WO1993021945A1 - PROCEDES DE TRAITEMENT DE MALADIES VASCULAIRES AU MOYEN DE L'INHIBITION DE L'ACTIVITE DE STIMULATION DE L'ENDOTHELINE DE TGF$g(b) - Google Patents

PROCEDES DE TRAITEMENT DE MALADIES VASCULAIRES AU MOYEN DE L'INHIBITION DE L'ACTIVITE DE STIMULATION DE L'ENDOTHELINE DE TGF$g(b) Download PDF

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Publication number
WO1993021945A1
WO1993021945A1 PCT/US1993/003795 US9303795W WO9321945A1 WO 1993021945 A1 WO1993021945 A1 WO 1993021945A1 US 9303795 W US9303795 W US 9303795W WO 9321945 A1 WO9321945 A1 WO 9321945A1
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WIPO (PCT)
Prior art keywords
endothelin
decorin
tgfβ
cys
methods
Prior art date
Application number
PCT/US1993/003795
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English (en)
Inventor
Marvin R. Brown
John R. Harper
Original Assignee
The Regents Of The University Of California
La Jolla Cancer Research Foundation
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Publication date
Application filed by The Regents Of The University Of California, La Jolla Cancer Research Foundation filed Critical The Regents Of The University Of California
Publication of WO1993021945A1 publication Critical patent/WO1993021945A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

