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WO1996014060A1 - Stimulation de l'activite dismutase superoxyde au moyen d'agonistes de recepteurs - Google Patents

Stimulation de l'activite dismutase superoxyde au moyen d'agonistes de recepteurs Download PDF

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Publication number
WO1996014060A1
WO1996014060A1 PCT/IB1995/000979 IB9500979W WO9614060A1 WO 1996014060 A1 WO1996014060 A1 WO 1996014060A1 IB 9500979 W IB9500979 W IB 9500979W WO 9614060 A1 WO9614060 A1 WO 9614060A1
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Prior art keywords
sod
substance
receptors
cells
disease
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PCT/IB1995/000979
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English (en)
Inventor
Stefan L. Marklund
Pontus STRÅLIN
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Marklund Stefan L
Straalin Pontus
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Priority to AU37082/95A priority Critical patent/AU3708295A/en
Publication of WO1996014060A1 publication Critical patent/WO1996014060A1/fr

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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

Definitions

  • the present invention relates in particular to the use of a substance for the manufacture of a composition for stimulat- ing the release of EC-SOD from cells or stimulating the syn ⁇ thesis of EC-SOD.
  • SOD Superoxide dismutases
  • EC-SOD extracellular superoxide dismutase
  • EC-SOD has a high affinity for heparan sulfate and exists anchored to heparan sulfate proteoglycans in the glycocalyx of cell surfaces and in the tissue interstitial matrix (Karlsson et al. , 1988; Marklund and Karlsson, 1989; Sandstr ⁇ m et al. , 1993; Karlsson et al. , 1994) .
  • vascular walls are low, thus comparison with other tissues on a DNA content basis indica ⁇ tes intermediate to high average contents of both CuZn-SOD and Mn-SOD of cells in the vascular wall.
  • Analysis of EC-SOD by immunohistochemistry indicates an even distribution in the vessel wall, including large amounts in the arterial intima.
  • the EC-SOD concentration in the human arterial wall intersti- tium is high enough to prevent most putative pathophysiologi- cal effects of superoxide radicals, such as oxidation of LDL, with the exception of the formation of the deleterious per- oxynitrite under maximal rates of nitric oxide synthesis.
  • EC-SOD exists in the interstitium of all tissues and as shown in Table 1 particularly large amounts are found in man in the wall of arteries, bronchi (unpublished) , skin (unpublished) , uterus, thyroid gland and lung (Marklund, 1984) .
  • the carboxy- terminal heparan-sulfate-binding domain (Sandstr ⁇ m et al. , 1992) is highly susceptible to proteolytic truncation (Karls ⁇ son et al. , 1993), such cleavage results in rapid loss from the tissue interstitium and may be caused by proteolytic enzymes released/activated in the inflammatory response.
  • the arterial wall interstitium contains by far most EC-SOD, Table 2.
  • the enzyme is evenly distributed over the wall, Fig 1, including large amounts in the intimal layer.
  • an average EC-SOD activity of 15000-20000 U/ml in the interstitium can be calculated. This corresponds to about 110 ⁇ g/ml of CuZn-SOD.
  • concentrations are reported to strongly suppress a variety of pathophysiological effects of the superoxide radical in in vitro models.
  • the functions of EC-SOD in the wall should be to protect against deleterious effects of superoxide radical.
  • Sources of the radical in the vessels could be activated neutrophil leucocytes, monocytes, macrophages and other phagocytic leucocytes. There is also evidence for release by endothelial cells, smooth muscle cells, by not always well-defined mechanisms. There is evi ⁇ dence for formation by autoxidation of thiols (Heinecke et al. , 1987). Superoxide can also be released as a byproduct during synthesis of prostaglandins and leukotrienes (Kukreja et al. , 1986) .
  • LDL oxidation has been suggested to be a primary step in atherogenesis (Steinberg et al., 1989). After oxidation the LDL particles are taken up into macrophages through the scavenger receptor resulting in the eventual formation of foam cells. These reactions take place in the arterial inti- ma, which is the site of formation of the atherosclerotic plaque. In a variety of studies addition of SOD has been shown to retard the LDL oxidation (Steinbrecher et al. , 1990; Heinecke et al. , 1986; Kawamura et al. , 1994) suggesting involvement of the superoxide radical in the process. Sup- 4 pression of such reactions should be a primary physiological function of EC-SOD.
  • * NO produced by the endothelium is a major physiological vasodilator (Palmer et al., 1987; Ignarro et al., 1987) and also reduces adhesion of platelets (Radomski et al., 1987) and leukocytes (Gaboury et al. , 1993) to the vascular wall.
  • * N0 may also be formed by activated phagocytic cells, and its synthesis can be induced to occur in smooth muscle cells (Beasley et al. , 1991). *N0 reacts with superoxide at a nearly diffusion-limited rate to form the very toxic species, peroxynitrite (Beckman et al. , 1990; Huie et al.
  • Per- oxynitrite in itself oxidizing, may nitrate proteins (Ischi- ropoulos et al., 1992), may induce LDL oxidation (Darley- Usmar et al., 1992), and may decompose to other strongly oxidizing species (Koppenol et al., 1992).
  • the inter ⁇ action between superoxide and 'NO both reduces the physiolo ⁇ gical effects of * N0 and leads to the formation of toxic compounds.
  • the in vivo occurrence of peroxynitrite is indi ⁇ cated by the extensive nitration of protein tyrosines found in human atherosclerotic lesions (Beckman et al. , 1994).
  • 6.7 x IO 9 M “1 s "1 Huie et al. , 1993)
  • it can be calculated that about 19000 U/ml (EC-)SOD is needed to compete equally with 0.85 ⁇ M 'NO for superoxide radicals occurring in the vascular wall.
