WO2005094864A2 - Traitement de tumeurs qui secretent du wnt et du hedgehog avec des inhibiteurs de biogenese de particule de lipoproteine - Google Patents

Traitement de tumeurs qui secretent du wnt et du hedgehog avec des inhibiteurs de biogenese de particule de lipoproteine Download PDF

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WO2005094864A2
WO2005094864A2 PCT/EP2005/003326 EP2005003326W WO2005094864A2 WO 2005094864 A2 WO2005094864 A2 WO 2005094864A2 EP 2005003326 W EP2005003326 W EP 2005003326W WO 2005094864 A2 WO2005094864 A2 WO 2005094864A2
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hedgehog
lipophorin
tumor
protein
lipoprotein
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PCT/EP2005/003326
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WO2005094864A3 (fr
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Suzanne Eaton
Christoph Thiele
Daniela Panakova
Eric Marois
Hein Sprong
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Max-Planck Gesellschaft zur Förderung der Wissenschaften e.V.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates to the use of an inhibitor of Microsomal Triglyceride Transfer Protein (MTP), HMG-CoA reductase, DGAT and/or ACAT for the preparation of a pharmaceutical composition for the treatment of tumors.
  • MTP Microsomal Triglyceride Transfer Protein
  • HMG-CoA reductase HMG-CoA reductase
  • DGAT DGAT
  • ACAT ACAT
  • growth and/or progression of the tumor are caused by one or more protein of the Wnt or Hedgehog family.
  • Preferred tumors are esophageal tumor, biliary tract tumor, gastric tumor, pancreatic tumor, malignant melanoma, colorectal tumor, squamous cell carcinoma and cervical tumor.
  • Wnt and Hedgehog family proteins are secreted ligands that play multiple critical roles in the development of multicellular organisms. Alterations in the cellular signaling pathways that respond to these Wnt and Hedgehog family ligands also play causative roles in the initiation and progression of a variety of tumors (Xie and Abbruzzese (2003)). Mutations in Hedgehog signal transduction components give rise to tumors of the skin, muscle and cerebellum.
  • Wingless signal transduction components is a critical step in the development of colon cancer and is associated with a variety of other types of malignancies (reviewed in Giles et al. (2003)). While alterations in the downstream components of these pathways have been known for some time, recent studies have shown that tumorigenesis can depend on the unregulated production of the ligands themselves.
  • Wnt1 One of the earliest identified oncogenes is Wnt1, whose ectopic activation in mouse mammary cells is the basis for MMTV mediated tumorigenesis (Nusse and Varmus (1992)). Consistent with this, overproduction of a variety of Wnt family ligands has been observed in human breast cancers.
  • Wnt proteins in humans is also symptomatic of many gastric cancers, colorectal cancers, pancreatic cancers, esophageal cancers, squamous cell carcinomas, cervical cancers and malignant melanomas.
  • a causative role has been directly demonstrated for Wnt5a in promoting both cell motility and invasion of malignant melanoma cells (Weeraratna et al. (2002)).
  • Wnt overproduction contributes to malignant phenotypes is not yet characterized in all cases, the frequency with which mutations in Wnt signaling pathway components promote other neoplasias suggests that Wnt overproduction has tumorigenic consequences.
  • Hedgehog ligands Overproduction of Hedgehog ligands has been demonstrated to play a direct causal role in promoting growth of tumors of the gastrointestinal tract, including those of the esophagus, stomach biliary tract and pancreas (Berman et al. (2003); Thayer et al. (2003). These tumors are very aggressive and some of the most resistant to current therapy.
  • the relevance of Hedgehog signalling for cancer formation and maintenance has been reviewed in Pasca di Magliano and Hebrok (2003). Preventing secretion of Hedgehog and Wnt proteins should be an effective therapy for cancers that depend on the unregulated production of these ligands.
  • Wingless and Hedgehog are covalently modified by lipid, which is thought to mediate their interaction with the exoplasmic face of the plasma membrane. This observation raises perplexing questions about how Wingless and Hedgehog, having affinity for cell membranes, are released from the cells that make them and move through adjacent tissue. As yet, it is unclear how lipid-modified proteins leave the plasma membrane and move over many cell diameters.
  • the technical problem underlying the present invention was to provide novel means and methods for the treatment of Hedgehog- and/or Wnt-secreting tumors.
  • this invention relates to the use of an inhibitor of Microsomal Triglyceride Transfer Protein (MTP), HMG-CoA reductase, DGAT and/or ACAT for the preparation of a pharmaceutical composition for the treatment of tumors.
  • MTP Microsomal Triglyceride Transfer Protein
  • HMG-CoA reductase HMG-CoA reductase
  • DGAT DGAT
  • ACAT ACAT
  • MTP Microsomal Triglyceride Transfer Protein
  • apoB apolipoprotein B
  • VLDL very low density lipoproteins
  • chylomicrons apolipoprotein B
  • LDL low density lipoproteins
  • inhibitors of HMG-CoA reductase, DGAT or ACAT which are enzymes involved in lipid biosynthesis affect the levels of lipoproteins.
  • the term "inhibitor” designates a compound lowering the activity of a target molecule, preferably by performing one or more of the following effects: (i) the transcription of the gene encoding the protein to be inhibited is lowered, (ii) the translation of the mRNA encoding the protein to be inhibited is lowered, (iii) the protein performs its biochemical function with lowered efficiency in presence of the inhibitor, and (iv) the protein performs its cellular function with lowered efficiency in presence of the inhibitor.
  • the inhibitor in particular with regard to inhibition of HMG-CoA reductase, is a statin.
  • Compounds falling in class (i) include compounds interfering with the transcriptional machinery and/or its interaction with the promoter of said gene and/or with expression control elements remote from the promoter such as enhancers.
  • Compounds of class (ii) comprise antisense constructs and constructs for performing RNA interference well known in the art (see, e.g. Zamore (2001) or Tuschl (2001)).
  • Compounds of class (iii) interfere with molecular function of the protein to be inhibited, in case of MTP with its enzymatic activity, in particular with the protein disulfide isomerase activity.
  • active site binding compounds in particular compounds capable of binding to the active site of any protein disulfide isomerase, are envisaged. More preferred are compounds specifically binding to an active site of MTP. Also envisaged are compounds binding to or blocking substrate binding sites of MTP as are compounds binding to or blocking binding sites of MTP for other interaction partners. An example for such an interaction partner would be apolipoproteinB (apoB). The latter group of compounds blocking binding sites of MTP may be fragments or modified fragments with improved pharmacological properties of the naturally occurring binding partners.
  • Class (iv) includes compounds which do not necessarily directly bind to MTP, but still interfere with MTP activity, for example by binding to and/or inhibiting the function or inhibiting expression of members of a pathway which comprises MTP. These members may be either upstream or downstream of MTP within said pathway.
