US20230293551A1 - Use of oxygenated cholesterol sulfates for treating autoimmune conditions - Google Patents

Use of oxygenated cholesterol sulfates for treating autoimmune conditions Download PDF

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US20230293551A1
US20230293551A1 US18/008,582 US202118008582A US2023293551A1 US 20230293551 A1 US20230293551 A1 US 20230293551A1 US 202118008582 A US202118008582 A US 202118008582A US 2023293551 A1 US2023293551 A1 US 2023293551A1
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25hc3s
hydroxycholesterol
sulfate
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Shunlin Ren
Yaping Wang
WeiQi Lin
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United States Represented By Department Of Veterans Affairs Washington DC AS
Virginia Commonwealth University
Durect Corp
US Department of Veterans Affairs VA
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Virginia Commonwealth University
Durect Corp
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    • 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
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/32—Alcohol-abuse
    • A—HUMAN NECESSITIES
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Definitions

  • the invention was made, in part, with government support under VA Merit Review Grant, Grant No. 1I01BX003656 awarded by Veterans Affairs. The government has certain rights in the invention.
  • Oxysterols have long been believed to be ligands of nuclear receptors such as liver x receptor (LXR), and they play an important role in lipids homeostasis and immune system, where they are involved in both transcriptional and post-transcriptional mechanisms.
  • Oxysterols are the oxidized form of cholesterol. In vivo, enzymatic transformation of sterols to oxysterols is for biosynthesis of important biological products such as steroid hormones, bile acids, and vitamin D in cells, blood, and tissues. Oxysterols participate in many biological processes including cholesterol homeostasis, triglyceride metabolism, inflammatory responses, cell proliferation, platelet aggregation, and apoptosis.
  • Oxysterols have also been implicated in many diseases such as metabolic syndrome and neurodegenerative diseases.
  • Oxysterols can be sulfated by sulfotransferase 2B 1b (SULT2B 1b) at the 3-position of the ring A of cholesterol to be oxysterol 3-sulfates including 5-cholesten-3 ⁇ -25-diol-3-sulphate (25HC3S), 5-cholesten-3 ⁇ -24-diol-3-sulphate (24HC3S), 5-cholesten-3 ⁇ -27-diol-3-sulphate (27HC3S) as well as Xol3S (cholesterol 3-sulfate).
  • cholesterol metabolite decreases lipid biosynthesis and increases cholesterol secretion and degradation, and may be useful for the treatment and prevention of hypercholesterolemia, hypertriglyceridemia, and conditions related to fat accumulation and inflammation (e.g., non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, and atherosclerosis).
  • NASH non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • AKI acute kidney injury
  • psoriasis psoriasis
  • atherosclerosis e.g., atherosclerosis
  • Oxysterols have also been implicated in several diseases such as metabolic syndrome. Oxysterols can be sulfated, and the sulfated oxysterols act in different direction: they decrease lipid biosynthesis, suppress inflammatory responses, and promote cell survival.
  • the present disclosure provides methods for treating at least one autoimmune condition, such as at least one of hepatitis, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis.
  • at least one autoimmune condition such as at least one of hepatitis, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis.
  • the at least one autoimmune condition is associated with Epstein-Barr virus infection.
  • oxysterol active agent compound selected from 25-hydroxycholesterol-3-sulfate (25HC3S), 25-hydroxycholesterol-disulfate (25HCDS), 27-hydroxycholesterol-3-sulfate (27HC3S), 27-hydroxycholesterol-disulfate (27HCDS), 24-hydroxycholesterol-3-sulfate (24HCDS), and 24,25-epoxycholesterol-3-s
  • aspects of the disclosure include:
  • a method of treating at least one autoimmune condition in a subject in need thereof comprising:
  • the at least one autoimmune condition comprises at least one of hepatitis, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis.
  • administering comprises at least one of oral administration, enteric administration, sublingual administration, transdermal administration, intravenous administration, peritoneal administration, parenteral administration, administration by injection, subcutaneous injection, and intramuscular injection.
  • administering comprises administering a pharmaceutical composition comprising the at least one compound and a physiologically acceptable excipient, diluent, or carrier.
  • the at least one autoimmune condition is optionally associated with Epstein-Barr virus infection.
  • At least one compound for use of aspect 33, wherein the method is a method according to any one of aspects 1 to 30.
  • FIGS. 1 A- 1 D Synthesis and enzyme kinetic studies of Xol3S, 25HC3S, and 27HC3S.
  • the biosynthesis of Xol3S, 25HC3S, and 27HC3S in the cells is shown in FIG. 1 A .
  • HPLC profiles of purified 25HC3S; Xol3S; and 27HC3S are shown in FIG. 1 B .
  • the concentration dependent, 0-0.001 M (10 points) effects of 25HC3S, Xol3S, and 27HC3S on the enzyme activities is shown in FIG. 1 C .
  • Comparison of 25HC with 25HC3S, cholesterol with Xol3S, and 27HC with 27HC3S is shown in FIG. 1 D .
  • FIGS. 2 A- 2 F Effects of 25HC3S on DNA methylation in hepatocytes by global methylation sequencing analysis.
  • Huh-7 cells were cultured in HG media for 72 hours and treated with ethanol (vehicle) and 25 mM 25HC3S in ethanol for 4 hours.
  • the levels of global methylation were estimated by LINE-1 assay.
  • Four CpG sites in promoter regions of LINE-1 element were chosen as the target positions as shown in FIG. 2 A .
  • WGBS Circos maps of DMR distribution in chromosomes is shown in FIG.
  • FIG. 2 B the first circle shows the distribution of hypermethylation DMRs; the second shows transposable element (TE) density; and the third shows the distribution of hypomethylation DMRs.
  • Venn diagrams of hypomethylated DMR-associated genes (DMGs) in 25HC3S and Vehicle libraries under CG, CHG, and CHH contexts of whole genome (Up) and promoter regions (Low) are shown in FIG. 2 C .
  • KOBAS software was used to test the statistical enrichment of DMR related genes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. DNA methylation levels in different genomic functional regions of the whole genome in FIG.
  • FIG. 2 D where the x-axis represents the different genomic regions (CGI, CGI-shore, promoter, UTR 5, exon, intron, UTR 3, and repeat), and the y-axis represents the methylation levels in 25HC3S and vehicle libraries under CG, CHG, and CHH contexts.
  • High enrichment of hypomethylated DMRs in whole genome in KEGG pathways is shown in FIG. 2 E .
  • High enrichment of hypomethylated DMRs in promoter regions in KEGG pathways is shown in FIG. 2 F .
  • the detailed KEGG pathways are shown in Table 1.3.
  • FIGS. 3 A- 3 D Expression of key genes related to signaling pathways.
  • Huh-7 cells were cultured in HG media for 72 hours and treated with 25HC3S at 6.25 ⁇ M,12.5 ⁇ M, 25 ⁇ M, and 50 ⁇ M for 1 hour, 2 hours, 4 hours, 6 hours, and 8 hours.
