EP1038178A1 - Methode diagnostique de detection de metalloprotease matricielle-9 et utilisation de ladite methode - Google Patents

Methode diagnostique de detection de metalloprotease matricielle-9 et utilisation de ladite methode

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Publication number
EP1038178A1
EP1038178A1 EP99953097A EP99953097A EP1038178A1 EP 1038178 A1 EP1038178 A1 EP 1038178A1 EP 99953097 A EP99953097 A EP 99953097A EP 99953097 A EP99953097 A EP 99953097A EP 1038178 A1 EP1038178 A1 EP 1038178A1
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EP
European Patent Office
Prior art keywords
mmp
antibody
solid support
pro
biological sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99953097A
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German (de)
English (en)
Inventor
Alison Joyce
Lisa F. Winchell
Laura Demeo
Gail Provuncher
Thomas E. Miller
Craig M. Sorensen
David E. Hill
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EMD Chemicals Inc
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Calbiochem Novabiochem Corp
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Publication date
Application filed by Calbiochem Novabiochem Corp filed Critical Calbiochem Novabiochem Corp
Publication of EP1038178A1 publication Critical patent/EP1038178A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • the present invention relates to assay methods for quantitation of metalloproteinase 9 (MMP-9) in biological samples using immunocapture techniques.
  • the assay is designed to quantify both free MMP-9 and MMP-9 complexed with the tissue inhibitor of metalloproteinase 9, TIMP-1.
  • this invention relates to methods and techniques for specifically selecting and isolating an MMP-9 from a biological sample, followed by the measurement of the amount of MMP-9, by using an antibody.
  • the assay method is useful in the diagnosis and prognosis of diseases and disorders relating to MMP-9, including cancer.
  • ECM extracellular matrix
  • the MMPs are members of a multigene family of metal-dependent enzymes. These proteases have been classified into four broad categories originally based on substrate specificity. These specific enzymes are the collagenases (MMP-1/EC3.4.24.7; MMP- 8/EC3.4.24.34;MMP-13), the gelatinases A(MMP-2/EC3.4.24.24) and B(MMP- 9/EC3.4.24.35), the stromelysins (MMP-3/EC3.4.24.17:MMP-10/EC3.4.24.22; MMP- 11/EC3.4.24.7) including a metalloelastase (MMP-12) and the membrane MMPs (MMP- 14).
  • MMP-12 metalloelastase
  • MMP- 14 membrane MMPs
  • MMPs are a key component of a system of "balanced proteolysis" wherein a finely tuned equilibrium exists between the amount of active enzyme and its proteinase inhibitor(s).
  • TIMPS tissue Inhibitor of MetalloProteinase
  • Matrix metalloproteinase-9 (MMP-9; 92-kDa gelatinase B) is an important member of the MMP family involved in tumor invasion and metastasis. Woodhouse, et al. Cancer. 80 (Suppl. 8):1529-1537, 1997; Chambers, et al. J. Natl. Cancer Inst. 89:1260-1270, 1997; Coussens, et al. Chem. Biol. 3:895-904, 1996; Yu, et al. Drugs Aging. 11 :229- 244, 1997; Kohn, et al. Cancer Res. 55:1856-1862, 1995.
  • MMP-9 is a collagenase with specificity for type IV collagen, which makes up the backbone of the basement membrane.
  • the proteolytic degradation of the ECM is an important aspect of many physiological and pathological conditions associated with alterations in connective tissue proteins such as embryo implantation, morphogenesis, wound healing, ovulation, cell migration, tissue involution, angiogenesis, and tumor invasion. DeClerk, et al. Adv. Exp. Med. Biol. 425:89-97, 1997.
  • MMP-9 is secreted from stimulated macrophages, neutrophils and transformed cells in a latent form.
  • TIMPs tissue inhibitors of metalloproteinases
  • the MMP-9 protein has four structural domains: an amino-terminal propeptide, a catalytic domain, a fibronectin type-II like domain within the catalytic domain and a hemopexin-like domain at the carboxyl-terminal.
  • Massova et al. FASEB J. 12:1075- 1095, 1998. Cleavage of the propeptide results in zymogen activation.