Definitions

  • the present invention was supported in part by
  • the present invention generally relates to the treatment of vascular disorders. More specifically, the invention provides methods of inhibiting the endothelin stimulatory activity of TGFB as a means to control the deleterious release of endothelin which can result in various vascular disorders.
  • Endothelin-1 is a 21 amino acid peptide synthesized by vascular endothelial cells. The peptide has been shown to stimulate vascular, cardiac and bronchial smooth muscle cell growth and contraction.
  • a C-terminal extended form of ET-1 referred to as ET-1 1 " 39 , is also synthesized by endothelial cells and is believed to serve as a precursor for ET-1. In contrast to ET-1, ET-1 1 " 39 exhibits low biological potency.
  • Two other ET-related peptides have been characterized, ET-2 and ET-3; however, neither of these peptides are produced by vascular endothelial cells.
  • Endothelin is constitutively released and anatomically distributed throughout the entire vascular system. Only modest increases of plasma concentrations of endothelin have been observed under a variety of physiological and pathophysiological conditions. Based on its anatomic distribution and biological actions, however, endothelin has been hypothesized to play an important role in the regulation of vascular smooth muscle tone and growth.
  • Endothelin release following vascular injury may cause the reduction of blood flow and changes of smooth muscle growth that result in the impairment of normal organ function.
  • the administration of endothelin to animals has been shown to result in an increase of vascular resistance and arterial pressure.
  • endothelin is believed to participate in the development of hypertension and abnormalities of vascular wall growth.
  • TGF ⁇ has been identified as the most potent stimulator of endothelin synthesis and release by vascular endothelial cells.
  • vascular segmental production of TGF ⁇ ⁇ by platelets, cardiac myocytes or smooth muscle cells may mediate changes of endothelin production resulting in changes of vascular smooth muscle tone or growth.
  • endothelin is believed to participate as a paracrine regulator of vascular function, the physiologic role of the peptide is unknown.
  • pharmacologic method that inhibits the endothelin stimulatory activity of TGF ⁇ .
  • a need exists to regulate the endothelin stimulatory activity of TGF ⁇ in order to prevent or treat vascular disorders characterized by the deleterious effects of endothelin.
  • the present invention satisfies this need and provides related advantages as well.
  • TGF ⁇ stimulates the production and release of endothelin and that by blocking this stimulatory activity of TGF ⁇ , the deleterious effects of endothelin release can be controlled.
  • the present invention thus provides methods for preventing or treating a disorder characterized by a deleterious release of endothelin by administering an effective amount of an agent that inhibits the endothelin stimulatory activity of TGF ⁇ .
  • the inhibitory agent can be an anti-TGF ⁇ antibody, decorin or a functional equivalent of decorin.
  • the present methods are particularly useful for treating or preventing cardiovascular diseases.
  • compositions containing an inhibitory agent and a pharmaceutically acceptable carrier are also provided.
  • the compositions are useful in the methods provided by the present invention.
  • Figure 1 shows the dose-dependent inhibition of decorin on TGF ⁇ -induced endothelin release from vascular endothelial cells in culture.
  • Figure 2 shows the inhibitory effects of a constant dose of decorin (6 j/g/ml) on differing doses of TGF ⁇ -induced endothelin release.
  • the present invention generally relates to methods of controlling the synthesis and release of endothelin by vascular endothelial cells. The ability of
  • TGF ⁇ to regulate the release of endothelin provides a mechanism for controlling such endothelin release. It has now been discovered that blocking the endothelin stimulatory activity of TGF ⁇ with antagonists results in inhibiting the release of endothelin. Thus, the inhibition of TGF ⁇ 's action on endothelial cells can prevent or reduce the production of endothelin release and attenuate the deleterious changes of vascular function that can occur following vascular injury.
  • the present invention accordingly provides methods for preventing or treating a disorder characterized by the deleterious release of endothelin in an individual.
  • the methods can be accomplished by administering an effective amount of an agent that inhibits the endothelin stimulatory activity of TGF ⁇ to the individual.
  • cardiovascular disorders are characterized by the deleterious release of endothelin and can, therefore, be treated or prevented by the methods of the present invention.
  • the present methods can be used to prevent or treat various cardiovascular disorders such as vasospasm associated with coronary artery disease, vasospasm and smooth muscle proliferation following vascular angioplasty procedures, cerebrovascular spasm associated with stroke and low perfusion states, decreased blood flow to ischemic extremities following surgical procedures such as replant surgery, or deleterious cardiovascular responses to burn injury and edema formation.
  • cardiovascular responses include all types of reperfusion, ischemic or occlusion disorders that relate to vasorestriction of smooth muscles.
  • the responses can also be caused by grafts, implants, angioplasty or stroke.
  • the methods can also be used for special cases, such as hypertension, hypertension of pregnancy, raynauds, congestive heart disease or burn injury for example.
  • disorders associated with or characterized by the deleterious release (i.e., overexpression) of endothelin can be readily determined by methods known in the art or by following the guidance set forth herein.
  • Agents that inhibit the endothelin stimulatory activity of TGF ⁇ include, for example, anti-TGF ⁇ antibodies, decorin and functional equivalents of decorin.
  • Other agents having the desired inhibitory activity can also be readily determined by those skilled in the art by testing the agent's ability to block the endothelin stimulatory activity of TGF ⁇ as set forth in the examples.
  • Anti-TGF ⁇ antibodies useful in the methods of the present invention can be produced by any method known in the art.
  • the production of monoclonal and polyclonal (i.e. antiserum) antibodies are well known in the art as described, for example, in Antibodies; A Laboratory Manual (Cold Spring Harbor Laboratory 1988) which is incorporated herein by reference.
  • Other known methods of producing antibodies or antibody-like compounds are also contemplated.
