WO2001092874A1 - Method for the identification of agents and genes influencing cardiovascular function - Google Patents
Method for the identification of agents and genes influencing cardiovascular function Download PDFInfo
- Publication number
- WO2001092874A1 WO2001092874A1 PCT/EP2001/005749 EP0105749W WO0192874A1 WO 2001092874 A1 WO2001092874 A1 WO 2001092874A1 EP 0105749 W EP0105749 W EP 0105749W WO 0192874 A1 WO0192874 A1 WO 0192874A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- teleost
- heart
- alevin
- contractility
- heart beat
- Prior art date
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Classifications
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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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
Definitions
- the present invention relates to a method for the identification of agents influencing cardiovascular function utilizing teleost alevin.
- Said method is also applicable for the identification of genes regulating heart function.
- WO 99/42606 discloses a method for screening an agent for angiogenesis activity or cell death activity (viz. the agent to be tested is administered to a teleost (e.g., zebrafish) and a response in the teleost is detected); and U.S. Patent No. 5,565,187 discloses a method for studying capillary circulation on living salmonid or other teleosts with yolk sac attached (viz. a fluorescent dye complex and a tracer is injected into the yolk sac and the fluorescence is tracked in the systemic circulation).
- a teleost e.g., zebrafish
- step (b) or simultaneous with step (b) a pharmaceutically active agent, preferably having an adverse effect (relative to the desired effect of the agents to be tested) on heart beat rate, rhythm of the heart, contractility of the heart and/or blood flow, is added to the medium with teleost alevin; and (3) a method as defined in (1) and (2) above, which is suitable for identification of genes involved in cardiovascular action.
- Fig. 1 shows the effects of cardiac pharmaceuticals (at a concentration of 100 ⁇ M) on the heart beat rate of 2d old zebrafish larvae.
- Fig. 2 shows the effect of increasing concentrations of the Ca 2+ channel blockers nifedipine and nimodipine on the heart beat rate of zebrafish larvae (2d post fertilization).
- Fig. 3 shows the reversibility of the nifedipine-induced decrease in heart beat rate of 4d old zebrafish larvae by simultaneous addition of the Ca 2+ channel agonist Bay K 8644.
- Fig. 4 shows the effect of the Bay K 8644 on the heart beat rate of 4d old zebrafish larvae.
- Fig. 5 shows re-animation of 3 d old zebrafish larvae with nifedipine- induced heart arrest by addition of Bay K 8644.
- Fig. 6 shows the reversibility of the nifedipine-induced decrease of heart contractility of 3d old zebrafish larvae.
- the present invention provides a method by which new agents influencing cardiovascular function can be found.
- “Fertilized teleost alevin” in accordance with the present invention means fertilized teleost eggs (hereinafter also shortly referred to as “teleost larvae”).
- "Teleosts” in accordance with the present invention include zebrafish and medaka. The preferred teleost is zebrafish.
- the preparation of a medium in which fertilized teleost alevin is bathed in accordance with steps (a) of embodiment (1) of the invention comprises the provision of a suitable medium, adding the teleost alevin (preferably zebrafish larvae), and incubating the teleost alevin, preferably for 2 to 7 days at 22 to 28°C.
- suitable media for raising teleost alevin are known in the art and include low salt, buffer solutions (e.g., solutions containing less than 10 mM salts (alkaline and earth alkaline salts) and less than 20 mM buffer substance).
- buffer solutions e.g., solutions containing less than 10 mM salts (alkaline and earth alkaline salts) and less than 20 mM buffer substance.
- the most preferred medium is the so-called "embryo medium” comprising:
- step (b) compounds to be tested are added to the medium prepared in step (a), preferably 2 to 5 d after fertilization of the teleost alevin.
- the compound to be tested is preferably added at concentration of 100 nM to 100 ⁇ M, most preferably at 1-10 ⁇ M.
- heart beat rate, rhythm and contractility
- blood flow of the teleost is visually monitored, e.g., via a microscope such as a dissecting microscope.
