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WO2008006007A2 - Régulateur de la conductance transmembranaire de la mucoviscidose (cftr) défectueux provoquant une synthèse accrue de sphingolipide - Google Patents

Régulateur de la conductance transmembranaire de la mucoviscidose (cftr) défectueux provoquant une synthèse accrue de sphingolipide Download PDF

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WO2008006007A2
WO2008006007A2 PCT/US2007/072801 US2007072801W WO2008006007A2 WO 2008006007 A2 WO2008006007 A2 WO 2008006007A2 US 2007072801 W US2007072801 W US 2007072801W WO 2008006007 A2 WO2008006007 A2 WO 2008006007A2
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ceramide
agent
synthase
cells
synthesis
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PCT/US2007/072801
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WO2008006007A3 (fr
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Tilla Worgall
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Columbia University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered

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  • the present invention describes certain aspects of abnormal sphingolipid metabolism in cystic fibrosis, and provides therapeutic methods for treating cystic fibrosis and P. aeruginosa infections.
  • Cystic fibrosis is the most frequent severe autosomal recessive disease in Caucasians where it occurs in one of 2500 live births 8 . It is caused by mutations in CFTR, a chloride channel. l The protein is part of a large family of ABC transporters that is activated by ATP and phosphorylated by AMP. The most frequent disease causing mutation, ⁇ F508, is in the ATP binding domain and leads to two types of functional defects: a trafficking defect causing retention in the endoplasmic reticulum and a gating defect resulting in decreased time in the open state. There are more than 1000 known mutations that differ in severity 1 .
  • the cftr gene is expressed in epithelial lung and intestinal cells and at low levels in lung fibroblasts, cultured cells of lymphocytic lineage and freshly isolated blood mononuclear cells, monocytes, neutrophils and alveolar macrophages 9"11 .
  • the mechanism by which mutations in the gene encoding the CFTR protein lead to the complex clinical phenomenon associated with cystic fibrosis are unclear. Explanations must take into account that this apical chloride channel is involved in fluid secretion and that increased viscosity of ductal fluids contributes to the pathology. However, a number of findings in cystic fibrosis are less obviously related to chloride channel function, such as the increased adherence of P.
  • aeruginosa to lung epithelial cells, a susceptibility to chronic lung infections, and an excessive inflammatory state present in cystic fibrosis children already at an early age.
  • Pseudomonas aeruginosa infections are the major cause of morbidity and mortality.
  • Increased phospho lipase A2 (PLA2) activity and NF-Kappa beta mediated interleukin-8 (IL-8) secretion are only two contributors that lead to an increased inflammatory state in CF.
  • Other clinical findings show abnormal plasma fatty acid profiles and low plasma concentrations of high-density lipoprotein (HDL) cholesterol.
  • HDL high-density lipoprotein
  • ceramide-1 -phosphate is an obligatory regulator of PLA2 ⁇ that is highly increased in CF, and the glycosphingo lipid GMl functions as part of a receptor complex for P. aeruginosa 3'7 .
  • Certain aspects of the invention are directed to methods for treating cystic fibrosis in an animal, by administering an agent that inhibits the synthesis of one or more sphingolipids in cells from the animal in an amount that ameliorates one or more symptoms of cystic fibrosis.
  • Some agents are enzyme inhibitors that inhibit the activity of an enzyme that catalyzes part of the de novo sphingo lipid pathway, including serine-palmitoyl transferase, ceramide synthase, sphingosine kinase, UDP-glucose ceramide glucosyltransferase and glucosylceramide synthase.
  • enzymes that can be inhibited to treat cystic fibrosis include an enzyme that is a member selected from the group: SPTLCl serine palmitoyltransferase, long chain base subunit 1, Degsl degenerative spermatocyte homo log 1, SGMSl sphingomyelin synthase 1, Sphingomyelin synthase 2, ASAH2 N-acylsphingosine amidohydrolase (non- lysosomal ceramidase) 2, LASS6 LAGl homolog, ceramide synthase 6, LASS5 LAGl homolog, ceramide synthase 5, LASS4 LAGl homolog, ceramide synthase 4, LASS3 LAGl homolog, ceramide synthase 3, LASS2 LAGl homolog, ceramide synthase 2, and LASS2 LAGl homolog, ceramide synthase 1.
  • SPTLCl serine palmitoyltransferase long chain base subunit
  • Some therapeutic enzyme inhibitors that can be used in this aspect of the invention include (a) myriocin; (b) cycloserine; (c) Fumonisin Bl; (d) PPMP; (e) compound D609; (f) methylthiodihydroceramide; (g) propanolol; and (h) resvaratrol.
  • the agent is a member of the group by ceramide, dihydroceramide, fenretinide, 4-oxo-HPR or a derivative, variant or fragment thereof, and an unsaturated fatty acid that is a member of the group by linoleic acid, linolenic acid, cholesterol DHA, eicosapentaenoic acid, oleic acid, and arachidonic acid.
  • de novo sphingolipid synthesis is reduced using a therapeutic agent that is an isolated antisense nucleic acid or small interfering RNA that is sufficiently complementary to the sense strand of the gene or to an mRNA encoding an enzyme in the sphingolipid synthesis pathway to permit hybridization to the sense strand of the gene or to the mRNA of the respective enzyme, and wherein the hybridization event prevents expression of the enzyme thereby decreasing the amount of sphingolipids produced in the cell.
  • a therapeutic agent that is an isolated antisense nucleic acid or small interfering RNA that is sufficiently complementary to the sense strand of the gene or to an mRNA encoding an enzyme in the sphingolipid synthesis pathway to permit hybridization to the sense strand of the gene or to the mRNA of the respective enzyme, and wherein the hybridization event prevents expression of the enzyme thereby decreasing the amount of sphingolipids produced in the cell.
  • the agent is administered to achieve a serum level of between about 1 nanogram per milliliter and about 10 micrograms per milliliter in the patient, or from between about 1 nanogram per milliliter and about 7 micrograms per milliliter.
  • the daily dose of the agent is between about 0.1 nanograms per kilogram body weight per day and about 20 milligrams per kilogram body weight per day, or between about 1 nanogram per kilogram body weight per day and about 10 milligrams per kilogram body weight per day.
  • a therapeutic amount of an agent that reduces sphingolipid synthesis in a biological sample taken from the animal is determined by : a. taking a first biological sample from the animal before administering the agent, b. determining a sphingolipid level in the first sample, c. administering an amount of the agent, d. taking a second biological sample from the animal after administering the agent, e. determining a sphingolipid level in the second sample, f. if the sphingolipid level in the second sample is significantly lower than in the first sample, then concluding that the amount of the agent is a therapeutic amount, and g. if the sphingolipid level in the second sample is not lower than the level in the first sample, then repeating steps a-f.
  • the level of sphingolipid synthesis in the biological sample can be determined using any method known in the art, including by measuring the level of sphingolipid mass using mass spectrometry or the amount of an individual sphingolipid that is a member of the group by sphinganine, C 16, C 18, C20, C22, C24, C 26, dihdyroceramide, sphingosine, sphingosine-s-phosphate, and sphingomyelin.
  • the animal is a human and the biological sample is peripheral blood mononuclear cells, bronchial lavage or lung epithelial cells.
  • P. aeruginosa infections in an animal are treated as described above for cystic fibrosis by administering an agent that inhibits the synthesis of one or more sphingo lipids in cells from the animal in an amount that ameliorates one or more of the symptoms of the disease.
  • Certain other aspects are directed to methods for identifying enzyme inhibitors that reduce the synthesis of a compound in the sphingo lipid synthesis pathway.
  • a method of identifying serine palmitoyl transferase inhibitors in an animal cell-based assay using cells expressing defective CFTR or no CFTR and overexpressing serine palmitoyl transferase by: a) providing test cells overexpressing serine palmitoyl transferase; b) contacting the test cells with a test compound; c) determining the level of ketosphinganine or sterol-regulatory element-binding protein produced by the test cells, d) comparing the determined level in the test cells to a level of ketosphinganine or sterol- regulatory element-binding protein in control cells that are not exposed to the test compound, and e) determining that the test compound is a serine palmitoyl transferase inhibitor if the level of ketosphinganine or sterol-regulatory element-bind
  • the expression of sterol-regulatory element-binding protein is indicated by a sterol-regulatory element reporter gene, including luciferase, green fluorescent protein and lacz.
  • the cell used in the drug screening assay is a member selected from the group including an IB3 cell, an epithelial cell from an animal having cystic fibrosis, a cell transduced to express defective CFTR or no CFTR, an A549 cell.
  • compositions including any combination of agent described above for decreasing sphingo lipid synthesis through de novo synthesis or recycling pathways, including ceramide and DHA, ceramide and EPA and ceramide plus EPA and DPA; ceramide and a compound selected from the group by fenretinide and 4-oxo- HPR; ceramide and a compound selected from the group by (a) myriocin; (b) cycloserine; (c) Fumonisin Bl; (d) PPMP; (e) compound D609; (f) nethylthiodihydroceramide; (g) propanolol; and (h) resvaratrol.
  • agent described above for decreasing sphingo lipid synthesis through de novo synthesis or recycling pathways including ceramide and DHA, ceramide and EPA and ceramide plus EPA and DPA; ceramide and a compound selected from the group by fenretinide and 4-oxo- HPR; ceramide
  • compositions include an isolated nucleic acid that is a member of the group by an antisense DNA, antisense RNA, and small interfering RNA, which nucleic acid is sufficiently complementary to the gene or mRNA encoding an enzyme in the sphingolipid de novo synthesis pathway to permit specific hybridization to the gene or mRNA, respectively.
  • Other aspects include methods to treat cystic fibrosis using an agent that reduces NF-kappa B and phospholipase A2, including fenretinide and ceramide and other agents known in the art.
  • FIG. 1 is a diagram of sphingo lipid synthesis through de novo and recycling pathways.
  • FIG. 2 shows the metabolic pathway caused by defective CFTR. Defective
  • FIG. 3(a) shows the increased synthesis of ceramide from 3 H-serine that reflects de-novo synthesis by SPT, and 3(b) ceramide synthesis from 3 H-sphinganine that reflects synthesis through the recycling pathway; standard deviations shown are of measurements in triplicates.
  • FIG. 4 (a) shows that SRE -mediated gene transcription is increased in the
  • Fig. 5 shows that ceramide and Doxosahexaenoic acid (DHA) increase linoleic acid mass in C38 control cells and in IB3 cells expressing mutant CFTR.
  • DHA Doxosahexaenoic acid
  • FIG. 6 shows mass spectrometry data confirming that sphingo lipid synthesis and mass is increased in cells expressing defective CFTR.
  • FIG. 7 shows the time and a concentration curve of fenretinide in CHO fibroblast cells.
  • FIG. 8 shows the effect of fenretinide and palmitic acid on SRE -mediated gene expression and transcription in IB3 cells (expressing ⁇ F508, the most prevalent (-90%) mutation in CF) compared to C38 control cells.
  • FIG. 9 shows that fenretinide decreases SRE -mediated gene transcription in cells that express no CFTR to levels seen in cells that express normal CFTR.
  • FIG. 10 shows that fenretinide and ceramide increased cellular linoleic acid levels in control C38 cells (black bar) and in Ib3 cells expressing defective CFTR.
  • FIG. 11 shows that inhibition of de-novo sphingolipid synthesis by treatment with ceramide, EPA or myriocin decreased P. aeruginosa-GFP adhesion to CF-model lung epithelial cells.