  • Key Genes and their targeting genes expression were determined by RT-PCR analysis.
  • the expressions of DUSP8 (Dual Specificity Phosphatases 8), DUSP7 (Dual Specificity Phosphatases 7), and MAPK1 (Mitogen-activated protein kinase 1) in MAPK signaling pathway are shown in FIG.
  • FIG. 3 A their target genes, CREB (cAMP responsive element binding protein), PRDX6 (peroxiredoxin 6), and BAD (BCL2 Associated Agonist Of Cell Death) are shown in FIG. 3 B ; Key genes, CACNA family (calcium voltage-gated channel subunits), in calcium-AMK pathway are shown in FIG. 3 C ; their targeting genes PGC1A (PPARG co-activator 1 alpha), HMGR (3-hydroxy-3-methylglutaryl-CoA reductase), and FAS (fatty acid synthase) are shown in FIG. 3 D .
  • PGC1A PARG co-activator 1 alpha
  • HMGR 3-hydroxy-3-methylglutaryl-CoA reductase
  • FAS fatty acid synthase
  • FIGS. 4 A- 4 F Effect of 25HC3S on transcription levels in hepatocytes.
  • HepG-2 cells were cultured in HG media and treated with 25 ⁇ M of 25HC3S for 2 hours, 4 hours, and 8 hours.
  • the up-regulated genes (>1.6 fold) are shown in FIG. 4 A .
  • Enrichment of up-regulated genes (8 hours) to Gene ontological (GO) groups are shown in FIG.
  • NRAP negative regulation of apoptotic process
  • NRPCD negative regulation of programmed cell death
  • RS regulation of signaling
  • SP regulation of phosphorylation
  • NRCD negative regulation of cell death
  • RES response to stress
  • NRPP negative regulation of protein phosphorylation
  • CRCS cellular response to chemical stimulus
  • NRP negative regulation of phosphorylation
  • ST signal transduction
  • FIG. 5 Sulfation of 25HC as an epigenetic regulatory pathway.
  • 25HC is an endogenous agonist of DNMT-1 that methylates CpG in promoter regions and subsequently silences gene expression, resulting in cell death and lipogenesis.
  • 25HC can be sulfated to 25HC3S, which acts as an endogenous ligand and inhibits activities of DNMTs.
  • 25HC3S demethylates 5m CpG in promoter regions, and successively increases gene expression.
  • the eminent pathways regulated by the sulfation of oxysterol are involved in energy and lipids metabolisms, MAPK-ERK, and calcium-AMPK.
  • 25HC3S significantly increases Dual-specificity phosphatases (DUSPs) and CREB expression, which activate MAPK/ERK pathway, including CREB, BAD, and ERK, and subsequently regulate cell survival and death.
  • 25HC3S decreases lipid biosynthesis and reduces lipid accumulation by demethylating 5m CpG in promoter regions, increasing expression of key genes involved in calcium channels and AMPK, and activating corresponding signaling pathways, which result in increased oxidation of free fatty acids (FFA), and decreased biosynthesis of cholesterol and FFA.
  • FFA free fatty acids
  • the global regulation by sulfation of oxysterol suggests the physiological and pathophysiological significance of this regulatory mechanism.
  • FIG. 6 Mechanisms of sulfation and metabolic pathways of oxysterol sulfates.
  • FIG. 7 Regulatory pathway of oxysterol sulfation.
  • the present disclosure provides methods for treating at least one autoimmune condition, such as at least one of hepatitis, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis
  • at least one autoimmune condition such as at least one of hepatitis, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis
  • the at least one autoimmune condition is associated with Epstein-Barr virus infection.
  • a subject e.g., human subject
  • the compound 25-hydroxycholesterol-3-sulfate (25HC3S) refers to a compound having the chemical structure:
  • 25HCDS 25-hydroxycholesterol-disulfate
  • the compound 27-hydroxycholesterol-3-sulfate refers to a compound having the chemical structure:
  • the compound 27-hydroxycholesterol-disulfate refers to a compound having the chemical structure:
  • 24-hydroxycholesterol-3-sulfate refers to a compound having the chemical structure:
  • 24-hydroxycholesterol-disulfate refers to a compound having the chemical structure:
  • the compound 24,25-epoxycholesterol-3-sulfate refers to a compound having the chemical structure:
  • Oxysterols can be sulfated by sulfotransferase 2B 1b (SULT2B 1b) at the 3-position of the ring A of cholesterol to be oxysterol 3-sulfates including 25HC3S, 24HC3S, 27HC3S as well as Xol3S (cholesterol 3-sulfate) as summarized in FIG. 6 .
  • the oxysterol sulfate can be further sulfated by sulfotransferase 2A1 (SULT2A1) to be oxysterol disulfates.
  • 25-hydroxycholesterol 3-sulfate can be further sulfated by SULT2A1 to 5-cholesten-3 ⁇ , 25-diol1-disulfate (25HCDS).
  • 25HC3S and 25HCDS are the only oxysterol sulfates that have been identified in vivo in hepatocyte nuclei while 27HC3S in human sera and 24HC3S in urine.
  • 25HC3S and 25HCDS are also potent regulators but function in a different direction from their precursor 25HC.
  • Cholesterol can be hydroxylated by CYP27A1 to 25HC or 27HC in the mitochondria, and hydroxylated to 25HC by CYP3A4, or by cholesterol 25-hydroxylase (CH25HL) in endoplasmic reticulum. Cholesterol can also be hydroxylated by cholesterol 24-hydroxylase to 24HC in brain tissue. This cholesterol precursor can also be used for synthesis of desmosterol via a shunt of the mevalonate pathway. The desmosterol can be oxygenated by CYP46A1 to form 24, 25-epoxycholesterol (24,25EC).
  • 25HC, 27HC, 24HC, and cholesterol can be subsequently sulfated at the 3 ⁇ -position by SULT2B1b to form 25HC3S, 27HC3S, 24HC3S, and Xol3S, respectively.
  • 24, 25EC can be sulfated to be 24, 25EC3S.
  • cytosine (5-methylcytosine, 5m C) in DNA promoter regions is an important epigenetic modification that regulates gene expression and other functions of the genome. Cytosine methylation of CpG in promoter regions is inversely correlated with transcriptional activity of associated genes as it causes chromatin condensation and gene silencing. Dysregulation of CpG methylation and gene expression affect metabolism, tissue function, and the metabolic state.
  • Cytosine methylation is catalyzed by DNA methyltransferases (DNMT-1, 3a/3b), which in some cases play a role in the regulation of DNA methylation/demethylation.
  • 25HC and 25HC3S are ligands of DNA methyltranferase-1 (DNMT-1).
  • the oxysterol active agent compounds described herein are cellular regulatory molecules that epigenetically regulate lipid metabolism, cell survival/death, and inflammatory responses via DNA CpG methylation and 5m CpG demethylation.