  • the high concentra- tions of EC-SOD found in the human arterial wall interstitium should be sufficient to compete with basal concentrations of • NO encountered in the vascular wall, but may allow signifi ⁇ cant formation of peroxynitrite from superoxide under maximu agonist stimulation and when "NO is formed by inflammatory cells and by smooth muscle cells after nitric oxide synthase induction.
  • the rate of endogenous formation of superoxide radical (and thus potentially of peroxynitrite) in the vascular wall is not known. Under some pathophysiological circumstances, how ⁇ ever, the rate of superoxide formation must approach that of nitric oxide, as indicated by the results of administration of large amounts of SOD.
  • the EC-SOD content of the arterial intimas is very high, it may under high nitric oxide synthesis allow significant formation of deleterious peroxynitrite, or con ⁇ versely when there is increased superoxide formation be barely sufficient to preserve nitric oxide for its physio- logical functions such as vasodilation (reduction of blood pressure) and deactivation of platelets (anticoagulant ef ⁇ fect) and phagocytic leukocytes (antiinflammatory effect) .
  • vasodilation reduction of blood pressure
  • platelets anticoagulant ef ⁇ fect
  • phagocytic leukocytes antiinflammatory effect
  • the phenotype has been shown to be caused by the same nucleotide exchange in all investigated individuals in all three popula ⁇ tions. It has previously been shown that an EC-SOD truncation mutant (Sandstr ⁇ m et al.
  • EC-SOD in the arterial wall is apparently the smooth muscle cells which secrete large amounts of EC-SOD as measured by ELISA, Fig 1.
  • EC-SOD synthesis by fibroblast has previously been shown to be influenced by inflammatory cytokines, IFN-7 induces whereas TNF ⁇ , II-lo. and particularly TGF / 3 repress the synthesis (Marklund, 1992) .
  • TNF ⁇ , II-lo. and particularly TGF / 3 repress the synthesis (Marklund, 1992) .
  • it was not possible to show any specific effect on synthesis of a va ⁇ riety of types of oxidant stress (Stralin and Marklund, 1994) .
  • SMC human arterial smooth muscle cells
  • glucose at concentrations relevant to diabetes, autoxidizes under formation of oxygen free radicals (Hunt et al., 1988).
  • Glucose glycates amino groups in proteins (amino terminal, lysine) .
  • Measurement of protein glycation is routinely used in management of diabetes pa ⁇ tients, and has proved to correlate strongly with complica- tions such as retinopathy and nephropathy.
  • Glycated proteins autoxidize easily (much more so than glucose itself) under formation of superoxide radical (Arai et al.
  • pancreatic B-cells show exceptional susceptibility to superoxide, e.g. formed by alloxan (Grankvist et al. , 1979; Grankvist et al. , 1981) , and injection of polyethylene-substituted SOD has been shown to suppress insulitis in NOD mice (Horio et al. , 1994) . This suggests that SOD might halt progression of loss of ⁇ - cell function in diabetes.
  • enhanced EC-SOD activity should preferably be accomplished by small molecules enhan ⁇ cing EC-SOD synthesis. Enhancement of synthesis in several cell types, e.g. smooth muscle cells, fibroblasts and glia cells, may be advantageous.
  • bronchi contain large amounts of EC-SOD.
  • the source in the bronchi are likely, as in the blood vessels, the smooth muscle cells. These cells are likely to respond like the SMC's of the vessels to simi ⁇ lar substances and to factors specially influencing bronchial reactions such as tonus. "NO is known to be formed in bronchi and to exert a dilating function. Peroxynitrite could like in blood vessels be formed, particularly in inflammatory states.
  • the EC-SOD content is high and also accounts for an unusually large proportion of the total SOD activity, Table 1. Variation of the EC-SOD content may influence dis ⁇ eases in the skin, especially when inflammation is involved. Enhanced EC-SOD may also be important for wound healing. Superoxide radicals are formed by activated phagocytis leuko ⁇ cytes and e.g. as a by-product upon synthesis of prostaglan ⁇ dins and leukotrienes (Kukreja et al. , 1986). Fibroblasts are a likely source of EC-SOD in the skin, but synthesis by other cell types is conceivable.
  • the basic idea here is to increase (or decrease) the synthesis of EC-SOD in skin by various effector molecules, especially among the inflammatory cyto- kines, growth factors, steroids, prostaglandins, phorbol esters, protein kinase C activators, etc.
  • the uterus also contains large amounts of EC-SOD, Table 1.
  • the source in uterus should be the smooth muscle cells of the myometrium. They should respond in a similar manner as the blood vessel smooth muscle cells to factors described below. Given the hormonal control of uterus, sexual hormones should also influence EC-SOD synthesis.
  • neutrophil leukocytes Boor et al., 1973
  • monocytes Johnston et al. , 1974
  • macrophages Johnston et al., 1978
  • eosinophil leukocytes de Chatelet et al., 1977
  • superoxide and secondary products formed from it seem to be of great importance for the bacteriocidal and cytotoxic actions of the cells.
  • Other cell types can also be stimulated to release superoxide al ⁇ though the amount is smaller than that of the granulocytes; endothelial cells (Gryglewsky et al.
  • mice carry ⁇ ing a targeted disruption of the EC-SOD gene have been gene ⁇ rated (Carlsson et al. , 1995).
  • EC-SOD null mutants were found to develop normally and have remained healthy for over a year.
  • other systems such as ascorbate and ceruloplasmin may compensate for the loss of EC-SOD.