  • Fig. 12 DHA and EPA alone or in combination decrease interleukin-8 secretion in immortalized human in lung epithelial cells (IHAEo).
  • FIG. 13 Assessment of the de-novo (a) and reverse or recycling pathways of (b) sphingolipid synthesis pathways in human peripheral blood mononuclear cells.
  • 3 H-serine was used to evaluate de-novo sphingolipid synthesis (a).
  • 3 H-sphingosine was used to assess sphingolipid synthesis through recycling pathways, (b).
  • De-novo sphingolipid synthesis correlates inversely with low plasma HDL cholesterol ( ⁇ 40 mg/dl).
  • FIG. 14 The results show that incubation in the presence of 10 ⁇ M fenretinide significantly (p ⁇ 0.05) decreases phosphatidylcholine specific PLA2 activity .
  • FIG. 15 Results demonstrate that NF -Kappa B is increased in control condition (1% BSA) and that Fenretinide decreases NF-Kappa B activity in IB3 cells comparably to Parthenolide which is known to decrease NF-Kappa B activity in CFTR defective cells
  • FIG. 16 Results demonstrate that inhibition of dihydroceramide desaturase using the specific Dihydroceramide desaturase inhibitor GT 11 decreases SRE -mediated gene transcription in CHO fibroblasts .
  • a therapeutically effective amount of a protein or polypeptide (i.e., an effective dosage) or nucleic acid (such as antisense nucleotides), is an amount that achieves the desired therapeutic result.
  • a therapeutically effective amount includes an amount that ameliorates one or more symptoms of the disease, or produces at least one effect selected from the group comprising a reduction of sphingolipid levels, gml gangliosides, a reduction of expression or activity of one or more of the enzymes serine palmitoyl transferase, sphingosine kinase, ceramide synthase/desaturase, or glucosylceramide synthase or a reduction of P. aeruginosa infection in cystic fibrosis patients or other patients having P. aeruginosa infections.
  • sphingolipid de novo synthesis includes, without limitation, (i) inhibiting sphingolipid de novo synthesis without inhibiting all other synthetic pathways, (ii) inhibiting sphingolipid de novo synthesis more than most or any other synthetic pathway, and/or (iii) inhibiting sphingolipid de novo synthesis without inhibiting any other synthetic pathway.
  • protein or “polypeptide” or “peptide” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). All “proteins” or “polypeptides” or “peptides” described herein include variants, derivatives and fragments and modifications thereof.
  • Peptide or protein variants means polypeptides that may contain one or more substitutions, additions, deletions and/or insertions such that the therapeutic, antigenic and/or immunogenic properties of the peptides encoded by the variants are not substantially diminished, relative to the corresponding peptide.
  • modifications may be readily introduced using standard mutagenesis techniques, such as oligonucleotide directed site-specific mutagenesis as taught, for example, by Adelman et al. (DNA, 2:183, 1983).
  • the antigenicity or immunogenicity of a peptide variant is not substantially diminished.
  • Variants also include what are sometimes referred to as "fragments.” Fragments also include peptides that may contain one or more amino acid substitutions, additions, deletions and/or insertions, such that the therapeutic, antigenic and/or immunogenic properties of the peptide variants are not substantially diminished, relative to the corresponding peptide.
  • Protein derivatives means that a protein has been derivatized.
  • Derivatization is a technique used in chemistry which transforms a chemical compound into a product of similar chemical structure, called a derivative.
  • a specific functional group of the compound participates in the derivatization reaction and transforms the educt to a derivate of deviating reactivity, solubility, boiling point, melting point, aggregate state, or chemical composition. Resulting new chemical properties can be used for quantification or separation of the educt or can be used to optimize the compound as a therapeutic agent.
  • Derivatives include the protein modifications described herein.
  • reporter gene any gene which encodes a product whose expression is detectable.
  • a reporter gene product may have one of the following attributes, without restriction: fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., lacZ or luciferase), or an ability to be specifically bound by a second molecule (e.g., biotin or an antibody-recognizable epitope).
  • protein activity or “biological activity” is meant the functional activity of a given protein in a standardized quantity of tissue or cells.
  • the activity of a protein, as a whole, in such a sample can be modified as a result of a change in the quantity of the given protein present (e.g., as a result of change in gene expression) or as a result of a change in the function of each protein molecule present in the sample (e.g., as a result of an alteration in amino acid sequence).
  • a “mature form” protein is meant the protein form that results from complete, eukaryotic, post-translational processing.
  • nucleic acid refers to both RNA and DNA, including cDNA, genomic DNA, mRNA and synthetic (e.g., chemically synthesized, non- naturally occurring sequences) DNA including antisense nucleic acids, small interfering RNA and isolated nucleic acids.
  • the nucleic acid can be double-stranded or single-stranded (i.e., a sense or an antisense single strand).
  • cDNA means a single-stranded DNA synthesized in the laboratory using messenger RNA as a template and the enzyme reverse transcriptase.
  • isolated nucleic acid refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a mammalian genome.
  • isolated as used herein with respect to nucleic acids also includes any non-naturally-occurring nucleic acid sequence, since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.
  • An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent.
  • an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences.
  • an isolated nucleic acid includes an engineered nucleic acid such as a DNA molecule that is part of a hybrid or fusion nucleic acid.
  • the term "subject” includes mammals, e.g., humans, dogs, cows, horses, kangaroos, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
  • the preferred subject is a human.
  • Bio samples include solid and body fluid samples.
  • the biological samples of the present invention may include tissue, organs, cells, protein or membrane extracts of cells, blood or biological fluids such as blood, serum, bronchial lavage, ascites fluid or brain fluid (e.g., cerebrospinal fluid).
  • Preferred samples are peripheral blood mononuclear cells and lung epithelium.
  • AA arachidonic acid (20:4); ACC, acetyl-CoA carboxylase; Ad, Adenovirus; aGMl, asialylated GM1;CF, cystic fibrosis; CFTR, cystic fibrosis transduction regulator; DHA, docosahexaenoic acid (22:6); EPA, eicosapentaenoic acid (20:5); FAS; fatty acid synthase; GC; gas-chromatography, GFP, green fluorescent protein; HDL, high- density lipoprotein; SPT, serine-palmitoyl transferase; IL-8, interleukin-8; LC-MS/MS; liquid chromatography tandem mass spectrometry, P.
  • aeruginosa Pseudomonas aeruginosa
  • PLA2 Phospholipase A2
  • SRE Sterol regulatory element, SREBP
  • SRE -binding protein sphingolipid, sphingolipid; sphingolipid synthesis, sphingolipid synthesis; SMS, sphingomyelin synthase; SK, sphingosine kinase; siRNA, small interfering RNA; TLC, thin-layer chromatography.
  • Sphingolipid uptake and synthesis are known to be tightly regulated and their metabolites are relevant to CF.
  • sphingolipid synthesis sphingolipid synthesis
  • sphingolipid synthesis through the de-novo pathway is abnormally high (about two times normal levels) in cells expressing defective CFTR.
  • Increased sphingolipid synthesis leads to a 50 % higher sphingolipid mass.
  • This increased sphingolipid synthesis can lead to increases in GmI gangliosides that facilitate the attachment of P. aeruginosa to lung epithelial cells.
  • ceramide-1 -phosphate that is known to be an activator of phospholipase A2, which in turn generates arachidonic acid (AA).
  • AA is a key precursor of inflammatory eicosanoids.
  • Increased NF -kappa B is a hallmark of cystic fibrosis that leads to increased IL-8 secretion.
  • cells expressing defective CFTR with abnormally high sphingolipid synthesis can be treated with various enzyme inhibitors or other agents/drugs that decrease sphingolipid synthesis causing it to return to levels seen in cells expressing wild-type CFTR.
  • These same agents can be administered therapeutically to cystic fibrosis patients to normalize sphingolipid.
  • Certain embodiments of the present invention are directed to methods for treating a cystic fibrosis patient by administering one or more agents that inhibit enzymes in the sphingolipid de novo synthesis pathway, including but not limited to: serine palmitoyl transferase (such as myriocin (0.1-10 ⁇ M), cycloserine (0.5-5 mM)), sphingosine kinase (such as dimethyl sphingosine), ceramide synthase/desaturase (such as fumonisin Bl (0.1-40 ⁇ M)), ceramidase, and glucosylceramide synthase (such as PPMP or n-butyldeoxynojirimycin), and UDP-glucose ceramide glucosyltransferase.
  • enzymes can also be inhibited by antisense nucleotides and si RNA.
  • Other methods for treating a cystic fibrosis patient include administering an amount of one or more agents that decrease sphingo lipid synthesis through an end product feedback mechanism, such as ceramide (including c6 ceramide and dihydro ceramide) (2-20 ⁇ M), 4-HPPv (fenretinide, 1-10 ⁇ M), glucosylceramide synthase inhibitors PPMP, Dihydroceramide desaturase inhibitors (i.e. GTl 1, 1-4 ug/ml) (NOTE: I had this in the other patent with SPvEBP already), including derivatives, variants, fragments or modifications thereof.
  • ceramide including c6 ceramide and dihydro ceramide
  • 4-HPPv fenretinide, 1-10 ⁇ M
  • glucosylceramide synthase inhibitors PPMP glucosylceramide synthase inhibitors
  • Dihydroceramide desaturase inhibitors i.e. GTl 1, 1-4 ug/ml
  • Unsaturated fatty acids such as oleic acid, AA, DHA, EPA, cholesterol, oleic acid, linoleic acid, linolenic acid or combinations thereof can also be used to decrease sphingo lipid synthesis by negative feedback.
  • Other embodiments include treating a cystic fibrosis patient with antisense nucleic acids or small interfering PvNAs that reduce sphingolipid synthesis by inhibiting expression of one or more of the relevant enzymes, including serine palmitoyl transferase, sphingosine kinase, (dihydro)ceramide synthase/desaturase, UDP-glucose ceramide glucosyltransferase, and glucosylceramide synthase to decrease sphingolipid synthesis.
  • the nucleic acid sequences for the cDNA for these enzymes and some others are set forth in the Sequence Listing at the end of the application with SEQ ID NOs. 1-14. They are:
  • SEQ ID NO. 1 Serine palmitoyltransferase Long Chain Base Subunit 1, Amino Acid Sequence, Homo sapiens;
  • SEQ ID NO. 2 Serine palmitoyltransferase Long Chain Base Subunit 1, Nucleotide Sequence, Homo sapiens;
  • SEQ ID NO. 3 Serine palmitoyltransferase Amino Acid Sequence Long Chain Base Subunit 2, Homo sapiens;
  • SEQ ID NO. 4 Serine palmitoyltransferase Amino Acid Sequence Long Chain Base Subunit 2, Homo sapiens;
  • SEQ ID NO. 5 Sphingosine kinase 1, Homo sapiens, Amino Acid Sequence
  • SEQ ID NO. 6 Sphingosine kinase 1, Homo sapiens, Nucleic Acid Sequence
  • SEQ ID NO. 7 Sphingosine kinase 2, Homo sapiens, Amino Acid Sequence
  • SEQ ID NO. 8 Sphingosine kinase 2, Homo sapiens;
  • SEQ ID NO. 11 Fatty acid desaturase (ceramide synthase) Homo sapiens, Gene Name DEGS2, Amino Acid Sequence;
  • SEQ ID NO. 12 Fatty acid desaturase (ceramide synthase), Homo sapiens, Gene Name DEGS2, Nucleotide sequence;
  • SEQ ID NO. 14 Fatty acid desaturase, Gene Name DEGSl, Nucleic Acid Sequence, Homo Sapiens.