- a main advantage of teleost is that the larvae can be raised in a large number and that they are transparent facilitating the microscopic inspection of the heart beat. Due to the prominent location of the heart just beneath the skin, agents acting on the heart rapidly reach their target. Responses to the cardiac pharmaceuticals tested are similar between mammals and fish. Thus, the teleosts are extremely well suited organisms for the study of compounds acting on heart function, e.g. by performing high-throughput screening (HTS) assays.
- HTS high-throughput screening
- teleosts can be used to screen a large number of compounds for their effects on heart beat. For example, using 24 well format and manual techniques (addition of the drug, pipetting of the larvae, microscopy) about 300 substances per day and person can be tested for their effects on the heart (2 concentrations per agent to be tested, each well containing about 10 zebrafish larvae).
- One particular advantage of the present invention in comparison to cell-free or even-cell based conventional in vitro HTS assays, is that the agents tested act on an intact heart integrated in the whole-body physiology. In particular, drugs influencing heart beat rate, contractility, and blood flow could be found with the developed method.
- hits arising from usual industrial high-throughput screening assays could be prioritized using teleosts in advance to experiments with mice or rats. Those hits might be substances with an expected effect on the heart function but also hits with other targets than the heart, in order to study potential side-effects (e.g., arrhythmia, bradycardia, tachycardia, cardiac failure) of these compounds.
- arrhythmia-inducing drugs belonging to different pharmacological classes (including, but not limited to, astemizole, terfenadine, cisapride, thioridazine, haloperidol, droperidol, pimozide) very promptly induced an arrhythmic heart beat in nearly 100 % of larvae, (studied by inspecting anaesthetisized larvae with a dissecting microscope). Most often the arrhythmia observed resembles an atrioventricular block, the atrium beating twice as often as the ventricle. In some cases, dependent on the drug and its concentration the heart beat is not periodical but rather arrhythmic without an atrio-ventricular block. All drugs studied decreased the heart beat rate.
- the present invention also provides a simpler, more reliable, faster and cheaper assay for HERG-binding substances.
- the advantage is that this method enables the early identification of proarrhythmic drugs and thus minimizes the risk that at a late stage drug development has to be stopped.
- a pharmaceutically active agent for example Ca 2+ channel blockers or agents causing arrhythmia, in order to find substances reverting the effects of the sensitizing agent and thereby rescue the larvae.
- heart beat rate and contractility is fast and strong, such that an increase in contractility and/or beat rate is rather difficult to detect by microscopic inspection.
- Ca 2+ channel blockers e.g. nifedipine, which lowers heart beat rate and contractility, prior to or simultaneous with an agent-containing medium, may be used to detect those agents which lead to an increase in contractility of the teleost heart (putative drugs for the treatment of congestive heart failure).
- putative anti-arrhythmic agents can be identified by studying heart beats of teleost larvae which were bathed prior to or simultaneous with the addition of the agents in a solution of an arrhythmia-inducing drug.
- the drug-treated teleost present the first whole-animal disease model, which is suited for high- throughput screenings.
- nifedipine e.g. 50 ⁇ M
- an arrest of the heart beat can be induced in teleost larvae, thereby offering the possibility to screen for compounds which can re-animate the heart to beat, potential drugs for the treatment of cardiac infarction.
- the method of the present invention e.g., application of Ca 2+ channel blockers to the teleost larvae, is moreover suitable for the identification of special genes involved in cardiovascular function. These genes are expected to encode especially those proteins, which will, after applying specific antagonists, lead to an increase in cardiac contractility. Up to now, no method exists, which could yield a similar number of new genes of this specific type. By performing high-throughput screening assays with these new proteins, antagonists (medicaments) for the treatment of congestive heart failure could be found.
- teleost larvae preferably zebrafish larvae carrying hetero- or homozygous mutations are sensitized by adding for example Ca 2+ channel blocker (step (a) of the screening method).
