WO1998053831A1 - Utilisation de facteur d'inhibition hypothalamique pour traiter l'hypoxie - Google Patents

Utilisation de facteur d'inhibition hypothalamique pour traiter l'hypoxie Download PDF

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Publication number
WO1998053831A1
WO1998053831A1 PCT/US1998/010889 US9810889W WO9853831A1 WO 1998053831 A1 WO1998053831 A1 WO 1998053831A1 US 9810889 W US9810889 W US 9810889W WO 9853831 A1 WO9853831 A1 WO 9853831A1
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hif
hypoxia
cell
host
tissue
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PCT/US1998/010889
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English (en)
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Garner T. Haupert, Jr.
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The General Hospital Corporation
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Priority to AU76034/98A priority Critical patent/AU7603498A/en
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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin

Definitions

  • hypothalamic inhibitory factor HIF
  • This hypothalamic inhibitory factor, HIF had been shown to have biological and biochemical properties similar to, but not identical to, those of ouabain (Haupert, G.T., Jr., In: The Sodium
  • HIF is a potent inhibitor of the Na pump in renal tubular cells (Cantiello, H.F., et al . , Am. J. Physiol . 255 :F574-F580, (1988)), has positive inotropic activity in cardiac myoctyes (Hallag, H.A. and G.T. Haupert, Jr., Proc. Natl . Acad. Sci . USA
  • inotropic activity in cardiac yocytes occurred at concentrations approximately three orders of magnitude less than for ouabain, with evidence for less toxicity in the cardiac cells (Hallaq, H.A. and G.T. Haupert, Jr., Proc.
  • the present invention relates to a method for conserving cellular adaptive responses to hypoxia in a mammalian host (e.g., human) comprising administering to the host in need thereof an effective amount of hypothalamic inhibitory factor (HIF) .
  • the present invention also relates to a method for treating hypoxia in a mammalian host comprising administering to the host in need thereof an effective amount of HIF.
  • the present invention can be used to treat or prevent hypoxia insult.
  • Also encompassed by the present invention is a method for conserving adenosine triphosphate in a mammalian cell comprising introducing an effective amount of HIF to the cell (e.g., a brain cell, a cardiac cell, a renal cell).
  • a mammalian cell comprising introducing an effective amount of HIF to the cell (e.g., a brain cell, a cardiac cell, a renal cell).
  • Figure 1 is a schematic representation of the study protocol described in the exemplification, wherein rats were kept both in regular cages (21%, 0 2 , left) and in hypoxic sealed cages (10% 0 2 , right) ; brain was removed and half of each midbrain was subsequently incubated in vi tro at either 95% or 4% 0 2 ; supernatants were collected and new medium rich in NaCl was added to study further release of Na + -K + -ATPase inhibitor; the same protocol was applied to adrenal tissue.
  • Figure 2A is a bar graph of sodium pump inhibition in midbrain tissue slices from CHP20P chromatography under various in vivo and in vi tro oxygen conditions; shaded portion shows area of elution of bovine HIF under identical chromatographic conditions.
  • Figure 2B is a bar graph of sodium pump inhibition in adrenal tissue slices from CHP20P chromatography under various in vivo and in vi tro oxygen conditions; shaded portion shows area of elution of bovine HIF under identical chromatographic conditions.
  • the present invention is based in part on the unexpected discovery that hypoxia is a potent stimulus for the release of hypothalamus inhibitory factor (HIF) and that HIF is involved in energy conserving cellular adaptive responses to hypoxia insult through adenosine triphosphate (ATP) conservation.
  • the present invention relates to methods for conserving cellular adaptive responses to hypoxia in a mammalian host comprising administering to the mammalian host in need thereof an effective amount of hypothalamic inhibitory factor.
  • the present invention also relates to a method for conserving ATP in a mammalian cell (e.g., brain cell, cardiac cell, renal cell) comprising introducing an effective amount of HIF to the cell.
  • a method for preventing and/or treating adverse consequences of hypoxia (hypoxic insult) in a mammalian host comprising administering to the host in need thereof an effective amount of hypothalamic inhibitory factor.
  • the mammalian host can be any mammal which is in need of increased cellular or tissular ATP levels in the face of hypoxia, and includes, for example, human, canine, feline, bovine and murine hosts.
  • An endogenous inhibitor of Na + -K + -ATPase has been isolated from bovine hypothalamus and human plasma and structurally characterized as an isomer of the plant cardiac glycoside, ouabain (Tymiak, A. . , et al . , Proc . Natl . Acad. Sci . , USA 90:8189-8193, (1993); and Zhao, ⁇ ., et al., Biochemistry 34:9893-9896, (1995)).
  • This hypothalamic inhibitory factor (HIF) inhibits Na + -K + -ATPase with high affinity in cardiovascular and renal tissues consistent with physiologic regulation in vivo . Stimulus (i) for the release of HIF from tissue are unknown.