  • oxysterol active agent compounds disclosed herein de-methylates 5m CpG in these promoter regions, increases gene expression, and up-regulates these signaling pathways.
  • the oxysterol active agent compound regulates the signaling pathways in an opposite direction from precursor 25HC.
  • the one or more oxysterol active agent compounds regulate cell signaling pathways in response to stress responses.
  • the one or more oxysterol active agent compounds affect protein phosphorylation, inositol phosphorylation, and sphingosine phosphorylation in regulating cellular functions. In certain cases, the one or more oxysterol active agent compounds regulate gene expression at transcriptional levels. An illustrative mechanism is depicted in FIG. 7 .
  • the one or more oxysterol active agent compounds in certain cases decrease lipid accumulation, anti-inflammatory response, and anti-apoptosis by increasing gene expression through demethylation of 5m CpG in promoter regions of the key genes involved in MAPK-ERK and Calcium-AMPK signaling pathways, such as CREB5 (CAMP Responsive Element Binding Protein 5), BAD (BCL2 Associated Agonist of Cell Death), and ERK (Mitogen-activated protein kinase 1).
  • CREB5 CAMP Responsive Element Binding Protein 5
  • BAD BCL2 Associated Agonist of Cell Death
  • ERK Mitogen-activated protein kinase
  • LMP1 is expressed in most EBV-associated autoimmune disorders, and it critically contributes to pathogenesis and disease phenotypes, such as but not limited to hepatitis, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis.
  • EBV LMP1 directly induces promoter activity of DNMT1, resulting in hypermethylation and silencing of E-cadherin gene expression.
  • LMP1 also upregulates the expression of PD-L1 via activation of MAPK/NF-kB pathway. While not being bound by theory, inhibition of DNMT by the compounds of the present application may be useful in treating EBV-associated autoimmune disorders.
  • the term “treat” is used herein to refer to administering at least one oxysterol active agent compound selected from 25-hydroxycholesterol-3-sulfate (25HC3S), 25-hydroxycholesterol-disulfate (25HCDS), 27-hydroxycholesterol-3-sulfate (27HC3S), 27-hydroxycholesterol-disulfate (27HCDS), 24-hydroxycholesterol-3-sulfate (24HC3S), 24-hydroxycholesterol-disulfate (24HCDS), and 24,25-epoxycholesterol-3-sulfate, or salt thereof to a human subject that: (1) already exhibits at least one symptom of at least one autoimmune condition, such as at least one of hepatitis, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis; and/or (2) is diagnosed as having at least one autoimmune condition, such as at least one of hepatitis, multiple sclerosis, systemic
  • treatment involves the lessening or attenuation, or in some instances, the complete eradication, of at least one symptom of the at least one autoimmune condition, such as at least one of hepatitis, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis that was present prior to or at the time of administration of the at least one oxysterol active agent compound selected from 25-hydroxycholesterol-3-sulfate (25HC3S), 25-hydroxycholesterol-disulfate (25HCDS), 27-hydroxycholesterol-3-sulfate (27HC3S), 27-hydroxycholesterol-disulfate (27HCDS), 24-hydroxycholesterol-3-sulfate (24HC3S), 24-hydroxycholesterol-disulfate (24HCDS), and 24,25-epoxycholesterol-3-sulfate, or salt thereof.
  • 25HC3S 25-hydroxycholesterol-disulfate
  • 25HCDS 25-hydroxy
  • treatment according to the present disclosure is sufficient to improve clinical indicators in the subject.
  • the improvement in the clinical indicators in the subject is such that the subject is considered to no longer have the at least one autoimmune condition, such as at least one of hepatitis, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis.
  • oxysterol active agent compound selected from 25-hydroxycholesterol-3-sulfate (25HC3S), 25-hydroxycholesterol-disulfate (25HCDS), 27-hydroxycholesterol-3-sulfate (27HC3S), 27-hydroxycholesterol-disulfate (27HCDS), 24-hydroxycholesterol-3-sulfate (24HCDS), and 24,25-epoxycholesterol-3-s
  • the oxysterol active agent compound is administered to the subject at a dosage of from 0.00001 mg/kg/day to 500 mg/kg/day, such as from 0.00005 mg/kg/day to 450 mg/kg/day, such as from 0.0001 mg/kg/day to 400 mg/kg/day, such as from 0.0005 mg/kg/day to 350 mg/kg/day, such as from 0.001 mg/kg/day to 300 mg/kg/day, such as from 0.005 mg/kg/day to 250 mg/kg/day, such as from 0.01 mg/kg/day to 200 mg/kg/day, such as from 0.05 mg/kg/day to 150 mg/kg/day, and including from 0.001 mg/kg/day to 100 mg/kg/day.
  • the oxysterol active agent compound is administered to the subject at a dosage of from 0.001 mg/kg/day to 100 mg/kg/day. In certain cases, the oxysterol active agent compound is administered to the subject at a dosage of from 0.1 mg/kg/day to 100 mg/kg/day. In certain cases, the oxysterol active agent compound is administered to the subject at a dosage of from 1 mg/kg/day to 100 mg/kg/day.
  • the amount of each daily dose of the at least one oxysterol active agent compound, such as 25-hydroxycholesterol-3-sulfate or 25-hydroxycholesterol-3-sulfate sodium, administered to the individual is from 0.5 mg to 5 mg, 5 mg to 10 mg, 10 mg to 15 mg, 15 mg to 20 mg, 20 mg to 25 mg, 20 mg to 50 mg, 25 mg to 50 mg, 50 mg to 75 mg, 50 mg to 100 mg, 75 mg to 100 mg, 100 mg to 125 mg, 125 mg to 150 mg, 150 mg to 175 mg, 175 mg to 200 mg, 200 mg to 225 mg, 225 mg to 250 mg, 250 mg to 300 mg, 300 mg to 350 mg, 350 mg to 400 mg, 400 mg to 450 mg, or 450 mg to 500 mg.
  • the amount of oxysterol active agent compound in the effective amount administered to the individual is in the range of from 0.5 mg to 500 mg, such as from 1 mg to 450 mg, such as from 2 mg to 400 mg, such as from 5 mg to 300 mg, such as from 10 mg to 200 mg, or such as from 20 mg to 100 mg.
  • the oxysterol active agent compound may be administered to the subject once per day or more, such as twice per day or more, such as three times per day or more, and including four times per day or more.
  • the oxysterol active agtent compound may be administered twice a day, once a day, once every other day, once every three days, once a week, or once a month.
  • the oxysterol active agent compound is administered to the subject once per day.
  • the oxysterol active agent compound is administered to the subject twice per day.
  • the oxysterol active agent compound is administered to the subject once or twice per day in a cycle for a duration of ranging from 1 day to 10 days, 1 day to 30 days, 7 days to 30 days, 7 days to 90 days, 10 days to 180 days, or 30 days to 1 year, 30 days to 5 years, 90 days to 5 years, or 1 year to 10 years.
