EP1232247A2 - Modulation de transduction de signal - Google Patents

Modulation de transduction de signal

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Publication number
EP1232247A2
EP1232247A2 EP00991029A EP00991029A EP1232247A2 EP 1232247 A2 EP1232247 A2 EP 1232247A2 EP 00991029 A EP00991029 A EP 00991029A EP 00991029 A EP00991029 A EP 00991029A EP 1232247 A2 EP1232247 A2 EP 1232247A2
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Prior art keywords
retinoid
biological effect
polypeptide
cell
metabolite
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German (de)
English (en)
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EP1232247A4 (fr
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Gerhart Graupner
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Graupner Gerhart
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Individual
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/203—Retinoic acids ; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells

Definitions

  • the field of the invention is modulation of signal transduction.
  • drugs that directly interfere with a physiological and pathophysiological condition include enzyme inhibitors (e.g., penicillin inhibits bacterial transpeptidases, or MevinolinTM inhibits 3-hydroxy-3-methylglutaryl coenzyme A reductase) or antisense nucleic acids (e.g., antisense DNA inhibit translation of viral genes).
  • enzyme inhibitors e.g., penicillin inhibits bacterial transpeptidases, or MevinolinTM inhibits 3-hydroxy-3-methylglutaryl coenzyme A reductase
  • antisense nucleic acids e.g., antisense DNA inhibit translation of viral genes.
  • drugs that modulate signal transduction pathways may be employed, and numerous compositions and methods are known in the art to interfere with signal transduction pathways.
  • a signaling molecule e.g., a cytokine or insulin
  • Adding signaling molecules is often advantageous, especially where exogenous addition to a system lacking the signaling molecule reconstitutes the physiologically normal level of the signaling molecule.
  • addition of exogenous signaling molecules is frequently problematic, especially where the molecules are immunogenic peptides or peptide preparations with impurities and/or inhomogeneities.
  • receptors for the signaling molecules may be blocked or otherwise rendered functionally inactive.
  • a beta-blocker competitively inhibits binding of the natural signal adrenalin to beta-adrenergic receptors in the nervous system.
  • Receptor blockers generally exhibit strong inhibition of their target receptors, however, they tend to inhibit non-target receptors, especially where the non-target receptors belong to the same family as the target receptor (e.g., various beta-blockers tend to block non-target beta-2 receptors).
  • elements within a signaling cascade may inhibit or prevent a signal from being transduced to the target compartment of a cell.
  • a particularly promising element in a signaling cascade is the vascular endothelial growth factor (VEGF) receptor kinase, which specifically phopsphorylates its substrate in dependence of binding of VEGF to the VEGF receptor, and it has recently been shown, that VEGF kinase inhibitors effectively inhibit signaling in VEGF kinase associated pathways [Drevs J. et al.
  • elements and processes at the end-point of a signaling cascade may inhibit or prevent the signal from being translated into a regulatory or other function in the cell.
  • end-point inhibition is the use of antisense nucleic acids that hybridize with a transcription product that is being formed in a response to the signal, or that form triple helices with a target sequence that is activated by the signal.
  • the present invention is directed to compositions and methods of cell-specifically modulating a signal transduction pathway in a system.
  • the signal transduction pathway is identified as functionally including a cellular polypeptide that binds a retinoid or retinoid metabolite, wherein the cellular polypeptide is not a retinoic acid receptor (RAR), retinoid X receptor (RXR), a vision pigment, nor a cellular retinoic acid binding protein (CRABP), and wherein binding of the retinoid or retinoid metabolite results in modulation of a biological effect modulated by the signal transduction pathway.
  • the retinoid or retinoid metabolite is administered to the system (e.g., a mammal, cell-, or tissue culture) in a concentration effective to modulate the biological effect.
  • the retinoid has a cis-configuration and is preferably a 9-cis-retinoid, a 4-hydroxyphenyl-retinamide, or a 4-hydroxyphenyl-retinamide analog.
  • retinoids and retinoid metabolites include various stereoisomers of retinylinositides, lipid-conjugated retinoids, sulfur-containing retinoids, and especially contemplated retinoid metabolites include a sulfated retinoid and S-adenosylretinoid.
  • the cellular polypeptide comprises an ion channel, preferably with a specificity for K + , Ca 2+ , Na + , or Cl ⁇ and it is particularly preferred that the ion channel functionally cooperates with a sulfonylurea receptor (SUR). It is further especially contemplated that the ion channel comprises an adenosine triphosphate (ATP) gated potassium channel complex.
