WO1999059562A9 - Methode de traitement ou de prevention d'une croissance cellulaire cardiaque anormale par inhibition du chemin de la 12-lipoxygenase - Google Patents

Methode de traitement ou de prevention d'une croissance cellulaire cardiaque anormale par inhibition du chemin de la 12-lipoxygenase

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WO1999059562A9
WO1999059562A9 PCT/US1999/011115 US9911115W WO9959562A9 WO 1999059562 A9 WO1999059562 A9 WO 1999059562A9 US 9911115 W US9911115 W US 9911115W WO 9959562 A9 WO9959562 A9 WO 9959562A9
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lipoxygenase
hete
compound
cardiac
group
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PCT/US1999/011115
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English (en)
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WO1999059562A3 (fr
WO1999059562A2 (fr
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Jerry L Nadler
Yeshao Wen
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Hope City
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Publication of WO1999059562A2 publication Critical patent/WO1999059562A2/fr
Publication of WO1999059562A3 publication Critical patent/WO1999059562A3/fr
Publication of WO1999059562A9 publication Critical patent/WO1999059562A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/04—Nitro compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4152—1,2-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. antipyrine, phenylbutazone, sulfinpyrazone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4166—1,3-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. phenytoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole

Definitions

  • This invention relates to a method for preventing or treating abnormal cardiac cell growth.
  • the treatment or prevention is achieved by blockade of the 12-lipoxygenase pathway in the affected or potentially affected cells.
  • Cardiac hypertrophy is an important indicator and often on early clinical sign of significant pathology in the heart. It is an adaptational state to prior hypertension and is a major risk factor associated with heart failure.
  • the cardiac muscle has a large capacity for protein and nucleic acid synthesis since a high degree of turnover of these structural building blocks is necessary for maintenance of the cardiac muscle tissue.
  • the normal process of continuous catabolism of heart proteins and their replacement allows the heart to more rapidly adjust to changes in the demands on the heart.
  • excessive compensation by the heart in the form of surplus protein and nucleic acid synthesis can lead to cardiac enlargement and compromised contractile function of the heart. Cardiac enlargement may be genetically influenced or may be caused by overworking the heart secondary to disease, pharmacological agents or exercise, and eventually may lead to cardiac failure.
  • the first signs of cardiac hypertrophy usually are increases in protein and nucleic acid synthesis, as well as other changes in heart metabolism. Because the myocytes of the adult heart rarely undergo mitosis, enlargement of the heart generally is manifested by increases in cardiac muscle cell size rather than cell number. Some of the major symptoms of abnormal cardiac cell growth which can be detected easily in the laboratory include protein content increases, increase in cell size, and accumulation of fibrillar collagen in the extracellular space. Although cardiac muscle cells do not divide, often the connective tissue cells in the heart do increase in number in cardiac enlargement. This increase, and the accumulation of collagen and fibrillar fibronectin in the extracellular matrix lead to myocardial stiffness and ventricular dysfunction.
  • Lipoxygenases are enzymes which produce active products, including 12 (S) -hydroxyeicosatetraenoic acid (12(S)-HETE) from arachidonic acid through stereospecific oxygenation. .
  • 12 (S) -hydroxyeicosatetraenoic acid (12(S)-HETE) from arachidonic acid through stereospecific oxygenation.
  • 12-lipoxygenase (12-LO) the enzyme which catalyzes the oxygenation of arachidonic acid to 12(S)-HETE and (S) -12-hydroperoxyeicosatetraenoic acid (12 (S) -HPETE)
  • 12-lipoxygenase (12-LO) the enzyme which catalyzes the oxygenation of arachidonic acid to 12(S)-HETE and (S) -12-hydroperoxyeicosatetraenoic acid (12 (S) -HPETE
  • 12(S)-HETE has direct mitogenic effects in a Chinese hamster ovary (CHO) fibroblast cell line overexpressing the rat vascular type la angiotensin II (AT la ) receptor.
  • AT la rat vascular type la angiotensin II
  • 12(S)-HETE effects mimicked the angiotensin II (All) mitogenic effects in these cells and led to a sustained increase in DNA synthesis as well as cell number.