  • the oxysterol active agent compound is administered to the subject once per day for a duration of from 1 day to 30 days, such as once per day for a duration of from 1 day to 28 days, from 1 day to 21 days, from 7 days to 14 days.
  • the oxysterol active agent compound is administered to the subject twice per day for a duration of from 1 day to 30 days, such as twice per day for a duration of from 1 day to 28 days, from 1 day to 21 days, from 7 days to 14 days. In some cases, the oxysterol active agent compound is administered to the subject three times per day for a duration of from 1 day to 30 days, such as three times per day for a duration of from 1 day to 28 days, from 1 day to 21 days, from 7 days to 14 days.
  • the dosing is administered in cycles of administration of the oxysterol active agent compound.
  • the cycle is 1 day or more, such as 2 days or more, such as 3 days or more, such as 4, days or more, such as 5 days or more, such as 6 days or more, such as 7 days or more, such as 14 days or more, such as 21 days or more, such as 28 days or more, and in some instances the cycle is 30 days or more.
  • the cycles of drug administration may be repeated for 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosage cycles, for a total period of 6 months, 1 year, 2 years, 3 years, or 4 years or more.
  • the administration of each pharmaceutical composition can be extended over an extended period of time (such as during maintenance therapy), such as from a month up to seven years.
  • the oxysterol active agent compound may be administered over a period of about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 48, 60, 72, or 84 months. In other cases, the oxysterol active agent compound is administered for the rest of the subject’s lifetime.
  • Implementation of the methods generally involves identifying (e.g., diagnosing) patients suffering from or at risk of at least one autoimmune condition, such as at least one of hepatitis, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis.
  • 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.
  • the dose will vary with the route of administration, the bioavailability, and the particular formulation that is administered, as well as according to the nature of the malady that is being prevented or treated.
  • each dosage of the oxysterol active agent compound is administered to the subject over duration of from 0.1 hours to 12 hours (e.g., by intravenous administration) such as from 0.5 hours to 10 hours, such as from 1 hour to 8 hours, and including over a duration of from 2 hours to 6 hours.
  • Administration may be oral or parenteral, including intravenously, intramuscularly, subcutaneously, intradermal injection, intraperitoneal injection, etc., or by other routes (e.g., transdermal, sublingual, rectal and buccal delivery, inhalation of an aerosol, intravaginally, intranasally, topically, as eye drops, via sprays, etc.).
  • the oxysterol active agent compound isadministered to the subject by one or more of oral administration, enteric administration, sublingual administration, transdermal administration, intravenous administration, peritoneal administration, parenteral administration, administration by injection, subcutaneous injection, and intramuscular injection.
  • the route of administration will depend on the nature or the condition that is treated, e.g., on the type or degree of the disease, and whether the treatment is prophylactic or intended to effect a cure. Further, administration of the compound by any means may be carried out as a single mode of therapy, or in conjunction with other therapies and treatment modalities, e.g., diet regimens, etc.
  • compositions are administered in conjunction with other treatment modalities such as various pain relief medications, anti-arthritis agents, chemotherapeutic agents, antibiotic agents, anti-neurodegeneration agents, anti-addiction agents, steroids, anti-inflammatory agents, anti-IL-1 biologics, anti-TNF biologics (TNF inhibitors), anti-IL-6 biologics, anti-CD20 biologics, B cell growth factor targeting biologics, anti-IL-17 biologics, anti-IL-23 biologics, anti-IL-12/23 biologics, anti-IL-5 biologics, anti-IL-4/IL-13 biologics, anti-IgE biologics, JAK inhibitors and the like, depending on the malady that is afflicting the subject.
  • “In conjunction with” refers to both administration of a separate preparation of the one or more additional agents, and also to inclusion of the one or more additional agents in a composition of the present disclosure.
  • the oxysterol active agent may be administered in conjunction with at least one of prednisone, methylprednisolone, dexamethasone, colchicine, hydroxychloroquine, sulfasalazine, dapasone, mycophenolate mofetil, azathioprine, sirolimus, cyclosporine, methotrexate, cyclophosphamide, etanercept, abatacept, secukinumab, ixekizumab, brodalumab, guselkumab, ustekinumab, mepolizumab, depilumab, omalizumab, vendolizumab, belimumab, infliximab, adalimumab, golimumab, certolizumab, tocilizumab, sarilumab, anakinra, canakinumab, rilonacept, e
  • the oxysterol active agent 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 oxysterol active agent 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.
  • compositions e.g., injectable compositions
  • 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 component at least one oxysterol active agent
  • the vehicular “carrier” will constitute 1% to 99% of the composition.
  • the pharmaceutical compositions of the present disclosure 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 at least one oxysterol active agent compound.
  • Additional suitable agents that may be co-administered or co-formulated also include other agents, including but not limited to: metabolites of the methionine and/or glutathione biosynthetic pathways such as S-adenosylhomocysteine (SAH), S-methylmethionine (SMM), cystine, betaine, etc., or various forms and/or salts thereof, e.g., acetylcysteine (e.g., intravenous N-acetylcysteine), various neutraceuticals, etc.
  • SAH S-adenosylhomocysteine
  • SMM S-methylmethionine
  • cystine betaine
  • salts thereof e.g., acetylcysteine (e.g., intravenous N-acetylcysteine), various neutraceuticals, etc.
  • compositions may include one or more pharmaceutically acceptable carriers.
  • Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.
  • the one or more excipients may include sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, or calcium carbonate, a binder (e.g., cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, poly(ethylene glycol), sucrose or starch), a disintegrator (e.g., starch, carboxymethylcellulose, hydroxypropyl starch, low substituted hydroxypropylcellulose, sodium bicarbonate, calcium phosphate, or calcium citrate), a lubricant (e.g., magnesium stearate, light anhydrous silicic acid, talc, or sodium lauryl sulfate), a flavoring agent (e.g., citric acid, menthol, glycine, or orange powder), a preservative (e.g., sodium benzoate, sodium, sodium
  • compositions of interest include an aqueous buffer.
  • Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers varying in strengths from 5 mM to 100 mM.
  • the aqueous buffer includes reagents that provide for an isotonic solution. Such reagents include, but are not limited to, sodium chloride; and sugars, e.g., mannitol, dextrose, sucrose, and the like.
  • the aqueous buffer further includes a non-ionic surfactant such as polysorbate 20 or 80.
  • compositions of interest further include a preservative.
  • Suitable preservatives include, but are not limited to, a benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In many cases, the composition is stored at about 4° C. Formulations may also be lyophilized, in which case they generally include cryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol, and the like. Lyophilized formulations can be stored over extended periods of time, even at ambient temperatures.
  • compositions include other additives, such as lactose, mannitol, corn starch, or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatins; with disintegrators, such as corn starch, potato starch, or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives, and flavoring agents.
  • additives such as lactose, mannitol, corn starch, or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatins
  • disintegrators such as corn starch, potato starch, or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium stearate
  • compositions may be formulated by dissolving, suspending, or emulsifying the oxysterol active agent compound in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives.