  • the null mutant mice displayed a considerable reduction in survival time compared to wild type mice and an earlier onset of severe lung edema.
  • the basic idea of this invention is to alter, e.g. increase, the level of EC-SOD in blood vessels, bronchi, lung, kidney, skin, gut, uterus, cornea, joints, central nervous system, possibly other organs such as the heart by altering, e.g. enhancing, the endogenous synthesis of EC-SOD, using drugs as outlined above. It is evident that for some drugs, the level may be altered in only one of the above-mentioned tissues whereas other drugs may alter the level in several or all of them. It may even be possible that the alteration is diffe ⁇ rent in the different tissue, e.g. that in some tissues the level will be increased whereas in other tissues it is decreased. Such situations are within the scope of the pre ⁇ sent invention as long as a overall beneficial effect is obtained.
  • the present invention relates to the use of a substance for the manufacture of a composition for stimulating the release of EC-SOD from cells or stimulat ⁇ ing the synthesis of EC-SOD in cells.
  • the cells are smooth muscle cells.
  • the present invention relates to use according to the invention, wherein the substance exhibits agonist activity on a receptor selected from the group con ⁇ sisting of adenosin receptors, adrenoceptors, angiotensin receptors, atrial natriuretic peptide receptors, bradykinin receptors, calcitonin gene-related peptide receptors, Ca ++ channels, dopamine receptors, endothelin receptors, fibro- blast growth factor, growth hormone, histamine receptors, 5- hydroxytryptamine receptors, interferon y , interleukin-l, interleukin-4, interleukin-8, interleukin-10, interleukin-13, leukotriene receptors, muscarinic receptors, neuropeptide Y receptors, nitric oxide receptors, platelet derived growth factor receptors, prostanoid receptors, P 2 purinoceptors, 5-
  • the substance is a vasoactive fac ⁇ tor.
  • a vasoactive factor is defined as a substance which has an effect on blood vessels, e.g. exhibits agonist activity on a vasoactive re ⁇ ceptor.
  • An agonist is defined as a molecule such as a drug, an enzyme activator, or a hormone, that enhances the activity of another molecule or receptor site whereas a receptor is a target site at the molecular level to which a substance be ⁇ comes bound as a result of a specific interaction.
  • the site may be on the cell wall, on the cell membrane, or on an intracellular enzyme or another protein with regulatory function, and the substance bound may be a hormone or a drug where the binding interaction will trigger the release of EC-SOD or stimulate the synthesis of EC-SOD.
  • a full agonist is a ligand that binds to a receptor and causes a maximum biological response whereas a partial ago ⁇ nist is a ligand that binds to a receptor and causes a less than maximum biological response even when it occupies all of the available receptor sites. Both full and partial agonists are within the scope of the present invention.
  • stimulating the transcription of the EC-SOD gene stimulating the trans ⁇ lation of EC-SOD messenger RNA or enhancing stability of mRNA, and/or stimulating the secretion of EC-SOD after pro ⁇ tein synthesis from the cells.
  • receptor systems which are involved in the regulation of the EC-SOD synthesis. Examples of potential such receptors as well as of agonists/antagonists of the spe ⁇ cific receptor types are outlined in the following:
  • Nomenclature A A 2A A 2B A 3 agonists N 6 cyclopentyl-adenosine CGS21680 APNEA 2-Cl-N 6 cyclopentyl-adenosine PAPA-APEC antagonists DPCPX(8.3-9.3) CP66713(7.7) I-ABOPX( ⁇ .l) 8-cyclopentyltheophylline(7.4) KF17837
  • adrenaline noradrenaline
  • noradrenaline procaterol BR 37344 antagonists betaxolol(8.5) butaximine(6.2) pindolol atenolol(7.0)
  • Angiotensin receptors :
  • angiotensin 2 endogenous agonists: angiotensin 2, angiotensin 3
  • Atrial natriuretic peptide receptors Atrial natriuretic peptide receptors:
  • bradykinin endogenous agonists: bradykinin, kallidin, T-kinin
  • endogenous agonists endothelin 1, endothelin 2, endothelin 3
  • neuropeptide Y neuropeptide Y
  • peptide YY pancreatic polypeptide
  • Nomenclature agonists [Pro ,3H4 NPY NPY 13-36 antagonists
  • Nomenclature DP FP IP TP agonists BW245C fluprostenol cicaprost U46619 antagonists BWA869C(9.3) GR32191(8.8)
  • Tachykinin receptors
  • vasopressin vasopressin
  • oxytocin vasopressin
  • PDGF-AA endogenous agonists
  • PDGF AB endogenous agonists
  • PDGF AB endogenous agonists
  • PDGF BB receptors PDGFR- ⁇
  • PDGFR-/3 antagonists neomysin: trapidil, neutralising antibodies, soluble receptor fragments 22
  • endogenous agonists acidic FGF, basic FGF receptors: FGFR-1, FGFR-2, FGFR-3, FGFR-4 antagonists: neutralising antibodies, soluble receptor fragments
  • Interferon 7 receptors IFN7R antagonists: neutralising antibodies, soluble receptor fragments
  • TNF ⁇ endogenous agonists: TNF ⁇
  • TNF/S endogenous antagonists soluble type I TNF-R
  • soluble type II TNF-R receptors TNF-RI
  • TNF-RI1 antagonists neutralising antibodies, soluble receptor fragments
  • endogenous agonists Il-l ⁇ , 11-13 endogenous antagonists:" endogenous II- 1 receptor antagonist" receptors:Il-l-R antagonists: neutralising antibodies, soluble receptor fragments
  • endogenous agonists 11-4 receptors:Il-4-R antagonists: neutralising antibodies, soluble receptor fragments
  • endogenous agonists 11-8, GRO ⁇ , NAP-2 receptors: 11-8 A , I1-8 B antagonists: neutralising antibodies, soluble receptor fragments
  • endogenous agonists 11-10 receptors:Il-10-R antagonists: neutralising antibodies, soluble receptor fragments
  • GH endogenous antagonist soluble GHR receptors: GHR antagonists: neutralising antibodies, soluble receptor fragments
  • thrombin receptor agonist receptors antagonists: neutralising antibodies, soluble receptor fragments agonists: thrombin receptor related polypeptides
  • Heparin receptors, receptors for other sulfated glycosaminoglycans are included in the following sulfated glycosaminoglycans.