- Such mutations can, e.g., be induced by ethylnitrosurea (P. Haffter et al., Development 1996, 123, 1-36) or insertion niutagenesis (A. Amsterdam et al., Genes Dev., 13(20):2713-24 (1999)).
- phenotypes which show resistance to the drug applied can be identified. This means, that the heart beat and blood flow is less influenced by the Ca 2+ channel blocker than in wildtypes. It is believed that these phenotypes carry mutations in genes encoding proteins, which will, after applying specific antagonists, lead to an increase in cardiac contractility. The reason for this feature is, that the underlying mutations are quite likely "loss of function” mutations ("gain of function" mutations are quite rare). Thus, with this kind of screen new targets (for which antagonists could be developed) for the treatment of congestive heart failure can be found, e.g. after positional cloning of the mutations.
- Genotypes with the above mentioned mutations do not show easily identifiable phenotypes if not previously incubated with, for example, Ca 2+ channel blockers, and are likely to be overlooked in conventional genetic screens for the heart beat (zebrafish, medaka, mice screens), because contractility is near the optimum.
- the described invention represents a major further development of conventional screens for contractility, which would yield mainly targets for agonists (for the treatment of heart failure).
- nifedipine or nimodipine for example nifedipine or nimodipine (Sigma)
- the contractility of the heart and blood flow was monitored, and the heart rate was determined by counting the heart beat with the aid of a stereo-microscope.
- ⁇ -adrenergic blocker propanolol and the Ca 2+ channel blockers verapamil (Fig. 1), nifedipine and nimodipine (Fig. 2), induced a decrease in heart beat rate, contractility and blood flow, similar to rodents and humans.
- the drugs propafenone (sodium channel blocker) and amiodarone (potassium channel blocker) have similar effects as the drugs mentioned above, indicating similarities between mammalian and teleost cardiac channels (Fig. 1).
- Zebrafish larvae of the F 3 or F 2 generation carrying heterozygous or homozygous mutations are raised according to the procedures described in Example 1.
- a genetic screen was performed by incubating about 25 zebrafish larvae of the F 2 (dominant screen) or F 3 (recessive screen) generation per crossing for 2-7 d at 22-28°C in petri dishes filled with 30 ml embryo medium.
- 25 larvae were incubated with 30 ml embryo medium, 1.5 ml MESAB solution and 30 ⁇ l of the respective drug added from a 1000-fold stock solution prepared with DMSO.
- the contractility of the heart, heart rate and blood flow were monitored with the aid of a stereo-microscope.
- the parents will be outcrossed to another zebrafish line (WIK) and subsequently the mutations will be cloned, e.g. by positional cloning.
- WIK zebrafish line
- Fig. 2 shows dose-response curves for nimodipine and nifedipine at 2d postfertilization. The effect was reversible after washout of the drug. Even zebrafish with an arrest of the heart beat for 30 min (induced by adding 50 ⁇ M nifedipine for 90 min) could be re-animated by washout of the drug, such that the heart beat rate and contractility were nearly the same as in controls (Fig. 6).