  • methods according to the present disclosure are directed to treatment of a subject based on a cellular response when the subject is administered the oxysterol active agent compound.
  • epigenetic modification plays a role in the regulation and coordination of gene expression.
  • Methylation at position 5 of cytosine (5-methylcytosine, 5mC) in DNA is an important epigenetic modification that regulates gene expression among other functions of the genome.
  • cytosine methylation of CpG in the promoter region is inversely correlated with transcriptional activity of associated genes as it causes chromatin condensation and thus gene silencing.
  • Dysregulation of CpG methylation and gene expression can affect tissue function and metabolic state. Cytosine methylation is catalyzed by DNA methyltransferase (DNMT-1, 3a/3b), which also functions in the regulation of DNA methylation.
  • DNMT-1 DNA methyltransferase
  • the major epigenetic regulation includes DNA and histone methylation, demethylation, acetylation, and deacetylation.
  • the enzymes involved in the process are DNA and histone methyltransferases/demethylases, and acetyltransferases/deacetylases.
  • administering one or more of the oxysterol active agent compounds is sufficient to act as an epigenetic modulator of one or more of DNMT1, DNMT3a, DNMT3b, GCN3 (Giant congenital nevi), p300 (histone acetyl transferase), Pcaf (KAT2B lysine acetyltransferase 2B), HDAC1 (histone deacetylase 1), HDAC2 (histone deacetylase 2), HDAC3 (histone deacetylase 3), HDAC6 (histone deacetylase 6), HDAC10 (histone deacetylase 10), and KDM6B-JMJD3 (lysine demethylase 6B), such as where 25HC3S, 27HC, and 27HC3S, or cholesterol (Xol) and cholesterol-3-sulfate (Xo13S) are their endogenous ligand(s) to one or more of DNMT1, DNMT3a
  • the one or more administered oxysterol active agent compounds inhibits DNMT-1, 3a, and 3b, which demethylated 5m CpG in promoter regions, increased gene expression and up-regulated master signaling pathways such as MAPK, Calcium, AMPK, and CREB signaling pathways.
  • the one or more oxysterol active agent compounds regulate cell signaling pathways at transcriptional levels in nuclei.
  • the one or more oxysterol active agent compounds are administered in an amount sufficient to affect protein phosphorylation, inositol phosphorylation, and/or sphingosine phosphorylation in regulating cellular functions.
  • the addition of one or more of the subject oxysterol active agent compound to human hepatocytes is sufficient to reverse methylation induced by HG, increase hypomethylated CpG in promoter regions of the key genes and increase targeting gene expression.
  • CpG demethylation by the oxysterol active agent compound is the mechanism for its function of global regulation: decreasing lipid accumulation, anti-inflammatory responses, anti-oxidants, and anti-cell death.
  • the DUSP family is a subset of protein tyrosine phosphatases, many of which dephosphorylate mitogen-activated protein kinases (MAPKs) and hence are referred to as MAPK phosphatase.
  • DUSP8 a unique member of DUSP family, plays an important role in signal transduction of the phosphorylation-mediated MAPK pathway, which regulates responses to oxidative stress and cell death signals in various human diseases.
  • administering the one or more oxysterol active agent compounds is sufficient to demethylate 5m CpG in promoter regions of DUSP genes, including DUSP8, DUSP1, and DUSP7, and their downstream genes, CREB5, PRDX, BAD, and ERK, and increase their expression.
  • the transcribed proteins from these genes are responsible for cell survival and proliferation.
  • the effects of the at least one oxysterol active agent compound on promoting cell survival/proliferation and alleviating oxidative stress occur through inhibiting DNMTs and increasing expression of the DUSP family, especially DUSP8 and their downstream elements.
  • a method of treatment involves modulating at least one gene selected from ABCC4, AC005264.2, ADCY1, ADCY4, ADCY5, ADH6, ADRB, ADRB1, AFDN, AGTR1, AKAP12, AL671762.1, ALAD, ANKRD1, ANKRD43, ATF3, ATP1A3, BAD, BIRC3, C11orf96, CACNA1A, CACNA1C-AS1, CACNA1D, CACNA1H, CACNB2, CACNG8, CELSR2, CREB5, CTB-186G2.1, CXCL2, CYB5B, CYP24A1, CYP51A1, CYR61, DDIT3, DRD5P2, DUSP genes, DUSP8, DUSP1, DUSP7, CREB5, EDNRB, EDN1, EHHADH, ELOVL6, ERK, FABP1, FDFT1, FRMD3, FMC1, FSTL3, GABBR1, GABBR2, GADD45B,
  • a method of treatment involves modulating at least one pathway selected from cAMP signaling pathway, cGMP-PKG signaling pathway, circadian entrainment, glutamatergic synapse, adrenergic signaling in cardiomyocytes, gap junction, Type II diabetes mellitus, endocytosis, calcium signaling pathway, dilated cardiomyopathy, vascular smooth muscle contraction, MAPK signaling pathway, cholinergic synapse, Rap1 signaling pathway, dopaminergic synapse, Adherens junction, arrhythmogenic right ventricular cardiomyopathy, pathways in cancer, GnRH signaling pathway, oxytocin signaling pathway, transcriptional misregulation in cancer, estrogen signaling pathway, insulin secretion, retrograde endocannabinoid signaling, long-term depression, colorectal cancer, insulin signaling pathway, axon guidance, alcoholism, platelet activation, amphetamine addiction, herpes simplex infection, tight junction, thyroid hormone signaling pathway,
  • molecular weight is weight average molecular weight
  • temperature is in degrees Celsius
  • pressure is at or near atmospheric.
  • average is meant the arithmetic mean.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
  • Cell culture reagents and supplies were purchased from GIBCO BRL (Grand Island, NY); Huh-7 cells were obtained from American Type Culture Collection (Rockville, MD).
  • the reagents for real time RT-PCR were from AB Applied Biosystems (Warrington, UK).
  • the chemicals used in this research were obtained from Sigma Chemical Co. (St. Louis, MO) or Bio-Rad Laboratories (Hercules, CA). All solvents were obtained from Fisher (Fair Lawn, NJ) otherwise indicated.
  • Huh-7 and HepG-2 cells were cultured in DMEM media supplemented with 10% heat-inactivated fetal bovine serum (FBS), high glucose (HG, 4.5 g/L) at 37° C. in a humidified atmosphere of 5% CO 2 .
  • FBS heat-inactivated fetal bovine serum
  • HG high glucose
  • RNA expression was quantified with the comparative cycle threshold (Ct) method using the primer set shown in Table 1.1 and was expressed as 2 - ⁇ Ct .
  • the cartridge was successively washed with the loading buffer (15 ml), water (15 ml), methanol (15 ml), 50% methanol (15 ml), 5% ammonia hydroxide in 10% methanol (15 ml), and 5% ammonia hydroxide in 50% methanol (15 ml).