  • the inhibitor is a low molecular weight compound.
  • Low molecular weight compounds are compounds of natural origin or chemically synthesized compounds, preferably with a molecular weight between 100 and 1000, more preferred between 200 and 750, and even more preferred between 300 and 600.
  • the efficiency of the inhibitor can be quantitized by comparing the level of activity in the presence of the inhibitor to that in the absence of the inhibitor.
  • an activity measure may be used: the change in amount of mRNA formed, the change in amount of protein formed, the change in amount of substrate converted or product formed, and/or the change in the cellular phenotype or in the phenotype of an organism.
  • the level of activity is less than 90%, more preferred less than 80%, 70%, 60% or 50% of the activity in absence of the inhibitor. Yet more preferred are inhibitors lowering the level down to less than 25%, less than 10%, less than 5% or less than 1 % of the activity in absence of the inhibitor.
  • Hedgehog and Wingless are released from cells on Lipoprotein particles. It has been speculated that these particles (which were termed "Argosomes”) represent a new type of particles. It further has been shown that these particles are required for the signaling activity of Wingless and Hedgehog (Panakova et al, in preparation).
  • the single insect lipoprotein organized by Apolipophorin I /II is similar in biosynthesis, structure and function to vertebrate lipoprotein particles like LDL and VLDL; it transports lipid and cholesterol from the gut to the fat body (an organ analogous to the liver and adipose tissue), and distributes lipid and sterol to peripheral tissues.
  • Apolipophorin is highly homologous to ApolipoproteinB. Therefore, drugs that inhibit the formation of lipoprotein particles in humans are expected to be useful for the treatment of tumors that secrete proteins of the Hedgehog and Wnt families.
  • MTP Microsomal Triglyceride Transfer Protein
  • Lipoprotein particles the transport of lipid-modified proteins. Lipid modifications like those present in Wingless, Hedgehog and gpi-anchored proteins, which target proteins to the exoplasmic membrane leaflet, would be of the correct length and topology to insert into the phospholipid monolayer of a Lipoprotein particle.
  • the present inventors have shown that Wingless, Hedgehog and gpi-anchored proteins co-purify and co- immunoprecipitate with Drosophila Lipophorin. Futhermore, Wingless and Hedghog co-localize extensively with Lipoprotein particles in tissue. Therefore, the particles previously termed "argosomes" are actually morphogen-bearing Lipoprotein particles.
  • lipid-linked proteins of the exoplasmic face of the membrane associate with Lipoproteins. These include many gpi-linked proteins with diverse functions, as well as the lipid-linked morphogens Wingless and Hedgehog. The mechanism allowing long-range dispersal of lipid-linked proteins is not yet understood. The finding that these proteins exist in both membrane-associated and Lipoprotein-associated forms suggests reversible binding to Lipoprotein particles as a plausible mechanism for intercellular transfer, and the consequences of lowering lipoprotein levels in Drosophila larvae supports this idea.
  • Lipophorin knock down narrows the range of both Wingless and Hedgehog signalling. Hedgehog accumulates to an abnormally high level in cells near the source of production and long-range signaling is inhibited; short-range target genes, however, are expressed normally. These data suggest that Hedgehog does not move as far when Lipophorin levels are low. The range over which Hedgehog moves is normally restricted by Patched-mediated endocytosis. In discs from Lipophorin RNAi larvae, accumulated Hedgehog co-localizes with Patched in endosomes, suggesting that it is more efficiently sequestered by Patched.
  • the disclosed data is consistent with the idea that Lipophorin is continuously needed for movement, rather than required only for the release of morphogens. If Lipophorin were important only for Hedgehog secretion, one would expect Lipophorin RNAi to decrease the amount of Hedgehog found in receiving tissue; this seems not to be the case. Furthermore, altered Hedgehog trafficking in receiving tissue is consistent with a model in which Lipophorin is required at each step of intercellular transfer. Without being bound by a theory, the inventors favour the idea that reversible association of Hedgehog with Lipophorin particles facilitates its transfer from the plasma membrane of one cell to that of the next.
  • argosomes are exosome-like particles with an intact membrane bilayer, and that lipid-linked morphogens needed to be assembled on these particles to be secreted by producing cells. Instead, the present invention discloses that argosomes are exogenously derived lipoproteins that facilitate that movement of morphogens through the epithelium. Many questions remain as to how morphogens become associated with argosomes, and how the spread and cell- interactions of these particles are regulated. Clearly, heparan sulfate proteoglycans are essential for the movement of Hedgehog and Wingless into receiving tissue 35 ' 36 .
  • HSPG's facilitate morphogen movement through Lipoprotein binding.
  • the inventors find many gpi-linked proteins, including the HSPG's Dally and Dally-like (unpublished data), on Lipoprotein particles themselves. These associated proteins have the potential to modulate the cellular affinities or trafficking properties of Lipoproteins and the morphogens they carry.
  • Lipophorin particles not only mediate intercellular transfer of Hedgehog, but may also be endocytosed together with the morphogen.
  • LDL receptor related proteins Arrow and Megalin have demonstrated roles in Wingless signaling and Hedgehog endocytosis, respectively 39"41 . It is intriguing to speculate that these receptors might be important for interaction with the Lipoprotein-associated form of the morphogen.
  • Cholesterol has the potential to modulate the activity of the Hedgehog pathway at many different points 3 ' 42-44 . Whether changes in the level of cellular cholesterol normally play a role in regulating the activity of the pathway is unclear. Here it is shown that Hedgehog interacts with the particle that delivers sterol to cells. This observation raises the possibility that internalization of Hedgehog is linked to sterol uptake, and suggests new mechanisms to link nutrition, growth and signalling during development. All known Hedgehog signal transduction pathways in vertebrates and invertebrate act by regulating the processing of GLI-family transcription factors, and ectopic activation of GLI proteins is a common feature of many tumors ⁇ Ruiz i Altaba, 2002 #674 ⁇ .
  • Lowering Lipophorin levels also inhibits the growth of imaginal discs by mechanisms that do not depend on morphogen signalling.
  • the inventors have shown that sterol delivery itself is essential for growth; lowering Lipophorin levels, or removing sterol from the diet blocks growth primarily at the level of cell division. Like insect cells, many human cell types do not synthesize their own sterol but rely on LDL-mediated uptake. The existence of a sterol-dependent growth control checkpoint indicates that LDL-lowering drugs should block the proliferation of tumors derived from such tissues. The inventors therefore propose to utilize drugs that lower LDL levels in humans to treat tumors derived from neurons and steroidogenic cells.
  • an alternative embodiment relates to the use of an inhibitor of lipoprotein secretion for the preparation of a pharmaceutical composition for the treatment of tumors.