  • SUR sulfonylurea receptor
  • contemplated biological effects are amplified by binding of the retinoid or retinoid metabolite, and particularly contemplated biological effects include cell division, insulin secretion, cell growth, and arrythmia.
  • contemplated retinoids have a bimodal effect, administration of the retinoid or retinoid metabolite at a first concentration modulates a first biological effect, and administration of the retinoid or retinoid metabolite at a second concentration modulates a second biological effect.
  • Figures 1 A- ID depict exemplary retinoids and retinoid metabolites.
  • Figure 2 depicts further exemplary structures of retinoids and retinoid metabolites.
  • Figures 3 A-3D show confocal laser micrographs depicting co localization of apoptosis in cardiac conductance cells after administration of 9-cis-retinoic acid, and control.
  • Figures 4A-4D are graphs depicting expression levels of various enzymes and signal transduction markers after administration of 9-cis-retinoic acid compared to the corresponding control signal after administration of carrier.
  • retinoids interact in various signal transduction pathways via binding to the retinoid acid receptor (RAR) and/or the retinoid X receptor (RXR) [see e.g., Vitamins and Hormones, 49, 327-382 (1994)].
  • RAR retinoid acid receptor
  • RXR retinoid X receptor
  • binding of retinoids to RAR and/or RXR has been demonstrated in signal transduction pathways involved in apoptosis [see e.g., Kastner, P. et al., The role of nuclear retinoid acid receptors in the regulation of gene expression; Vitamin A in health and disease, Marcel Decker, Inc. New York 189-238 (1994)].
  • the RAR and/or RXR act as nuclear transcription activators as dimers with a further RAR and/or RXR molecule or other nuclear transcription activator.
  • retinoids and/or retinoid metabolites also bind to cellular polypeptides other than RAR and/or RXR, and that binding of the retinoids and/or retinoid metabolites to such cellular polypeptides results in a modulation of the biological effect in signal transduction pathways that functionally include such cellular polypeptides.
  • a method of cell-specifically interfering with a signal transduction pathway that controls a biological effect in a system may include one step in which the signal transduction pathway is identified as functionally including a cellular polypeptide that binds a retinoid or retinoid metabolite, wherein contemplated cellular polypeptides are polypeptides other than RAR, RXR a visual pigment, or CRABP, and binding of the retinoid or retinoid metabolite to such polypeptides results in a modulation of the biological effect.
  • the retinoid or retinoid metabolite is administered to the system in a concentration effective to modulate the biological effect.
  • binding to a cellular polypeptide means that the cellular polypeptide retains a bound substance with a dissociation constant of less than 10 "3 /Mol, however, specifically excludes binding of a retinoid to a catalytically active site of an enzyme converting the retinoid to a retinoid metabolite.
  • the inventor discovered that in vivo administration of 9-cis retinoic acid to mice resulted in reduced growth of cardiomyocytes, and specifically lead to apoptosis in cardiac neurons and cardiac conductive cells due to signal amplification in the corresponding mitochondrial signal transduction pathways in the cardiac neurons and cardiac conductive cells (see experimental data, infra).
  • cardiac neurons and cardiac conductive cells, as well as cardiomyocytes contain significant amounts of RXR and RAR [see, e.g., Georgiades P, Brickell PM, Regulation of retinoid X receptor-gamma gene transcript levels in rat heart cells; Cell Biol lnt 1998;22(6):457-63, and Kastner et al, Vitamin A deficiency and mutations of RXRalpha, RXRbeta and RARalpha lead to early differentiation of embryonic ventricular cardiomyocytes; Development 1997 Dec;124(23):4749-58].
  • RXR and RAR see, e.g., Georgiades P, Brickell PM, Regulation of retinoid X receptor-gamma gene transcript levels in rat heart cells; Cell Biol lnt 1998;22(6):457-63, and Kastner et al, Vitamin A deficiency and mutations of RXRalpha, RXRbeta and RARalpha lead to early differentiation of embryonic ventricular cardio
  • modulation of the apoptosis signal transduction pathway is not mediated by binding of the 9-cis retinoid to the RAR and/or RXR, and/or a heterodimeric complex including RXR, but by binding of the 9-cis retinoid to an alternative binding target.