  • the retained sulphated sterol was eluted with 5% ammonia hydroxide in 80% methanol (10 ml), respectively. After dilution with 10 times volume of acetonitrile, the solvents were evaporated to dryness under nitrogen gas stream, and the sterol sulphates were obtained in white powder form.
  • the substrate solution 0.001 mg/ml Poly(dI-dC):Poly(dI-dC) in 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 5 mM DTT, 1 mM PMSF, 5% glycerol, 0.01% Brij35, 1% DMSO was used.
  • DNMT3a/3b activity assay 0.0075 mg/ml Lambda DNA in 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 5 mM DTT, 1 mM PMSF, 5% glycerol, 1% DMSO, was used.
  • the indicated DNMT1, DNMT3a, or DNMT3b was added to the appropriate substrate solution and gently mixed.
  • Amounts of cholesterol (Xol), 25HC, 27HC, Xol3S, 25HC3S, or 27HC3S ranging from 5.08E-09 to 0.0001 M in DMSO were added to the reaction mixture by using Acoustic Technology (Echo 550, LabCyte Inc.
  • PCR reaction and product purification were performed as per the manufacturer’s protocol (GE Healthcare Life Sciences).
  • the PCR products, 10 ⁇ l, were sequenced by Pyrosequencing on the PSQ96 HS System following the manufacturer’s instructions (Pyrosequencing, Qiagen).
  • the methylation status of each CpG site was determined individually as an artificial C/T SNP using QCpG software (Pyrosequencing, Qiagen).
  • the methylation level at each CpG site was calculated as the percentage of the methylated alleles divided by the sum of all methylated and unmethylated alleles.
  • the mean methylation level was calculated using methylation levels of all measured CpG sites within the targeted region of each gene.
  • Each experiment included non-CpG cytosines as internal controls to detect incomplete bisulfite conversion of the input DNA.
  • a series of unmethylated and methylated DNA were included as controls in each PCR assay.
  • PCR bias testing was performed by mixing unmethylated control DNA with in vitro methylated DNA at different ratios (0%, 5%, 10%, 25%, 50%, 75%, and 100%), followed by bisulfite modification, PCR, and Pyro-sequencing analysis.
  • the library preparations were sequenced on an Illumina Hiseq 2500/4000 or Novaseq platform and 125 bp/150 bp paired-end reads were generated. Image analysis and base calling were performed with Illumina CASAVA pipeline. Trimmomatic (Trimmomatic-0.36) software was used for quality control. Bismark software (version 0.16.3; Krueger F, 2011) was used to perform alignments of bisulfite-treated reads to a reference genome (-X 700 --dovetail). DSS software (23) was used to identify differentially methylated regions (DMRs). KOBAS software was used to test the statistical enrichment of DMR related genes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.
  • KEGG Kyoto Encyclopedia of Genes and Genomes
  • 25HC3S 25-Hydroxycholesterol-3-Sulfate Specifically Inactivate DNMT Activities
  • 25HS3S, Xol3S, and 27HC3S ( FIG. 1 A ) were synthesized and purified to more than 95% purity using triethylamine sulphate complex methods as shown in FIG. 1 B .
  • 25HC3S 25-Hydroxycholesterol-3-Sulfate
  • LINE-1 analysis was first performed to estimate global demethylation. Methylation usually occurs in repetitive elements, such as LINE elements. There are ⁇ 500,000 LINE elements and 750 million copies in total human genome. Human LINE-1 is a retro-transposable region (promoter region) and has only 700,000 copies, which correlates to ⁇ 17% of the human genome.
  • the specific sequence includes four CpG dinucleotides (Pos 1, 2, 3, and 4), which serve as methylation/demethylation targets in LINE-1.
  • HG high glucose media
  • Pos 3 and Pos 4 had higher methylation
  • all 4 Pos increased methylation after culturing cells in ethanol control.
  • Reduction of methylation (demethylation) at Pos1(-5%), Pos3 (-10%), and Pos 4 (-5.6%) occurred after incubating cells with 25HC3S for 4 hours.
  • the results indicate that 25HC3S significantly reduce 5m CpG methylation in promoter regions induced by HG or ethanol.
  • CpG methylation and demethylation are well documented to related with gene expression.
  • DMRs differential methylated regions
  • DMGs differential methylated genes
  • FIG. 2 B The hypomethylated genes were highly enriched in 75 KEGG pathways (p ⁇ 0.05) (Table 1.3). The top 20 pathways (from the most significance, p ⁇ 10 -9 ) were shown in FIGS. 2 E and 2 F .
  • MAPK-ERK and calcium-cAMP signaling are believed as the master pathways regulating cell survival, anti-oxidants, anti-apoptosis, energy metabolism, and lipid homeostasis.
  • the pathways identified from whole genome are shown in FIG. 2 E
  • those identified from promoter regions are shown in FIG. 2 F . Both sets of pathways, from whole genome or from promoter regions, are very similar. All pathways identified from promoter regions were hypomethylated without any hypermethylated CpGs in their promoter regions, indicating up-regulated gene expressions.
  • DNA methylation levels in whole genome and differential methylated regions are shown in FIG. 2 B .
  • DMRs differential methylated regions
  • CHG H represents adenosine or thymidine residues
  • CHH contexts are shown in FIG. 2 B .
  • a total of 6,923 differentially methylated genes (DMGs) were screened out among the two libraries.
  • 1,510 were 20 identified under CG context, 420 under CHG context, and 3,359 under CHH context.
  • HG high glucose incubation
  • 25HC3S demethylated 5m CpG in promoter regions of 23 genes in MAPK signaling pathway (Table 1.4), 19 genes in Calcium pathway (Table 1.5), and 28 genes in cAMP pathway (Table 1.6).
  • No hypermethylated DMR was found in the genes involved in the signaling pathways.
  • the chromosome and sequence location of the hypermethylated 5m CpG by HG and the hypomethylated CpG by 25HC3S in promoter regions are compared in the tables.
  • DNA methylation levels generally show a varied distribution across different functional regions of the genome.
  • the methylation levels in the CGI (CG island), CGI-shore (up to 2k bp away from the CGI), promoter (upstream 2k bp sequence from transcription starting site), 5′untranslated region (UTR5), exon, intron, 3′untranslated region (UTR3), and repeat were 10 significant different between vehicle and 25HC3S treated groups. It is interesting that 25HC3S treatment resulted in significantly higher hypomethylation levels than vehicle ( FIG. 2 D ).
  • the genes under CG context were highly enriched in 120 KEGG pathways (69 hypomethylated and 51 hypermethylated).
  • the genes under CHG context were enriched in 48 pathways (33 hypomethylated and 15 hypermethylated), while those under CHH context, enriched in 136 pathways (101 hypomethylated and 35 hypermethylated).
  • DMGs in promoter regions were highly enriched in 114 (31 hypermethylated and 83 hypomethylated) pathways, of which 75 (0 hypermethylated and 75 hypomethylated) under CG context, 13 (13 hypermethylated and 0 hypomethylated) under CHG context, and 26 (18 hypermethylated and 8 hypomethylated) under CHH context (Table 1.3).