  • genes in the sphingolipid synthesis pathway that are targets for enzyme inhibitors and antisense or siRNA inhibition include:
  • Changes in sphingolipids can be monitored by determining sphingolipid mass or the amount of individual sphingolipids (including sphinganine, C16/C18/C20/C22/C24/C 26, (Dihdyro) ceramide, sphingosine, sphingosine -s-phosphate, and sphingomyelin) in a biological sample taken from the patient as is described in the examples.
  • Preferred biological samples include peripheral blood mononuclear cells or lung and intestinal epithelium.
  • Certain other embodiments of the present invention are directed to in vitro drug screening methods to identify compounds that: inhibit serine palmitoyl transferase, (dihydro)ceramide synthase/desaturase, ceramidase, glucosylceramide synthase, sphingosine kinase and/or sphingomyelinase; or inhibit the production or accumulation of sphingolipids, GmI gangliosides, or ceramide- 1 -phosphate in a cell, preferably a cell expressing defective CFTR or no CFTR.
  • the term "inhibit” as used herein means to decrease or reduce enzyme activity or enzyme expression compared to pretreatment levels, or decrease synthesis of a substance being determined.
  • Cystic fibrosis patients exhibit a state of severe and chronic inflammation, characterized by increased phospholipase A2 and N-kB activity as well as chronic lung infection, particularly with P. aeruginosa.
  • a patient preferably a cystic fibrosis patient, preferably a human
  • administering a therapeutic amount of one (or more) agents that decrease sphingolipid synthesis, in an amount that ameliorates one or more of the symptoms of cystic fibrosis and/or the P.
  • Sphingo lipids are ubiquitous and form a large family of lipid second messengers, sphingolipid are synthesized de-novo or through recycling pathways of end products (FIG. 1).
  • SPT serine-palmitoyl transferase
  • SPT is the rate limiting enzyme of de-novo sphingolipid synthesis.
  • Ceramide is a central intermediary that is metabolized to sphingosine, ceramide-1 -phosphate, sphingo-myelin or glucosylceramides.
  • An equally important synthetic pathway occurs through recycling of long chain bases derived from these end products (recycling pathway).
  • Ceramide is metabolized to sphingomyelin, glucosylceramides, ceramide-1 -phosphate or sphingosine.
  • cystic fibrosis are the two sphingolipid metabolites (1) ceramide-1 -phosphate, which directly activates cPLA2.
  • sphingolipid synthesis is inhibited by its end-product ceramide, glucosylceramide, sphingosine, sphinganine, and by unsaturated fatty acids.
  • Unsaturated fatty acids regulate sphingolipid synthesis through both the de novo and recycling pathways.
  • Saturated fatty acids i.e. palmitic acid increase de-novo sphingolipid synthesis.
  • Laura acid (12:0) a substrate for reverse ceramidases, and sphingosine, increase sphingolipid synthesis through recycling pathways 47 .
  • SREBP sterol-regulatory element-binding protein
  • cystic fibrosis patients have altered fatty acid profiles, low plasma HDL-cholesterol and increased cholesterol synthesis.
  • Our lab showed that increased sphingolipid synthesis increased the levels of ceramide-1 -phosphate and glucosylceramide, which are both intrinsically related to increased phospholipase A2 (PLA2) activity increased P. aeruginosa adherence to epithelial cells and increased NF-Kappa B activity.
  • PPA2 phospholipase A2
  • mutant CFTR While cells that express normal CFTR take up long base sphingoid bases that lead to feedback inhibition of sphingolipid synthesis (FIG. 2a), expression of mutant CFTR correlates with decreased uptake of sphingoid base phosphates 2 (FIG. 2b), causing a lack of end-product inhibition, for example by sphingoid base phosphates, which in turn leads to increased sphingolipid synthesis. Thus sphingolipid mass is increased in cells expressing defective, decreased or no CFTR.
  • IB3 cells (ATCC CRL- 2777) that express mutant CFTR ( ⁇ F508/W1282X), and C38 control cells (ATCC CRL-2778) that were derived from the IB3 cells by transfection with episomal copies of wild-type CFTR 51 ;
  • 16HBE cells that are a human bronchial epithelial cell line that maintains tight junctions and stably expresses episomes encoding CFTR in the sense or antisense orientation.
  • Kube D Sontich U
  • Fletcher D Davis PB.
  • the cells expressing the CFTR antisense construct show the expected absence of CFTR function and fail to secrete chloride (Cl " ) in response to reagents that stimulate cAMP production 52 ; and (3) A549 cells derived from a human pulmonary adenocarcinoma cell line that does not express CFTR 53 ' 54 .
  • a 549 cells were transduced with Ad-CFTR to evaluate the effect of CFTR expression. Details of cell culturing are set forth in Example 1.
  • C38 and IB3 cells were grown in 6-well plates and incubated for 1.5h in serine-free medium in the presence of either 3 H-serine, used to assess de-novo sphingolipid synthesis, or 3 H- sphinganine, used to determine sphingolipid synthesis through recycling pathways.
  • 3 H-serine used to assess de-novo sphingolipid synthesis
  • 3 H- sphinganine used to determine sphingolipid synthesis through recycling pathways.
  • sphingolipid synthesis correlates with SRE- mediated gene transcription 32 .
  • increased cellular ceramide decreases mSREBP protein levels and SRE -mediated gene transcription.
  • Inhibition of ceramide de novo synthesis decreases SRE-mediated gene transcription.
  • Worgall, T. S., Johnson, R. A., Seo, T., Gierens, H., and Deckelbaum, R. J. (2002) J Biol Chem 277, 3878-85 Worgall, et al, U/S. Application Serial No. 20050182020.
  • Both exogenous and endogenous ceramide exert a negative feed-back mechanism on its own synthesis. Therefore, SRE-mediated gene transcription is a good indirect and surrogate marker for sphingolipid synthesis that can be easily assessed using a promoter/reporter gene assay.
  • SRE-mediated gene transcription sphingolipid synthesis was increased in IB3 cells having defective CFTR compared to the C38 controls with normal CFTR (FIG. 4a). As expected, SRE-mediated gene expression was low in A549 cells that have CFTR expression (FIG. 4b). Addition of cholesterol or oleic acid to IB3 cells decreased SRE-mediated gene transcription, indicating that post-transcriptional regulatory mechanisms of SREBP were intact in all examined cell lines.
  • Cystic fibrosis is associated with altered fatty acid profiles that show a deficiency of essential fatty acids and increased ratios of saturated to unsaturated fatty acids.
  • the most significant differences compared to controls are: palmitic acid (16:0), which is a product of endogenous fatty acid synthase, and lauric acid (12:0).
  • Plasma levels of unsaturated fatty acids, in particular linoleic (18:2,n-6) and linolenic acid (18:3,n-3), are also decreased in individuals with cystic fibrosis compared to controls 25"27 . It has been shown, however, that individuals with cystic fibrosis absorb supplemented fatty acids as efficiently as controls 28 .
  • SREBPs sterol-regulatory element binding proteins
  • fatty acid profiles in the experimental cell lines reflect the abnormal plasma fatty acid profiles found in cystic fibrosis patients, and assesses whether inhibition of sphingolipid synthesis affects fatty acid profiles.
  • the level of linoleic and linolenic acid in cystic fibrosis patients is typically decreased over normal levels by about 50% or even less.
  • Certain embodiments of the invention are therefore directed to methods for treating cystic fibrosis in a human being by reducing sphingolipid synthesis, including by administering ceramide (including c6-ceramide or dihydroceramide, or fragments, variants or derivatives thereof), or ceramide combined with DHA.
  • DHA is presently used clinically for treating cystic fibrosis.
  • Other embodiments are directed to a pharmaceutical composition for use in treating cystic fibrosis or P. aeruginosa infections that includes a therapeutic amount of ceramide, or ceramide plus DHA. All of the therapeutic agents described herein can be modified to enhance uptake, prevent degradation, target delivery, or increase half life using methods known in the art and described herein. Therapeutic amounts or doses of ceramide and DHA and other therapeutic agents discussed herein are described below.
  • C6-Ceramide has been used for treating ovarian cancer in amounts of about 100 mg/kg administered intravenously either in aqueous solution or as a nanoparticle in biodegradable poly(ethylene oxide)-modified poly (epsilon-caprolactone). Cancer Ther. Paclitaxel and ceramide co-administration in biodegradable polymeric nanoparticulate delivery system to overcome drug resistance in ovarian cancer, Devalapally, et al. On Line 7 June 2007, incorporated herein by reference. Although ceramide has some adverse side effects, an attending physician can determine if the benefits outweight the adverse effects in treating a cystic fibrosis patient. A range of doses and frequency of administration can be varied to find the optimum therapy, as described below.
  • FIG. 6 shows mass spectrometry data confirming that sphingo lipid synthesis and mass are both increased in cells expressing defective CFTR.
  • No CFTR expression A549 vs A549 Ad-CFTR
  • decreased expression Sense/ Antisense
  • cells expressing the most prevalent mutation ⁇ F 508 C38/IB3
  • fenretinide a synthetic retinoid that has been used as an anticancer agent to treat neuroblastoma as well as other cancers.
  • Fenretinide was recently shown to be an inhibitor of dihydroceramide desaturate, that increases cellular dihydroceramide concentration in Dul45 cells. Zheng, W et al BBA 1758 (2006) 1864-1884. We have previously showed that, like ceramide, dihydroceramide decreases sphingolipid synthesis in regular fibroblasts (Worgall, JBC, 2002).
  • GTl 1 a specific inhibitor of dihydroceramide desaturase on SRE -mediated gene transcription in CHO- fibroblasts.
  • GTl 1 is a target for enzyme inhibition to decrease sphingolipid synthesis in cystic fibrosis and P. aeruginosa infections.
  • FIG. 7 shows that at six hours (lowest line) fenretinide decreased SRE-mediated gene transcription by as much as 33% in cystic fibrosis model cells compared to controls (1% BSA) in a dose dependent manner from 1-10 ⁇ M fenretidine. There was no major difference between 10 and 20 ⁇ M fenretinide.
  • fenretinide can be used therapeutically to treat cystic fibrosis patients by normalizing sphingolipid synthesis.
  • Certain embodiments of the invention are directed to treatment of cystic fibrosis by administering a therapeutic amount of fenretinide or of a combination of ceramide and fenretinide.
  • Other embodiments are directed to a pharmaceutical composition for use in treating cystic fibrosis or P. aeruginosa infections, that includes a therapeutic amount of ceramide plus fenretinide.
  • Retinoids are natural and synthetic derivatives of vitamin A (retinol), which modulate different cellular processes, including proliferation, differentiation and apoptosis.
  • Fenretinide or N-(4-hydroxyphenyl) retinamide (4-HPR) is a synthetic retinoid (an amide of all-trans retinoic acid), which, in preclinical models, proved to be less toxic than many other retinoids while maintaining a significant biological activity.
  • Fenretinide has been successfully used at a dose of about 200 mg PO daily for up to 5 years (two capsules, 100 mg each, at dinner) to treat stage I breast cancer or ductal carcinoma-in-situ. Journal of Clinical Oncology, VoI 19, Issue 6 (March), 2001 : 1664-167. These amounts can be administered to treat cystic fibrosis and P.