  • mitochondrial ATP-gated K + channels are involved in apoptotic processes by increasing K + influx and consequently increasing Ca 2+ fluxes.
  • the mitochondrial ATP-gated K + channels are regulated in a protein complex by the sulfonylurea receptor (SUR), which has markedly distinct binding affinities towards its substrates when expressed as type 2 on cardiomyocytes and when expressed as type 1 on cardiac neurons cardiac/conductive cells (Aguilar-Bryan L and Bryan J, Molecular biology of adenosine triphosphate sensitive potassium channels, Endocr.
  • SUR sulfonylurea receptor
  • Type 2 SUR on cardiomyocytes are known to have generally a lower affinity to various substrates (e.g., various sulfony) urea-based drugs) than type 1 SUR on cardiac neurons cardiac/conductive cells (Inagaki N et al., A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels, Neuron 16(5), 1011-1017, 1996). It has also been postulated, that cardiac conductive cells (i.e., cells that comprise the cardiac conductance system) contain type 1 SUR, and cardiomyocytes contain type 2 SUR.
  • retinoids and their metabolites specifically bind to SUR and/or the SUR-K + channel complex, and it is further contemplated that such binding modulates (e.g., up-regulates) the activity of the ATP-gated K + channel.
  • the system need not be limited to a mouse, and appropriate systems include in vivo and in vitro systems.
  • suitable in vitro systems include cell and tissue cultures, wherein the cells may be derived from a live specimen (e.g., biopsy), a secondary cell culture, or a thawed cell or tissue sample.
  • Particularly contemplated cells are mammalian cells, however, non-mammalian vertebrate and invertebrate cells are also contemplated.
  • Especially contemplated in vivo systems include mammals (and particularly human), non-mammal vertebrates, and invertebrates (e.g., yeasts).
  • retinoids are suitable for use in conjunction with the teachings presented herein, and exemplary retinoids are described in Chemistry and Biology of Synthetic Retinoids (Marcia Dawson, William H. Okamura (Editor), CRC Pr; ISBN: 0849347971), Retinoids: The Biochemical and Molecular Basis of Vitamin A and Retinoid Action (Heinz Nau (Editor), William S. Blaner (Editor); Springer Verlag; ISBN: 3540658920), or Retinoids (Maria A. Consa, Lester Packer (Editor); Marcel Dekker; ISBN: 0824787587), all of which are incorporated by reference herein.
  • the retinoid comprises at least one cis-configuration, and it is particularly contemplated that the cis-configuration is a 9-cis configuration (e.g., 9-cis retinoic acid).
  • exemplary retinoids are depicted in Figure 2, in which RpRis are independently selected from H, Halogen, alkyl, alkenyl, alkynyl, aryl, alkraryl, all of which may independently further comprise a halogen, a functional group (e.g., a CHO, COOH, NO 2 , NO, NH 2 , NH,
  • R ⁇ -R ⁇ 8 may further comprise heteroatoms, which may be in various positions of R ⁇ -R ⁇ 8 , including pending or in the backbone. Where stereoisomeric or chiral variations of contemplated retinoids exist, all chemically reasonable configurations (of R, S, cis, and trans) are contemplated.
  • suitable retinoids need not be limited to the depicted 9-cis configuration, but may also include all-trans, 9-cis- 1 l-cis-13-trans configuration, etc.
  • retinoid as used herein particularly excludes retro-retinoids as described in Retro-Retinoids in Regulated cell growth and death, O'Connel et al, J. Exp. Med 1996, 184: 549-555.
  • retinoid analogs are contemplated as suitable alternative retinoids.
  • the term "retinoid analog” as used herein refers to any molecule that displays an activity conventionally ascribed to retinoic acid derivatives as summarized in U.S. Pat. No. 6,034,242 to Vuligonda et al. (March 7, 2000), which is incorporated by reference herein.
  • An especially contemplated retinoid analog is 4-hydroxyphenyl-retinamide or a
  • retinoid analogs especially include known bicyclical spaced conformationally constrained ligands of RXR, and particularly include RXR agonists such as LG1069, and compounds described by Benoit et al.(RAR- independent RXR signaling induces t(15;17) leukemia cell maturation EMBO J (1999) 18(24), 7011- 7018).
  • the retinoid or retinoid analog may also be metabolized in one or more biochemical (e.g., enzymatic) or fhermo dynamical (e.g., thermal isomerization) conversion to form a retinoid metabolite or retinoid analog metabolite.