  • endogenous agonists heparin, heparan sulfate, other sulfated glycosaminoglycans, fragments of these synthetic: low-molecular weight heparin, synthetic other negatively charged saccharides, and polymers endogenous antagonists: heparitinases, heparinases
  • EGF endogenous agonists
  • HB-EGF HB-EGF
  • TGF- ⁇ receptor antagonists EGF-R
  • neutralising antibodies neutralising antibodies
  • Protein kinase C Protein kinase C
  • PKC activator phorbol, 12-myristate, 13-acetate, synthetic activators
  • PKC inhibitor staurosporine, synthetic inhibitors
  • Insulin
  • insulin receptor insulin receptor
  • Example 2 it is demonstrated how a potential vasoactive factor, growth factor or other factors influencing the syn ⁇ thesis of EC-SOD can be tested and selected.
  • the present in ⁇ vention relates in particular to a substance selected from the group consisting of angiotensin, bradykinin, endothelin, histamine, serotonin, thrombin, vasopressin and substances releasing nitric oxide such as Na-nitroprusside, glyceryl trinitrate, isosorbide dinitrate, isosorbide mononitrate and SIN-l.
  • the invention relates to substances related to inflammation, such as a substance selected from the group consisting of IFN7, IL-4, IL-8 and indomethacin.
  • the invention relates to the use according to the invention wherein the substance is a growth factor such as growth hormone or insulin. Moreover, the invention relates to the use according to the invention wherein the substance is he ⁇ parin, or other sulfated glycosaminoglycans or fragments thereof, or phorbol 12-myristate 13-acetate or other factors stimulating protein kinases C.
  • a further aspect of the invention thus relates to a method for determining the effect of a substance with respect to stimulating the release of EC-SOD from cells or stimulating the synthesis of EC-SOD in cells comprising
  • a further aspect of the invention is use of a substance, the effect of which with respect to stimulating the release of EC-SOD from cells or stimulating the synthesis of EC-SOD in cells has been established using the above method for the preparation of a pharmaceutical composition for stimulating the release of EC- SOD from cells or stimulating the synthesis of EC-SOD in cells.
  • the invention relates to a method for pre ⁇ venting, diminishing, controlling or inhibiting a disease or disorder connected with the presence or formation of su ⁇ peroxide radicals and other toxic intermediates derived from the superoxide radical comprising administering, to a patient in need thereof, an effective amount of a substance the effect of which for stimulating the release of EC-SOD from cells or stimulating the synthesis of EC-SOD in cells has been established using the above method.
  • the basic concept of the in ⁇ vention is to alter the EC-SOD content.
  • this invention is thus also a method for determining the effect of a substance with respect to decreasing the level of EC-SOD in blood vessels, bronchi, lung, kidney, skin, gut, uterus, cornea, joints, central nervous system, possibly other organs such as the heart by decreasing the endogenous synthesis of EC-SOD, using a test as outlined above, as well as substances selected by a such method, use of a such sub- stance and a method for preventing, diminishing, controlling or inhibiting a disease or disorder using a substance selec ⁇ ted by a method as outlined above and which decreases the level of EC-SOD.
  • the agonist or antagonist may be prepared as formulations in pharmaceutically acceptable media, for example, saline, phosphate buffered saline (PBS), Ringer's solution, dextro ⁇ se/saline, Hank's solution, and glucose.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like.
  • Additives may also include additio ⁇ nal active ingredients, e.g. bactericidal agents, or stabili ⁇ zers.
  • the amount administered to the patient will vary depen ⁇ ding upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the host, the manner of administration, and the like.
  • compositions are typically intended for oral, transdermal or parenteral administration, e.g. intra ⁇ venously, subcutaneously, or intramuscularly, or for delivery through inhalation, e.g. by means of a metered dose inhaler (MDI) or a dry powder inhaler (DPI) .
  • MDI metered dose inhaler
  • DPI dry powder inhaler
  • Orally administrative forms are desirable and can, if necessary, be provided by modifying the composition to bypass the stomach environment.
  • the composition can be used for prophylactic and/or thera- Treatment.
  • the pharmaceutical composi ⁇ tions can be administered intravenously.
  • the invention provides compositions which comprise an agonist substance dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being com ⁇ bined with a sterile aqueous carrier prior to administration.
  • the agonist may also be administered with a second biologi ⁇ cally active agent.
  • the pharmaceutical compositions are administered to a patient in an amount sufficient to produce the desired effect, defined as a "therapeutically effective dose".
  • the therapeutically effective dose of an agonist will vary according to, for example, the particular use for which the treatment is made, the manner of admini ⁇ stration, the health and condition of the patient, and the judgement of the prescribing physician.
  • the dose for continuous infusion will typically be in the range of about 500 ng to about 800 ⁇ g per day for a 70 kg patient, preferably between about 10 ⁇ g and about 300 ⁇ g.