  • JNK c-jun amino terminal kinase
  • the present invention provides a method of treating cardiac fibroblast cell growth and hypertrophy in a cell having an excess of
  • 12-lipoxygenase activity or 12-lipoxygenase products comprising contacting said cell with a compound selected from the group consisting of a 12(S)-HETE receptor blocker, a 12-lipoxygenase antisense nucleotide, a 12-lipoxygenase ribozyme and a
  • Another embodiment provides a method of preventing cardiac fibroblast cell growth and hypertrophy in a cell having an excess of 12-lipoxygenase activity or 12-lipoxygenase products comprising contacting said cell with a compound selected from the group consisting of a 12(S)-HETE receptor blocker, a 12-lipoxygenase antisense oligonucleotide, a 12-lipoxygenase ribozyme and a 12-lipoxygenase inhibitor in an amount effective to reduce or eliminate the cardiac fibroblast cell growth and hypertrophic effects of said excess of 12-lipoxygenase activity or 12-lipoxygenase products.
  • Yet another embodiment provides a method of reducing or eliminating increased protein content in cardiac fibroblasts due to an excess of 12-lipoxygenase activity or 12-lipoxygenase products, comprising contacting said fibroblasts with a compound selected from the group consisting of a 12(S)-HETE receptor blocker, a 12-lipoxygenase antisense oligonucleotide, a 12-lipoxygenase ribozyme and a 12-lipoxygenase inhibitor in an amount effective to reduce or eliminate the increased protein content resulting from said excess of 12-lipoxygenase activity or 12-lipoxygenase products .
  • a compound selected from the group consisting of a 12(S)-HETE receptor blocker, a 12-lipoxygenase antisense oligonucleotide, a 12-lipoxygenase ribozyme and a 12-lipoxygenase inhibitor in an amount effective to reduce or eliminate the increased protein content resulting from said excess of 12-lipoxygena
  • Figure 1 shows a Western blot analysis of cardiac fibroblasts overexpressing 12-LO cDNA.
  • Figure 2 shows levels of 12(S)-HETE released into the medium bathing 12-LO-transfected and mock-transfected cardiac fibroblasts.
  • Figure 3 provides data demonstrating 3 H-thymidine incorporation (DNA synthesis) and 3 H-leucine labeling (an indicator of protein synthesis) in
  • Figure 4 is a set of photomicrographs showing morphological changes in rat cardiac fibroblasts which have been transfected with 12-LO (Figure 4A) compared to mock-transfected rat cardiac fibroblasts ( Figure 4B) .
  • Figure 5 shows the forward scatter results of fluorescence-activated cell sorting comparisons of 12-LO-transfected and mock-transfected cells. In each case, 10 6 cells were sorted.
  • Figure 6 shows the effect of 12-lipoxygenase overexpression in cardiac fibroblasts on MAP kinases and PAK activities. Arrows on the left side of the Figure indicate the substrates used for measurement of the respective kinase activities indicated at the right.
  • Figure 7 illustrates the effect of SB202190, a specific p38 MAP kinase inhibitor, on the protein content in 12-LO-transfected and mock-transfected cardiac fibroblasts.
  • Figure 8 shows the effect of C 3 transferase pretreatment on 3 H-thymidine incorporation by 12-LO-transfected and mock-transfected cells.
  • Figure 9 shows Northern blot data indicating collagen Ic-j mRNA levels in 12-LO-transfected and mock-transfected cardiac fibroblasts.
  • Figure 10 provides data showing the level of soluble fibronectin released into the medium bathing 12-LO-transfected or mock-transfected cardiac fibroblasts and the increase of the fibrillar form of fibronectin resulting from 12-LO overexpression.
  • Figure 11 shows a significant reduction in cell growth induced by either All or 12(S)-HETE (O.l ⁇ M) by DuP654, a specific 12(S)-HETE receptor blocker.
  • Figure 12 shows the inhibitory effect on leucine incorporation of baicalein, a specific 12-LO enzyme inhibitor, in cardiac fibroblasts.
  • 12(S)-HETE has several biological effects linked to cellular growth in vascular smooth muscle, CHO AT la cells and cardiac fibroblasts (Natarajan et al.. Hypertension 23:1142-1147 (1994); Wen et al.. Am. J. Physiol. 211:C1212-C1220 (1996)), it is implicated in the etiology of several cardiovascular diseases, including cardiac hypertrophy.