  • hypoxia may be a stimulus for the elaboration of digitalis-like activity in humans
  • animal studies indicate that high NaCl concentration in the central nervous system stimulates ouabain-like activity.
  • the ability of low 0 2 tension in vivo and in vi tro to stimulate HIF release from midbrain and adrenal tissues in istar rats was examined.
  • a Na + -K + -ATPase inhibitor was recovered from supernatants of these tissues incubated in vi tro, and this activity co-chromatographed with bovine HIF and showed Na + -K + -ATPase inhibitory activity which was identical to the activity of pure HIF in two biological assays.
  • hypoxia stimulated a remarkable release of the inhibitor, and this release was further enhanced by subsequent exposure to 300 mM NaCl .
  • Plasma from rats exposed to hypoxia in vivo also showed increased levels of the purified inhibitory activity.
  • hypoxia is a potent stimulus for the release of HIF, and HIF is involved in energy conserving cellular adaptive responses to hypoxic or ischemic insult through ATP conservation.
  • HIF for use in the present invention can be obtained by purifying HIF from natural sources or chemically synthesizing HIF.
  • purified HIF is used.
  • Purified HIF refers to HIF which is substantially free of or isolated from other tissue or fluid protein components and contaminants .
  • Various procedures may be used to purify HIF from natural sources. For example, as described in U.S. patent application No.
  • HIF has been purified to homogeneity using an affinity chromatography method in which purified renal Na + , K + - ATPase (e.g., isolated from a canine) is coupled to paramagnetic particles through a glutaraldehyde bridge (Tymiak, A. . , et al . Proc . Natl . Acad. Sci . , USA, 50:8189- 8193 (1993)).
  • purified renal Na + , K + - ATPase e.g., isolated from a canine
  • the enzyme immobilizes bound HIF in the presence of Mg ++ and inorganic phosphorous with high affinity, and after washing away contaminating materials, HIF is eluted from the affinity column by chelating Mg ++ with EDTA. A subsequent HPLC step results in purification of HIF (Tymiak, A.A. , et al . Proc. Natl . Acad. Sci . , USA,
  • HIF has also been purified to homogeneity using an immunoaffinity chromatography method in which an antibody which binds to HIF is coupled to the resin of an immunoaffinity column.
  • the antibody recognizing HIF immobilizes bound HIF with high affinity, and after washing away contaminating materials, HIF is eluted from the affinity column.
  • a subsequent HPLC step can be used to further purify HIF. See U.S. patent application No. , filed May 30, 1997, Attorney Docket No. BION97-01, which is incorporated by reference.
  • pharmaceutically acceptable salts of HIF are also contemplated. Suitable salts include those well known to those of skill in the art.
  • an "effective amount” is an amount sufficient, when administered to the host, to result in a conserved cellular adaptive response to hypoxia in the mammalian host relative to the cellular adaptive response to hypoxia when an effective amount of HIF is not administered.
  • An "effective amount” of HIF as defined herein also refers to the amount of HIF which upon introduction into a mammalian tissue results in the conservation of ATP. Additionally, an “effective amount” also refers to an amount sufficient to ameliorate the effects of hypoxia.
  • the amount of HIF used to treat a host will vary depending on a variety of factors, including the size, age, body weight, general health, sex and diet of the host.
  • an effective amount of HIF will depend on the nature of the disease being treated, and can be determined by standard clinical techniques. The precise dose to be employed will also depend on the route of administration and the seriousness of the disease or disorder, and should be decided according to the judgement of the practitioner and each patient's circumstances.
  • suitable dosage ranges for intravenous or intraarterial administration are generally about 4.5 ug to about 20 ug per kilogram body weight. In one embodiment the dosage is about 4.5 ug per kilogram body weight, in another embodiment the dosage is about 12 ug per kilogram body weight, and in a further embodiment the dosage is about 20 ug per kilogram body weight.
  • Suitable dose ranges for oral administration of HIF are the same as for intravenous administration of HIF, but the HIF dosage is administered orally over several days. Effective doses may be extrapolated from dose response curves derived from in vitro or animal test models.
  • the HIF of the present invention can also be administered prophylactically to a host as a method of preventing the conditions described herein.
  • HIF can be administered therapeutically to a host as a method of treating an existing disease and/or condition in the host, and can result in amelioration or elimination of the disease and/or condition.
  • the formulation and route of delivery of HIF to the host can be accomplished in a variety of ways .
  • Routes of administration include intradermal, transdermal (e.g. slow release polymers) , intramuscular, intraperitoneal, intraarterial, intracardiac, intravenous, subcutaneous, oral, epidural, and intranasal routes. Any other convenient route of administration can be used, for example, infusion or bolus injection, or absorption through epithelial or mucocutaneous linings.