  • biochemical e.g., enzymatic
  • fhermo dynamical e.g., thermal isomerization
  • a metabolic conversion may include addition of a chemical function or group, including polar, charged, lipophilic or hydrophilic groups, sulfur, nitrogen, or oxygen containing groups, etc.
  • retinoid metabolites include a sulfated retinoid and S-adenosylretinoid, and various stereoisomeric forms of retinolinositides.
  • the cellular polypeptide to which the retinoid or retinoid metabolite binds need not be restricted to a SUR- ATP gated K + channel complex, and alternative polypeptides include ion channels with a selectivity for calcium ions, sodium ions, or chloride ions. It is especially contemplated that the ion channel functionally cooperates with an SUR.
  • Alternative further contemplated polypeptides include a complex comprising an inward rectifier potassium channel HERG with a member of the MinK family of regulatory transmembrane peptides.
  • the term "ion channel functionally cooperates with an SUR" as used herein means that the activity of the ion channel is directly or indirectly influenced by the SUR.
  • directly influenced means that the SUR is physically coupled to the ion channel, and that binding of the retinoid to the SUR and/or SUR-ion channel complex influences the activity of the ion channel.
  • directly influenced means that the SUR is not physically coupled to the ion channel, and that the activity of the ion channel may be modulated by at least one intermediary molecule between the SUR and the ion channel complex when the retinoid or retinoid metabolite binds to the SUR.
  • Still further contemplated alternative cellular polypeptides include membrane transport proteins for cellular detoxification, and particularly include members of the family of mgm and PgP.
  • alternative biological effects include all biological effects that are mediated by a signal transduction pathway, which includes an ion channel and/or an SUR.
  • a signal transduction pathway which includes an ion channel and/or an SUR.
  • insulin secretion of neuroendocrine cells is at least partly regulated by the activity of a K + ion channel and/or SUR (see e.g., Gribble F et al. Tissue specificity of sulfonylureas, Diabetes 47, 1412-1418, 1998).
  • various neuroendocrine disorders that are at least partly linked to activity of a K + ion channel and/or SUR are contemplated (see e.g.
  • contemplated diseases include cystic fibrosis, which is known to involve malfunction of a chloride ion channel in the pathogenesis.
  • contemplated biological effects may also include cell growth, cell division, and arrythmia, all of which are contemplated to be modulated by a signal transduction pathway that includes an ion channel and/or SUR (Malhi H et al, KATP channels regulate mitogenically induced proliferation in primary rat hepatocytes and human liver cell lines, J. Biol. Chem.
  • retinoid binding motif comprises an ATP binding cassette (ABC)
  • administration of the retinoid may include modulation of a drug resistant phenotype in cancer cells (e.g., via interaction with the ABC in PgP or mdm).
  • modulation of the biological effect may comprise amplification or reduction of the biological effect.
  • the retinoid may also bind to a cellular polypeptide other than an ion channel
  • suitable alternative polypeptides include transmembrane proteins, membrane associated proteins, cytosolic proteins, proteins associated with or located within cellular compartments, including mitochondria, endoplasmatic reticulum, endosomes, and the nucleus.
  • particularly contemplated alternative cellular polypeptides include inositide binding polypeptides such as PI 3 -kinase, protein kinase B, PLi-kinase, PL;,5- kinase, and inositol phosphate receptor isoforms.
  • P 2 X ion channels which are regulated by extracellular ATP, bcl-x and bcl-2.
  • ITP/IDP and GTP/GDP binding proteins are particularly preferred, and binding of cis-retinoids is especially preferred over binding of all-trans retinoids.
  • particularly excluded from appropriate alternative cellular proteins are RAR, RXR, and CRABP-I, CRABP-II, and retinoid isomerases in the vision pathway (Wald, G., Science 162, 230-239 (1968)). It should be recognized that alternative cellular polypeptides are not nuclear polypeptides that act alone or in combination with another proteins as a transcription factor.
  • a ligand-polypeptide complex that has a ligand comprising at least one of a retinoid and a retinoid metabolite, and that further has polypeptide other than a retinoic acid receptor, a retinoid X receptor, and a cellular retinoic acid binding protein, wherein the polypeptide binds the ligand with a dissociation constant of less than 10 " /Mol, and wherein binding of the ligand to the polypeptide results in a modulation of a biological effect in a signal transduction pathway.
  • a particular system will typically determine the particular administration.