  • Another alternative embodiment relates to the use of an inhibitor of lipoprotein formation for the preparation of a pharmaceutical composition for the treatment of tumors.
  • Yet another alternative embodiment relates to the use of an inhibitor of lipoprotein transport for the preparation of a pharmaceutical composition for the treatment of tumors.
  • a further embodiment relates to the use of an inhibitor of lipoprotein association with a target ceil for the preparation of a pharmaceutical composition for the treatment of tumors.
  • Targets that affect interaction of Lipoproteins with target cells are e.g. LDL receptors and LDL receptor family proteins, preferably LRP5 and 6, and the LRP Megalin which have been shown to be involved in wingless and hedgehog signalling.
  • Other targets affecting interaction of Lipoproteins with target cells are Heparan sulfate proteoglycans
  • a further embodiment relates to the use of an inhibitor of the association of a protein of the Wnt or Hedgehog family with lipoproteins for the preparation of a pharmaceutical composition for the treatment of tumors.
  • This class of inhibitors prevent association of Wingless or Hedgehog with Lipoproteins include any inhibitors of the acyl-transferases that acylate Wingless and Hedgehog (these are called Porcupine and Skinny Hedgehog, respectively).
  • Another inhibitor which is in accordance with the teaching of the present invention is the enzyme acyl protein thioesterase (APT-1), which will cleave the palmitate from the Wingless protein, preventing its association with Lipoproteins (Willert et al., Nature 423, p 448).
  • the Wnt pathway and the Hedgehog pathways are similar, evolutionary conserved signal transduction pathways playing a role, inter alia, in embryogenesis and tumorigenesis. Despite identity or similarity of several components of the two pathways, the Wnt and Hedgehog protein, although located at corresponding positions of the two pathways, are unrelated proteins.
  • the Hedgehog family of proteins includes Hedgehog (in Drosophila) and, in humans, Desert Hedgehog, Indian Hedgehog, and Sonic Hedgehog.
  • the Wnt family of proteins appears to be larger, comprising the Drosphila proteins Wingless, DWnt2, DWnt3/5, DWnt4, DWnt6, DWnt ⁇ and DWntlO. There are more than a dozen vertebrate Wnt proteins, from Wnt1 to Wnt16, however, not strictly sequentially numbered. Vertebrate Wnt1 is the orthologue of Drosophila Wingless.
  • said association of a protein of the Wnt or Hedgehog family with lipoproteins is inhibited by inhibiting the covalent lipid modification of said protein of the Wnt or Hedgehog family, thereby reducing its affinity for said lipoprotein.
  • the lipid modifications include palmitoylation and cholesterol modification.
  • Hedgehog proteins are palmitoylated and cholesterol modified; Wnt proteins are palmitoylated.
  • Any inhibitor of the lipid modification of Wnt or Hedgehog proteins is an inhibitor useful for the purpose of the present invention. Accordingly, use of such inhibitors for the preparation of the pharmaceutical composition according to the invention is envisaged.
  • said lipoprotein is very low density lipoprotein (VLDL) or low density lipoprotein (LDL).
  • Lipoprotein particles comprise a phospholipid monolayer surrounding a core of esterified sterol and triglycerides, and are organized by different Apolipoproteins.
  • Lipoprotein particles of different densities and apolipoprotein composition transport lipid, sterols and fat-soluble vitamins between the gut, liver and peripheral tissues. Insects have only one apolipoprotein, Apolipophorin, which is most similar to vertebrate ApoB. Physiological studies indicate that it plays a role analogous to that of vertebrate Lipoproteins, transporting sterol and diglyceride between tissues.
  • said tumor is a malignant tumor.
  • Malignant tumors are also referred to as cancers herein.
  • growth and/or progression of the tumor are caused by one or more protein of the Wnt or Hedgehog family.
  • the person skilled in the art can identify which tumors fall under this definition without undue burden.
  • said tumor over-expresses one or more protein of the Wnt or Hedgehog family.
  • the skilled person is aware of concrete tumors and malignant diseases comprised in this definition. Furthermore, means and methods are described below enabling the person skilled in the art to identify which tumors or malignant diseases are encompassed by the embodiment recited above.
  • over-expression denotes an expression level of an mRNA encoding a protein of the Wnt or Hedgehog family and/or of a protein of the Wnt or Hedgehog family, which is elevated in comparison to normal expression.
  • normal expression refers to a reference expression level determined in one or more samples from healthy individuals. These samples are preferably from healthy tissue corresponding to the tissue affected by the tumor under consideration. Samples may be drawn from a mixed population, from a fraction of the population, wherein the population has previously been stratified according to one or more parameters, or from healthy regions of the tissue affected by the tumor from the same patient. Statistical methods known in the art may be used in order to assign significance values and confidence intervals to the measured expression and over-expression data.
  • the expression level to be determined is the mRNA expression level.
  • Methods for the determination of mRNA expression levels are known in the art and comprise Real Time PCR, Northern blotting and hybridization on microarrays or DNA chips equipped with one or more probes or probe sets specific for transcripts encoding proteins of the Wnt or Hedgehog family.
  • the expression level to be determined is the protein expression level.
  • the skilled person is aware of methods for the quantitation of proteins. Amounts of purified protein in solution can be determined by physical methods, e.g. photometry. Methods of quantifying a particular protein in a mixture rely on specific binding, e.g of antibodies. Specific detection and quantitation methods exploiting the specificity of antibodies comprise immunohistochemistry (in situ) and surface plasmon resonance. Western blotting combines separation of a mixture of proteins by electrophoresis and specific detection with antibodies.
  • said protein of the Wnt family is Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt ⁇ A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt ⁇ A, Wnt ⁇ B, Wn9A, Wnt9B, WntlOA, WntlOB, Wnt11 and/or Wnt16 and/or said protein of the Hedgehog family is Desert Hedeghog, Indian Hedgehog and/or Sonic Hedgehog. A list of Wnt genes and pertinent information is maintained by Roel Nusse at http://www.stanford.edu/ ⁇ rnusse/wntwindow.html.
  • said tumor is selected from the group consisting of esophageal tumor, biliary tract tumor, gastric tumor, pancreatic tumor and malignant melanoma.
  • Wnt and/or Hedgehog family proteins have been shown to play a causative role in tumor growth or progression.
  • a further preferred embodiment relates to a tumor selected from the group consisting of gastric tumor, colorectal tumor, pancreatic tumor, esophageal tumor, squamous cell carcinoma, cervical tumor and malignant melanoma. These tumors have been shown to over-express one or more members of the Wnt and/or Hedgehog families of proteins.
  • the tumor or cancer is a tumor or cancer of muscle, cerebellum or breast.
  • MTP Microsomal Triglyceride Transfer Protein
  • HMG-CoA reductase statins
  • diacylglycerol acyl transferase and ACAT (acyl-CoA:cholesterol acyl-transf erase).
