 2022-07-12
US10844089B2 (en) 2020-11-24

Similar Documents

Publication Publication Date Title
US11384115B2 (en) Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
US9321802B2 (en) Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis
Fiorucci et al. Bile acids and their receptors in metabolic disorders
Slominski et al. Cytochrome P450scc-dependent metabolism of 7-dehydrocholesterol in placenta and epidermal keratinocytes
CN110437297B (zh) 法尼醇x受体的抑制剂和在医学中的用途
JP6125610B2 (ja) 代謝障害、高脂血症、糖尿病、脂肪性肝疾患及びアテローム性動脈硬化症の治療のための新規コレステロール代謝物、５−コレステン−３β，２５−ジオールジスルファート（２５ＨＣＤＳ）
Wang et al. Cholesterol metabolism pathways–are the intermediates more important than the products?
Ren et al. Sulfated oxysterol, 25HC3S, is a potent regulator of lipid metabolism in human hepatocytes
Landrier et al. Vitamin E decreases endogenous cholesterol synthesis and apo-AI-mediated cholesterol secretion in Caco-2 cells
Ren et al. Identification of a novel sulfonated oxysterol, 5-cholesten-3β, 25-diol 3-sulfonate, in hepatocyte nuclei and mitochondria
US20190201419A1 (en) Compound and method for the treatment and diagnosis of neurodegenerative conditions
Carino et al. Disruption of TFGβ-SMAD3 pathway by the nuclear receptor SHP mediates the antifibrotic activities of BAR704, a novel highly selective FXR ligand
Qiao et al. Metabolic regulatory effects of licorice: a bile acid metabonomic study by liquid chromatography coupled with tandem mass spectrometry
Wang et al. Farnesoid X receptor regulates SULT1E1 expression through inhibition of PGC1α binding to HNF4α
Hall et al. Overexpression of CYP27 in hepatic and extrahepatic cells: role in the regulation of cholesterol homeostasis
Ren et al. Identification of novel regulatory cholesterol metabolite, 5-cholesten, 3β, 25-diol, disulfate
Dolezelova et al. High soluble endoglin levels regulate cholesterol homeostasis and bile acids turnover in the liver of transgenic mice
Heo et al. Clusterin deficiency induces lipid accumulation and tissue damage in kidney
Bratoeff et al. Molecular interactions of progesterone derivatives with 5α-reductase types 1 and 2 and androgen receptors
Kim et al. Changes in cholesterol biosynthetic and transport pathways after excitotoxicity
Ying et al. EP4 emerges as a novel regulator of bile acid synthesis and its activation protects against hypercholesterolemia
Li et al. Integrated lipidomics and metabolomics study of four chemically induced mouse models of acute intrahepatic cholestasis
Watanabe et al. Genistein enhances NAD+ biosynthesis by upregulating nicotinamide phosphoribosyltransferase in adipocytes
Honda et al. Disrupted coordinate regulation of farnesoid X receptor target genes in a patient with cerebrotendinous xanthomatosis
Hirayama et al. Hypercholesterolemia in rats with hepatomas: increased oxysterols accelerate efflux but do not inhibit biosynthesis of cholesterol

Legal Events

Date Code Title Description
AS Assignment

Owner name: VIRGINIA COMMONWEALTH UNIVERSITY, VIRGINIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:REN, SHULIN;PANDAK, WILLIAM N.;REEL/FRAME:019586/0780

Effective date: 20070619

STCB Information on status: application discontinuation

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

AS Assignment

Owner name: VIRGINIA COMMONWEALTH UNIVERSITY, VIRGINIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:VIRGINIA COMMONWEALTH UNIVERSITY;REEL/FRAME:062318/0798

Effective date: 20211012

Owner name: THE UNITED STATES GOVERNMENT AS REPRESENTED BY THE DEPARTMENT OF VETERANS AFFAIRS, DISTRICT OF COLUMBIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:VIRGINIA COMMONWEALTH UNIVERSITY;REEL/FRAME:062318/0798

Effective date: 20211012