  • the catalytic domain contains two zinc ions and a calcium ion.
  • MMP-9 incorporates three repeats homologous to the type-II module of fibronectin into the catalytic domain. This region is also known as the gelatin binding domain and is involved in binding to denatured collagen or gelatin. Murphy, et al. J. Biol.
  • the hemopexin-like domain is highly conserved among MMPs and shows sequence similarity to the plasma protein, hemopexin.
  • the hemopexin like domain has been shown to play a functional role in substrate binding and/or in interactions with the tissue inhibitors of metalloproteinases (TIMPs).
  • TIMP-1 binds exclusively to the zymogen form of MMP-9 (Kd ⁇ 35 nM)
  • TIMP-2 binds to the zymogen form of MMP-2 (Kd ⁇ 5 nM). Murphy, et al. Matrix Biol. 15:511-518, 1997.
  • MMPs play an important role in normal and pathological process, including embryogenesis, wound healing, inflammation, arthritis and cancer, it would be desirable to have a highly specific and accurate assay to measure MMP levels in patient samples.
  • the present invention employs an improved immunoassay that detects all forms of MMP-9 (both the form complexed with TIMP and the "free" uncomplexed form) in biological samples including human serum and plasma.
  • the assay is preferably a capture assay using two antibodies to MMP-9 that do not compete with each other (i.e., bind to distinct epitopes). The epitopes that are bound to must be exposed in both the complexed MMP-9 as well as the free MMP-9.
  • a detector MMP-9 antibody and a capture anti- MMP-9 antibody.
  • serum pro-matrix metalloproteinase- 9 precursor of matrix metalloproteinase 9, pro-MMP-9, progelatinase B
  • pro-MMP- 9/TIMP1 complex levels [pro-MMP-9 noncovalently bound to tissue inhibitor of metalloproteinases (TIMP-1)] are elevated in cancer patients.
  • a monoclonal anti-MMP-9 antibody such as clone 6-6B (ORP product MMP9 (Ab-1), Cat #IMO9L) and a polyclonal antibody to MMP-9 which is available from BioDesign (Cat.# K90163C).
  • clone 6-6B ORP product MMP9 (Ab-1), Cat #IMO9L)
  • polyclonal antibody to MMP-9 which is available from BioDesign (Cat.# K90163C).
  • the improved immunoassay of the present invention has excellent clinical utility. For example, ninety percent of cancer sera read above normal samples and most of the pro- MMP-9 in cancer sera is found to be complexed with TIMP-1.
  • the present invention provides a method of determining the presence of an MMP-9 associated disorder, e.g., cancer, comprising the step of assaying a biological sample obtained from a patient for the presence of free MMP and MMP/TIMP complexes, such as MMP-9 and pro-MMP-9/TIMP-l complexes.
  • MMP-9 above normal limits correlates with the presence of cancer.
  • the measurement of the level of MMP-9 in a biological sample is useful in the diagnosis, prognosis, and treatment of cancer as well as monitoring of a patients response to therapy and staging of disease.
  • the present inventors have detected pro-MMP-9 protein complexed with TIMP-1 and uncomplexed MMP-9 in human sera and plasma samples and cell culture supernatants. Specificity was demonstrated by immunoaffmity extraction (inhibition of assay signal) of pro-MMP-9 positive samples by a specific pro-MMP-9 antibody and a specific TIMP-1 antibody. For instance, an MMP-9 antibody, which is not a component of the ELISA, extracted the MMP-9 which leads to loss of signal in the assay (almost all the signal was lost), while the control antibody (e.g., a non MMP- 9) did not affect the signal of the MMP-9 positive samples (see Figure 3).
  • TIMP-1 antibody extracted pro-MMP-9 activity detected by this immunoassay, but very little of the pro-MMP-9 activity detected by other traditional immunoassays which shows that the traditional assay detects mainly uncomplexed MMP-9 protein and therefore fails to accurately measure MMP-9 levels in patient samples. Addition of TIMP-1 and TIMP-2 recombinant proteins to both positive and negative samples does not change the level of MMP-9 detected.