  • the dose will typically be between 700 ng/kg/day and 16 ⁇ g/kg/day.
  • the concentration of the agonist in the pharmaceutical formu- lations can vary widely, i.e. from about 10 ⁇ g to about
  • a typical pharmaceu- tical composition for intravenous infusion could be made up to contain 250 ml of dextrose/saline solution and 2.5 g of the agonist.
  • non-toxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharma ⁇ ceutically acceptable non-toxic composition is formed by incorporating normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, an agonist substance, preferably 25-75%.
  • the agonist is preferably sup ⁇ plied in finely divided form along with a surfactant and propellant.
  • Typical percentages of agonist are 0.01-20% by weight, preferably 1-10%.
  • the surfactant must, of course, be non-toxic, and preferably soluble in the propellant.
  • esters or partial esters of fatty acids containing from 6 to 22 carbon atoms such as caproic, octanoic, lauric, palmitic, stearic, linoleic, lino- lenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as, for example, ethy ⁇ lene glycol, glycerol, erythritol, arbitol, mannitol, sorbi ⁇ tol, the hexitol anhydrides derived from sorbitol, and the polyoxyethylene and polyoxypropylene derivatives of these esters.
  • Mixed esters, such as mixed or natural glycerides may be employed.
  • the surfactant may constitute 0.1-20% by weight of the compo ⁇ sition, preferably 0.25-5%.
  • the balance of the composition is ordinarily propellant.
  • Liquified propellants are typically gases at ambient conditions, and are condensed under pres ⁇ sure.
  • suitable liquified propellants are the lower al- kanes containing up to 5 carbons, such as butane and propane; and preferably fluorinated or fluorochlorinated alkanes. Mix ⁇ tures of the above may also be employed.
  • a container equipped with a suitable valve is filled with the appropriate propellant, containing the finely divi ⁇ ded peptide(s) and surfactant. The ingredients are thus main ⁇ tained at an elevated pressure until released by action of the valve.
  • the agonist may be encapsula ⁇ ted, introduced into the lumen of liposomes, prepared as a colloid, or other conventional techniques may be employed which provide an extended lifetime of the peptides.
  • the agonist may be encapsulated in a liposome.
  • Another aspect of the present invention relates to the use of a substance for the manufacture of a composition for prophy ⁇ laxis or treatment of a disease or disorder connected with the presence or formation of superoxide radicals and other toxic intermediates derived from the superoxide radical, in particular to use according to the invention wherein the sub ⁇ stance alters the level of EC-SOD in blood vessels, bronchi, lung, skin, uterus, gut, joint, cornea, kidney, central ner ⁇ vous system, and/or other organs such as the heart by alter- ing the endogenous synthesis of EC-SOD, e.g.
  • dis ⁇ ease or disorder is alterated blood pressure, inflammation or formation of atherosclerotic lesions, reconstriction after arterial angioplasty, bronchial diseases involving inflamma ⁇ tion and constriction such as asthma, other lung disorders, diseases or disorder selected from conditions involving ischaemia followed by reperfusion, e.g.
  • infarctions such as heart, kidney, brain or intestine infarctions
  • inflammatory diseases such as rheumatoid arthritis, pancreatitis, in par ⁇ ticular acute pancreatitis, colitis, pyelonephritis and other types of nephritis, and hepatitis, keratitis, otitis media with effusion, autoimmune diseases, diabetes mellitus, dis ⁇ seminated intravascular coagulation, fatty embolism, adult respiratory distress, infantile respiratory distress, brain haemorrhages in neonates, burns, wound healing, adverse effects of ionizing radiation, and carcinogenesis. 31
  • a substance selected from the group consisting of PDGFaAA, PDGFBB, A-FGF, B-FGF, EGF and derivatives thereof for the manufacture of a composition for decreasing the release of EC-SOD from cells, e.g. smooth muscle cells, or decreasing the synthesis of EC-SOD in cells, in particular in a human, such as the use of such a substance for prophylaxis or treat ⁇ ment of atherosclerosis or other vascular disease.
  • the present invention relates to the use of a substance selected from the group consisting of inter- feron-7, prostaglandin E 2 , indomethacin, interleukin-1, TNF, TGF3 and derivatives thereof for the manufacture of a compo ⁇ sition for decreasing the release of EC-SOD from cells (e.g. smooth muscle cells) or decreasing the synthesis of EC-SOD in cells, in particular in a human.
  • a substance selected from the group consisting of inter- feron-7, prostaglandin E 2 , indomethacin, interleukin-1, TNF, TGF3 and derivatives thereof for the manufacture of a compo ⁇ sition for decreasing the release of EC-SOD from cells (e.g. smooth muscle cells) or decreasing the synthesis of EC-SOD in cells, in particular in a human.
  • the invention relates to a method of preventing, diminishing, controlling or inhibiting a disease or disorder connected with the presence or formation of superoxide radi ⁇ cals and other toxic intermediates derived from the superoxi- de radical in a patient who has been established to have a high risk of developing a such disease or disorder, or who has developed a such disease or disorder, the method compri ⁇ sing administering an effective amount of a substance which is capable of stimulating the release of EC-SOD from cells or stimulating the synthesis of EC-SOD in cells.
  • the invention relates to a method as outlined above wherein the disease or disorder is selected from the group consisting of alterated blood pressure, inflammation or formation of atherosclerotic lesions, proliferation of ar- terial intima, diabetes, bronchial diseases involving in ⁇ flammation and constriction such as asthma, conditions in ⁇ volving ischaemia followed by reperfusion, e.g.