  • Table 1.4- After culturing Huh-7 cells in DMEM medium with HG for 72 hours followed by treating with ethanol (vehicle) and 25 ⁇ M 25HC3S for 4 hours, genomic DNA from 5,000 cells were extracted using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). Each sample (6 ⁇ g) was used for analysis of the whole genome bisulfite sequencing (WGBS). The KEGG analysis shows that the demethylated genes are involved in MAPK signaling pathway (p 0.00087). Of the 257 total genes in the MAPK signaling pathway, 23 were demethylated by the 25HC3S treatment. Of these 23 genes, 10 were found to be methylated by a HG environment (shown in bold).
  • the first column represents the gene name
  • the second column shows the location of differential methylation region in the chromosome
  • the third column shows the methylation rates by high glucose (HG) and demethylation rates induced by 25HC3S.
  • the first column represents the gene name
  • the second column shows the location of differential methylation region in the chromosome
  • the third column shows the methylation rates by high glucose (HG) and demethylation rates induced by 25HC3S.
  • DUSP7,8 and MAPK1 key genes and their target genes CREB5, PRDX6, and BAD in the MAPK pathway, as well as the key genes CACNA1D(CaV1), CACNA1A (CaV2), and CACNA1H (CaV3) (encoding for calcium voltage-gated channel subunits) and their targeting genes (PGC1A, HMGR, and FAS) in the calcium-AMK pathway were determined by RT-PCR analysis.
  • the DUSP-MAPK signaling pathway is the major pathway involved in cell survival/death and anti-oxidization, and the calcium signaling pathway controls lipid and energy metabolism.
  • 25HC3S increased expression of DUSP8 by 5-fold and its targeting gene, CREB5, by up to 20-fold, which is the key element involved in cell survival and death ( FIGS. 3 A and 3 B ).
  • 25HC3S treatment significantly increased expression of key genes involved in the calcium signaling pathway, and its down-stream element, PGC1A, by 12-fold, while it decreased expression of HMGR and FAS genes by ⁇ 90%, which encode the key enzymes controlling energy metabolism in mitochondria, cholesterol biosynthesis, and fatty acid biosynthesis, as shown in FIGS. 3 C and 3 D .
  • FIGS. 4 A and B Genetic analysis of different GO processes, a collection of genes associated with a specific biological functional process, revealed that at 8 hours, the majority of up-regulated pathways are involved in cell survival ( FIGS. 4 A and B ); in contrast, majority of down-regulated genes are involved in lipid metabolism ( FIGS. 4 C and D ).
  • the up-regulated genes related with anti-apoptosis are listed in FIG. 4 E ; and the down-regulated genes related with lipid metabolism (decreased by 50% to 95%) are listed in FIG. 4 F .
  • the detailed individual up-regulated genes are listed in Table 1.7; the down-regulated genes are listed in Table 1.8.
  • the Calcium, AMPK, and PPAR signaling pathways are ones involved in regulation of energy, lipids, and carbohydrate metabolisms.
  • the Ca 2+ /calmodulin-dependent protein kinase (CaMKK) and AMPK signaling pathway increases expression and decreases acetylation of PGC-1 ⁇ , which regulates mitochondrial biogenesis and lipid metabolism.
  • CaMKK calcium-dependent protein kinase
  • mRNA level mRNA level
  • 25HC3S globally regulated metabolic pathways mainly via the Calcium-AMPK signaling pathway as shown in FIG. 5 .
  • 25HC and 25HC3S are potent modulators in regulating DNA methylation.
  • PGC-1 ⁇ is a key regulator of mitochondrial biogenesis, oxidative phosphorylation, and mitochondrial antioxidant defense, and it is also responsible for maintaining metabolic homeostasis.
  • PGC-1 ⁇ expression is up-regulated by the CREB protein and the AMPK signaling pathway.
  • 25HC3S up-regulates expression of CREB and AMPK via demethylating 5m CpG in their promoter regions, and subsequently increases intracellular PGC-1 ⁇ levels ( FIG. 3 ), which provides a detailed mechanism for how 25HC3S functions as proposed in FIG. 5 .
  • 25HC3S suppresses DNMTs activities and demethylates 5m CpG in the key promoter regions.
  • the demethylation up-regulates gene expression and increases MAPK-CREB signalings, which blocks cell apoptosis, induces cell proliferation.
  • the demethylation also up-regulates calcium-AMPK signaling, resulting in inhibition of SREBP-1 activity by which inhibits fatty acid and triglyceride biosynthesis, and inhibition of HMGCR expression, decreases in cholesterol biosynthesis, and increases in the levels of malnonyl-CoA as shown FIG. 5 .
  • the oxysterol sulfate, 25-hydroxycholesterol-3-sulfate (25HC3S) has been shown in this example to play an important role in lipid metabolism, inflammatory response, and cell survival.
  • Example 1 provides a study of the molecular mechanism by which 25HC3S functions as an endogenous epigenetic regulator.
  • high glucose induces lipid accumulation by increasing promoter CpG methylation of key genes involved in development of non-alcoholic fatty liver diseases (NAFLD).
  • NAFLD non-alcoholic fatty liver diseases
  • 25HC3S converted the 5mCpG to CpG in the promoter regions of 1074 genes involved in 79 KEGG pathways.
  • Messenger RNA array analysis showed that the up-regulated genes encoding for key elements in keeping cell survival and the down-regulated genes encoding for key enzymes in decreasing lipid biosynthesis.
  • the results shown in Example 1 indicate that the expression of these elements and enzymes are regulated by the demethylated signaling pathways, and 25HC3S DNA demethylation of 5mCpG in promoter regions is a potent regulatory mechanism.
  • the objectives of this study were to determine the plasma pharmacokinetics of [4- 14 C]-25HC3S-derived radioactivity in male Sprague Dawley rats, determine the routes of elimination and excretion mass balance of [4- 14 C]-25HC3S-derived radioactivity in male Sprague Dawley rats, determine the tissue distribution and tissue pharmacokinetics of [4- 14 C]-25HC3S-derived radioactivity using quantitative whole body autoradiography methods in male Sprague Dawley and Long Evans rats following a single intravenous (bolus) dose, and to provide plasma, urine, and fecal homogenate samples for metabolite profiling of [4- 14 C]-25HC3S-derived radioactivity.
  • mice Nine male Sprague Dawley rats (Group 1) were designated for the pharmacokinetic phase, 3 male Sprague Dawley rats (Group 2) for the excretion mass balance phase, and 7 male Sprague Dawley rats (Group 3) and 9 male Long Evans rats (Group 4) for the tissue distribution phase. All animals received a single intravenous dose of [ 14 C]-25HC3S at 10 mg/kg and a target radioactivity of 225 ⁇ Ci/kg. Blood samples were collected from all Group 1 animals at approximately 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours post-dose. Urine and feces were collected from all Group 2 animals periodically through 168 hours post-dose.