  • the present invention further provides a general method for the detection and quantitation of a MMP-9 in a biological fluid from a human.
  • the method comprising the steps of:
  • one antibody is polyclonal and the other is monoclonal.
  • the amount of MMP-9 in the biological sample is determined from the amount of detectably labeled antibody bound to the solid support.
  • the detectably labeled antibodies can be labeled with a detectable substance such as, for example, biotin, radioactive agents, chromophoric agents, enzymatic agents, chemiluminescent, fluorometric and haptenic.
  • the method of the invention for measuring MMP levels in biological samples contemplates the use of a capture antibody, which can be a monoclonal, polyclonal recombinant or chimeric antibody, and a detectably labeled antibody, which can also be a monoclonal, polyclonal recombinant or chimeric antibody.
  • the present invention further provides a kit useful for the detection of MMP-9 in a biological sample.
  • This kit comprises antibodies which specifically bind MMP-9 to capture the MMP-9 present in the sample and a detectably labeled antibody which specifically binds MMP-9, such as pro-MMP-9, to quantify the amount of MMP-9.
  • Figure 1 shows a standard curve: The mean signal of of each standard run in replicates of four in eight assays using two different lots of plates and two lots of detector antibody.
  • Figure 2 shows the sensitivity of one embodiment of the assay of the present invention:
  • the lower limit of detection (LLD) commonly used to define sensitivity, was measured by assaying four replicates of zero eight times using two different lots of plates and two different lots of detector antibody. The grand mean signal and pooled standard deviation of zero was calculated. The grand mean of each standard (run in replicates of four in the eight assays) was used for the standard curve ( Figure 1), and the response, mean signal of zero plus two standard deviations, read in dose from the standard curve is the LLD; that is, the smallest dose that is not zero with 95% confidence.
  • LLD lower limit of detection
  • Figure 3 shows the specificity of an assay of the present invention: Levels of MMP-9 detected by the ELISA after immunoaffinity extraction of MMP-9 positive samples (PMA treated HT1080 and HL-60 cells) with a MMP-9 antibody that is not used in the ELISA, a TIMP-1 antibody, a TIMP-2 antibody and a TIMP-3 antibody.
  • FIGS 4A-4D show levels of MMP-9 detected by the MMP-9 ELISA of the present invention after 2-aminophenylmercuric acetate (APMA) treatment.
  • APMA promotes the autocatalytic cleavage of the N-terminal prosequence of the latent 92-kDa enzyme to yield the active form of the enzyme.
  • Analysis of the samples by zymography showed both cleavage of the proenzyme by APMA and a good correlation with the levels of the proenzyme determined with the MMP-9 ELISA.
  • MMP-9 induction was used to generate positive samples such as treatment with 50 ng/ml amphiregulin (AR), 50 ng/ml epidermal growth factor (EGF), 1 ng/ml transforming growth factor beta (TGFb) and 25 ng/ml phorbol 12- myristate 13-acetate (PMA).
  • AR amphiregulin
  • EGF epidermal growth factor
  • TGFb transforming growth factor beta
  • PMA 25 ng/ml phorbol 12- myristate 13-acetate
  • Normal human sera (NHS) containing moderate levels of MMP-9 were as treated with APMA and analyzed.
  • FIG. 5 shows phorbol ester induced up-regulation of MMP-9 protein in HL-60 cells. It has been shown that the induction of MMP-9 gene expression is associated with macrophage differentiation. Xie, et al. J. Biol. Chem. 273:11576-11582, 1998. Phorbol 12-myristate 13-acetate (PMA) at all concentrations studied induced the synthesis of MMP-9 protein. This up regulation closely parallels the timing of PMA induced cell adhesion and spreading, a hallmark of macrophage differentiation.
  • PMA Phorbol 12-myristate 13-acetate
  • Figure 6 shows phorbol ester induced up-regulation of MMP-9 in HT1080.
  • PMA has been shown to increase the expression of MMP-9 by HT1080 cells. Morodomi, et al. Biochem. J. 285: 603-611, 1992; Lim, et al. J. Cell. Physiol. 167:333-340, 1996.