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NZ523247A NZ523247A (en) | 2000-05-27 | 2001-05-19 | Use of the teleost alevin system for the screening of antiarrythmic drugs |
AU2001265996A AU2001265996A1 (en) | 2000-05-27 | 2001-05-19 | Method for the identification of agents and genes influencing cardiovascular function |
CA002410564A CA2410564A1 (en) | 2000-05-27 | 2001-05-19 | Method for the identification of agents and genes influencing cardiovascular function |
EP01943412A EP1290437A1 (en) | 2000-05-27 | 2001-05-19 | Method for the identification of agents and genes influencing cardiovascular function |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP00111444.6 | 2000-05-27 | ||
EP00111444A EP1160570A1 (en) | 2000-05-27 | 2000-05-27 | Method for the identification of agents and genes influencing cardiovascular function |
EP01100298.7 | 2001-01-04 | ||
EP01100298 | 2001-01-04 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2001092874A1 true WO2001092874A1 (en) | 2001-12-06 |
Family
ID=26070995
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2001/005749 WO2001092874A1 (en) | 2000-05-27 | 2001-05-19 | Method for the identification of agents and genes influencing cardiovascular function |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1290437A1 (en) |
AU (1) | AU2001265996A1 (en) |
CA (1) | CA2410564A1 (en) |
NZ (1) | NZ523247A (en) |
WO (1) | WO2001092874A1 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006113914A3 (en) * | 2005-04-20 | 2007-04-19 | Hutchinson Fred Cancer Res | Methods, compositions and articles of manufacture for enhancing survivability of cells, tissues, organs, and organisms |
US7435870B2 (en) | 1998-02-23 | 2008-10-14 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
US7465848B2 (en) * | 2002-11-20 | 2008-12-16 | The General Hospital Corporation | Zebrafish assay |
US7482507B2 (en) | 1998-02-23 | 2009-01-27 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
US7767880B2 (en) | 2006-09-01 | 2010-08-03 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
US7993681B2 (en) | 2003-10-22 | 2011-08-09 | Fred Hutchinson Cancer Research Center | Methods, compositions and devices for inducing stasis in tissues and organs |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5565187A (en) * | 1993-06-01 | 1996-10-15 | Zikria; Bashir | Methods for studying capillary circulation using fish fry and tadpoles |
WO1998031787A1 (en) * | 1997-01-22 | 1998-07-23 | Eisai Co., Ltd. | In vivo apoptosis screening |
US5932418A (en) * | 1996-04-08 | 1999-08-03 | Naiad Systems, Inc. | Fish embryo screening test for genotoxic agents using three different developmental life stages |
WO1999042606A1 (en) * | 1998-02-23 | 1999-08-26 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
-
2001
- 2001-05-19 AU AU2001265996A patent/AU2001265996A1/en not_active Abandoned
- 2001-05-19 CA CA002410564A patent/CA2410564A1/en not_active Abandoned
- 2001-05-19 EP EP01943412A patent/EP1290437A1/en not_active Withdrawn
- 2001-05-19 NZ NZ523247A patent/NZ523247A/en not_active IP Right Cessation
- 2001-05-19 WO PCT/EP2001/005749 patent/WO2001092874A1/en not_active Application Discontinuation
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5565187A (en) * | 1993-06-01 | 1996-10-15 | Zikria; Bashir | Methods for studying capillary circulation using fish fry and tadpoles |
US5932418A (en) * | 1996-04-08 | 1999-08-03 | Naiad Systems, Inc. | Fish embryo screening test for genotoxic agents using three different developmental life stages |
WO1998031787A1 (en) * | 1997-01-22 | 1998-07-23 | Eisai Co., Ltd. | In vivo apoptosis screening |
WO1999042606A1 (en) * | 1998-02-23 | 1999-08-26 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7435870B2 (en) | 1998-02-23 | 2008-10-14 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
US7482507B2 (en) | 1998-02-23 | 2009-01-27 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
US7687682B2 (en) | 1998-02-23 | 2010-03-30 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
US7951989B2 (en) | 1998-02-23 | 2011-05-31 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
US8993834B2 (en) | 1998-02-23 | 2015-03-31 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
US7465848B2 (en) * | 2002-11-20 | 2008-12-16 | The General Hospital Corporation | Zebrafish assay |
US7993681B2 (en) | 2003-10-22 | 2011-08-09 | Fred Hutchinson Cancer Research Center | Methods, compositions and devices for inducing stasis in tissues and organs |
WO2006113914A3 (en) * | 2005-04-20 | 2007-04-19 | Hutchinson Fred Cancer Res | Methods, compositions and articles of manufacture for enhancing survivability of cells, tissues, organs, and organisms |
US7767880B2 (en) | 2006-09-01 | 2010-08-03 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
Also Published As
Publication number | Publication date |
---|---|
NZ523247A (en) | 2004-06-25 |
CA2410564A1 (en) | 2001-12-06 |
AU2001265996A1 (en) | 2001-12-11 |
EP1290437A1 (en) | 2003-03-12 |
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