  • [4- 14 C]-25HC3S and/or its metabolites were broadly distributed and detected by quantitative whole body autoradiography in all tissues except the eye (lens). Plasma concentrations were similar to those determined in the pharmacokinetics phase.
  • the whole blood C max was 8530 ng-equiv/g, and AUC last was 25,200 h*ng-equiv./g.
  • the T 1 ⁇ 2 was 44.3 hours in plasma and 52.2 hours in whole blood; differences in plasma T 1 ⁇ 2 between the PK phase and the QWBA phase are due to the difference in blood collection time points.
  • the C max and AUC last for [4- 14 C]-25HC3S-derived radioactivity were highest in the liver: up to 87,900 ng-equiv./g and 364,000 h.ng/g, respectively.
  • Kidney (all sections), small intestine (wall), lung, and adrenal gland concentrations ranged from 43,200 ng-equiv./g to 13,600 ng-equiv./g, higher than the maximum plasma concentration of 12,400 ng-equiv./g.
  • Thymus, bone (femur), uveal tract, fat, testes, and brain concentrations were lowest relative to the other tissues: ⁇ 5000 ng-equiv./g (around 1500 ng-equiv./g).
  • Remaining tissues had concentrations between 5000 and 10,800 ng-equiv./g.
  • the T max was most often 0.083 to 0.5 hours post-dose. Concentrations were below quantitation limit in all tissues except adrenal gland, harderian gland, liver, and small intestine by 168 hours post-dose.
  • the tissue:plasma ratios were high for liver and small intestine (wall) at 11.4 and 7.44, respectively. High liver and small intestine concentrations are consistent with extensive biliary (fecal) excretion following an intravenous dose. All other tissue:plasma ratios demonstrated limited affinity for remaining tissue types.
  • Plasma, urine, and feces from rats were analyzed for determination of 25HC3S related radiolabeled materials. Samples were profiled using high performance liquid chromatography with radiodetection and metabolic characterization was performed using mass spectrometry and tandem mass spectrometry analysis.
  • Plasma pools were made from Group 1 rats at the 0.083, 0.25, 0.5, and 1-hour collection time points. From these Group 1 sample pools and from a Group 3 0.083-hour plasma sample, the largest component present in the 0.083- and 0.25-hour collections was attributed to the parent 25HC3S representing about 58% to 92% of the radioactivity. Three metabolites present at > 10% of the radioactivity in the 0.5- and 1-hour collections were M14 (up to 15% relative observed intensity), M24 (up to 13% relative observed intensity), and M28 (up to 83% relative observed intensity).
  • Urine pools were prepared for Group 2 at 0 to 6 and 6 to 12 hours postdose. The largest component present was attributed to the parent 25HC3S representing about 78% to 93% of the radioactivity. A total of 4 metabolites were identified, although no metabolites were present at > 1.2% of dose or > 10% relative observed intensity. Four metabolites present at ⁇ 10% relative observed intensity in at least 1 sample were M7 ( ⁇ 5% relative observed intensity), M16 ( ⁇ 3% relative observed intensity), M19 ( ⁇ 6% relative observed intensity), and M25 ( ⁇ 5% relative observed intensity).
  • Feces pools were prepared for Group 2 at 0 to 12, 12 to 24, and 24 to 48 hours postdose.
  • metabolites A total of fourteen metabolites were identified. Four metabolites present at ⁇ 5% of dose were M1 (21% of dose and 23% to 30% relative observed intensity), M2 (7% of dose and 4% to 12% relative observed intensity), M3 (15% of dose and 13% to 23% relative observed intensity), and M4 (8% of dose and 6% to 12% relative observed intensity). Parent 25HC3S was present at 2% of dose (1% to 5% relative observed intensity).
  • the primary metabolic pathways involved oxidation of 25HC3S resulting in the conversion of the sulfate group to a hydroxyl group followed by further oxidation to form bile acid structures related to deoxycholic acid and cholic acid or their isomers.
  • glutathione conjugation of deoxycholic acid or an isomer of deoxycholic acid was suggested by the presence of a metabolite having the corresponding molecular weight for that structure.
  • desmosterol sulfate nor 25-hydroxycholesterol was detected in any of the plasma, urine, or feces samples.
  • Remaining tissues had concentrations between 3600 ng-equiv./g and 10,700 ng equiv./g.
  • the T max was 6 hours postdose or less.
  • tissue concentrations were near or below the quantitation limit in all tissues except adrenal gland and liver.
  • the tissue:plasma ratios were highest for the small intestine (wall, 15.4) followed by the liver and adrenal gland at 6.96 and 6.64, respectively. High liver and small intestine concentrations are consistent with oral administration and biliary (fecal) excretion. All other tissue:plasma ratios demonstrated limited affinity for remaining tissue types.
  • Radiolabeled components in plasma and feces extracts were profiled and identified using radio-high performance liquid chromatography (HPLC) and high performance liquid chromatography/mass spectrometry (HPLC/MS) methods.
  • HPLC radio-high performance liquid chromatography
  • HPLC/MS high performance liquid chromatography/mass spectrometry
  • Plasma pools were prepared for Group 1 (75 mg/kg, [ 14 C]-25HC3S) samples collected at 2, 4, and 6 hours postdose.
  • the primary radiolabeled component was parent 25HC3S which was present at 63% relative observed intensity (ROI) and a concentration of 2090 ng-equiv./g.
  • ROI relative observed intensity
  • One metabolite M29 was identified as 25-hydroxycholesterol with 37% ROI and a concentration of 1233 ng-equiv./g.
  • the plasma collections at 4 and 6 hours post-dose did not contain sufficient concentrations for radioprofiling.
  • Feces pools were prepared for Group 2 (75 mg/kg, [ 14 C]-25HC3S) samples collected from 0 to 24, 24 to 48, 48 to 72, 72 to 96, 96 to 120, 120 to 144, and 144 to 168 hours postdose. A total of eleven metabolites were identified. None of the metabolites were present at ⁇ 5% of dose.
  • Metabolites present at 2 - 5% of dose were M1 (4.5% of total dose and 1% - 69% ROI), M3 (4.6% of total dose and 1% - 44% ROI), M4 (2.0% of total dose and 0% - 10% ROI), M8 (3.1% of total dose and 1% - 46% ROI), M29 (1.9% of total dose and 0% - 2% ROI), and M30 (3.3% of total dose and 0% - 5% ROI).
  • the primary radiolabeled component was parent 25HC3S which was present at 71.1% of total dose (0% - 88% ROI).
  • Radiolabeled desmosterol sulfate was not found in any of the plasma or feces samples.
  • the primary metabolic pathways involved oxidation of 25HC3S, resulting in the conversion of the sulfate group to a hydroxyl group followed by further oxidation to form bile acid structures related to deoxycholic acid and cholic acid or their isomers and 25-hydroxycholesterol.

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