  • the MMP-9 ELISA of the present invention detects this increase in synthesis in a time dependent matter.
  • Figures 7A and 7B show detection of MMP-9 in Human Sera and Plasma: Levels of MMP-9 detected by the assay of the present invention are significantly elevated in cancer sera samples compared to normal sera and plasma samples (left figure is the data on a logarithmic scale and the right is a linear scale). The following samples were assayed: normal human sera (NHS), normal human plasma (NHP), sera from cervical cancer (CV), breast cancer (BR) and prostate cancer (PT) patients.
  • NHS normal human sera
  • NHS normal human plasma
  • CV cervical cancer
  • BR breast cancer
  • PT prostate cancer
  • Figure 8 shows the clinical utility of the MMP-9 Immunoassay of the present invention.
  • the assay has a 90% percent clinical sensitivity (ability to recognize affected individuals) at a 95 % clinical specificity (ability to recognize unaffected individuals).
  • Figure 9 shows parallelism using the assay of the present invention: The study tested dilution-recovery often positive samples. The dilutions were run in the MMP-9 ELISA and the "found" doses were plotted against the "expected” doses to determine linearity of dilution. The slope is not significantly different from one and the intercept is not significantly different from zero.
  • Ca Cancer
  • NHS Normal Human Sera
  • NHP Normal Human Plasma
  • PMA Phorbol 12-myristate 13-acetate
  • EGF Epidermal Growth Factor
  • AR Amphiregulin
  • Both EGF and AR
  • A 2 x 10 6 cells/ml
  • B 1.0 x 10 6 cells/ml.
  • Figure 10 shows the precision of the assay of the present invention:
  • the pooled coefficients of variation (according to the formula of Henry, R.J., Cannon, CD., and Winkleman, J.W., Clinical Chemistry, 1974 Harper and Row, New York, N.Y.) and between assay coefficients of variation are plotted against MMP-9 concentrations.
  • the pooled data were collected from samples run eight times using two different lots of plates and two different lots of detector antibody in replicates of four on two separate occasions.
  • the present invention provides an immunoassay, e.g., a "sandwich” enzyme immunoassay employing antibodies specific for both free and complexed MMP-9.
  • the epitopes need to be exposed in both the free and complexed form.
  • the detector antibody binds the pro-MMP-9 form.
  • the antibody, specific for the human MMP-9 protein is immobilized onto surface of a solid phase support, e.g., an immunoassay plate, (other solid supports such as beads, tubes or magnetic particles can be used as well).
  • a labeled e.g., biotinylated, detector antibody that is specific for pro-MMP-9 is added.
  • the detector antibody can be simultaneously pipetted into the wells and allowed to incubate for a period of time (e.g., two hours), during which any MMP-9 present binds to both the capture and detecting antibodies. Unbound material is washed away and, if the detector antibody is biotinylated, horseradish peroxidase-conjugated streptavidin is added, which binds to the detector antibody.
  • the horseradish peroxidase catalyzes the conversion of a chromogenic substrate (i.e., tetramethylbenzidine) from a colorless solution to a colored solution, the intensity of which is proportional to the amount of pro- MMP-9 protein in the sample.
  • the colored reaction product is quantified using a spectrophotometer.
  • the streptavidin-biotin- horseradish peroxidase labeled immunologically responsive substance such as radioactive agents, chemiluminescence agents, bioluminescence agents, fluorescence agents, or other chromophoric agents.
  • the detector antibody can be directly labeled or labeled indirectly with other molecules in place of biotin.
  • Quantitation is achieved by the construction of a standard curve using known concentrations of, e.g., the pro-MMP-9. By comparing the signal obtain from a sample containing an unknown amount of a pro-MMP-9 with that obtained from a known concentration of a pro-MMP-9, the concentration of the MMP-9 in the sample can be determined.
  • an ELISA uses two antibodies that bind to nonoverlapping epitopes on the same antigen and may utilize either two antibodies that recognize discrete sites on an antigen or one batch of affinity-purified polyclonal antibodies.
  • the assay of the present invention can employ a capture antibody specific for both free and complexed MMP-9 and a non-antibody system for detecting the captured protein.