  • the administration will typically comprise admixing a retinoid-containing solution to the cell or tissue culture.
  • suitable administrations include oral or parenteral administration, injection, infusion, topical or transdermal application, etc.
  • the schedule of administration will predominantly depend on the particular system and biological effect, and it is therefore contemplated that the schedule may vary considerably. For example, while in some cell cultures a single administration will produce the desired modulation of the biological effect, administrations to a human may require multiple administrations to obtain the desired modulation of the biological effect.
  • the dose of the retinoid or retinoid metabolite may vary substantially, and it is contemplated that the dose is generally within the range of several micrograms and several grams. However, it is preferred that the concentration of the retinoid in the system is between about 1 micromolar and about 10 millimolar. In further contemplated aspects of the inventive subject matter, it should be recognized that administration of contemplated retinoids or retinoid metabolites has a bimodal effect with respect to the amount of administered retinoids and retinoid metabolites.
  • retinoids and/or retinoid metabolites exhibit a bimodal activity.
  • a retinoid and/or retinoid metabolite at a first concentration has a modulation of a first biological effect
  • administration of the retinoid and/or retinoid metabolite at a second concentration has a modulation of a second biological effect.
  • first biological effects include reduction of growth in cardiomyocytes
  • second biological effect include induction of apoptosis in a cardiac neurons and cardiac conductive cells.
  • Pups were gavage-fed from a tuberculin syringe connected with a bent vein catheter.
  • the strong suckling reflex facilitated delivery of the viscous and odorous formulation.
  • pups were briefly rolled in cage stray after gavage to prevent triggering maternal infanticide behavior through unusual smell of the pups.
  • pups were carefully monitored to prevent maternal infanticide. Every 24h, the weight was recorded.
  • pups were sacrificed after 72h, hearts were rinsed in PBS, weighed and fixed in 4% para- formaldehyde in PBS before embedding in OCT for standard cryostat procedures. If pups were on the brink of death (typically between 36 and 72h after gavage), they were sacrificed, and hearts were collected for confocal microscopy or HPLC.
  • cryostat sections were first stained for apoptotic DNA strand breaks according to the TUNEL protocol (Apoptag kit, Oncor. Gaithersburg) and counterstained with rabbit anti-connexin37 antibody or rabbit anti-connexin40 antibody for conductance cells, or with rabbit anti-myosin antibody for cardiac myocytes.
  • TUNEL protocol Apoptag kit, Oncor. Gaithersburg
  • Immunodetection of connexin isoforms 40 and 43 has been established as a useful method to detect cells of the cardiac conductance system in rodents (Gourdie et al., The spatial distribution and relative abundance of gap-junctional connexin40 and connexin43 correlate to functional properties of components of the cardiac atrioventricular conducting system, J.
  • the connexin isoform 37 can be used as an equivalent marker for the conductance system in the mouse model (Willecke K et al, 1991; Willecke K, personal communication).
  • the combination of rabbit anti-Neuron-specific enolase with propidium iodide followed published procedures (Current Protocols in Molecular Biology) and was used to identify cardiac neurons with advanced apoptotic condensation of nuclei. Procedures of immunohistology and image analysis are further described in detail in Graupner, WO 00/53236.
  • the mechanism of apoptosis was investigated using commercially available primary antibodies of highest purity, at dilutions recommended by the manufacturer, against IGF-1, IGF- ⁇ , the entire set of IGFbinding proteins (IGFBP 1 -5), IGF-R, mitochondrial Mn- dependent superoxide dismutase Mn-SOD, and cytoplasmic Cu/Zn-dependent superoxide dismutase Cu/Zn-SOD.
  • Cellular phenotypes were identified with counterstain using cardiac myosin, actin, connexin37, connexin40, and connexin43 antisera. Confocal images were captured and processed for quantitative analysis by Zeiss LSM-300 software.
  • the pixel intensities per unit area amounting to channel background were determined for each antibody/seconday antibody combination and subtracted from the experimental pixel intensities. Pixel intensities per fluorescent channel in regions of high signal were normalized to pixel intensity per unit area, as were pixel intensities in control regions. At least five independent regions of similar size were analyzed per tissue type and retinoid treatment condition. Averaged pixel intensities per unit area upon 9-cis retinoid induction were expressed as multiples of averaged pixel intensities per unit area in the absence of 9-cis retinoid induction (fold induction +/- s.e.m.).