  • 4-HPR is sometimes complexed with the novel lipid matrix Lym-X-SorbTM (LXS) by Avanti.
  • LXS novel lipid matrix Lym-X-SorbTM
  • 4-oxo-HPR is a metabolite of 4-HPR that is more potent and that has a wider range of tumor cell growth inhibitory activity than the parent drug. It can also be used to treat cystic fibrosis or P. aeruginosa infections by reducing sphingolipid synthesis.
  • 4-oxo-HPR enhances the effects of fenretinide, therefore certain embodiments are directed to methods for treating cystic fibrosis (or P. aeruginosa infections) by administering a therapeutic amount of fenretinide or 4-oxo-HPR or both, and to a pharmaceutical compositions comprising fenretinide and ceramide; 4-oxo-HPR and ceramide; and fenretinide, 4-oxo-HPR and ceramide for the treatment of cystic fibrosis and PA.
  • Palmitic acid is a substrate for serine-palmitoyl transferase (the rate limiting enzyme of sphingolipid de-no vo synthesis) as well as a substrate for LASS 5 ceramide/dihydroceramide synthase.
  • serine-palmitoyl transferase the rate limiting enzyme of sphingolipid de-no vo synthesis
  • LASS 5 ceramide/dihydroceramide synthase the rate limiting enzyme of sphingolipid de-no vo synthesis
  • fenretinide (10 ⁇ M) again decreased SRE -mediated gene transcription in both control and IB3 cells to about 40 % of the level seen in the respective cells under control conditions. Unlike palmitic acid, fenretinide also decreased sphingolipid synthesis by about 50% in control C38 cells. [0080] We next looked at the effect of fenretinide (10 ⁇ M) on A549 human bronchoalveolar cells that do not express CFTR (No CFTR) in comparison to A549 cells that were transduced to express normal CFTR.
  • FIG. 10 shows the effect of ceramide (20 uM) and fenretinide (10 uM) on cellular linoleic (18:2) fatty acid content in control cells (C38, black bar) and cells expressing defective CFTR (IB3, white bar).
  • both agents increased linoleic fatty acid content in both control and in IB3 cells showing that both ceramide and fenretinide can be used to reduce sphingolipid synthesis and normalize fatty acid profiles in cystic fibrosis cells and in cystic fibrosis patients.
  • certain embodiments are directed to decreasing sphingolipid synthesis in cells expressing defective CFTR by inhibiting enzymes in the sphingolipid synthesis biosynthetic pathway.
  • serine palmitoyl transferase such as myriocin or cycloserine
  • sphingosine kinase such as dimethyl sphingosine
  • agents that have a negative feedback effect on sphingolipid synthesis through recycling pathways including ceramide, fenretinide, sphingosine, sphinganine, glucosylceramide, dihydroceramide and unsaturated fatty acids, including eicosapentaenoic acid (EPA),and docosahexaenoic acid (DHA), or combinations thereof.
  • Agents that decrease de novo sphingolipid synthesis can be administered together with agents that have a negative feedback effect on sphingolipid synthesis through recycling pathways.
  • the agents can be administered at the same time on the same day or at different times on the same day, or on different days. They can be formulated and administered as a single pharmaceutical composition.
  • a preferred route of administration for cystic fibrosis is by inhalation since the lungs are heavily involved in the disease. However, any route of administration may be used as is discussed in more detail below.
  • DHA and EPA decrease interleukin-8 expression in lung epithelial cells.
  • IL-8 a member of the neutrophil-specific CXC subfamily of chemokines, is a potent neutrophil chemotactic and activating factor. It has been reported that IL-8 levels are constituitively higher in non-infected cystic fibrosis infants compared to culture positive controls 43"45 . We have discovered that DHA and EPA decrease IL-8 secretion from 12 HAEo cells, which are an SV -40 transformed human airway cell line that constitutively expresses IL-8. Assessment of IL-8 secretion was carried out in low serum conditions because serum itself stimulates IL-8 secretion. Cells were preincubated for 16h in the presence of 0.1 % serum.
  • IL-8 secretion of IL-8 was determined in the supernatant using standard ELISA techniques and data were normalized to cell protein content. IL-8 concentration was decreased by both DHA or EPA ( Figure 12). Both DHA and EPA are presently used to improve fatty acid patterns in cystic fibrosis.
  • Certain embodiments of the invention are directed to methods for treating cystic fibrosis or ameliorating one or more of its symptoms in an animal by administering ceramide, including c6-ceramide, combined for example with EPA, or EPA plus DHA.
  • Other embodiments are directed to a pharmaceutical composition for use in treating cystic fibrosis or P. aeruginosa infections that includes a therapeutic amount of ceramide plus EPA, or EPA plus DHA, or ceramide plus both EPA and DPA.
  • cystic fibrosis patients have low plasma HDL cholesterol levels in cystic fibrosis patients 48 .
  • Low HDL cholesterol is an independent risk factor for coronary artery disease.
  • a major regulator of plasma HDL levels is the expression of the ABCAl protein at the plasma membrane. This protein functions as a cholesterol efflux receptor and is, like CFTR, part of the ABC transporter family.
  • ceramide or inhibition of glucosylceramide synthase increases ABCAl expression 49 ' 50 . It is not known whether sphingo lipid have a role in the modulation of plasma HDL-cholesterol in CF. Inhibition of Sphingolipid Synthesis decreases Phosphatidylcholine specific PLA2 activity, and NF-Kappa B activity in cells expressing defective CFTR
  • the inflammatory PLA2s are potent mediators of inflammatory processes and are highly expressed in serum and synovial fluids of patients with inflammatory disorders as well as in cystic fibrosis.
  • the secretory phospho lipase A2 (PtA2) superfamily comprises a number of heterogeneous enzymes whose common feature is to hydrolyze the sn-2 fatty acid acyl ester bond of phospholipids.
  • PtA2 secretory phospho lipase A2
  • phosphatidylcholine is a substrate for the PLA2 which is increased in cystic fibrosis.
  • Hydrolysis of phosphatidylcholine (PC) to lyso-PC affords a chemoattractant for circulating monocytes and arachidonic acid.
  • Arachidonic acid is the key eicosanoids precursor for the production of thromboxanes, prostaglandins and leukotrienes. Furthermore, leukotriene-B4 is known to function in a feedback loop which further increases PLA2 activity (Wijkander, J. et al. (1995) J. Biol. Chem. 270:26543-26549). [0086] We have discovered that inhibition of sphingolipid synthesis decreases phosphatidylcholine specific PLA2 activity and NF-Kappa B activity in cells expressing defective CFTR. FIG. 14 shows the effect of fenretinide on phosphatidylcholine specific PLA2 activity.
  • CFTR control (Sense) and CFTR minus (Antisense) cells were incubated for 16 h in the presence of Fenretinide (10 ⁇ M). Cells were harvested and lysed by passing through a 22 gauge needle. The cell extract was reacted with a phospho lipase A2 specific phosphatidylcholine probe (Echelon- inc, Gift from CEO Prestwich). Fluorescence of this substrate is quenched in the unreacted state. Fluorescence is emitted when arachidonic is cleaved at the sn-2 position. This probe is specific for phosphatidylcholine.
  • Cystic fibrosis is characterized by prolonged and excessive inflammatory responses in the lung and increased activation of NF- kappa B.
  • NF -kappa B is an ubiquitously expressed transcription factor that controls the expression of a diverse range of genes involved in inflammation, immune response, lymphoid differentiation, growth control and development.
  • NF- kappa B resides in the cytoplasm as an inactive dimer consisting of p50 and p65 subunits, bound to an inhibitory protein known as I kappa B. The latter becomes phosphorylated and degraded in response to various environmental stimuli, such as pro-inflammatory cytokines, viruses, lipopolysaccharides, oxidants, UV light and ionizing radiation.
  • NF-kappa B This allows NF-kappa B to translocate to the nucleus where it activates genes that play a key role in the regulation of inflammatory and immune responses, including genes that encode pro-inflammatory cytokines (TL-I. beta., TNF, GM-CSF, IL-2, IL-6, IL-I l, IL-17), chemokines (IL-8, RANTES, MIP- 1. alpha., MCP-2), enzymes that generate mediators of inflammation (NO synthetase, cyclo- oxygenase), immune receptors (IL-2 receptor) and adhesion molecules (ICAM-I, VCAM-I, E- selectin).
  • cytokines TL-I. beta., TNF, GM-CSF, IL-2, IL-6, IL-I l, IL-17
  • chemokines IL-8, RANTES, MIP- 1. alpha., MCP-2
  • enzymes that generate mediators of inflammation NO synthetas
  • NF-kappa.B NF-kappa.B related diseases including cystic fibrosis.
  • fenretinide show that reducing sphingolipid synthesis will in turn reduce NF- kappa.B.
  • the methods described herein for reducing sphingolipid synthesis through de novo and recycling pathways can be used to treat other diseases associated with elevated NF-kappa B besides cystic fibrosis and P. aeruginosa.
  • FIG. 15 shows the effects of fenretinide and parthenolide on NF -Kappa B activity in CFTR control (C38 ) and CFTR defective cells transfected with an NF-Kappa B reporter gene construct together with ⁇ -gal transfection control.
  • C38 CFTR control
  • CFTR defective cells transfected with an NF-Kappa B reporter gene construct together with ⁇ -gal transfection control.
  • parthenolide 15 ⁇ M
  • Fenretinide lO ⁇ M
  • Parthenolide is a sesquiterpene lactone derived from the plant feverfew, which has been used in folk medicine for anti-inflammatory activity. It has been used clinically to treat cystic fibrosis. Jain NK, Kulkarni SK. Antinociceptive and anti-inflammatory effects of Tanacetum parthenium L. extract in mice and rats. /J Ethnopharmacol/ 1999;68:251-259; Reuter U, Chiarugi A, Bolay H, Moskowitz MA. Nuclear factor-kappaB as a molecular target for migraine therapy. Ann Neural/ 2002;51 :507-516).
  • Parthenolide has been recently shown to inhibit the NF- B pathway, Saadane, A., et al.
  • the agents described herein for reducing sphingolipid synthesis, including fenretinide, can be combined with parthenolide to treat cystic fibrosis or other inflammatory diseases.
  • FIG. 16 shows the effect of the dihydroceramide synthase inhibitor Gl 1 on SRE- mediated gene transcription.
  • GTl 1 is the only described specific dihydoceramide desaturase inhibitor so far (GenBank note). Gemma Fabrias and Gerhild von Echten, Specificity of the dihydroceramide desaturate inhibitor GTl 1 In primary cultured cerebellar Neurons, Molecular Pharmacology 66:1671-1678, 2004).
  • CHO fibroblasts were incubated for 5 h in the presence of increasing concentrations of GTl 1. Luciferase activity was measured and normalized to cellular protein levels. The results demonstrate that inhibition of dihydroceramide desaturase decreases SRE -mediated gene transcription.
  • inhibitors such as Gl 1 or antisense nucleotides or siRNA can be used to treat cystic fibrosis or P. aeruginosa infections.