  • a capture antibody specific for both free and complexed MMP-9 and a non-antibody system for detecting the captured protein.
  • Antibodies which specifically bind MMP-9 can include human or animal (e.g., rabbit, mouse, rat, etc.) polyclonal or monoclonal or recombinant or chimeric antibodies. Methods used to prepare and purify polyclonal, monoclonal, recombinant and chimeric antibodies are known in the art.
  • anti-MMP-9 antibodies are also available commercially.
  • anti-MMP-9 antibodies include:
  • the detectably labeled antibody can be labeled using any of a variety of labels and methods of labeling known in the art.
  • types of labels encompassed by the present invention include, but are not limited to, radioisotopic labels (e.g., 125 1, 131 1, 35 S, 14 C, etc.), non-radioactive isotopic labels 55 Mn, 56 Fe, etc.), fluorescent labels (e.g., a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaldehyde label, a fluorescamine label, etc.), chemiluminescent labels, enzyme labels (e.g., alkaline phosphatase, horse radish peroxidase, etc.), protein labels, etc.
  • radioisotopic labels e.g., 125 1, 131 1, 35 S, 14 C, etc.
  • Biological samples include body fluids, for example, plasma, serum, saliva, urine, lung lavage fluid, cyst fluid, etc., and tissue extracts wherein the tissue pieces are removed and separated from the liquid extract.
  • the pro-MMP-9 present in a biological sample for example, plasma, serum, saliva, urine, lung lavage fluid, cyst fluid, tissue extract, etc.
  • a biological sample for example, plasma, serum, saliva, urine, lung lavage fluid, cyst fluid, tissue extract, etc.
  • the immunocapture assay can be performed in solution phase or on solid support as is well known in the art.
  • the anti-pro-MMP-9 antibodies are affixed onto a solid support by any stable interaction known to the art such as hydrophobic, electrostatic or covalent interaction.
  • solid supports are microtiter plates, magnetic particles, beads, sheets, membranes, chromatography resins, e.g., Sepharose, etc.
  • multiwell microtiter plates or nitrocellulose membranes are used, so that when the pro-MMP-9 is captured by the antibodies, the resultant immunological complex is immobilized and easily isolated, e.g., by rinsing away non-binding material remaining in the biological sample.
  • the method of this invention has utility in screening biological samples to detect malignancy through the measurement of high levels of MMP9 and pro-MMP-9, in the sample material.
  • This invention also contemplates the preparation of a kit useful for the detection of pro-MMP-9 in a biological sample.
  • a kit useful for the detection of pro-MMP-9 in a biological sample.
  • Such a kit comprises anti-pro-MMP-9 antibodies, adsorbed onto a solid support, preferably a microtiter plate having from approximately four to approximately ninety-six wells and a second anti-pro-MMP-9 antibody conjugated to a label; and, where necessary, a means for measuring said label.
  • the MMP-9 ELISA disclosed in this example is a "sandwich" enzyme immunoassay employing a mouse monoclonal antibody and a sheep polyclonal antibody.
  • An antibody, specific for the human MMP-9 protein, has been immobilized onto the surface of microtiter wells using standard techniques.
  • the sample to be assayed and biotinylated detector monoclonal antibody are pipetted into the wells and allowed to incubate for two hours, during which time any MMP-9 present binds to the capture and detecting antibodies. Unbound material is washed away and horseradish peroxidase-conjugated streptavidin is added, which binds to the detector antibody.
  • the detector antibody is anti-MMP-9 mouse monoclonal antibody clone 6-6B (ORP product MMP9 (Ab-1), Cat # IM09L).
  • the capture antibody is an anti-MMP-9 polyclonal raised in sheep and obtained from BioDesign of Kennebunk, Maine (Cat # K90163C).
  • the horseradish peroxidase catalyzes the conversion of the chromogenic substrate tetra-methylbenzidine (TMB) from a colorless solution to a blue solution (or yellow after the addition of stopping reagent), the intensity of which is proportional to the amount of MMP-9 protein in the sample.
  • TMB chromogenic substrate tetra-methylbenzidine
  • the colored reaction product is quantified using a spectrophotometer.
