CA2459265A1 - Affinity-assay for the human erg potassium channel - Google Patents
Affinity-assay for the human erg potassium channel Download PDFInfo
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- CA2459265A1 CA2459265A1 CA002459265A CA2459265A CA2459265A1 CA 2459265 A1 CA2459265 A1 CA 2459265A1 CA 002459265 A CA002459265 A CA 002459265A CA 2459265 A CA2459265 A CA 2459265A CA 2459265 A1 CA2459265 A1 CA 2459265A1
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- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
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Abstract
The invention relates to an assay to establish the affinity of compounds at the "ether-a-go-go" (ERG) potassium (K+) channel, in particular the human ERG
(hERG) potassium channel, using a labelled inwardly rectifying potassium channel (IKR) blocker. This assay is useful to identify compounds with undesirable effects on cardiac repolarisation in man, in particular the propensity to prolong the QT interval in the electrocardiogram.
(hERG) potassium channel, using a labelled inwardly rectifying potassium channel (IKR) blocker. This assay is useful to identify compounds with undesirable effects on cardiac repolarisation in man, in particular the propensity to prolong the QT interval in the electrocardiogram.
Description
Assay The invention relates to an assay to establish the affinity of compounds at the "ether-a-go-go" (ERG) potassium (K+) channel, in particular the human ERG (hERG) potassium channel, using a labelled rapid delayed rectifying potassium channel (IKR) blocker, for example [3H]-dofetilide or [3H]-MK-499. This assay is useful to identify compounds with undesirable effects on cardiac repolarisation in man, in particular the propensity to prolong the QT interval in the electrocardiogram, which may lead to Torsades de Pointes.
In recent years the development of some compounds proposed for therapeutic use has been abandoned in late phase drug development due to the detection of undesirable effects on cardiac repolarisation in man. The effects of these drugs are assessed in terms of the QT interval in the electrocardiogram (ECG). The QT interval is the portion of an ECG. that represents the time from the beginning of, ventricular depolarization to the .end of ventricular repolarisation. Because the QT interval can be attected by neart rate lengthening with a decrease in heart rate and shortening with an increase in heart rate, the _QT is: often "corrected" for .heart rate, resulting in the QTc interval. In rare cases the administration of some drug molecules results in a prolongation of the QT
interval of the ECG in man. The ECGs of these patients resemble those of individuals suffering from an. inherited disorder known as long QT syndrome. Drug-induced ventricular fibrillation; in these cases, can eventually lead to sudden death (Morganroth J et , al. (1993) Am J Cardiol. 72, 26B-31 B; De , Ponti . F. et , al., (2000) Eur J. . Clin.
Pharmacol. 56, 1-18). A number of drug molecules, including, E-4031,, cisapride and terfenadine, are all known to prolong the QT interval of. the electrocardiogram in man (Fuliki A, et al. (1994), Cardiovascular Pharmacol. 23: 374-378; Van Haarst AD
et al., (1998) Clin Pharmacol. Ther. 64: 542-546; Honig P.K. et al. (1993) J.A.M.A.
269; 1513-1518).
The launch of new drugs with undetected potentially cardiotoxic side effects could have hazardous consequences and could trigger lethal cardiac dysrhythmias in patients.
Late detection of QT prolongation, induced by compounds of pharmacological interest can impede drug discovery and development programs, and consequently have a profound impact on the outcome of a program. It is desirable, therefore, to test for the potential cardiotoxic side effects of compounds at an early stage of drug development.
According to the invention there is provided an assay that comprises, or consists of, the following steps:
a) incubation of cells expressing ERG or membranes derived from cells expressing ERG or membranes derived from tissue expressing ERG with labelled IKR
blocker in assay buffer in the presence or absence of different amounts of a test compound or a mixture of test compounds;
b) determination of specifically bound labelled IKR blocker;
c) calculation of the,inhibition of labelled IKR blocker binding by the test compound or mixture of test compounds.
The, assay is useful as a preclinical predictive indicator fo,r identification of compounds with a propensity to prolong the QT interval in man. The assay is a competitive binding assay that .measures the ability of a test compound or mixture of compounds to displace labelled IKR blocker from the ERG K+ channel (ether-a-go-go K+, channel, herein called .ERG). The assay can be performed.in a high throughput test-system. In conjunction with structure-activity relationships (SAR), ligand binding assays using labelled IKR blockers can be used to assist in the design of new drugs devoid of, or with reduced affinity to ERG, in particular human ERG (hERG). .
The assay buffer used is particularly important for optimising binding of the IKR blocker or test compounds) .to. ERG. It has been found that,optimal assay pertormance is achieved using a Tris based buffer (pH 7.2 to 7.6, preferably pH 7.4 at room temperature) containing potassium (K+) ions. Potassium ions in the assay ,buffer may be provided, for example as potassium choride (KCI). The concentration of potassium ions in the assay buffer determines the predictive value of the assay. Assays performed in assay buffer containing from 7.5 to 12.5mM KCI, preferably from 8.5 to 11.5mM KCI, most preferably lOmM KCI are particularly useful to provide an IC2o value predictive of onset of QT prolongation.