  • HMG-CoA reductase inhibitors include e.g.atorvastatin (lipitor) (Topliss et al., 2002), simvastatin (Topliss et al. 2002), lovastatin (Topliss et al. 2002), cerivastatin (Yasunobu et al., 1997), pravastatin (BMS product info sheet), fluvastatin (Suzumura et al., 1999), mevastatin (compactin) (Chakravarti et al., 2004), rosuvastatin (Bolego et al., 2002), pitavastatin (Bolego et al., 2002.
  • atorvastatin lipitor
  • simvastatin Topliss et al. 2002
  • lovastatin Topliss et al. 2002
  • cerivastatin Yasunobu et al., 1997)
  • Diacylglycerol acyl transferase -1 inhibitors include Gemfibrozil: 5-(2,5-dimethylphenoxy)-2,2-dimethylpentanoic acid (Ammazzalorso et al., 2002; Zhu et al., 2002).
  • said inhibitor of MTP is selected from the group consisting of diaminoindanes (derivatives of indane) such as 8aR or 19aR (Ksander et al. (2001)); citrus flavonoids (derivatives of flavone) such as naringenin (Borredaile et al. (2003)) or hesperetin; quercitin (Casaschi et al. (2002)); BMS- 197636 (Harrity et al. (1996)); BMS-201038 (Wetterau et al. (1998)); BMS-200150 (Jamil et al.
  • said inhibitor of HMG-CoA is selected from atorvastatin (lipitor), simvastatin, lovastatin, cerivastatin, pravastatin, fluvastatin, mevastatin (compactin), rosuvastatin, pitavastatin.
  • the Diacylglycerol acyl transferase -1 inhibitor is Gemfibrozil: 5-(2,5- dimethylphenoxy)-2,2-dimethylpentanoic acid.
  • compositions prepared according to the invention may further comprise a pharmaceutically acceptable carrier and/or diluent.
  • suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods.
  • These pharmaceutical compositions can be administered to the subject at a suitable dose.
  • Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and clinical factors.
  • dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • a typical dose can be, for example, in the range of 0.001 to 1000 ⁇ g; however, doses below or above this exemplary range are envisioned as well.
  • the dose is in the range of 0.01 to 100 ⁇ g, more preferred between 0.1 and 10 ⁇ g.
  • the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 ⁇ g to 10 mg units per day.
  • compositions prepared according to the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the invention may comprise further agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition.
  • the lipoprotein inhibitor is selected from siRNA, shRNA, ribozyme, antisense nucleic acid molecule, aptamer or inhibitory antibody.
  • the siRNA, shRNA, ribozyme and antisense nucleic acid molecules have a region of complementarity with at least one gene encoding a protein essential for lipoprotein secretion, formation, transport and/or association of lipoproteins with a target cell.
  • Said target may, for example, be selected from the group consisting of MTP, diacylglycerol acyltransferase, or enzymes in the cholesterol biosynthesis pathway such as HMGCoA reductase.
  • the target sequence of the ACAT gene (acyl-CoA:cholesterol acyl-transferase) may be any sequence selected from:
  • the target sequence of the ACAT2 gene (acyl-CoA:cholesterol acyl-transferase 2) may be any sequence selected from:
  • the target sequence of the diacylglycerol acyl transferase gene may be any sequence selected from:
  • the target sequence of the HMGCoA reductase gene may be any sequence selected from:
  • the target sequence of the MTP gene may be any sequence selected from:
  • siRNA means “short interfering RNA”
  • shRNA refers to "short hairpin RNA”.
  • siRNA small interfering RNAs
  • Transfection of cells with siRNAs can be achieved, for example, by using lipophilic agents (among them Oligofectamine ⁇ and Transit-TKOO) and also by electroporation.
  • the siRNA molecules are essentially double-stranded but may comprise 3' or 5' overhangs. They may also comprise sequences that are not identical or essentially identical with the target gene but these sequences must be located outside of the sequence of identity.
  • the sequence of identity or substantial identity is at least 14 and more preferably at least 19 nucleotides long (target sequence). It preferably does not exceed 23 nucleotides.
  • the siRNA comprises two regions of identity or substantial identity that are interspersed by a region of non-identity.
  • the term "substantial identity" refers to a region that has one or two mismatches of the sense strand of the siRNA to the targeted mRNA or 10 to 15% over the total length of siRNA to the targeted mRNA mismatches within the region of identity. Said mismatches may be the result of a nucleotide substitution, addition, deletion or duplication etc. dsRNA longer than 23 but no longer than 40 bp may also contain three or four mismatches.
  • the interference of the siRNA with the targeted mRNA has the effect that transcription/translation is reduced by at least 50%, preferably at least 75%, more preferred at least 90%, still more preferred at least 95%, such as at least 98% and most preferred at least 99%.
  • FIG. 22 A possible system for generating siRNAs would be the one outlined in Miyagishi et al., 2004 (Journal of gene medicine; 6: 715).
  • Figure 22 is adapted from this paper.
  • the 21 nucleotide-long "stem" is derived from the gene to be silenced and contains 3 or 4 C to U or A to G mutations in the sense strand. Intervening between the inverted repeats is a loop sequence derived from a naturally occuring microRNA. Any unique e.g. 21 base pair sequence from the enzymes of interest could be used to generate these constructs (the sequences of the enzymes are attached). For example:
  • a useful siRNA for MTP might be:
  • a useful siRNA for ACAT might be:
  • a useful siRNA for DGAT-1 might be:
  • a useful siRNA for HMG-CoA reductase might be:
  • the inhibitor can be an antisense nucleic acid molecule specifically hybridizing to a nucleic acid molecule (such as an mRNA) derived from gene encoding a protein essential for lipoprotein secretion, formation, transport and/or association of lipoproteins with a target cell.
  • a nucleic acid molecule such as an mRNA
  • the term "antisense nucleic acid molecule” refers to a nucleic acid molecule which can be used for controlling gene expression.
  • the underlying technique, antisense technology can be used to control gene expression through antisense DNA or RNA or through triple-helix formation. Antisense techniques are discussed, for example, in Okano, J. Neurochem.
  • the 5' coding portion of a polynucleotide that encodes a protein essential for lipoprotein secretion, formation, transport and/or association of lipoproteins with a target cell may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a gene region involved in transcription thereby preventing transcription and the production of the protein essential for lipoprotein secretion, formation, transport and/or association of lipoproteins with a target cell.
  • protein refers to proteins and (poly)peptides.
  • the term ((poly)peptide means refers to peptides and polypeptides.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the protein essential for lipoprotein secretion, formation, transport and/or association of lipoproteins with a target cell.