  • Quantitation is achieved by the construction of a standard curve using known concentrations of MMP-9. By comparing the absorbance obtained from a sample containing an unknown amount of MMP-9 with that obtained from the standards, the concentration of MMP-9 in the sample can be determined.
  • This example discloses the use of one embodiment of the assay of the present invention.
  • Standards should be assayed in duplicate. A standard curve must be performed on the same plate and at the same time as the samples.
  • the MMP-9 ELISA provides sufficient reagents to ran two sets of standard curves, and 41 samples (if assayed in duplicate all at once using one standard curve), or 34 samples (if assayed on two separate occasions using two standard curves).
  • MMP-9 STANDARD two vials containing lyophilized MMP-9 protein calibrated to recombinant MMP-9 protein. Reconstituted standards should be discarded after one use.
  • DETECTOR ANTIBODY Biotinylated monoclonal anti-human MMP-9 antibody.
  • PLATE SEALERS To cover plates during incubations. Sample Preparation For suspension cells: Pellet by centrifugation (1000 x g for 10 minutes, 4 °C) and remove supernatant for testing. Samples may be stored at -20 °C.
  • tissue culture media remove tissue culture media, centrifuge tissue culture media (1000 x g for 10 minutes), and remove supernatant for testing. Samples may be store at -20 °C.
  • Samples found to contain greater than 20 ng/ml MMP-9 must be diluted with Sample Diluent (provided), so that the MMP-9 concentration falls within the range spanned by the standard curve, and assayed again.
  • the MMP-9 ELISA is provided with removable strips of wells so the assay can be carried out on two separate occasions. Since conditions may vary, a standard curve MUST be determined each time the assay is performed. Standards should be assayed in duplicate. Disposable pipette tips and reagent troughs should be used for all transfers to avoid cross-contamination of reagents or samples.
  • FLOOD ENTIRE PLATE WITH dH 2 O Remove contents of wells by inverting over sink and tapping on paper towels.
  • the MMP-9 concentration must be multiplied by the dilution factor (ie., if the sample was diluted five-fold, then the MMP-9 value obtained from the standard curve must be multiplied by five).

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Abstract

La présente invention met en oeuvre une analyse immunologique permettant de détecter toutes les formes de MMP-9 ( à la fois la forme complexée avec TIMP et la forme 'libre' non complexée) dans des prélèvements biologiques du type sérum et plasma. La mise en oeuvre de cette méthode immunologique permet un niveau de détection et de quantification bien supérieur aux analyses précédentes. Cette méthode est de préférence un dosage en captivité mettant en oeuvre deux anticorps dirigés contre les MMP-9 qui ne se font pas concurrence (c'est à dire qu'ils se lient à des épitopes distincts). Les épitopes engagés dans une liaison doivent être exposés à la fois dans la MMP-9 complexée et dans la MMP-9 libre.
EP99953097A 1998-10-08 1999-10-07 Methode diagnostique de detection de metalloprotease matricielle-9 et utilisation de ladite methode Withdrawn EP1038178A1 (fr)

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US16852698A 1998-10-08 1998-10-08
US168526 1998-10-08
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FI990888A0 (fi) * 1999-04-20 1999-04-20 Medix Biochemica Ab Oy Menetelmä ja testikittejä respiratorisen alueen tulehduksen läsnäolon ja vaikeusasteen arvioimiseksi
US7071164B2 (en) 2001-08-16 2006-07-04 Kimberly-Clark Worldwide, Inc. Anti-cancer and wound healing compounds
US6906036B2 (en) 2001-08-16 2005-06-14 Kimberly-Clark Worldwide, Inc. Anti-aging and wound healing compounds
US7186693B2 (en) 2001-08-16 2007-03-06 Kimberly - Clark Worldwide, Inc. Metalloproteinase inhibitors for wound healing
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US7189700B2 (en) 2003-06-20 2007-03-13 Kimberly-Clark Worldwide, Inc. Anti-chrondrosarcoma compounds
DE602004026488D1 (de) 2004-07-23 2010-05-20 Ct De Rech Public De La Sante Überwachung von herzversagen
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