  • the HIF can be administered together with other components or biologically active agents, such as pharmaceutically acceptable surfactants (e.g., glycerides) , carriers (e.g., saline, buffered saline, dextrose, water, glycerol, ethanol) , excipients (e.g., lactose), diluents and vehicles and combinations thereof.
  • pharmaceutically acceptable surfactants e.g., glycerides
  • carriers e.g., saline, buffered saline, dextrose, water, glycerol, ethanol
  • excipients e.g., lactose
  • the composition can also include, if desired, minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides .
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the HIF is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous or intraarterial administration to the mammalian host (e.g., a human) .
  • HIF for intravenous administration can be a solution in sterile isotonic aqueous buffer.
  • the HIF composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent.
  • composition is administered by injection
  • an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the invention also relates to the use of HIF in the manufacture of medicaments for the treatment or prevention of hypoxia, hypoxic insult and/or conserving ATP in a mammalian cell or tissue.
  • the invention can be used to conserve cellular adaptive responses to hypoxia in a mammalian host.
  • the invention can be used to prevent and/or treat a patient with hypoxia.
  • the release of an endogenous Na + -K + - ATPase inhibitor in response to hypoxia at the cellular level can be used to save energy adaptive responses in certain tissues.
  • down-regulation of ion- channel activity in turtle brain with resulting lower demand for depleted ATP stores, has been postulated to account in part for this species' remarkable resistance to anoxic brain injury (Lutz, P.L., Ann . Rev. Physiol . 54:601-
  • Rats were anaesthetized using sodium pentobarbital (6.5 mg/lOOg) .
  • Blood samples were collected from each rat in tubes containing sodium heparin, and adrenals and brain were removed and washed twice in a chilled buffer solution (HEPES lOmM, NaCl 114mM, KCl 5mM, MgS0 4 1.15mM, d-glucose lOmM, NaHC0 3 25mM, CaCl 2 2.5mM, NaH 2 P0 4 ImM, pH 7.4).
  • Brains were divided into two halves, cortex was removed and the midbrain dissected free.
  • group 3 15 supernatants from midbrain tissue of rats kept in special cages with 10% 0 2 and incubated with 95% 0 2
  • group 4 15 supernatants from midbrain tissue of rats kept in special cages with 10% 0 2 and incubated with 4% 0 2
  • - groups 5-8 15 supernatants from midbrain tissue treated respectively as groups 1-4, but after the incubation with 300 Mm NaCl
  • group Al 3 supernatants from adrenal tissue of rats kept in regular cages with 21% 0 2 and incubated with 95% 0 2
  • group A2 3 supernatants from adrenal tissue of rats kept in regular cages with 21% 0 2 and incubated with 4% 0 2
  • group A3 3 supernatants from adrenal tissue of rats kept in special cages with 10% 0 2 and incubated with 95% 0 2
  • group A4 3 supernatants from adrenal tissue of rats kept in special cages
  • erythrocytes were washed and suspended to 50% cells in a HEPES buffer pH 7.4 (HEPES 20 mM, CaCl 2 1 mM, MgS0 4 1 mM, NaH 2 P0 4 5 mM, NaCl 138 mM, Glucose 11 mM) .
  • HEPES buffer pH 7.4 HEPES 20 mM, CaCl 2 1 mM, MgS0 4 1 mM, NaH 2 P0 4 5 mM, NaCl 138 mM, Glucose 11 mM
  • One ml aliquots of each chromatographic fraction were dried and reconstituted with 50 ⁇ l of buffer and incubated at 37°C with 50 ⁇ l of erythrocyte suspension and 4 ⁇ Ci/ml 86 RbCl for 90 min.
  • Figures 2A-2B shows the chromatographic profiles for Na + pump inhibitory activity from midbrain (Fig. 2A) and adrenal (Fig. 2B) supernatants under the various oxygenation conditions .
  • Inhibitory activity under all circumstances chromatographed with a retention time from seventy-six to ninety-six minutes (10 collected fractions, flow rate 2ml/min) .
  • This retention corresponded exactly with that found for bovine HIF (shaded region) suggesting but not proving that the inhibitor recovered from brain and adrenal supernatants is the same as the bovine hypothalamic inhibitor structurally characterized as an isomer of ouabain (Tymiak, A. . , et al . , Proc . Natl . Acad. Sci . , USA
  • adrenal tissue ultimately O 98/53831
  • hypoxia is a potent stimulus for the release of HIF from both midbrain and adrenal tissues in vitro, and this release is in general further stimulated by high NaCl concentration.
  • the substance co-chromatographs with and has Na + -K + -ATPase inhibitory activity in two bioassays parallel to HIF, a mammalian isomer of ouabain with demonstrated regulation of Na + pump activity in cardiovascular and renal cells .
  • the present invention is based, in part, on the finding that the mammalian response to hypoxia is release of HIF.
  • the invention encompasses the administration of an effective amount of HIF in a method for treating hypoxia in a mammalian host.
  • Table 1 the characteristics of each group are specified, i.e. 21-95% 0 2 describes rats kept at 21% 0 2 in vivo and tissue slices incubated at 95% 0 2 in vitro.
  • Sodium chloride (final concentration 300 mM) was added into the media of the groups labeled +Na + .
  • GROUP (adrenal) 50 ⁇ l 100 ⁇ l 200 ⁇ l p VALUES