  • Antibody-like compounds can be, for example. single chain antibodies .or fragments of anti-TGF ⁇ antibodies, such as Fab' or F(ab) ' 2 . In general, fragments of such antibodies contain a TGF ⁇ binding region that is capable of inhibiting the ability of TGF ⁇ to induce endothelin release.
  • TGF ⁇ x antiserum The usefulness of antibodies and antibody-like compounds as the agent has been demonstrated by the administration of a TGF ⁇ x antiserum.
  • the antiserum has been shown to prevent serum-induced endothelin release from bovine vascular endothelial cells in culture as disclosed in Brown et al.. Endocrinology 129:2355-2360 (1991), which is incorporated herein by reference.
  • one microliter of anti-TGF ⁇ ⁇ antiserum completely prevented the increase of endothelin induced by 0.1 ng TGF ⁇ .
  • the results of these studies thus demonstrate that the administration of TGF ⁇ j antiserum to endothelial cells in culture attenuated endothelin release induced by TGF ⁇ x .
  • the antiserum did not attenuate the release of the C-terminal extended forms of ET-1, specifically ET-1 1 " 38 (human) and ET-1 1 " 39 (porcine) . This finding suggests that the antiserum is specific for inhibiting only the biologically active forms of endothelin, and in particular ET-1.
  • the agent can be decorin or a functional equivalent of decorin.
  • decorin or a functional equivalent of decorin.
  • decorin refers to a proteoglycan having substantially the structural characteristics described in Krusius &
  • Human fibroblast decorin has substantially the amino acid sequence described in Krusius & Ruoslahti, supra.
  • Decorin also refers to the native composition, the core protein and to modifications of the native proteoglycan that substantially retain the functional characteristics.
  • Decorin core protein refers to decorin that no longer is substantially substituted with glycosaminoglycan and is included within the definition of decorin.
  • Decorin can be rendered glycosaminoglycan-free by enzymatic treatment, mutation or other means, such as by producing decorin in recombinant host cells in which the cells are incapable of attaching glycosaminoglycan chains to a core protein.
  • Functional equivalents of decorin include modifications of decorin that retain its functional characteristics and molecules that are homologous to decorin, such as the decorin family members biglycan and fibromodulin, for example, that have the similar functional activity of decorin.
  • Such functional equivalents can also include fragments of decorin having similar functional activities of decorin in their ability to inhibit TGF ⁇ - induced endothelin release. The fragments can be made by recombinant DNA methods or synthetical using methods known in the art.
  • Modifications of decorin can include, for instance, the addition of one or more side chains or point mutations that do not interfere with the functional activity of the decorin core protein.
  • both decorin and biglycan are about 80% homologous and contain a leucine-rich repeat of about 24 amino acids in which the arrangement of the leucine residues is conserved. As defined, each repeat generally contains at least two leucine residues and can contain five or more. Proteoglycans and other peptides having structural similarity to decorin, such as fibromodulin, for instance, would be expected to have similar activity and are thus also considered functional equivalents of decorin.
  • the present invention further provides pharmaceutical compositions containing an agent that inhibits the endothelin stimulatory activity of TGF ⁇ , such as those described above, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers include, for example, hyaluronic acid and aqueous solutions such as bicarbonate buffers, phosphate buffers. Ringer's solution and physiological saline supplemented with 5% dextrose or human serum albumin, if desired. Other pharmaceutical carriers known to those skilled in the art or later developed are also contemplated.
  • the pharmaceutical compositions can also include other reagents that are useful for the prevention or treatment of the various disorders. Those skilled in the art can readily identify such reagents. In treating specific vascular disorders, various reagents, such as angiotensic converting enzyme inhibitors, heparin, aspirin, ⁇ -adrenic receptor antagonists or nitrates, can be included in the pharmaceutical compositions.
  • compositions can be used in the methods of the present invention.
  • Such compositions or other formulations containing the TGF ⁇ antagonists (agents) can be administered to a patient in a number of ways, including, for example, intravenously or orally if using stabilized analog.
  • Bovine aortic endothelial cells were prepared, cloned, and maintained by methods previously described in Gospodaraowicz et al., J. Cell Phvsiol. 122:323-332 (1985), and in Gospodaraowicz et al.. Endocrinology 118:82-90 (1986), both incorporated herein by reference. Cells were grown to confluence in 24 well dishes and 1 ml HEPES (25 mM) buffered Delbeco's Modified Eagles Media (DMEM) supplemented with 10% calf serum. Prior to addition of test substances, cells were washed twice with DMEM and the culture media was replaced with DMEM-containing 0.1% bovine serum albumin.
  • HEPES 25 mM
  • DMEM Delbeco's Modified Eagles Media
  • FIGS. 1 and 2 Data are presented as mean + SEM. Group differences were determined using one-way analysis of variance followed by the tests of Dunnett and Duncan. As shown in the figures, decorin produced a dose-dependent inhibition of TGF ⁇ -induced endothelin release, with 6 ⁇ g/ml of decorin resulting in a near complete inhibition of TGF ⁇ (0.1 ng)-induced endothelin release.
  • ET concentrations in 50 ⁇ l samples of tissue culture fluid were measured using a specific radioimmunoassay described in Hexum et al., Biochem. Biophys. Res. Commun. 167:294-300 (1990), incorporated herein by reference.
  • I 125 -endothelin was prepared using the conventional chloramine-T method and subsequently purified on Bond Elute C 18 columns (Analytichem International, Harbor City, CA) as described in Example IV.
  • Radioimmunoassays were performed using a buffer containing 0.05 M sodium phosphate, 0.15 M sodium chloride, 0.25 M sodium EDTA, 0.25% BSA, and 0.1% sodium azide at pH 7.4.
  • Serum containing endothelin was purified on Bond Elut C 18 columns containing 500 mg sorbent. Columns were wetted using 50% 2-propanol/50% TEAF (11.5 ml 88% formic acid/liter glass-distilled water, pH adjusted to 3.0 with triethylamine) . Samples were mixed with an equal amount of TEAF and applied to the column. No greater than 1 ml serum was applied to each column, columns were washed with 3 ml TEAF, and endothelin-releasing activity was eluted with 1 ml 50% 2-propanol/50% TEAF to the column. After collection, samples were lyophilized and reconstituted in 100 ⁇ l DMEM.