  • ribozyme refers to RNA molecules with catalytic activity (see, e.g., Sarver et al, Science 247:1222-1225 (1990)); however, DNA catalysts (deoxyribozymes) are also known. Ribozymes and their potential for the development of new therapeutic tools are discussed, for example, by Steele et al. 2003 (Am. J. Pharmacogenomics 3: 131-144) and by Puerta-Fernandez et al. 2003 (FEMS Microbiology Reviews 27: 75- 97).
  • ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs for translation of the protein essential for lipoprotein secretion, formation, transport and/or association of lipoproteins with a target cell
  • trans-acting hairpin or hammerhead ribozymes is preferred.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
  • the construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).
  • Ribozymes may be composed of modified oligonucleotides (e.g.
  • DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of other nucleic acid molecules.
  • a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is generally required for efficiency.
  • Ribozyme-mediated RNA repair is another therapeutic option applying ribozyme technologies (Watanabe & Sullenger 2000, Adv. Drug Deliv. Rev. 44: 109-118) and may also be useful for the purpose of the present invention.
  • catalytic group I introns can be employed in a trans-splicing reaction to replace a defective segment of target mRNA in order to alleviate a mutant phenotype.
  • said essential protein of the lipoprotein particle is selected from the group consisting of apolipophorin, Apolipoprotein B100 and Apolipoprotein B48.
  • the aptamer and the inhibitory antibody are specific for a protein essential for lipoprotein secretion, formation, transport and/or association of lipoproteins with a target cell.
  • aptamer refers to RNA and also DNA molecules capable of binding target proteins with high specificity, comparable with the specificity of antibodies.
  • Methods for obtaining or identifying aptamers specific for a desired target are known in the art. Preferably, these methods may be based on the "systematic evolution of ligands by exponential enrichment” (SELEX) process (Ellington and Szostak, Nature, 1990, 346: 818-822; Tuerk and Gold, 1990, Science 249: 505-510; Fitzwater & Polisky, 1996, Methods Enzymol. 267: 275-301).
  • said aptamers may be specific for any of the (poly)peptides listed under (1) to (19).
  • antibody refers to monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, or a fragment thereof. Preferably the antibody is specific for the protein essential for lipoprotein secretion, formation, transport and/or association of lipoproteins with a target cell.
  • the antibodies may be bi-specific antibodies, humanized antibodies, synthetic antibodies, antibody fragments, such as Fab, F(ab 2 )', Fv or scFv fragments etc., or a chemically modified derivative of any of these, all comprised by the term "antibody”.
  • Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Kohler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals with modifications developed by the art.
  • antibodies or fragments thereof specific for the aforementioned proteins can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1998.
  • surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies Swhich bind to an epitope of the peptide or polypeptide to be analyzed (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
  • An inhibitory antibody is an antibody which upon binding interferes with secretion, formation, transport and/or association of lipoproteins with a target cell.
  • FIG. 1 (A) Schematic picture of CD63:GFP fusion protein. GFP is attached to the C terminus of the protein.
  • B CD63:GFP (green) expressed in the wing imaginal disc under the apGAL4 driver, membranes are stained with FM4-64 (red). CD63:GFP is localized to the plasma membrane and large internal vesicles in producing cells, in non-expressing tissue it is found 1 to 3 cell diameters away from producing cells.
  • C CD63:GFP (green) colocalizes with lysotracker (red) in enlarged endocytic compartments.
  • D CD63:GFP (green) colocalizes with CFPRab ⁇ (blue) in non- expressing tissue, membranes are stained with FM4-64 (red).
  • FIG. 2 (A) schematic picture of Drosophila lipophorin. Lipophorin is synthesized as a large precursor, Proapolipophorin, which is then proteolyticaly cleaved into Lipophorinl; ApoLI (220kD) and Lipophorinll; ApoLII (70kD).
  • B Different tissues from 3 rd instar larvae were examined for the presence of ApoLII.
  • anti-ApoLII antibody recognizes doublet of expected size in imaginal discs, brain and fat body, in addition a higher molecular weight band is recognized in the fat body that corresponds to unprocessed proapolipophorin.
  • C Different tissues from 3 rd instar larvae were examined for the presence of ApoLI. anti-ApoLI antibody recognizes a single band of appropriate size in all tissues examined.
  • FIG. 3 Differential centrifugation of homogenates of wild-type larvae or larvae that expressed different GFP fusion proteins.
  • Plasma membrane markers including Thickvein, Cadherin, GFPCD8, GFPCD63 are completely pelleted after 3 hours at 120,000g.
  • Majority of Lipophorin together with soluble cytoplasmic GFP end up in supernatant.
  • Most of gpi-linked proteins including Fasciclin I (Fasl), Connectin, Klingon, acetylcholineasterase (AchE) and lipid-linked morphogens Wingless and Hedgehog are present in the 120,000g pellet.
  • FIG. 4 Isopycnic centrifugation in KBr of S120-proteins.
  • A SDS-PAGE analysis of fractionated KBr gradient of larval homogenate on 10% Coomassie-blue stained gel.
  • B Lipophorin is present in the top, low-density fraction as well as many other gpi-linked proteins, indicating their association with lipidic particles. Upon PI-PLC treatment gpi-linked proteins shift to the high-density fraction, showing that this association is gpi-anchor dependent.
  • Wing imaginal discs are capable of producing lipidic particles.
  • A ApoLII:myc expressed in wing imaginal discs is secreted and endocytosed by non- producing cells (middle). ApoLI is over-accumulated in the same cells in which ApoLl myc is over-expressed (left). Close-up (right) shows ApoLI (red) colocalization with ApoLILmyc (green) in non-expressing tissue. Lipidic particles colocalize with Wingless or Hedgehog.
  • B,C ApoLII:myc (green) expressed either in Wingless or Hedgehog-producing cells, strongly colocalizes with Wingless (red) (B, left) or Hedgehog (red) (C). Note that CD63:GFP (green) expressed in Wingless producing cells does not colocalize with Wingless (red) (B, right)
  • FIG. 6 Lipophorin RNAi.
  • A ApoLII levels of the whole larvae extract are reduced by approximately 80% after induction of RNAi comparing to the wild type.
  • B Images show overaccumulation of neutral lipids (green) and expansion of lipid droplets (green) in the posterior midgut of lipophorin RNAi larvae (right) comparing to the wild type (left), membranes are stained red.
  • C Lipophorin RNAi discs (right) show no dramatic increase in caspase-3 activity comparing to the control (left).
  • Figure 7 Lipophorin depletion reduces the efficiency of Wingless signaling.
  • Dll lower right
  • levels are strongly reduced in lipophorin RNAi wing imaginal discs (see also lower right in B) comparing to the wild type (upper right). Wingless continues to be produced (middle). Merged images are shown to left, dll in green, wg in red.