  • Certain embodiments of the invention are directed to cell-based and non-cell based methods of drug screening to identify candidate agents that:
  • enzyme inhibitors or reduce the activity or expression of certain enzymes in an the de novo sphingolipid synthesis pathway in an animal cell expressing defective CFTR including serine palmitoyl transferase, ceramide synthase, sphingosine kinase, ceramidase, UDP-glucose ceramide glucosyltransferase , and glucosylceramide synthase, also referred to herein as "the various proteins;" and
  • Non-cell based assays for identifying enzyme inhibitors or agents that affect gene expression are very well known. They generally involve (a) contacting a transformed or recombinant cell that has a mutant of a native allele encoding a reporter of gene expression of one (or more) of the various proteins, wherein the expression of the reporter is under the control of the native gene expression regulatory sequences of the native allele, with a candidate agent under conditions whereby but for the presence of the agent, the reporter is expressed at a first expression level; and, (b) determining the expression of the reporter to obtain a second expression level, wherein a difference between the first and second expression levels indicates that the candidate agent modulates expression of one of the gene.
  • the mutant may result from replacement of a portion of the native allele with a sequence encoding the reporter.
  • the cell may be a progeny of a genetic knock-in cell made by homologous recombination of the native allele with a transgene comprising a sequence encoding the reporter flanked by flanking sequences capable of effecting the homologous recombination of the transgene with the native allele, a positive selectable marker positioned inside the flanking sequences and a negative selectable marker positioned outside the flanking sequences.
  • P. aeruginosa to cystic fibrosis lung epithelial cells, the formation of bio films and failure of the host to eliminate P. aeruginosa 7 ' 37 .
  • P. aeruginosa binds to the GalNAc ⁇ l-4 Gal moiety of gangliosides and flagella activate airway cells through aGMl and toll-like receptors 2 and 5 38 .
  • P. aeruginosa is among many bacteria that bind to glucosylceramide derived glycosphingolipids 39 .
  • Gangliosides are glucosylceramide-derived sialylated or fucosylated acid glycosphingolipids, found on the outside of cells ( Figure 1).
  • CFTR mutations are associated with the presence of an increased number of a GMl gangliosides 7 .
  • P. aeruginosa type IV pili and fimbriae bind to aGMl and stimulate the expression of IL-8 by lung epithelial cells 40 .
  • glycosphingolipid depletion therapy with PPMP has been experimentally shown to decrease the bacterial as well as viral load in several studies 42 .
  • There are many strains of P. aeruginosa that differ with regard to expression of pili, other fimbriae and flagella. We used the well-characterized laboratory strain PAOl 46 .
  • Example 5 The well-characterized laboratory strain PAOl 46 . Example 5.
  • c6-ceramide is not a substrate for metabolism to differentiated sphingolipid, but instead decreases sphingolipid synthesis through an end-product feedback mechanism.
  • concentrations can be used, including from about 5-100 micromolar can be used; (2) Eicosapentaenoic acid (EPA) (20:6, 0.3 mM), an unsaturated fatty acid that decreases sphingolipid synthesis; and (3) Myriocin (l ⁇ M), a specific inhibitor of SPT, the rate limiting enzyme of de-novo sphingolipid synthesis.
  • Confluent cells were incubated for Ih with 50 ⁇ l of either the experimental GFP-labeled P. aeruginosa or the control P.
  • ceramide decreased P. aeruginosa adhesion in both cell lines. Specifically, ceramide reduced bacterial adhesion in C38 control cells expressing normal CFTR by about 40%, and in antisense 16HBE cells (no CFTR) by about 64%. EPA and myriocin had no significant effect on P. aeruginosa adhesion in cells expressing normal CFTR because sphingolipid synthesis is not increased in these cells, but dramatically reduced adhesion by about 64% (EPA) and about 80% (myriocin) in cells expressing no CFTR.
  • some embodiments of the invention are directed to a method for decreasing P. aeruginosa infection in a human or animal, preferably in a cystic fibrosis patient, by administering a therapeutic amount of an agent that decreases sphingolipid synthesis, particularly in lung epithelium.
  • agents that decreases sphingolipid synthesis particularly in lung epithelium.
  • agents that decreases sphingolipid synthesis include fenretinide, unsaturated fatty acids, GT 11 , EPA, myriocin, dihydroceramide, ceramide, or a combination thereof.
  • Any agent with acceptable toxicity that reduces sphingolipid synthesis (through either de novo or recycling pathways) in an animal expressing no CFTR or defective CFTR can be administered alone or in combination with other agents to treat or prevent P. aeruginosa infection.
  • the preferred route of administration is by inhalation since the main site of P. aeruginosa infection in cystic fibrosis patients is in the lungs.
  • T Sphinganine T: Saturated Fatty acids 3 H-Sphingomyelin T: Sphingosine 3 H-Glucosylceramide
  • bioactive agent or "exogenous compound” as used herein includes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, lipid, etc., or mixtures thereof, with the capability of directly or indirectly altering the bioactivity of one of the various proteins. Some of the bioactive agents can be used therapeutically. Generally a plurality of assay mixtures is run in parallel with different bioactive agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
  • Bioactive agent agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Bioactive agent agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The bioactive agent agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Bioactive agent agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
  • Bioactive agent agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety or organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterif ⁇ cation, amidification to produce structural analogs.
  • the bioactive agent is a protein.
  • protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • the protein may be made up of naturally occurring amino acids and peptide bounds, or synthetic peptidomimetic structures.
  • amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo- phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
  • Amino acids also includes imino acid residues such as proline and hydroxyproline.
  • the side chains may be in either the (R) or the (S) configuration.
  • the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
  • the bioactive agent is a naturally occurring protein or fragment or variant of a naturally occurring protein.
  • cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts may be used.
  • libraries of prokaryotic and eukaryotic proteins may be made for screening against one of the various proteins. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
  • the bioactive agent are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred.
  • the peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased” random peptides.
  • randomized or grammatical equivalents herein is meant that each peptide consists of essentially random amino acids. Since generally these random peptides are chemically synthesized, they may incorporate any amino acid at any position.
  • the synthetic process can be designed to generate randomized proteins, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized bioactive agent bioactive proteinaceous agents.
  • the library is fully randomized, with no sequence preferences or constants at any position.
  • the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
  • the amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, pralines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
  • the bioactive agent is an isolated nucleic acid, preferably antisense, siRNA, or cDNA" that binds to either the gene encoding the protein of interest, or its mRNA to block gene expression or mRNA translation, respectively.
  • nucleic acid or “oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together.
  • a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al, Tetrahedron 49)10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); SRocl et al., Eur. J. Biochem. 81 :579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc.
  • nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxathine, isocytosine, isoguanine, etc.
  • nucleic acid bioactive agents may be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes may be used as is outlined above for proteins.
  • the bioactive agents are obtained from combinatorial chemical libraries, a wide variety of which are available in the literature.
  • combinatorial chemical library herein is meant a collection of diverse chemical compounds generated in a defined or random manner, generally by chemical synthesis. Millions of chemical compounds can be synthesized through combinatorial mixing.
  • the bioactive agent is labeled, and binding determined directly. For example, this may be done by attaching all or a portion of one of the various proteins to a solid support, adding a labeled bioactive agent (for example a bioactive agent comprising a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support.
  • a labeled bioactive agent for example a bioactive agent comprising a fluorescent label
  • washing off excess reagent for example a fluorescent label
  • Various blocking and washing steps may be utilized as is known in the art.
  • labeled herein is meant that the bioactive agent is either directly or indirectly labeled with a label which provides a detectable signal, e.g.
  • a radioisotope such as H 3 , C 14 , P 32 , P 33 , S 35 , or I 125
  • a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine, or luciferin
  • an enzyme such as alkaline phosphatase, beta- galactosidase or horseradish peroxidase
  • antibodies such as magnetic particles, or specific binding molecules, etc.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc.
  • the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above.
  • the label can directly or indirectly provide a detectable signal.
  • only one of the components is labeled.
  • more than one component may be labeled with different labels.
  • the binding of the bioactive agent is determined through the use of competitive binding assays.
  • the competitor is a binding moiety known to bind to the target molecule (i.e. one of the various proteins), such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding as between the bioactive agent and the binding moiety, with the binding moiety displacing the bioactive agent.
  • the bioactive agent is labeled. Either the bioactive agent bioactive agent, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present. Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 degrees Centigrade and 40 degrees Centigrade. Incubation periods are selected for optimum activity, but may also optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
  • Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 degrees Centigrade and 40 degrees Centigrade. Incubation periods are selected for optimum activity, but may also optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding
  • the competitor is added first, followed by the bioactive agent bioactive agent.
  • Displacement of the competitor is an indication that the bioactive agent is binding to one of the various proteins and thus is capable of binding to, and potentially modulating, its activity.
  • either component can be labeled.
  • the presence of label in the wash solution indicates displacement by the agent.
  • the bioactive agent is labeled, the presence of the label on the support indicates displacement.
  • the bioactive agent is added first, with incubation and washing, followed by the competitor.
  • the absence of binding by the competitor may indicate that the bioactive agent is bound to one of the various proteins with a higher affinity.
  • the bioactive agent is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate that the bioactive agent is capable of binding to one of the various proteins.
  • the methods comprise differential screening to identify bioactive agents that are capable of modulating the activity of one of the various proteins.
  • the methods comprise combining a protein and a competitor in a first sample.
  • a second sample comprises a bioactive agent, a protein and a competitor. Addition of the bioactive agent is performed under conditions which allow the modulation of one of the various proteins.
  • the binding of the competitor is determined for both samples, and a change or difference in binding between the two samples indicates the presence of an agent capable of binding to one of the various proteins and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to one of the various proteins.
  • Positive controls and negative controls may be used in the assays.
  • Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non- specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radio label is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.
  • a variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
  • Screening for agents that modulate the activity of one of the various proteins may also be done.
  • methods for screening for a bioactive agent capable of modulating the activity of one of the various proteins comprise the steps of adding a bioactive agent to a sample of one of the various proteins, as above, and determining an alteration in the biological activity of one of the various proteins.
  • “Modulating the activity of one of the various proteins” includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present.
  • the bioactive agent should both bind to the protein (although this may not be necessary), and alter its biological or biochemical activity as defined herein.
  • the methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of one of the various proteins.
  • the invention thus provides methods for treating cystic fibrosis and P. aeruginosa infections in an animal.
  • the method involves administering a therapeutic agent that includes compounds that decrease sphingolipid synthesis including ceramide, dihydroceramide, dihydroceramide synthase inhibitors such as fenretinide or GTl 1 (Triola et al, Molecular Pharmacology 2004, 1671-1678), inhibitors of glucosylceramide synthase , sphingomyelin synthesis, sphingosine synthesis, given that these substances increase ceramide and thus inhibit de-no vo synthesis, and enzyme inhibitors or antisense nucleic acids or si RNA that reduce the expression or activity of serine-palmitoyl transferase, dihydroceramide desaturase, sphingomyelin synthase, sphingosine synthase, glucosylceramide synthase in an amount sufficient to treat cystic fibro
  • the invention encompasses use of the polypeptides, nucleic acids, small molecules, antisense nucleic acids and si RNA and other therapeutic agents described herein formulated in pharmaceutical compositions to administer to a subject or to target cells or tissues, preferably to the lung, in a subject whose cells express abnormal amounts of CFTR or defective CFTR, such as one of the mutations in CFTR in cystic fibrosis patients.
  • the therapeutic agents also referred to as "active compounds” can be incorporated into pharmaceutical compositions suitable for administration to a subject, preferably a human.