Abstract

L'invention se rapporte à des procédés servant à la prévention ou au traitement d'une maladie caractérisée par une libération délétère d'endothéline au moyen de l'administration d'une quantité efficace d'un agent inhibant l'activité de stimulation de l'endothéline de TGFβ. L'agent inhibiteur peut être un anticorps anti-TGFβ, de la décorine ou un équivalent fonctionnel de la décorine. Les procédés décrits par l'invention sont particulièrement efficaces dans le traitement ou la prévention de maladies cardiovasculaires. Des compositions pharmaceutiques contenant un agent inhibiteur et un véhicule pharmaceutiquement acceptable sont également décrites.
PCT/US1993/003795 1992-04-23 1993-04-21 PROCEDES DE TRAITEMENT DE MALADIES VASCULAIRES AU MOYEN DE L'INHIBITION DE L'ACTIVITE DE STIMULATION DE L'ENDOTHELINE DE TGF$g(b) WO1993021945A1 (fr)

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US87267692A 1992-04-23 1992-04-23
US07/872,676 1992-04-23

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995013827A1 (fr) * 1993-11-19 1995-05-26 The University Of Sydney Procede de prevention ou de reduction de la cataracte
WO1996025178A1 (fr) * 1995-02-15 1996-08-22 The University Of Utah THERAPIE GENIQUE ANTI-FACTEUR DE CROISSANCE TRANSFORMANT $g(b)
WO1997013844A1 (fr) * 1995-10-06 1997-04-17 Cambridge Antibody Technology Limited Elements de fixation specifiques destines au facteur beta humain de croissance transformant, materiaux et procedes associes
WO1998024482A2 (fr) * 1996-12-04 1998-06-11 Schering Aktiengesellschaft Utilisation de conjugues d'endothelines en therapie, nouveaux conjugues d'endothelines, agents les contenant, et leurs procedes de production
US7151169B2 (en) 1999-04-30 2006-12-19 Cambridge Antibody Technology Limited Specific binding members for TGFβ1
US7368111B2 (en) 1995-10-06 2008-05-06 Cambridge Antibody Technology Limited Human antibodies specific for TGFβ2
EP2832362A1 (fr) * 2013-08-01 2015-02-04 Fraunhofer Gesellschaft zur Förderung der angewandten Forschung e.V. Protéine glycosylée d'une matrice extracellulaire destinée à être utilisée dans un procédé de traitement d'un c'ur ischémique d'un sujet humain ou animal qui en a besoin

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991010727A1 (fr) * 1990-01-22 1991-07-25 La Jolla Cancer Research Foundation Inhibiteurs de facteurs regulateurs cellulaires

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991010727A1 (fr) * 1990-01-22 1991-07-25 La Jolla Cancer Research Foundation Inhibiteurs de facteurs regulateurs cellulaires

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ENDOCRINOLOGY vol. 129, no. 5, November 1991, BALTIMORE, US pages 2355 - 2360 M.R. BROWN ET AL. 'TRANSFORMING GROWTH FACTOR-BETA: ROLE IN MEDIATING SERUM-INDUCED ENDOTHELIN PRODUCTION BY VASCULAR ENDOTHELIAL CELLS.' cited in the application *
NATURE. vol. 344, no. 6262, 8 March 1990, LONDON GB page 114 T. WATANABE ET AL. 'ENDOTHELIN IN MYOCARDIAL INFARCTION.' *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995013827A1 (fr) * 1993-11-19 1995-05-26 The University Of Sydney Procede de prevention ou de reduction de la cataracte
WO1996025178A1 (fr) * 1995-02-15 1996-08-22 The University Of Utah THERAPIE GENIQUE ANTI-FACTEUR DE CROISSANCE TRANSFORMANT $g(b)
US5824655A (en) * 1995-02-15 1998-10-20 The University Of Utah Anti-transforming growth factor-β gene therapy
WO1997013844A1 (fr) * 1995-10-06 1997-04-17 Cambridge Antibody Technology Limited Elements de fixation specifiques destines au facteur beta humain de croissance transformant, materiaux et procedes associes
AU702049B2 (en) * 1995-10-06 1999-02-11 Cambridge Antibody Technology Limited Specific binding members for human transforming growth factor beta; materials and methods
US7368111B2 (en) 1995-10-06 2008-05-06 Cambridge Antibody Technology Limited Human antibodies specific for TGFβ2
WO1998024482A2 (fr) * 1996-12-04 1998-06-11 Schering Aktiengesellschaft Utilisation de conjugues d'endothelines en therapie, nouveaux conjugues d'endothelines, agents les contenant, et leurs procedes de production
WO1998024482A3 (fr) * 1996-12-04 1999-04-01 Schering Ag Utilisation de conjugues d'endothelines en therapie, nouveaux conjugues d'endothelines, agents les contenant, et leurs procedes de production
US7151169B2 (en) 1999-04-30 2006-12-19 Cambridge Antibody Technology Limited Specific binding members for TGFβ1
EP2832362A1 (fr) * 2013-08-01 2015-02-04 Fraunhofer Gesellschaft zur Förderung der angewandten Forschung e.V. Protéine glycosylée d'une matrice extracellulaire destinée à être utilisée dans un procédé de traitement d'un c'ur ischémique d'un sujet humain ou animal qui en a besoin

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