  • infarctions such as heart, kidney, brain or intestine infarctions
  • in ⁇ flammatory diseases such as rheumatoid arthritis, pancreati- tis, in particular acute pancreatitis, enteritis, colitis, 32 pyelonephritis and other types of nephritis, and hepatitis, keratitis, otitis media with effusion, autoimmune diseases, central nervous system degenerative disorders such as ALS, Parkinson's disease, Alzheimer's disease, diabetes mellitus, disseminated intravascular coagulation, fatty embolism, adult respiratory distress, other lung disorders, infantile re ⁇ spiratory distress, brain haemorrhages in neonates, burns, adverse effects of ionizing radiation, and carcinogenesis.
  • ALS ALS
  • Parkinson's disease Alzheimer's disease
  • diabetes mellitus disseminated intravascular coagulation
  • fatty embolism fatty embolism
  • adult respiratory distress
  • a particular embodiment of the above is a method wherein the patient is a patient who has been established to have a high risk of developing a disease or disorder connected with the presence or formation of superoxide radicals and other toxic intermediates derived from the superoxide radical by having a high-risk-indicating score of a serum or plasma marker for said disease such as high content of glycated hemoglobin (diabetes, diabetes complications) , high content of lipid hydroperoxide in plasma (atherosclerosis, diabetes) , altered amount of EC-SOD (such as reduced amount because of the person being e.g.
  • Figure 1 shows immunostaining of EC-SOD in nondiseased thora ⁇ cic aorta from a 40 year old man, collected 48 hours post mortem.
  • the anti EC-SOD antibody used (1.4 ⁇ g/ml) (A) , was raised against the recombinant enzyme in rabbit.
  • Nonimmunized rabbit IgG was used as a negative control (2.4 ⁇ g/ml) (B) .
  • Figure 2 is a graph showing the results of the EC-SOD analy- sis when the synthesis regulation experiments described in Example 2 were performed using endothelin 1 as an active substance on smooth muscle cells.
  • Figure 3 is a graph showing the results of the EC-SOD analy ⁇ sis when the synthesis regulation experiments described in Example 2 were performed using angiotensin 2, vasopressin or endothelin 2 as an active substance on smooth muscle cells.
  • Figure 4 is a graph showing the results of the EC-SOD analy ⁇ sis when the synthesis regulation experiments described in Example 2 were performed using trombin or bradykinin as an active substance on smooth muscle cells.
  • Figure 5 is a graph showing the results of the EC-SOD analy ⁇ sis when the synthesis regulation experiments described in Example 2 were performed using histamine or serotonin as an active substance on smooth muscle cells.
  • Figure 6 is a graph showing the results of the EC-SOD analy ⁇ sis when the synthesis regulation experiments described in Example 2 were performed using Na-nitroprusside, SIN-1 or growth hormone as an active substance on smooth muscle cells.
  • Figure 7 is a graph showing the results of the EC-SOD analy- sis when the synthesis regulation experiments described in Example 2 were performed using heparin or EGF as an active substance on smooth muscle cells.
  • Figure 8 is a graph showing the results of the EC-SOD analy ⁇ sis when the synthesis regulation experiments described in Example 2 were performed using insulin as an active substance on smooth muscle cells.
  • Figure 9 is a graph showing the results of the EC-SOD analy ⁇ sis when the synthesis regulation experiments described in Example 2 were performed using phorbol ester (PMA) as an active substance on smooth muscle cells.
  • PMA phorbol ester
  • Figure 10 is a graph showing the results of the EC-SOD analy ⁇ sis when the synthesis regulation experiments described in Example 2 were performed using phorbol ester (PMA) as an active substance on fibroblasts.
  • PMA phorbol ester
  • Figure 11 is a graph showing the results of the EC-SOD analy ⁇ sis when the synthesis regulation experiments described in Example 2 were performed using IFNy as an active substance on a glia cell line.
  • Figure 12 is a graph showing the results of the EC-SOD analy- sis when the synthesis regulation experiments described in
  • Example 2 were performed using TGF3 as an active substance on a malignant glioma cell line.
  • Macroscopically normal pieces (0.5-1.5 g) of human left ante ⁇ rior descending coronary artery (LAD) , proximal thoracic aorta, saphenous vein, bronchi and skin were cut out at autop ⁇ sy within 48 h after death. Thoracic aortas from the other mammals were collected within a few hours after death. The pieces were kept at -80°C prior to analysis.
  • LAD left ante ⁇ rior descending coronary artery
  • frozen pieces were pulverized in a Braun Microdismembrator II (B Brown Biotech Inc, Allentown, PA) and the frozen powder added to 10 volumes of 50 mM potassium phosphate, pH 7.4, with 0.3 M KBr, and a set of antiproteolytic agents (phenyl- methylsulfonylfluoride 0.5 mM, diethylenetriamine pentaacetic acid 3 mM, aprotinin 90 mg/1, pepstatin 10 mg/1, chymostatin 10 mg/1 and leupeptin 10 mg/1) .
  • the homogenates were then sonicated and finally extracted for 30 min at 4°C.
  • the ex- tracts were then centrifuged (20.000 g for 15 min). Unless analysed immediately, the supernatants were stored at -80°C. 060
  • SOD enzymatic activity was determined using the direct spec- trophotometric method employing K0 2 (Marklund, 1976) as modi- fied (Marklund, 1985) .
  • 3 M cyanide was used to distinguish between the cyanide-sensitive isoenzymes CuZn-SOD and EC-SOD and the resistant Mn-SOD.