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Abstract

L'invention concerne une méthode pour conserver les réponses cellulaires adaptatives à l'hypoxie chez un mammifère (p. ex. un humain). Cette méthode consiste à administrer au sujet nécessitant un tel traitement une quantité efficace de facteur d'inhibition hypothalamique (HIF). L'invention porte en outre sur une méthode de traitement de l'hypoxie chez un mammifère, comprenant l'administration au sujet nécessitant le traitement, d'une quantité efficace de HIF. L'invention porte en outre sur le traitement ou la prévention des accidents hypoxiques. Une méthode pour conserver l'adénosine triphosphate dans une cellule de mammifère, consistant à introduire une quantité efficace de HIF dans la cellule (p. ex. un neurone cérébral, une cellule cardiaque, une cellule rénale) est également décrite.
PCT/US1998/010889 1997-05-30 1998-05-28 Utilisation de facteur d'inhibition hypothalamique pour traiter l'hypoxie WO1998053831A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7396658B2 (en) 1998-12-24 2008-07-08 The General Hospital Corporation Methods for screening HIF like ouabain-resistant Na+—K+-ATPase agents

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0433074A1 (fr) * 1989-12-13 1991-06-19 The General Hospital Corporation Méthode de traitement de malfonction cardiaque

Patent Citations (1)

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EP0433074A1 (fr) * 1989-12-13 1991-06-19 The General Hospital Corporation Méthode de traitement de malfonction cardiaque

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C. DE ANGELIS ET AL.: "EFFECTS OF HIGH ALTITUDE EXPOSURE ON PLASMA AND URINARY DIGOXIN-LIKE IMMUNOREACTIVE SUBSTANCE", AMERICAN JOURNAL OF HYPERTENSION, vol. 5, no. 9, 1992, pages 600 - 607, XP002081419 *
C. DE ANGELIS ET AL.: "HYPOXIA PROMOTES IN VITRO RELEASE OF THE HYPOTHALAMIC NA+/K+- ATPase (NKA) INHIBITOR FROM RAT MIDBRAIN", J. AM. SOC. NEPHROL., vol. 4, no. 3, 1993, pages 436, XP002081418 *
C. DE ANGELIS ET AL.: "HYPOXIA TRIGGERS RELEASE OF AN ENDOGENOUS INHIBITOR OF NA+-K+-ATPase FROM MIDBRAIN AND ADRENAL", AMERICAN JOURNAL OF PHYSIOLOGY, vol. 43, no. 1, January 1998 (1998-01-01), pages F182 - F188, XP002081417 *
C. FERRI ET AL.: "PLASMA ENDOGENOUS DIGOXIN-LIKE SUBSTANCE LEVELS ARE DEPENDENT ON BLOOD O2 IN MAN", CLINICAL SCIENCE, vol. 87, no. 4, 1994, pages 447 - 451, XP002081422 *
C.T. CARILLI ET AL.: "HYPOTHALAMIC FACTOR INHIBITS (NA,K)ATPase FROM THE EXTRACELLULAR SURFACE", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 260, no. 2, 1985, pages 1027 - 1031, XP002081423 *
N. ZHAO ET AL.: "NA,K-ATPase INHIBITORS FROM BOVINE HYPOTHALAMUS AND HUMAN PLASMA ARE DIFFERENT FROM OUABAIN: NANOGRAM SCALE CD STRUCTURAL ANALYSIS", BIOCHEMISTRY, vol. 34, 1995, pages 9893 - 9896, XP002081420 *
S. VARSANO ET AL.: "ENDOGENOUS DIGOXIN-LIKE IMMUNOREACTIVE FACTOR IS ELEVATED IN ADVANCED CHRONIC RESPIRATORY FAILURE", CHEST, vol. 101, no. 1, 1992, pages 146 - 149, XP002081421 *
TYMIAK A A ET AL: "PHSICOCHEMICAL CHARACTERIZATION OF A OUABAIN ISOMER ISOLATED FROM BOVINE HYPOTHALAMUS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 90, no. 17, 1 September 1993 (1993-09-01), pages 8189 - 8193, XP002074993 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7396658B2 (en) 1998-12-24 2008-07-08 The General Hospital Corporation Methods for screening HIF like ouabain-resistant Na+—K+-ATPase agents

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