  • B Levels of hnd (lower middle) are reduced in sensory organ precursors, some of which are Wingless dependent. Notably, hnd continues to be expressed in non-Wingless- dependent sensory organ precursors. Merged images together with dll are shown to the left, hnd in red, dll in green.
  • Figure 8 Lipid-linked proteins cofractionate with Lipophorin. Western blots of fractionated extracts probed with antibodies to indicated proteins.
  • A,B larval S120's and indicated proportions of larval P120's.
  • AchE Acetylcholinesterase.
  • Top fraction 1.14 g/cm 3
  • bottom fraction 1.4 g/cm 3 .
  • G,H S120's from wild-type or fusion protein-expressing larvae immunoprecipitated with pre-immune, anti-ApoLII, or anti-GFP serum.
  • A-C disc stained with anti-Wingless (A and B red) after 20-minute incubation with Alexa488Lipophorin (B green and C).
  • D-F disc expressing CD63:GFP (B green and F) in Wingless-producing cells stained with anti- Wingless (D and E red).
  • G-l disc stained with anti-Hedgehog (G and H red) after 20- minute incubation with Alexa488Lipophorin (H green and I).
  • J-L disc expressing CD63:GFP (K green and L) in Hedgehog-producing cells stained with anti-Hedgehog (J and K red).
  • FIG. 10 Lipophorin-RNAi perturbs lipid transport.
  • YeIlow:Neutral lipid. Red:Plasma membrane. Scale bar 40 ⁇ (B,C) or 10 ⁇ (D-G).
  • A-C dpplacZ/+ disc 4 days AHS stained for LacZ (A,B red) and Collier (B green.C).
  • D-F hs- flippase/+;dpplacZ/+;Tubulin:GAL4/UAS:dsRNA disc 4 days AHS stained for LacZ (D,E red) and Collier (E green, F).
  • G-l wild-type disc 4 days AHS stained for Hedgehog (G,H red) and Patched (H green, I).
  • Figure 12 Hedgehog signalling is unaffected by lipid-depletion.
  • A,B discs from fully fed (A) or lipid-starved (B) larvae.
  • C,D Wings from fully fed (C) or lipid-starved (D) flies.
  • Scale bar 250 ⁇ .
  • FIG. 13 Lipophorin-RNAi narrows the range of Wingless signaling.
  • A-D Apical (A,B) and basolateral (C,D) sections of wild type (A,C) and Adh:GAL4/+; UASdsRNA + (B,D) wing discs 5 days AHS stained for extracellular Wingless.
  • E,F Distalless protein accumulation in wild type (E) and hs-flippase/+;UAS dsRNA/ TubulinGAL4 (F) wing discs 5 days AHS.
  • E) Released CD63 (green) in receiving tissue is present in early endosomes that are marked by CFP:Rab5 (blue). The plasma membrane has been stained red with FM4-64.
  • Figure 15 Hedgehog and Fasciclin I in low density gradient fractions co- immunoprecipitate with Lipophorin
  • the top fraction of a KBr gradient derived from larval S120 was desalted and immunoprecipitated with either pre-immune or anti- ApoLII serum, then probed with antibodies to ApoLII, Hedgehog or Fas-1.
  • Hedgehog and Fas-1 co-immunoprecipitate with ApoLII.
  • FIG. 16 ApoLI levels are decreased by Lipophorin-RNAi.
  • Figure 17 Lipophorin-RNAi does not elevate cell death or prevent Insulin signaling.
  • A,B confocal images of living imaginal discs from either wild type (A) or Lipophorin RNAi (B) larvae that ubiquitously produce a Plekstrin homology domain:GFP fusion protein.
  • C,D confocal images of imaginal discs from either wild type (C) or Lipophorin RNAi (D) larvae stained with an antibody to activated Caspase3.
  • Figure 18 Hedgehog accumulates with Patched in endosomes in Lipophorin-RNAi larvae.
  • A-D. confocal image of an imaginal disc from a Lipophorin RNAi larva stained with antibodies to Patched (A green and C) and Hedgehog (A blue and D). Endosomes were labeled by red dextran uptake (A red and B).
  • Figure 19 Individual traces of Distalless staining intensity for 5 wild type and 5 Lipophorin RNAi discs.
  • Figure 20 Rescue of Hedgehog accumulation in dissected Lipophorin RNAi discs by in vitro Lipophorin addition. All scale bars are 10 ⁇ m.
  • A-C Projection of confocal sections comprising the apical-most 2 ⁇ m of a Lipophorin RNAi imaginal disc incubated for 2 hours in Graces medium. The disc was stained for Hedgehog (A and C red) and Patched (B and C blue).
  • D-F Projection of confocal sections comprising the apical-most 2 ⁇ m of a Lipophorin RNAi imaginal disc incubated for 2 hours with purified Lipophorin particles.
  • the disc is stained for Hedgehog (D and F red) and Patched (E and F blue).
  • Discs were stained in parallel and imaged under identical conditions on the same day. Addition of Purified Lipophorin particles at approximately 1/10 the estimated concentration in hemolymph reduces the accumulation of both Patched and Hedgehog in endosomes in the anterior compartment (D-F) compared to those in mock-treated discs (A-C).
  • FIG. 21 Wing imaginal discs from wild type or lipophorin RNAi larvae were stained with antibodies to Hedgehog (redO and Patched(green, lower panel), and with an antibody that detects only the full length unprocessedform of cubutus Interruptus (Ci) (green in lower panel). Inhibiting Lipophorin production prevents cleavage of Ci throughout the anterior compartment. This uncleaved Ci does not activate Patched.
  • Figure 22 Schematic representation of a proposed siRNA-expression system (adapted from Figure 5 of Miyagishi et al., 2004 Journal of gene medicine; 6: 715).
  • Example 1 A fraction of gpi-linked proteins, Wingless and Hedgehog does not associate with membranes
  • Lipophorin is a Lipoprotein containing two protein subunits, Lipophorinl and Lipophorinll that are generated by cleavage of a larger precursor, Apolipophorin ( Figure 2A). Since Apolipophorin is produced mainly in the fat body, we wished to determine whether Lipophorin particles accumulated in imaginal discs at all. To address this question, we probed Western blots containing different tissues from equal numbers of larvae with antibodies to Lipophorins I and II.
  • Anti- Lipophorin I recognizes a single band of the appropriate size on Western blots from a variety of larval tissues, including imaginal discs (arrow in Figure 2C).
  • Anti-Lipophorinll recognizes a doublet of the expected size in discs and brain (arrow in Figure 2B).
  • An additional higher molecular species is present in extracts from fat body, and its size corresponds to that of the unprocessed Proapolipophorin.
  • the presence of Proapolipophorin only in fat body is consistent with its being the primary source of Lipophorin production. Nevertheless, almost equivalent levels of mature Lipophorin are found in many other tissues including imaginal discs, brain and gut.