  • One unit in the assay is defined as the activity that brings about a decay of 0 2 — concentration at a rate of 0.1 s "1 in 3 ml of buffer. It corresponds to 8.3 ng of human CuZn-SOD, 6.3 ng of bovine CuZn-SOD, 8.6 ng of human EC-SOD and 65 ng of bovine Mn-SOD.
  • the "K0 2 -assay” is carried out at pH 9.5 and at relatively high superoxide concentration. In comparison, the xanthine oxidase-cytochrome C SOD assay (McCord and Fridovich, 1969) is carried out under more physiological conditions, i.e. neutral pH and low super ⁇ oxide concentration.
  • One unit in the "K0 2 -assay” corresponds to about 0.024 units of CuZn-SOD and EC-SOD and 0.24 units of Mn-SOD, respectively in the "xanthine oxidase” assay.
  • the “K0 2 -assay” is thus about 10 times more sensitive for CuZn- SOD and EC-SOD activity than Mn-SOD activity.
  • Vessels for immunostaining were obtained at autopsy within 48 hours of death or immediately at vessel surgery. Cryostat sections were fixed for 45 minutes in 1 % paraformaldehyde solution. An avidin- biotin-horseradish-peroxidase system (DACOPATTS, Glostrup, Denmark) was used for immunostaining. Anti-EC-SOD antibodies, raised against recombinant human protein (Tibell et al., 1987) in goat and rabbit, and purified by adsorption/desorp- tion on EC-SOD immobilized on CNBr-activated sepharose, were used at concentrations 0.7-8.6 ⁇ g/ml. As negative controls, primary antibodies were substituted with non-immunised goat/- rabbit IgG (2.4-11.6 ⁇ g/ml). Serial sections were stained by eosin for histological orientation.
  • LAD thoracic aorta and saphenous vein
  • CuZn-SOD, Mn-SOD and EC-SOD were determined as described in Methods above. Where ng values are given for EC-SOD, the level were determined with ELISA, in other cases with the
  • the vessel walls were homogenized and the extracts were as ⁇ sayed for contents of CuZn-SOD and Mn-SOD activity, EC-SOD protein, total protein and DNA.
  • the results for EC-SOD pro ⁇ tein converted to activity units are also presented.
  • the con ⁇ version factor 8.6 ng per unit was used.
  • the cyanide-sensitive SOD activities were subtracted with these calculated EC-SOD units. Note that the SOD activity assay used (Marklund, 1976; Marklund, 1985) is 10 times less sensitive to Mn-SOD activity than to CuZn-SOD and EC-SOD activity.
  • the results are presented as means ⁇ S.D.
  • CuZn-SOD is the predominant SOD iso- enzyme, with Mn-SOD being about half as abundant, Table 1 and Marklund, 1984.
  • EC-SOD normally accounts for less than 10% of the total SOD activity.
  • the human blood vessel wall is found to contain exceptionally large amounts of EC-SOD which is comparable in abundance to CuZn-SOD, Table 2.
  • Human vascular smooth muscle cell lines were initiated from uterine artery media collected at hysterectomy of women suffering from uterine myoma, using Waymouth MB 752/1 with 15% fetal calf serum (FCS) , 72 ⁇ g/ml benzylpenicillin, 100 ⁇ g/ml, 2 mM glutamine and ImM Na-pyruvate as medium. The lines were used between the 5th and the 8th passages.
  • FCS fetal calf serum
  • the cells were seeded into 12-well culture plates, bottom area 3.80 cm 2 , and grown into near confluence.
  • FCS fetal calf serum
  • BSA bovine serum albumin
  • the medium was exchanged twice to medium with 1% BSA about 20 h before the start of the experiments.
  • the experiments were started by exchange to 0.5 ml medium with 15% FCS or 1% BSA containing indicated concentrations of active substances or only medium with 15% FCS or 1% BSA (controls) . Every 24 hours the media were collected and replaced with fresh media containing active substances.
  • Human skin fibroblast lines were initiated from skin punch biopsy specimens obtained from healthy volunteers. Cells were grown using Ham's F10 with 10% fetal calf serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 25 mM HEPES and 2 mM glutamine as medium. The lines were used between the 10th and the 20th passages.
  • the cells were mostly seeded into 12-well culture plates, bottom area 3.80 cm 2 , and grown into near confluence.
  • the medium was exchanged twice to medium with 0.5% fetal calf serum about 20 hours before the start of the experiments.
  • the experiments were started by exchange to 0.5 ml medium with 0.5% fetal calf serum containing indicated concentrations of active factors or only medium with 0.5% fetal calf serum (controls). Every 24 hours the media were collected and replaced with fresh media containing active factors.
  • the media were collected and the wells were washed 3 times with 0.15 M NaCl.
  • EC-SOD protein was determined in cell culture media and cell homogenates with a double antibody sandwich ELISA.
  • Microtiter plates Nunc; Roskilde; Denmark
  • 100 ⁇ l/well of a solution containing 16 ⁇ g/ml of polyclonal rabbit anti-human EC-SOD IgG antibodies (raised using puri ⁇ fied recombinant EC-SOD as antigen) in 50 mM Na 2 C0 3 , pH 9.6.
  • the wells were washed and then blocked overnight with 300 ⁇ l of blocking buffer (lOmM Na phosphate, pH 7.4, 140 mM NaCl, 0.1% wt/vol Tween 20, and 0.5% BSA).
  • the assay was standardized with human umbilical cord EC-SOD C.
  • the limit of sensitivity was about 0.25 ng/ml.
  • Coomassie Brilliant Blue G-250 was em ⁇ ployed, standardized with human serum albumin.