  • the mechanism of apoptosis was investigated using commercially available primary antibodies of highest purity, at dilutions recommended by the manufacturer, against IGF-1, IGF-H, the entire set of IGFbinding proteins (IGFBP1-5), IGF-R, mitochondrial Mn- dependent superoxide dismutase Mn-SOD, and cytoplasmic Cu/Zn-dependent superoxide dismutase Cu/Zn-SOD.
  • Cellular phenotypes were identified with counterstain using cardiac myosin, actin, connexin37, connexin40, and connexin43 antisera. Confocal images were captured and processed for quantitative analysis by Zeiss LSM-300 software.
  • paired confocal laser micrographs from perinatal mouse heart sections after administration of 20 mg/kg 9cis-RA, or after administration of carrier are presented in Figures 3A-3D.
  • apoptotic events were identified in the transmitted light mode of the confocal microscope, DIC images were recorded, and matching images were scanned in the fluorescent mode, permitting accurate overlay images.
  • Figure 3A illustrates the occurrence and distribution of apoptotic cells in the outflow tract region of a mouse heart after treatment with 20 mg/kg 9cis-RA.
  • Figure 3B is the corresponding fluorescence image that shows the staining of apoptotic cells with the mouse conductance cell marker connexin37.
  • Figure 3C illustrates the absence of apoptotic cells in the outflow tract region of a mouse heart after treatment with carrier.
  • Figure 3D is the corresponding fluorescence image that shows the presence of cells stained with the mouse conductance cell marker connexin37 in the same section.
  • FIG. 4A-4D Further examples of quantitative confocal analysis of perinatal mouse heart sections are presented in graph form in Figures 4A-4D. Pixel intensities (per unit area) amounting to channel background were determined for each antibody/seconday antibody combination. This background signal was subtracted from pixel intensities per unit area observed under different experimental conditions, or under control conditions. If substantially different levels of marker expression were observed in distinct areas of a supposedly identical cellular environment under the same experimental condition, such signals were grouped and presented in a separate bar (e.g. in Fig. 4B for signals from Cu/Zn-SOD levels, and in Fig. 4C for IGF-Receptor levels).
  • Fig. 4A averaged pixel intensities per unit area upon 9cis retinoid induction were expressed as multiples of averaged pixel intensities per unit area in the absence of 9cis retinoid induction (fold induction).
  • Figure 4A quantifies the up regulation of immunoreactive Mn-dependent Superoxide Dismutase (Mn-SOD) in mitochondria of cells in the cardiac outflow tract after administration of 20 mg/kg 9cis-RA as fold induction of the corresponding control signal after administration of a carrier.
  • Figure 4B shows no significant change in expression of Cu/Zn-dependent Superoxide Dismutase in perinatal murine cardiac myocytes and in cells of the perinatal murine cardiac outflow tract after administration of 20 mg/kg 9cis -RA in comparison to the corresponding control signal after administration of the carrier; two markedly different levels of Cu/Zn SOD expression can be observed in either retinoid-treated or I control hearts.
  • Mn-SOD immunoreactive Mn-dependent Superoxide Dismutase
  • Figure 4C shows that two different levels of IGF-1 receptor expression can be observed in perinatal murine ventricular myocytes after administration of 20 mg/kg 9cis -RA in comparison to the corresponding control signal after administration of the carrier; one compartment of cardiomyocytes contains IGFR levels that are the same as in control animals, another compartment of cardiomyocytes contains significantly elevated levels of IGFR.
  • Figure 4D describes the marginal increase in IGF-binding protein 5 expression in perinatal murine cardiomyocytes after administration of 20 mg/kg 9cis -RA in comparison to the corresponding control signal after administration of the carrier.
  • Tissues collected for retinoid analysis were dispersed by two rounds of sonication.
  • the slurry was doted with a known amount of synthetic retinoid to correct for variable extraction efficiencies between different samples. Extraction steps followed published procedures (see e.g. Biesalski HK, Comparative assessment of the toxicology of vitamin A and retinoids in man, Toxicology 57, 117-161, 1989).
  • the crude retinoid fraction was resuspended in HPLC buffer, injected on a RPC column and eluted with decreasing polarity in an isocratic gradient.
  • the elution profiles were recorded and analyzed with system software. The positions of retinoic acid isoforms were identified, and absolute amounts of individual retinoids per mg tissue could be calculated.