  • Wingless:GFP, Hedgehog and gpi-linked proteins including Fasciclinl, GFP:Dally, Connexin, Klingon and Acetylcholineasterase
  • Wingless:GFP, Hedgehog and gpi-linked proteins were present in the 120,000g pellet; this was not unexpected, because these proteins can be found on the plasma membrane or in membrane compartments like endosomes.
  • Example 2 Gpi-linked proteins, Wingless and Hedgehog are present on lipidic particles
  • Figure 4A shows the Coomasie-stained proteins present in the fractions from such a gradient.
  • Western blotting shows that, under these conditions, Lipophorin moves to low density fractions at the top of the gradient whereas soluble proteins like secreted GFP are present in higher density fractions (compare the first two panels of Figure 4B).
  • Gpi-linked proteins are found almost entirely in the low-density fraction with Lipophorin, indicating that they are present on a low-density particle ( Figure 4B).
  • Figure 4C shows that virtually all the Wingless and Hedgehog present in the S120 co-migrates with Lipophorin in the top fraction of density gradients. This suggests that a fraction of Wingless and Hedgehog is associated with low-density particles.
  • Lipophorin is actually transcribed and translated by imaginal disc cells and associates with Wingless or Hedgehog in the biosynthetic pathway.
  • lipophorin RNA was detected only in cells of the fat body (data not shown), consistent with previous reports.
  • the uncleaved precursor, Apolipophorin is detected only in the fat body ( Figure 2B). This indicates that the Lipophorin synthesized in the fat body is taken up and accumulates in the brain, gut and imaginal discs.
  • Lipophorin can transfer its lipid cargo without being degraded and is often recycled by insect cells.
  • imaginal discs could construct lipidic particles from exogenously derived Lipophorin protein.
  • Tissues from myc:Lipophorinll-expressing larvae were homogenized, and the S120 was subjected to isopycnic density centrifugation.
  • ApoLlhmyc was expressed in the fat body, the majority of the protein was incorporated into low-density particles ( Figure 4E).
  • Lipophorin particles normally contain both protein subunits: Lipophorin I and Lipophorin II. Since ApoLILmyc appeared to be incorporated into low density particles by imaginal disc cells, we knew whether these particles might also contain endogenous Lipophorin I. To address this question, we examined the distribution of Lipophorins I and II in imaginal discs expressing ApoLILmyc under the control of patchedGAL4. The over-produced myc fusion protein is detected in pafcfre -expressing cells. It is also found in punctate structures in cells outside the expressing region, indicating that it is secreted and possibly endocytosed by surrounding tissue (Figure 5A).
  • RNA interference reduces but does not eliminate lipid traffic between gut, fat body and imaginal discs.
  • CD63 is a tetraspanin that localizes to internal vesicles of multivesicular endosomes, and is released on exosomes 17,18.
  • CD63:GFP localizes to late endosomes in producing cells, consistent with vertebrate studies ( Figure 14A- D).
  • the S120 contains both free soluble proteins and lipoprotein particles. To separate them, we performed isopycnic density centrifugation. In these gradients, Lipophorin moves to the top low-density fraction whereas soluble proteins are present in higher density fractions (first two panels of Figure 8C). Gpi-linked proteins are found almost entirely in the top fraction with Lipophorin. Treating the S120 with Phosphatidylinositol-specific Phospholipase C (PI-PLC) prior to density centrifugation shifts their migration to higher density fractions (Figure 8C). This suggests that gpi- linked proteins associate with low-density particles via their gpi-anchor.
  • PI-PLC Phosphatidylinositol-specific Phospholipase C
  • Larvae of the latter genotype made only 50% of the wild type level of ApoLII, even in the absence of heat shock; basal activity of the heat shock promoter in the fat body causes HcRed excision in approximately 50% of fat body cells, although excision strictly depends on heat shock in other larval tissues (data not shown). Although they survive less frequently, these flies have no obvious phenotype.
  • Wild type fat body contains both small and large lipid droplets (Figure 10D). Fat bodies of Lipophorin-RNAi larvae are reduced in size and have fewer small lipid droplets (Figure 10E), although larger droplets appeared normal. These data suggest that Lipophorin delivers lipid to the fat body. Lipid droplets in discs from Lipophorin-RNAi larvae are fewer and smaller than in wild type (compare Figure 10F and G). Their discs are also reduced in size, particularly in the wing pouch (data not shown). Thus, discs require Lipophorin for accumulation of lipid droplets and for growth. Neither Caspase3 activation nor membrane PIP3 accumulation is altered in Lipophorin-RNAi discs ( Figure 17), suggesting their small size is not due to cell death or reduced Insulin signalling 29.
  • Hedgehog Hedgeghog expressed in the posterior compartment moves across the anterior-posterior (AP) compartment boundary and activates transcription of short and long-range target genes.
  • Cells closest to the source respond by activating the transcription of collier ( Figure 11 B,C) and patched ( Figure 11 H,I).
  • Hedgehog activates transcription of decapentaplegic ( Figure 11A,B) 1 ,30,31.
  • dpplacZ decapentaplegic reporter construct
  • Discs from Lipophorin RNAi larvae activate collier at least as efficiently as those of wild type (compare Figure 11C and F).
  • the range of activation of dppLacZ is significantly narrowed in lipophorin RNAi discs.
  • dppLacZ is expressed up to 11 cells away from the AP boundary in wild type discs ( Figure 11A, B), but only up to 6 cells away in Lipophorin RNAi larvae ( Figure 11 D, E,M).
  • Hedgehog To ask whether Hedgehog trafficking was altered, we stained discs for Hedgehog and Patched.
  • Hedgehog moves into the anterior compartment where it is found in endosomes, often with Patched 32,34 ( Figure 11 G-l). Patched- mediated endocytosis is thought to sequester Hedgehog and limit its spread 32,33. Hedgehog is most abundant up to 5 cell rows away from the AP boundary; although Hedgehog signals over a wider range, specific staining there cannot be distinguished from background.
  • Hedgehog In Lipophorin-RNAi discs, Hedgehog (Figure 11J,K) accumulates to abnormally high levels in the first 5 rows of anterior cells.
  • Lipophorin RNAi disc shown in Figure 11 J contains 1208 Hedgehog spots in the same region. Most accumulated Hedgehog colocalizes with Patched ( Figure 11 K,L) in endosomes ( Figure 18). Furthermore, Patched co-accumulates more extensively with Hedgehog in endosomes than it does in wild type ( Figure 11 H, K). These data indicate that Lipophorin RNAi either increases the susceptibility of Hedgehog to Patched-mediated endocytosis, or prevents subsequent degradation of the protein.
  • Lipophorin-depletion might affect Hedgehog trafficking indirectly by preventing release of a needed co-factor from some other larval tissue.