  • Applicants have discovered that 12-LO participates in a previously unknown growth promoting pathway in the heart by discovering that overexpression of 12-LO causes cardiac fibroblast cell growth.
  • the present invention takes advantage of the 12-LO pathway effects which mediate cardiac hypertrophy to provide a new method of treating cardiac fibroblast cell growth and hypertrophy by blocking the expression, activity and/or products of 12-LO.
  • 12(S)-HETE receptor blockers include 13 (S) -hydroxyoctadecadienoic acid, 2- phenylmethyl-1-naphthol, 2-N-butyl-4-chloro-5- hydroxymethyl-1- [ (2 ' - (lH-tetrazol-5-yl) biphenyl-4- yl)methyl] imidazole, pertussis toxin, 12(S)-HETE analogs, antibodies to the 12(S)-HETE receptor and the like.
  • Inhibitors of the 12-LO enzyme include panaxynol, phenidone, pioglitazone, substituted (carboxyalkyl) benzyl ethers, cinnamyl-3, 4-dihydroxy- ⁇ - cyanocinnamate and the like. Compounds which serve as a structural analog for the enzyme may be useful. Some of these compounds have been described. Gorins et al . (J. Med. Chem. 39:4871-4878 (1996)). The harmful effects of 12-LO and it products also may be ameliorated by reducing or eliminating 12-LO expression using antisense or ribozyme methods.
  • Ribozy es which cleave the 12-LO mRNA, preventing its expression, are useful in the invention, and may be constructed according to known methods.
  • Antisense oligonucleotides which bind the 12-LO gene likewise are effective in preventing or reducing 12-LO expression and activity. These oligonucleotides may be constructed using methods known in the art.
  • 12-LO was overexpressed using the calcium phosphate method in fibroblast cells from neonatal rat heart (kindly provided by Dr. Ping H. Wang, University of California, Irvine) .
  • Mouse leukocyte type 12-lipoxygenase cDNA was stably transfected into cardiac fibroblast cells (ML12-L0 cells) .
  • Cardiac fibroblast cells were maintained in DME medium with 10% FBS containing 20 mM HEPES, pH 7.4, penicillin and streptomycin at 37°C in 5% C0 2 and 95% air.
  • cardiac fibroblasts were seeded at a density of 1 X 10 6 cells per 100 mm 2 dish.
  • pcDNAl/MLl2-LO vector and pPUR vector a plasmid conferring resistance to puromycin
  • the 12-LO overexpressing cell line is known as M4.7 cells.
  • Cells transfected with the empty vector pcDNA 1 (Invitrogen) without the 12-LO cDNA insert (P3 cells) were used as a negative, mock-transfected control.
  • Vectors were purified using an Endofree plasmid kit (Qiagen Co.). Forty-eight hours after transfection, the cells were split 1:15. Selection was then initiated with 2 ⁇ g/ml of puromycin to select cells expressing resistance to this marker. Individual resistant clones were isolated 2-3 weeks later and expanded into cell lines. Transfected cells were maintained in medium containing 10% FBS, and 2 ⁇ g/ml puromycin.
  • Immunoblots were used to analyze the expression of the 12-LO protein.
  • a polyclonal antibody against a peptide comprising amino acids 646-662 of porcine leukocyte 12-LO was raised in rabbits. The antibody showed excellent cross-reactivity with murine leukocyte 12-LO.
  • cells were lysed in PBS buffer (pH 7.4) containing 1% Triton X-100, 0.1% SDS and a standard protease inhibitor cocktail according to known methods . Lysates were sedimented and the supernatants were collected for assay. Protein concentration was determined by the Bradford method for this assay and all assays disclosed here.
  • the protein (20 ⁇ g) was resolved by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide gel) and subsequently transferred to a polyvinylidine difluoride membrane. After the membrane was incubated in blocking buffer (Tropix Inc., Bedford) overnight, 12-LO antibody was added at a 1:1000 dilution. After incubation, an alkaline phosphatase coupled goat anti-rabbit secondary antibody was added at a 1:10,000 dilution. The protein bands were visualized using a chemiluminescence substrate and the Western Light Chemiluminescent detection system (Tropix Inc., Bedford). Figure 1 shows the Western blot analysis of these transfected cells. The results indicate several positive clones.