  • DNA was deter ⁇ mined with fluorimetry as a complex with (2- [2- (4-hydroxy- phenyl) -6-benzimidazolyl-6- (1-methyl-4-piperazyl)benzimidazol 3HC1 (Hoechst 33258) using calf thymus DNA as standard.
  • Inflammation Direction: Level of change: (-fold) IFN ⁇ (5000 U/ml) + 4* IL-4 (15 ng/ml) + 2.5* IL-8 (1 ⁇ g/ml) + 2* TNF ⁇ (30 ng/ml) 1/3 TGF0 (5 ng/ml) 1/6 Il-l ⁇ + /- variable
  • PDGF-BB 50 ng/ml
  • 1/2 acid FGF 120 ng/ml
  • basic FGF 120 ng/ml
  • Nitric oxide prevents leukocyte adhe ⁇ rence: role of superoxide. Am. J. Physiol . 265:H862-H867.
  • the B lymphocyte a newly recognized source of reactive oxygen species with immunoregulatory potential. Free Rads . Res . Comms . 8:143-148.
  • Atte ⁇ nuated coronary relaxation after reperfusion Effects of superoxide dismutase and TxA 2 inhibitor U 63557A. Am. J. Physiol . 257.-H1240-H1246.
  • Ni ⁇ tric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524- 526.

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Abstract

La présente invention concerne l'utilisation d'une substance pour produire une composition stimulant la libération de la dismutase superoxyde extracellulaire ou 'EC-SOD' des cellules ou stimulant la synthèse de la EC-SOD dans les cellules. En l'occurrence, l'invention concerne l'utilisation d'une substance pour produire une composition de prophylaxie ou de traitement d'une affection ou d'un trouble lié à la présence ou la formation de radicaux superoxyde et d'autres intermédiaires toxiques dérivés du radical superoxyde. L'invention concerne également un procédé permettant de déterminer l'effet non seulement d'une substance sur la stimulation de la libération de la EC-SOD des cellules ou la stimulation de la synthèse de la EC-SOD dans les cellules, mais aussi de substances sélectionnées au moyen dudit procédé. L'invention concerne en outre un procédé permettant de prévenir, atténuer, enrayer ou restreindre une affection ou un trouble lié à la présence ou la formation de radicaux superoxyde et d'autres intermédiaires toxiques dérivés du radical superoxyde, chez un patient pour lequel il est établi qu'il présente un risque élevé de développer une telle affection ou un tel trouble, ou chez un patient qui a déjà développé une telle affection ou un tel trouble. Le procédé consiste en l'administration d'une quantité suffisante d'une substance capable de stimuler la libération de la EC-SOD des cellules ou de stimuler la synthèse de la EC-SOD dans les cellules.
PCT/IB1995/000979 1994-11-04 1995-11-03 Stimulation de l'activite dismutase superoxyde au moyen d'agonistes de recepteurs WO1996014060A1 (fr)

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AU37082/95A AU3708295A (en) 1994-11-04 1995-11-03 Use of receptor agonists to stimulate superoxide dismutase activity

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999043339A1 (fr) * 1998-02-24 1999-09-02 A+ Science Invest Ab Preparation pharmaceutique comprenant un agoniste du recepteur de l'angiotensine ii type 2 et son utilisation
EP1014995A4 (fr) * 1997-06-18 2005-02-16 Aderis Pharmaceuticals Inc Compositions et procedes prevenant les restenoses consecutives a des interventions de revascularisation
US20070110788A1 (en) * 2005-11-14 2007-05-17 Hissong James B Injectable formulation capable of forming a drug-releasing device
US7414036B2 (en) 2002-01-25 2008-08-19 Muscagen Limited Compounds useful as A3 adenosine receptor agonists
CN110662549A (zh) * 2017-05-01 2020-01-07 路易斯安娜州立大学监测委员会，农业和机械学院 用于治疗眼病的组合物和方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
H.A. LEHR ET AL.: "Superoxide-dependent stimulation of leukocyte adhesion by oxidatively modified LDL in vivo.", ARTERIOSCLEROS. THROMBOS., vol. 12, no. 7, pages 824 - 829 *
S.L. MARKLUND: "Regulation by cytokines of extracellular superoxide dismutase and other superoxide dismutase isoenzymes in fibroblasts.", J. BIOL. CHEM., vol. 267, no. 10, pages 6696 - 6701 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1014995A4 (fr) * 1997-06-18 2005-02-16 Aderis Pharmaceuticals Inc Compositions et procedes prevenant les restenoses consecutives a des interventions de revascularisation
WO1999043339A1 (fr) * 1998-02-24 1999-09-02 A+ Science Invest Ab Preparation pharmaceutique comprenant un agoniste du recepteur de l'angiotensine ii type 2 et son utilisation
AU755949B2 (en) * 1998-02-24 2003-01-02 Pharmacore Ab A pharmaceutical preparation comprising an angiotensin II type 2 receptor agonist, and use thereof
US7414036B2 (en) 2002-01-25 2008-08-19 Muscagen Limited Compounds useful as A3 adenosine receptor agonists
US20070110788A1 (en) * 2005-11-14 2007-05-17 Hissong James B Injectable formulation capable of forming a drug-releasing device
CN110662549A (zh) * 2017-05-01 2020-01-07 路易斯安娜州立大学监测委员会，农业和机械学院 用于治疗眼病的组合物和方法
EP3618850A4 (fr) * 2017-05-01 2021-01-27 The Board of Supervisors of Louisiana State University and Agricultural and Mechanical College Compositions et méthodes de traitement de pathologies oculaires
US11759438B2 (en) 2017-05-01 2023-09-19 The Board Of Supervisors Of Louisiana State Compositions and methods for treating ocular pathologies

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