  • Lipophorin acts directly in imaginal discs to control Hedgehog trafficking, although it is still possible that its effects on signalling are indirect.
  • Example 8 Methods used in Examples 3 to 7
  • DNA encoding amino acids 195-509 or 891-1070 was amplified from GH 18004 (Resgen) and cloned into pQE30. His- tagged fusion proteins (Qiagen, Valencia, CA) were used to immunize rats or rabbits.
  • RNA-interference was induced by expressing inverted repeats derived from two different regions of the Pro-apolipophorin cDNA (607 bp ending 47 bp from stop codon, and 500 bp starting at ATG). The first was amplified and inserted into pENTR2B (Invitrogen, Leek, The Netherlands). Using the Gateway system, we inserted it twice in inverted orientation into pFRIPE.
  • pFRIPE is derived from pUAST; downstream of the UAS are two Gateway insertion sites flanking an FLP cassette containing the HcRed gene and a transcription termination sequence.
  • the second fragment was amplified and cloned as an inverted repeat into pUhr.
  • pUhr was derived from pUAST by inserting an HcRed-containing FLP cassette between the UAS and the multiple cloning site.
  • Imaginal discs were fixed and stained as described 13 .
  • Antibodies were diluted as follows: anti-Wg 46 , 1 :200; anti-Hh 47 , 1 :500; 1 :100; anti-Ptc 48 1 :50; anti- ⁇ gal (Promega Z378A) 1:100; anti-Col 31 1:200.
  • tissues were stained in parallel and imaged under identical conditions with an LSM Zeiss or Leica confocal microscope.
  • Hedgehog-positive spots in wild type and Lipophorin-RNAi discs were quantified in 10 confocal sections 1 ⁇ apart. Signal threshold was adjusted to 130 and images were despeckled using ImageJ. Grids were overlaid on the processed image and spots were counted manually.
  • Lipophorin particles were fluorescently labeled with Alexa Fluor 488 (Molecular Probes) according to manufacturer's instructions. Conjugate was separated from un- reacted label using Sephadex G-25 PD-10 columns (Amersham Pharmacia Biotech) and eluted with 100 mM Na-Phosphate, pH7.4, 100 mM NaCl, 10% sucrose. Incubation of dissected discs with Lipophorin particles
  • Hepatocyte apoB-containing lipoprotein secretion is decreased by the grapefruit flavonoid, naringenin, via inhibition of MTP-mediated microsomal triglyceride accumulation. Biochemistry. 42, 1283-1291.
  • Hedgehog patterning activity role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell. 86, 21-34.
  • MTP inhibitor decreases plasma cholesterol levels in LDL receptor-deficient WHHL rabbits by lowering the VLDL secretion.
  • Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature 425, 851-856.
  • Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature. 423, 448-452.
  • RNA interference listening to the sound of silence. Nat Struct Biol 8, 746-750.
  • Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448-52 (2003).
  • mice Micchelli, C. A., The, I., Selva, E., Mogila, V. & Perrimon, N. Rasp, a putative transmembrane acyltransferase, is required for Hedgehog signaling. Development 129, 843-51 (2002). 9. Lee, J. D. et al. An acylatable residue of Hedgehog is differentially required in Drosophila and mouse limb development. Dev Biol 233, 122-36 (2001).
  • Torroja, C, Gorfinkiel, N. & Guerrero, I. Patched controls the Hedgehog gradient by endocytosis in a dynamin-dependent manner, but this internalization does not play a major role in signal transduction. Development 131, 2395-408 (2004).

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Abstract

L'invention concerne l'utilisation d'un inhibiteur de protéine de transfert de triglycéride microsomale (MTP), de réductase HMG-CoA, de DGAT et/ou de ACAT utilisé dans la préparation d'une composition pharmaceutique destinée au traitement de tumeurs. Dans un mode de réalisation préféré, la croissance et/ou la progression de la tumeur sont entraînées par une ou plusieurs protéines de la famille Wnt ou Hedgehog. Les tumeurs préférées sont la tumeur oesophagienne, la tumeur du tractus biliaire, la tumeur gastrique, la tumeur pancréatique, le mélanome malin, la tumeur colorectale, l'épithélioma spinocellulaire et la tumeur cervicale.
PCT/EP2005/003326 2004-03-30 2005-03-30 Traitement de tumeurs qui secretent du wnt et du hedgehog avec des inhibiteurs de biogenese de particule de lipoproteine WO2005094864A2 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008131354A2 (fr) * 2007-04-20 2008-10-30 The Curators Of The University Of Missouri Phytoestrogènes en tant que régulateurs de signalisation hedgehog et procédés d'utilisation dans le traitement du cancer
WO2009067397A2 (fr) * 2007-11-19 2009-05-28 Ore Pharmaceuticals Inc. Traitement de tumeurs solides
WO2010037715A1 (fr) * 2008-10-01 2010-04-08 Novartis Ag Antagonisme smoothened pour le traitement de troubles liés à la voie de signalisation hedgehog
US20110251144A1 (en) * 2008-09-16 2011-10-13 Massachusetts Institute Of Technology Molecular modulators of the wnt/beta-catenin pathway
CN107523638A (zh) * 2017-10-11 2017-12-29 安徽省立医院 Wnt7b在监测促进胃癌细胞迁移中的应用
US9861622B2 (en) 2004-03-05 2018-01-09 The Trustees Of The University Of Pennsylvania Methods for treating disorders or diseases associated with hyperlipidemia and hypercholesterolemia while minimizing side-effects
WO2018154064A1 (fr) 2017-02-24 2018-08-30 Bioactor Bv Flavanones destinées à être utilisées dans le traitement de l'hypersensibilité viscérale

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5145839A (en) * 1988-06-23 1992-09-08 Mirko Beljanski Pharmaceutical composition and method of use
WO1998003174A1 (fr) * 1996-07-24 1998-01-29 Bristol-Myers Squibb Company Procede de traitement de tumeurs ayant des besoins eleves en lipoproteine de basse densite (ldl) a l'aide d'inhibiteurs de la proteine microsomale de transfert des triglycerides (mtp)
US20030054357A1 (en) * 2001-09-20 2003-03-20 Charles Young Methods and compositions for inhibiting the proliferation of prostate cancer cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5145839A (en) * 1988-06-23 1992-09-08 Mirko Beljanski Pharmaceutical composition and method of use
WO1998003174A1 (fr) * 1996-07-24 1998-01-29 Bristol-Myers Squibb Company Procede de traitement de tumeurs ayant des besoins eleves en lipoproteine de basse densite (ldl) a l'aide d'inhibiteurs de la proteine microsomale de transfert des triglycerides (mtp)
US20030054357A1 (en) * 2001-09-20 2003-03-20 Charles Young Methods and compositions for inhibiting the proliferation of prostate cancer cells

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