  • the TCA-insoluble material was washed twice with 95% ethanol. Fixed cellular material was solubilized in 0.1 N NaOH at 24 °C for 2 hours. The sample was divided into 6 wells (3 wells for incorporated and protein content measurements; 3 wells for cell counting) . The 3 H-isotope incorporation was determined by liquid scintillation spectrometry . Cells were counted with a Coulter Counter. The data was normalized as cpm/10 6 cells or ⁇ g protein/10 6 cells and expressed as fold increase over mock-transfected controls. The data presented in Figure 2 show that 12-LO transfected cardiac fibroblasts release into the medium about 4 times the level of 12(S)-HETE than mock-transfected cells. 12(S)-HETE concentrations were measured by a specific radioimmunoassay with a sensitivity of 10 pg/ml and intraassay variation of 8%.
  • the cell size increase in 12-LO transfected cells was confirmed using FACS analysis.
  • the left panel of Figure 5 illustrates the forward scatter histogram of control and 12-LO transfected cells.
  • the right panel of Figure 5 shows both 12-LO overexpressing and mock-transfected cells redrawn using a computer program. The average size of the 12-LO overexpressing cells was shifted to the right compared to that of control cells.
  • the effect of 12-LO overexpression on MAP kinases and PAK was evaluated since these signaling enzymes have been linked to cell growth, cell apoptosis and focal adhesion activity.
  • ERK1/2, JNK-1 and p38 activity was measured with an immune complex kinase assay according to methods known in the art. Growth of the cells was arrested by incubation in depletion DME medium containing 0.2% BSA, 0.4% FCS, 20 mM HEPES, pH
  • lysate 1 mM Na 3 V0 4 and the protease inhibitors PMSF, leupeptin and aprotinin.
  • the lysate was sedimented at 14,000 xg at 4°C for 10 minutes.
  • the lysate protein 50 ⁇ g was immunoprecipitated with JNK, p38 or ERK antibodies in lysis buffer and the mixture incubated under rotation at 4°C overnight, followed by addition of the solution to 60 ⁇ l protein A Sepharose beads. After 1 hour incubation with protein A Sepharose at 4°C, the beads were washed 4 times with buffer and pelleted.
  • the pelleted beads were resuspended in 60 ⁇ l kinase buffer containing substrates as follows: 2 ⁇ g GST-c-Jun(aa 1-79) for the JNK assay, 2 ⁇ g ATF-2 for the p38 assay or 2 ⁇ g MBP for the ERK assay and 20 ⁇ M ATP containing 5 ⁇ Ci [ ⁇ - 32 P]ATP. After 30 minutes at 30°C, the reaction was stopped and samples were resolved on 12% SDS-polyacrylamide gels followed by autoradiography .
  • Figure 7 presents data showing the effect of SB202190, a specific p38 MAP kinase inhibitor, on the protein content of cardiac fibroblast cells. Comparing the data for the 12-LO transfected cells and mock-transfected controls shows that the overexpression of 12-LO results in increases in protein content of about 2 fold.
  • the p38 inhibitor had no significant effect on protein content in mock-transfected cells, implying that p38 MAP kinase activation may be important for 12-LO-induced protein content increases, but completely blocked the protein content increase induced by 12-LO overexpression.
  • the compound PD58059 an inhibitor of MEK, had no effect on leucine incorporation either in 12-LO-transfected or mock-transfected cells (data not shown) .
  • FIG. 9 illustrates the results. As shown in the top panel, the Northern Blot detected two collagen bands at 4.6 kb and 5.6 kb. This is consistent with the published data for this probe. The lower two panels show the densities of these two bands indicating that the overexpression of 12-LO increased mRNA content of these two bands about 4 fold over that in mock-transfected cells .
  • fibrillar fibronectin is more closely related to cardiac hypertrophy, measurements were taken not only for fibronectin released into medium (soluble fibronectin), but also 1% deoxycholate insoluble fibronectin (fibrillar fibronectin) .