  • the myocardial growth stunting seen at lower concentrations of 9cis-RA approached complete growth arrest around 10 mg/kg 9cis-RA, in contrast to the limited effect (less than 30% growth reduction) caused by substantially higher doses (40 mg/kg) of at RA.
  • IGF pathways known to be of primary significance in muscle cell growth regulation, in cardiac cell types reveals no indication for a connection between interruption of upstream IGF signaling and either myocardial growth stunting or tissue-specific apoptosis of conductance cells.
  • IGF ligand levels, IGF receptor levels, and modulatory binding protein levels were examined. While IGFBP5, a known negative regulator of IGF -1 function, is marginally up-regulated in cardiac myocytes after 9cis-RA treatment ( Figure 4C), the scope of the response is unlikely to account for the full picture of the growth arrest.
  • the level of IGF-Receptor was found to be unchanged in one population of the myocardial myocytes, and even significantly up-regulated in another population of cardiomyocytes (Figure 4D).
  • the amount of gas released was too high to be stored in, or produced by, conductance cells which are of very low abundance compared to myocytes.
  • very high up-regulation of MnSOD protein signal was detected in connexin-positive cells of the cardiac outflow tract; the signal of 3.4fold up- regulation localizes to the region of the atrium surrounding the sino-atrial node; the signal of 1 lfold up-regulation localizes most likely to the area of the sino-atrial node proper.
  • apoptosis occurs in cells that have initiated a program to counteract the damaging effects of reactive oxygen species through up-regulation of MnSOD at the protein level.
  • cardiac myocytes survive well with high levels of endogenous peroxidase activity and without elevated levels of enzymes protecting against reactive oxygen species.
  • retinoid-induced apoptosis and enzymatic pathways producing and eliminating reactive oxygen are separate phenomena not causatively linked in the execution of programmed cell death in perinatal cardiac tissue.
  • contemplated methods particularly lend themselves to development and improvement of anti-diabetic drugs with reduced cardiac side-effects by selecting retinoid and retinoid analogs/derived compositions (which are preferably devoid of RXR agonist activity) that bind to SURl, but not to SUR2.
  • retinoids and retinoid analogs/derived compositions to selectively modulate the function of SUR 2 and thus generate novel compositions of channel-selective anti-arrythmic compounds.
  • Another contemplated use of retinoids and retinoid analogs/derived compositions is the selective modulation of transport proteins for cellular detoxification, with the purpose to increase the susceptibility of highly drug-resistant tumors to antiproliferative therapy.

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Abstract

Selon l'invention, un polypeptide est identifié comme étant fonctionnellement compris dans une voie de transduction du signal présentant un effet biologique. Ces polypeptides sont différents du récepteur d'acide rétinoïque, du récepteur de rétinoïde X, ou d'une protéine de liaison d'acide rétinoïque cellulaire, cependant, la liaison du rétinoïde ou du métabolite rétinoïde module l'effet biologique. Dans des procédés de la présente invention, un rétinoïde ou un métabolite rétinoïde est administré à une cellule ou à un mammifère en concentration suffisante à moduler cet effet biologique.
EP00991029A 1999-11-23 2000-11-22 Modulation de transduction de signal Withdrawn EP1232247A4 (fr)

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US16743899P 1999-11-23 1999-11-23
US167438P 1999-11-23
PCT/US2000/042233 WO2001038344A2 (fr) 1999-11-23 2000-11-22 Modulation de transduction de signal

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Publication number Priority date Publication date Assignee Title
EP2277516A1 (fr) 2004-06-23 2011-01-26 ReVision Therapeutics, Inc. Dérivés de rétinyle pour le traitement de troubles ophtalmiques
CA2584396A1 (fr) * 2004-11-04 2006-05-18 Sirion Therapeutics, Inc. Modulateurs de la formation d'un complexe transthyretine (ttr)-proteine de liaison retinol-retinol (rbp)
CN101072555B (zh) * 2004-12-08 2011-06-29 矫正诊疗公司 治疗与视黄醇有关的疾病的方法、分析和组合物

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of WO0138344A2 *
SUCOV H M ET AL: "RETINOIC ACID AND RETINOIC ACID RECEPTORS IN DEVELOPMENT" MOLECULAR NEUROBIOLOGY, HUMANA PRESS, US, vol. 10, no. 2/3, 1995, pages 169-184, XP002949771 ISSN: 0893-7648 *

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