  • Such compositions typically comprise the nucleic acid molecule, protein, modulator (enzyme inhibitor), or small molecule and a pharmaceutically acceptable carrier. It is understood however, that administration can also be to cells in vitro as well as to in vivo model systems such as human or non-human transgenic animals.
  • compositions of the present invention are used in its broadest sense and includes any method of introducing the compositions of the present invention into a subject; preferably inhalation therapy that delivers the agent to the lung.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds or therapeutic agents can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral including by inhalation, which is a method that is preferred for treating lung infections with P. aeruginosa and cystic fibrosis, transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diamante tetra acetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • compositions of the present invention for treatment of atherosclerosis or the other elements of metabolic syndrome can be formulated and administered systemically or locally. Techniques for formulation and administration can be found in "Remington: The Science and Practice of Pharmacy” (20.sup.th edition, Gennaro (ed.) and Gennaro, Lippincott, Williams & Wilkins, 2000).
  • the agent can be contained in enteric forms to survive the stomach or further coated or mixed to be released in a particular region of the GI tract by known methods.
  • the active compound can be incorporated with excipients and used in the form of tab lets, troches, or capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. [0124] If appropriate, the compounds can also be prepared in the form of suppositories
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. [0126] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • a therapeutically effective amount of protein or polypeptide or nucleic acid [0127] A therapeutically effective amount of protein or polypeptide or nucleic acid
  • antisense or si RNA i.e., an effective dosage
  • This amount typically varies and can be an amount sufficient to achieve serum therapeutic agent levels typically of between about 1 nanogram per milliliter and about 10 micrograms per milliliter in the subject, or an amount sufficient to achieve serum therapeutic agent levels of between about 1 nanogram per milliliter and about 7 micrograms per milliliter in the subject. Expressed as a daily dose, this amount can be between about 0.1 nanograms per kilogram body weight per day and about 20 milligrams per kilogram body weight per day, or between about 1 nanogram per kilogram body weight per day and about 10 milligrams per kilogram body weight per day.
  • a therapeutic amount can be determined as an amount that ameliorates one or more symptoms of the disease, or that decreases sphingolipid synthesis in a biological sample taken from the patient, such as peripheral blood mononuclear cells (described in the Examples) or lung epithelium.
  • a biological sample taken from the patient such as peripheral blood mononuclear cells (described in the Examples) or lung epithelium.
  • certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the condition, previous treatments, the general health and/or age of the subject, and other disorders or diseases present.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or nucleotide can include a single treatment or, preferably, can include a series of treatments.
  • the effective dosage of, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from monitoring the level of sphingo lipids in a biological sample. If the therapeutic agent is administered by inhalation, higher amounts of the agent may be administered than if the agent were to be administered systemically. In studies by others C6-ceramide has been administered at a dose of about 100mg/kg by intravenous injection; the associated benefits were considered to outweigh the adverse effects. Other drugs like myriocin that have toxic side effects can be used. As with anticancer agents that have adverse side effects and high toxicity, localized delivery is advantageous.
  • Myriocin has been used either intraperitoneally or orally at doses around 1 mg/kg ( in mice ).
  • the drug FTY720 a derivative of myriocin, has been used at doses of 0. 5 -2.5 mg/dose in human adults orally.
  • Fumonisin Bl has been given subcutaneously at 2.5 mg/kg in mice.
  • Fenretinide is used from 200 mg/day orally or at higher doses.
  • An agent may, for example, be a small molecule.
  • small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1 ,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • antisense nucleic acids either DNA or RNA
  • small interfering RNA to inhibit expression of the various enzymes associated with sphingolipid synthesis in cells expressing defective CFTR, including serine palmitoyl transferase, sphingosine kinase, ceramide synthase/fatty acid desaturase, UDP- glucose ceramide glucosyltransferase and glucosylceramide synthase (hereafter "the various enzymes”).
  • the SEQ ID NOs. for these enzymes are set forth below.
  • the antisense nucleic acid can be antisense RNA, antisense DNA or small interfering RNA.
  • nucleic acids that interfere with transcription or translation of a gene product including these various enzymes include DNA that stops transcription of the sense strand of the gene encoding the targeted enzyme, such as antisense DNA. Identification of antisense nucleotides (RNA or DNA) is straightforward since the gene sequence encoding the enzymes in humans is known. RNA nucleic acids that stop or otherwise interfere with efficient transcription or translation of the gene or messenger RNA for the various enzymes can also be used therapeutically to interfere with expression; these include antisense RNA or small interfering RNA.
  • the sequence of messenger RNA for the various enzymes can be determined form the gene sequence, and antisense nucleic acid s that would bind to the messenger RNA and block translation can be determined using routine methods.
  • Antisense technology and small interfering RNAs are well known in the art and are described in detail below.
  • Antisense-RNA and anti-sense DNA have been used therapeutically in mammals to treat various diseases. See for example Agrawal, S. and Zhao, Q. (1998) Curr. Opi. Chemical Biol. Vol. 2, 519-528; Agrawal, S and Zhang, R. (1997) CIBA Found. Symp. Vol. 209, 60-78; and Zhao, Q, et al, (1998), Antisense Nucleic Acid Drug Dev. VoI 8, 451-458; the entire contents of which are hereby incorporated by reference as if fully set forth herein.
  • Antisense oligodeoxyribonucleotides can base pair with a gene, or its transcript.
  • An antisense PS-oligodeoxyribonucleotide for treatment of cytomegalovirus retinitis in AIDS patients is the first antisense oligodeoxyribonucleotiede approved for human use in the US. Anderson, K.O., et al., (1996) Antimicrobiol. Agents Chemother. Vol. 40, 2004-2011, the entire contents of which are hereby incorporated by reference as if fully set forth herein.
  • U.S. Patent No. 6, 828, 151 by Borchers, et al. entitled Antisense modulation of hematopoietic cell protein tyrosine kinase expression describes methods for making and using antisense-nucleic acid s and their formulation.
  • the entire contents of U.S. Patent No. 6, 828, 151 are hereby incorporated by reference as if fully set forth herein.
  • antisense nucleic acid s complementary to the gene for glutamine synthetase mRNA in Mtb effectively enter the bacteria, complex with the mRNA and inhibit glutamine synthetase expression, the amount of the poly-L-glutamate/glutamine component in the cell wall, and bacterial replication in vitro. Harth, G., et al, PNAS Jan. 4, 2000, Vol. 97, No. 1, P 418-423, the entire contents of which are hereby incorporated by reference as if fully set forth herein.
  • target nucleic acid encompass DNA encoding the various enzymes, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA.
  • RNA including pre-mRNA and mRNA
  • cDNA derived from such RNA.
  • the specific hybridization of a nucleic acid oligomeric compound with its target nucleic acid interferes with the normal function of the target nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as "antisense".
  • the functions of DNA to be interfered with include replication and transcription.
  • RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, and catalytic activity which may be engaged in or facilitated by the RNA.
  • the overall effect of such interference with target nucleic acid function is modulation of the expression of the various enzymes .
  • modulation means (inhibition) in the expression of the genes or mRNA of the various proteins (enzymes).
  • inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.
  • the targeting process includes determination of a site or sites within the target gene (or mRNA) encoding the various enzymes for the antisense interaction to occur to achieve the desired inhibitory effect.
  • a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon,” the "start codon” or the "AUG start codon”.
  • translation initiation codon can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene.
  • a translation termination codon (or "stop codon”) of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively).
  • start codon region and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon.
  • stop codon region and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon.
  • Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding nucleotides on the gene.
  • 5'UTR 5' untranslated region
  • 3'UTR 3' untranslated region
  • variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon.
  • Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA.
  • Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA.
  • One specific type of alternative stop variant is the "polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites.
  • nucleic acids are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect of inhibiting gene expression and transcription.
  • hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • nucleic acid and the DNA or RNA are considered to be complementary to each other at that position.
  • the nucleic acid and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the nucleic acid and the DNA or RNA target.
  • an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
  • An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.
  • Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target are identified through routine experimentation, and the sequences of these compounds are herein below identified as preferred embodiments of the invention.
  • the target sites to which these preferred sequences are complementary are herein below referred to as "active sites” and are therefore preferred sites for targeting. Therefore another embodiment of the invention encompasses compounds which hybridize to these active sites.
  • Antisense nucleic acid drugs, including ribozymes have been safely and effectively administered to humans and numerous clinical trials are presently underway.
  • nucleic acid s can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans, for example to regulate expression of the various enzymes involved in sphingo lipid synthesis in cystic fibrosis patients.
  • Nucleic acid in the context of this invention includes “oligonucleotide”, which refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • Nucleic acid in the context of this invention includes "oligonucleotide”, which refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • mimetics oligonucleotide
  • This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly.
  • antisense nucleic acids are a preferred form of antisense compound
  • the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics.
  • the antisense compounds in accordance with this invention preferably comprise from about 8 to about 50 nucleobases (i.e. from about 8 to about 50 linked nucleosides).
  • Particularly preferred antisense compounds are antisense nucleic acids, even more preferably those comprising from about 12 to about 30 nucleobases.
  • Antisense compounds include ribozymes, external guide sequence (EGS) nucleic acid s (oligozymes), and other short catalytic RNAs or catalytic nucleic acid s which hybridize to the target nucleic acid and modulate its expression.
  • EGS external guide sequence
  • the antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare nucleic acid s such as the phosphorothioates and alkylated derivatives. [0147]
  • the antisense compounds of the invention are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules.
  • the compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat.
  • the antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits.
  • an animal preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of the various enzymes, such as cystic fibrosis, is treated by administering antisense compounds in accordance with this invention.
  • the compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier.
  • Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay P. aeruginosa infection.
  • the present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention that are administered to return the level of sphingo lipid synthesis in cystic fibrosis patients to normal, especially in lung and colon epithelial cells.
  • the pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • RNA interference double-stranded RNA
  • dsRNA double-stranded RNA
  • RNAi RNA interference
  • mammalian cells dsRNA that is 30 base pairs or longer can induce sequence-nonspecific responses that trigger a shut-down of protein synthesis and even cell death through apoptosis.
  • RNA fragments are the sequence-specific mediators of RNAi (Elbashir et al., 2001).
  • siRNA small interfering RNA
  • RNA nucleic acid s In mammalian cell culture, a siRNA-mediated reduction in gene expression has been accomplished by transfecting cells with synthetic RNA nucleic acid s (Caplan et al., 2001; Elbashir et al., 2001).
  • the 20040023390 application provides methods using a viral vector containing an expression cassette containing a pol II promoter operably-linked to a nucleic acid sequence encoding a small interfering RNA molecule (siRNA) targeted against a gene of interest.
  • RNAi is the process of RNA interference.
  • a typical mRNA produces approximately 5,000 copies of a protein.
  • RNAi is a process that interferes with or significantly reduces the number of protein copies made by an mRNA.
  • a double- stranded short interfering RNA (siRNA) molecule is engineered to complement and match the protein-encoding nucleotide sequence of the target mRNA to be interfered with.
  • the siRNA molecule associates with an RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the siRNA-associated RISC binds the target mRNA (such as mRNA encoding "the various enzymes" the sequences of which are set forth herein) through a base-pairing interaction and degrades it.
  • the RISC remains capable of degrading additional copies of the targeted mRNA.