  • the supernatants were assayed for released fibronectin. Washed cell layers were extracted with 1% deoxycholate. Deoxycholate extractions were performed in 0.02 M Tris buffer, pH 8.3, containing 2 mM phenylmethylsulfonilfluoride (PMSF), 2 mM ethylenediaminetetraacetic acid (EDTA) , 2 mM ethylmaleimide and 2 mM iodoacetic acid. For sequential extraction, cell layers were scraped into 1% deoxycholate and sedimented at 17,000 rpm for 20 minutes . Deoxycholate-insoluble material was solubilized at 70°C in 4% SDS.
  • PMSF phenylmethylsulfonilfluoride
  • EDTA ethylenediaminetetraacetic acid
  • iodoacetic acid 2 mM iodoacetic acid
  • the fibronectin in supernatants was classified as soluble fibronectin, while the 1% deoxycholate-insoluble material was classified as fibrillar.
  • Fibronectin in all samples was determined by a double-antibody sandwich enzyme-linked immunosorbent assay using the methods provided by the manufacturer (DACO Corp., Carpinteria, CA) .
  • a polyclonal rabbit anti-human fibronectin (1:1000) was used as the coating antibody, and the detection antibody was a peroxidase-conjugated rabbit anti-human fibronectin (1:2000).
  • 12-LO causes both cardiac fibroblast cell hypertrophy and matrix production, and demonstrate that 12-LO participates in a heretofore unrecognized growth-promoting pathway in the heart.
  • the present invention therefore provides a method of treating or preventing abnormal cardiac cell growth by inhibiting the effects of 12-LO and its products.
  • a 12(S)-HETE receptor blocker such as 13 (S) -hydroxyoctadecadienoic acid, 2-phenylmethyl-l-naphthol (DuP654)
  • a 12-LO enzyme inhibitor for example, panaxynol, a polyacetylene compound isolated from ginseng (Powell et al., Science 215:186-188 (1989)), phenidone, 5, 6, 7-trihydroxyflavone (baicalein) , pioglitazone, substituted
  • Ribozymes designed to specifically cleave 12-LO mRNA are provided by the present invention. Methods known in the art may be used to construct ribozymes which bind and cleave 12-LO mRNA. He cardiac cell growth and hypertrophic effects of 12-LO are thereby reduced or eliminated.
  • dosage forms for administration to humans for use in the present invention is within the ordinary skill in the art. Such dosage forms include tablets, capsules, syrups, suspensions, drops, injectable solutions, lozenges, implants, transdermal patches, and other dosage forms well known in the art for enteral or parenteral administration.
  • a dose of between about 0.5 and about 30 mg/kg/day would be effective in blocking 12(S)-HETE receptors in humans in vivo, and preferably from about 1 to about 10 mg/kg/day. Similar dosages may be given when 12-LO inhibitors are used.
  • Methods of administering ribozymes or antisense oligonucleotides may be designed according to any suitable known method.
  • Example 1 Reduction of cell growth induced by All or 12 (S)-HETE.
  • DuP654 a selective 12(S)-HETE receptor antagonist, significantly reduced cell growth induced by either All or 12(S)-HETE at a concentration of 0.1 mM in CHO-AT la cells in vitro. Complete inhibition of 12(S)-HETE induced mitogenic effects was seen. See Figure 11.
  • Baicalein a specific inhibitor of 12- lipoxygenase, inhibited leucine incorporation in cardiac fibroblasts.
  • the inhibition was both highly significant and dose-dependant (P ⁇ 0.005 at l ⁇ M; P ⁇ 0.001 at 10 ⁇ M) . See Figure 12.

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Abstract

L'invention concerne une méthode de traitement ou d'inhibition d'une croissance cardiaque anormale. Notamment, l'invention concerne l'inhibition du mécanisme d'action de la 12-lipoxygénase ou de l'action de produits à base de 12-lipoxygénase, de manière à réduire ou éliminer les effets d'hypertrophie cardiaque induits par le mécanisme d'action de la 12-lipoxygénase au niveau cellulaire.
PCT/US1999/011115 1998-05-20 1999-05-20 Methode de traitement ou de prevention d'une croissance cellulaire cardiaque anormale par inhibition du chemin de la 12-lipoxygenase WO1999059562A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU39055/99A AU3905599A (en) 1998-05-20 1999-05-20 Method of treating or preventing abnormal cardiac cell growth by inhibiting the 12-lipoxygenase pathway

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US8614798P 1998-05-20 1998-05-20
US60/086,147 1998-05-20

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