  • Other forms of RNA can be used such as short hairpin RNA and longer RNA molecules. Longer molecules cause cell death, for example by instigating apoptosis and inducing an interferon response. Cell death was the major hurdle to achieving RNAi in mammals because dsRNAs longer than 30 nucleotides activated defense mechanisms that resulted in non-specific degradation of RNA transcripts and a general shutdown of the host cell. Using from about 20 to about 29 nucleotide siRNAs to mediate gene-specific suppression in mammalian cells has apparently overcome this obstacle. These siRNAs are long enough to cause gene suppression but not of a length that induces an interferon response.
  • Uncarboxylated osteocalcin can be modified according to known methods in medicinal chemistry to increase its stability, half-life, uptake or efficacy.
  • Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA
  • N-terminal amino group can be accomplished using a hydrophilic compound, such as hydroorotic acid or the like, or by reaction with a suitable isocyanate, such as methylisocyanate or isopropylisocyanate, to create a urea moiety at the N- terminus.
  • a hydrophilic compound such as hydroorotic acid or the like
  • a suitable isocyanate such as methylisocyanate or isopropylisocyanate
  • Other agents can also be N-terminally linked that will increase the duration of action of the SRIF analog as known in this art.
  • Reductive amination is the process by which ammonia is condensed with aldehydes or ketones to form imines which are subsequently reduced to amines.
  • reductive amination is a potentially useful method for conjugation to PEG.
  • Covalent linkage of poly(ethylene glycol) (PEG) to drug molecules results in water-soluble conjugates with altered bioavailability, pharmacokinetics, immunogenic properties, and biological activities.
  • reductive amination is a potentially useful method for conjugation to PEG.
  • Proteins like osteocalcin can also be modified to create peptide linkages that are susceptible to proteolytic enzymes. For instance, alkylation of cysteine residues with P- haloethylamines yields peptide linkages that are hydrolyzed by trypsin. Such modifications are well-known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins—Structure and Molecular Properties, 2nd ed., T. E. Creighton, W. H. Freeman and Company, New York (1993).
  • polypeptides are not always entirely linear.
  • polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of post-translation events, including natural processing events and events brought about by human manipulation which do not occur naturally.
  • Circular, branched and branched circular polypeptides may be synthesized by non-translational natural processes and by synthetic methods.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. Blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally- occurring and synthetic polypeptides. For instance, the amino terminal residue of polypeptides made in E. coli, prior to proteolytic processing, almost invariably will be N-formylmethionine. [0159] The modifications can be a function of how the protein is made. For recombinant polypeptides, for example, the modifications will be determined by the host cell posttranslational modification capacity and the modification signals in the polypeptide amino acid sequence.
  • a polypeptide when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell. Insect cells often carry out the same posttranslational glycosylations as mammalian cells, and, for this reason, insect cell expression systems have been developed to efficiently express mammalian proteins having native patterns of glycosylation. Similar considerations apply to other modifications.
  • the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain more than one type of modification.
  • Isolated osteocalcin can be purified from cells that naturally express it, e.g., osteoblasts, purified from cells that naturally express it but have been modified to overproduce osteocalcin, e.g., purified from cells that have been altered to express it (recombinant), synthesized using known protein synthesis methods, by modifying cells that naturally encode osteocalcin to express it, or from bacteria modified to overexpress it.
  • osteoblasts purified from cells that naturally express it but have been modified to overproduce osteocalcin, e.g., purified from cells that have been altered to express it (recombinant), synthesized using known protein synthesis methods, by modifying cells that naturally encode osteocalcin to express it, or from bacteria modified to overexpress it.
  • Example 1 Cell cultures of C38 and IB3 cells [0163] C38 and IB3 cells were grown in 6-well plates and incubated for 1.5h in serine- free medium in the presence of 3 H-serine to assess de-novo sphingo lipid synthesis or 3 H- sphinganine to determine sphingo lipid synthesis through recycling pathways. At the end of the incubation period, lipids were extracted with chlorofornrmethanol and separated by thin-layer chromatography (TLC). Ceramide spots were analyzed by scintillation counting. Dpm associated with ceramide reflect synthesis. The synthesis of ceramide from H-serine reflects de-novo synthesis by SPT.
  • TLC thin-layer chromatography
  • Example 2 CFTR and SRE-mediated gene transcription in C38/IB3 and A549 cells
  • CFTR expression was confirmed by Western analysis (FIG. 4c). Forty-eight hours later, cells were incubated for 16h in the presence of 1 % BSA, the sterol-free control medium known to increase SRE-mediated gene transcription. This condition was used to compare SRE- mediated gene transcription between cell lines. We also incubated cells in the presence of cholesterol and oleic acid, known inhibitors and direct regulators of SREBP, to assess whether regulation of SREBP is physiologically intact in our cell lines.
  • C38/IB3 cells were transduced with Ad-SRE-luc/ ⁇ gal (FIG. 4a).
  • A549 cells, that do not express CFTR were transduced with SRE-luc/ ⁇ gal together with Ad-CFTR or the control vector Ad-null (b).
  • Western analysis in A549 demonstrates CFTR expression in A549 cells transduced with Ad-CFTR (c).
  • Cells were incubated for 48h in growth medium to allow for transgene expression. Then, cells were incubated for 16h in sterol depleted control medium (1 % BSA) to compare the effect of CFTR expression on SRE-mediated gene transcription.
  • Cholesterol and oleic acid significantly (p ⁇ 0.05) decreased SRE- mediated gene transcription in all conditions, reflecting intact regulation of SREBP.
  • Bl was used to decrease sphingo lipid synthesis in general.
  • NOE inhibits ceramidases in fibroblasts, decreases de-novo synthesis and promotes synthesis of sphingomyelin and glucosylceramide.
  • DMS inhibits sphingosine kinase by increasing sphingolipid synthesis through recycling pathways by promoting synthesis of ceramide using sphingosine as a substrate.
  • PPMP and NB-DNJ inhibit glucosylceramide synthase.
  • PPMP also increases ceramide levels and decreases de-novo sphingolipid synthesis. 59 .
  • SPT activity can be decreased by siRNA-mediated knock-down. All expression plasmids are available in the laboratory.
  • control plasmid Lc is a scrambled version of L394.
  • LC394, LCl 176 and LC control were also looked at.
  • Lipofectamine was used to transiently trans feet cells with these plasmids; this is a standard approach.
  • Lipofectamine was used to transiently trans feet cells with these plasmids; this is a standard approach.
  • Sphingolipid synthesis was determined using direct radiotracer incorporation, pulse-chase. Microsomal assays well non in the art can also be used. Cells were plated in 12-well plates, grown to confluency and incubated for 2-16h with inhibitors and stimulators of sphingolipid synthesis ( Figure 9, Tables 1 and 2). Endpoint for all experiments was lipid extraction and separation by TLC (see analysis of lipid synthesis, below). For example, cells were incubated in the presence of palmitic acid to increase de-novo sphingolipid synthesis or lauric acid to increase recycling pathways. Or, cells were incubated for 6h in the presence of myriocin or C6-ceramide to decrease de-novo sphingolipid synthesis.
  • Microsomal assays SPT activity can also be assessed in standard microsomal assays. For example, to prepare microsomes, 75 x 10 6 cells will be pelleted, washed with cold PBS and resuspended in homogenization buffer (300 ⁇ l, HEPES, pH 7.4, EGTA, NAF, leupeptin, trypsin inhibitor). Cells will be disrupted with a tissue homogenizer, lysates centrifuged (800 x g, 5 min), postnuclear supernatant centrifuged (250,000 x g, 30 min) and the microsomal membrane pellet will be resuspended in homogenization buffer.
  • homogenization buffer 300 ⁇ l, HEPES, pH 7.4, EGTA, NAF, leupeptin, trypsin inhibitor.
  • SPT activity will be assessed by dissolving 100-300 ⁇ g of microsomal protein in 0.1 ml of reaction buffer (HEPES, DTT, EDTA, pyridoxal phosphate, palmitoyl-CoA) in the presence of 3 H-serine or 3 H-palmitoyl- CoA (1 ⁇ Ci) for 10 min at 37°C.
  • Controls will be microsomes extracts of palmitoyl-CoA (positive) or myriocin (negative) treated cells. Radioactivity associated with sphinganine and ceramide will be evaluated by TLC and scintillation counting.
  • Lipid mass analysis We will use enzymatic methods and mass spectrometry to measure lipid mass. Ceramide mass was assessed by the diacyl-glycerol (DAG) kinase assay. Extracted lipids were solubilized by bath-sonication (30 min, 37°C) in the presence of di-oleoyl- phosphatidylglycerol and ⁇ -octylglucoside (825 mM) to generate micelles, incubated for 30 min with 32 P- ⁇ ATP (0.1 ⁇ l, 3000 Ci/mmol), DAG kinase (1 U) and carrier ATP (50 mM), followed by extraction and separation on TLC (chloroform:methanol:acetic acid (130:30:10)).
  • DAG diacyl-glycerol
  • Ceramide standards (0.5-20 nmol) were run in parallel. In this solvent system, ceramide stays at the origin and ceramide- 1 -phosphate migrates up. Mass was quantified by densitometry of radioactive spots and correlation to known standards. Sphingomyelin mass was determined in a quantitative high-throughput 96-well assay 68 . After incubation of cells in the presence or absence of sphingomyelinase (10 ⁇ U/ml, 30 min) to generate phosphorylcholine, lipids were extracted, subjected to alkaline phosphatase (lU/ml) and choline oxidase (lO ⁇ U/ml) to generate hydrogen peroxide.
  • Fatty acid and triglyceride synthesis will assessed by incubation for 4h in the presence of H- acetyl-CoA (0.5 ⁇ Ci/ml, fatty acids and triglycerides) or 3 H-oleic acid (triglycerides only) tracers and in the presence and absence of cerulenin, an inhibitor of fatty acid synthesis (500 ⁇ M,). All lipids were extracted with hexane:isopropanol (3:2), dried under N 2 , re-suspended, spotted on TLC plates, separated in hexane:ethyl-ether:glacial acetic acid (70:30:1), and visualized in iodine vapors.
  • H- acetyl-CoA 0.5 ⁇ Ci/ml, fatty acids and triglycerides
  • 3 H-oleic acid triglycerides only
  • Desirable concentrations of the labeled organisms are 10 6 -10 8 cfu/ml.
  • P. aeruginosa-GPF was cultured in MEM9 medium containing piperacillin (200 ⁇ g/ml) until late logarithmic growth is reached.
  • P. aeruginosa controls were cultured in MEM9 alone.
  • Logarithmic phase was determined by measurement of the optical density at ⁇ 600 nm. Numbers of bacteria were confirmed by determination of the cfu of diluted aliquots on MacConkey agar plates.
  • glucosylceramide synthesis To differentiate between the dependence on glucosylceramide synthesis and sphingolipid synthesis, cells were preincubated with conditions that decrease de-novo synthesis (i.e. myriocin or unsaturated fatty acids) together with conditions that increase glucosylceramide synthesis such as acetylsphingosine or sphingomyelinase.
  • de-novo synthesis i.e. myriocin or unsaturated fatty acids
  • acetylsphingosine or sphingomyelinase To assess whether sphingolipid synthesis alone, independent of CFTR affects P. aeruginosa adherence, we will use A549 cells that do not express CFTR, modulate sphingolipid synthesis and measure P. aeruginosa-adherence. To evaluate whether inhibition of CFTR function affects P.
  • aeruginosa adherence cells that express functional CFTR was incubated in the presence or absence of the CFTR inhibitor GlyH-101, followed by assessment of P. aeruginosa adherence.
  • GlyH-101 the CFTR inhibitor
  • P. aeruginosa adherence we will assess ceramide and glycosphingolipid synthesis using radioactive tracer incorporation and TLC separation as outlined in d.l.b.
  • 35 S-labeled P. aeruginosa to assess internalization and differentiate between intracellular and extracellular organisms.
  • cells were plated in 35 mm cell Matek microscopy culture dishes. Confluent monolayers were incubated with P. aeruginosa- GFP for the desired time. At the end of the experiment, cells were washed and fixed for 15 min in 3% paraformaldehyde. Cells will then be mounted and wide field images were collected on a DMIRB inverted Leica microscope with standard GFP optics. Images were collected on a Zeiss LSM510 laser scanning confocal unit equipped with an image capture device.
  • Excitation was produced with a 24m W-Argon laser (excitation ⁇ 488 nm, emission ⁇ 505 nm, long pass filter).
  • cells were visualized with transmitted light to ensure that they are intact.
  • fluorescence was visualized and intensity was determined using MetaMorph image analysis software (Universal Imagine).
  • MetaMorph image analysis software Universal Imagine.
  • In the second methods we will assess uptake using 35 S-labeled bacteria. Confluent monolayers were incubated at the end of the incubation period with cidal concentrations (> 50 ⁇ g/ml) of gentamicin to kill extracellular bacteria. The cells will then be washed extensively and lysed using 0.1 NaOH. Aliquots of cell lysates were analyzed by scintillation counting.
  • 3 H-serine was used to assess de-novo sphingolipid synthesis.
  • 3 H-sphingosine was used to assess sphingolipid synthesis though recycling pathways.
  • lipids were extracted by chloroform/methanol, separated by TLC and dpm associated with ceramide were quantified using a scintillation counter.
  • the data show an inverse relationship of de-novo sphingolipid synthesis and plasma HDL cholesterol levels (FIG. 13a) but no correlation with reverse sphingolipid synthesis (FIG. 13b). Recycling pathways do not correlate with plasma HDL levels.
  • Nucleic Acid sequence 1689 nucleotides atgcggccggagcccggaggctgctgctgccgccgcacggtgcgggcgaatggctgcgtg gcgaacggggaagtacggaacgggtacgtgaggagcagcgccgcagccgcagccgcagcccgcggccagatccatcatgttacacaaaatggaggactatataaaagaccgtttaat gaagcttttgaagaaacaccaatgctggttgctgtgctcacgtatgtggggtatggcgta ctcaccctctttggatcttcgagatttctttgaggtattggagaattgaaagtgtcac catgcaacagaaag
  • SEQ ID NO. 5 Sphingosine kinase 1 Homo sapiens Amino Acid Sequence
  • QKPLCSLP AGSGNAL AASLNHYAGYEQVTNEDLLTNCTLLLCRRLLSPMNLLSLHTASG
  • SEQ ID NO. 6 Sphingosine kinase 1 Homo sapiens Nucleic Acid Sequence atggatccagtggtcggttgcggacgtggcctctttggttttgttttctcagcgggcggc ccccggggcgtgctggtgctgaacccgcgcggcggc aagggcaaggccttgcagctcttccggagtcacgcccctttggctgaggctgaa atctccttcacactgagcggcggaaccacgcgcgggagctggtgcggtcg gaggagctgggccgctgggtggtggtggtcatg
  • SEQ ID NO. 8 Sphingosine kinase 2 Homo sapiens atgaatggacaccttgaagcagaggagcagcaggaccagaggccagaccaggagctgacc gggagctggggccacgggcctaggagcaccctggtcagggctaaggccatggccccgccccaccgccactggctgccagcaccccgctcctccatggcgagtttggctcctacccagcc cgaggcccacgctttgccctcacccttacatcgcaggccctgcacatacagcggctgcgc cccaaacctgaagccaggccccccggggtggtggtggtggcccgtggccgaggtctcaggctgc tgc cccaaacctgaa
  • SEQ ID NO. 11 Fatty acid desaturase (ceramide synthase) Homo sapiens Gene Name DEGS2 Amino Acid Sequence
  • SEQ ID NO. 12 Fatty acid desaturase (ceramide synthase) Homo sapiens Gene Name DEGS2 Nucleotide sequence atgggcaacagcgcgagccgcagcgacttcgagtgggtctacaccgaccagccgcacacg cagcggcgcaaggagatactggccaagtacccggccatcaaggccctgatgcggccagac ccgctcaagtgggcggtgctggtgctggtgcagatgctgacctgctggctg gtgcgcgtgtgtgtgtgtgtgtgtgtgtgtgtgaccactcgctgacgctggccatccacgacatctcgcacaacgc
  • SEQ ID NO. 14 Fatty acid desaturase Gene Name DEGSl Nucleic Acid Sequence Homo Sapiens atggggagccgcgtctcgcgggaagacttcgagtgggtctacaccgaccagccgcacgcc gaccggcgccgggagatcctggcaaagtatccagagataaagtccttgatgaaacctgat cccaatttgatatggattataattatgatggttctcacccagttgggtgcattttacata gtaaaagacttggactggaaatgggtcatatttggggcctatgcgtttggcagttgcatt aaccactcaatgactctggctattcatgagattgcccacaatgctgcctttggcaactgc aaagcaatgtggaatctggtgg
  • a novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Cell. 1991;65: 1043-1051.
  • Cystic fibrosis epithelial cells have a receptor for pathogenic bacteria on their apical surface. Proc Natl Acad Sci USA. 1995;92:3019-3023.
  • CFTR is a pattern recognition molecule that extracts Pseudomonas aeruginosa LPS from the outer membrane into epithelial cells and activates NF -kappa B translocation. Proc Natl Acad Sci USA. 2002;99:6907-6912.
  • Kelley TJ al-Nakkash L
  • Drumm ML CFTR-mediated chloride permeability is regulated by type III phosphodiesterases in airway epithelial cells. Am J Respir Cell MoI Biol. 1995;13:657-664.
  • Sullards MC Analysis of sphingomyelin, glucosylceramide, ceramide, sphingosine, and sphingosine 1-phosphate by tandem mass spectrometry. Methods Enzymol. 2000;312:32- 45.
  • Lysophosphatidic acid is constitutively produced by human peritoneal mesothelial cells and enhances adhesion, migration, and invasion of ovarian cancer cells. Cancer Res. 2006;66:3006-3014.
  • Radin NS Preventing the binding of pathogens to the host by controlling sphingo lipid metabolism. Microbes Infect. 2006;8:938-945.

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L'invention concerne des procédés et des compositions pour le traitement de la mucoviscidose et des infections P. aeruginosa par administration d'une quantité thérapeutique d'un ou de plusieurs agents inhibant la nouvelle synthèse de sphingolipide ou les voies de recyclage.
PCT/US2007/072801 2006-07-03 2007-07-03 Régulateur de la conductance transmembranaire de la mucoviscidose (cftr) défectueux provoquant une synthèse accrue de sphingolipide WO2008006007A2 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
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WO2009001097A2 (fr) * 2007-06-27 2008-12-31 Isis Innovation Limited Thérapie par réduction de substrat
ITFE20080032A1 (it) * 2008-10-23 2010-04-24 Azienda Ospedaliera Istituti Ospit Alieri Di Vero Effetto anti infiammatorio del miglustat cellule dell'epitelio bronchiale
TWI405579B (zh) * 2008-09-26 2013-08-21 Univ Nat Taiwan 伏馬鐮毒素b1用於製備治療病毒性出血熱的藥學組成物的用途
WO2016011535A1 (fr) * 2014-07-25 2016-01-28 Laurent Pharmaceuticals Formulation orale solide de fenrétinide
EP2906570A4 (fr) * 2012-10-15 2016-06-08 Yeda Res & Dev Utilisation de bases à chaînes longues sphingoïdes et de leurs analogues dans le traitement et la prévention d'infections bactériennes
US10406127B2 (en) 2014-07-25 2019-09-10 Laurent Pharmaceuticals Solid oral formulation of fenretinide
WO2020016894A1 (fr) 2018-07-19 2020-01-23 Yeda Research And Development Co. Ltd. Analogues de sphingosine et leur utilisation contre des infections pulmonaires bactériennes
WO2020011745A3 (fr) * 2018-07-11 2020-02-20 Roche Innovation Center Copenhagen A/S Oligonucléotides antisens ciblant cers6
WO2020011744A3 (fr) * 2018-07-11 2020-02-27 Roche Innovation Center Copenhagen A/S Oligonucléotides antisens ciblant cers5
WO2020112967A1 (fr) * 2018-11-29 2020-06-04 University Of Massachusetts Modulation de sptlc1 par l'intermédiaire de vecteurs adéno-associés recombinés

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070197656A1 (en) * 2004-03-02 2007-08-23 Stan Kubow Compositions and methods for preventing or treating an inflammatory response

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070197656A1 (en) * 2004-03-02 2007-08-23 Stan Kubow Compositions and methods for preventing or treating an inflammatory response

Cited By (15)

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WO2009001097A2 (fr) * 2007-06-27 2008-12-31 Isis Innovation Limited Thérapie par réduction de substrat
WO2009001097A3 (fr) * 2007-06-27 2009-07-02 Isis Innovation Thérapie par réduction de substrat
US8557844B2 (en) 2007-06-27 2013-10-15 The Chancellor, Masters And Scholars Of The University Of Oxford Substrate reduction therapy
US9428541B2 (en) 2007-06-27 2016-08-30 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Substrate reduction therapy
TWI405579B (zh) * 2008-09-26 2013-08-21 Univ Nat Taiwan 伏馬鐮毒素b1用於製備治療病毒性出血熱的藥學組成物的用途
ITFE20080032A1 (it) * 2008-10-23 2010-04-24 Azienda Ospedaliera Istituti Ospit Alieri Di Vero Effetto anti infiammatorio del miglustat cellule dell'epitelio bronchiale
EP2906570A4 (fr) * 2012-10-15 2016-06-08 Yeda Res & Dev Utilisation de bases à chaînes longues sphingoïdes et de leurs analogues dans le traitement et la prévention d'infections bactériennes
WO2016011535A1 (fr) * 2014-07-25 2016-01-28 Laurent Pharmaceuticals Formulation orale solide de fenrétinide
US10406127B2 (en) 2014-07-25 2019-09-10 Laurent Pharmaceuticals Solid oral formulation of fenretinide
US10512619B2 (en) 2014-07-25 2019-12-24 Laurent Pharmaceuticals Solid oral formulation of fenretinide
WO2020011745A3 (fr) * 2018-07-11 2020-02-20 Roche Innovation Center Copenhagen A/S Oligonucléotides antisens ciblant cers6
WO2020011744A3 (fr) * 2018-07-11 2020-02-27 Roche Innovation Center Copenhagen A/S Oligonucléotides antisens ciblant cers5
WO2020016894A1 (fr) 2018-07-19 2020-01-23 Yeda Research And Development Co. Ltd. Analogues de sphingosine et leur utilisation contre des infections pulmonaires bactériennes
US11471430B2 (en) 2018-07-19 2022-10-18 Yeda Research And Development Co. Ltd. Sphingosine analogs and use thereof against bacterial lung infections
WO2020112967A1 (fr) * 2018-11-29 2020-06-04 University Of Massachusetts Modulation de sptlc1 par l'intermédiaire de vecteurs adéno-associés recombinés

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