The assay buffer of the invention preferably comprises or consists of Tris.Cl and KCI.
Optionally, MgCl2 may be included in the assay buffer.
In recent years the development of some compounds proposed for therapeutic use has been abandoned in late phase drug development due to the detection of undesirable effects on cardiac repolarisation in man. The effects of these drugs are assessed in terms of the QT interval in the electrocardiogram (ECG). The QT interval is the portion of an ECG. that represents the time from the beginning of, ventricular depolarization to the .end of ventricular repolarisation. Because the QT interval can be attected by neart rate lengthening with a decrease in heart rate and shortening with an increase in heart rate, the _QT is: often "corrected" for .heart rate, resulting in the QTc interval. In rare cases the administration of some drug molecules results in a prolongation of the QT
interval of the ECG in man. The ECGs of these patients resemble those of individuals suffering from an. inherited disorder known as long QT syndrome. Drug-induced ventricular fibrillation; in these cases, can eventually lead to sudden death (Morganroth J et , al. (1993) Am J Cardiol. 72, 26B-31 B; De , Ponti . F. et , al., (2000) Eur J. . Clin.
Pharmacol. 56, 1-18). A number of drug molecules, including, E-4031,, cisapride and terfenadine, are all known to prolong the QT interval of. the electrocardiogram in man (Fuliki A, et al. (1994), Cardiovascular Pharmacol. 23: 374-378; Van Haarst AD
et al., (1998) Clin Pharmacol. Ther. 64: 542-546; Honig P.K. et al. (1993) J.A.M.A.
269; 1513-1518).
The launch of new drugs with undetected potentially cardiotoxic side effects could have hazardous consequences and could trigger lethal cardiac dysrhythmias in patients.
Late detection of QT prolongation, induced by compounds of pharmacological interest can impede drug discovery and development programs, and consequently have a profound impact on the outcome of a program. It is desirable, therefore, to test for the potential cardiotoxic side effects of compounds at an early stage of drug development.
According to the invention there is provided an assay that comprises, or consists of, the following steps:
a) incubation of cells expressing ERG or membranes derived from cells expressing ERG or membranes derived from tissue expressing ERG with labelled IKR
blocker in assay buffer in the presence or absence of different amounts of a test compound or a mixture of test compounds;
b) determination of specifically bound labelled IKR blocker;
c) calculation of the,inhibition of labelled IKR blocker binding by the test compound or mixture of test compounds.
The, assay is useful as a preclinical predictive indicator fo,r identification of compounds with a propensity to prolong the QT interval in man. The assay is a competitive binding assay that .measures the ability of a test compound or mixture of compounds to displace labelled IKR blocker from the ERG K+ channel (ether-a-go-go K+, channel, herein called .ERG). The assay can be performed.in a high throughput test-system. In conjunction with structure-activity relationships (SAR), ligand binding assays using labelled IKR blockers can be used to assist in the design of new drugs devoid of, or with reduced affinity to ERG, in particular human ERG (hERG). .
The assay buffer used is particularly important for optimising binding of the IKR blocker or test compounds) .to. ERG. It has been found that,optimal assay pertormance is achieved using a Tris based buffer (pH 7.2 to 7.6, preferably pH 7.4 at room temperature) containing potassium (K+) ions. Potassium ions in the assay ,buffer may be provided, for example as potassium choride (KCI). The concentration of potassium ions in the assay buffer determines the predictive value of the assay. Assays performed in assay buffer containing from 7.5 to 12.5mM KCI, preferably from 8.5 to 11.5mM KCI, most preferably lOmM KCI are particularly useful to provide an IC2o value predictive of onset of QT prolongation.
The assay buffer of the invention preferably comprises or consists of Tris.Cl and KCI.
Optionally, MgCl2 may be included in the assay buffer.
The concentration of Tris.Cl in the assay buffer is preferably from 30mM to 100mM
Tris.Cl, more preferably from 30mM to 70mM Tris.Cl, yet more preferably from 40mM to 60mM Tris.Cl, further preferably from 45mM to 55 mM Tris.Cl, most preferably 50mM
Tris.Cl.
The concentration of KCI in the assay buffer is preferably from 5 to 20mM KCI, more preferably from .6 to l5mM KCI, yet more preferably from 7.5 to 12.5mM KCI, further preferably from 8.5 to 11.SmM KCI, most preferably lOmM KCI.
In a particularly preferred embodiment, the assay buffer comprises or consists of from 30 to 1 OOmM Tris.Cl and from 5 to 20mM KCI, preferably from 30 to 70mM or from 30 to 100mM .Tris.Cl and from 6 to l5mM KCI; yet more preferably from 40 to 60mM
Tris.Cl and from 7.5 to 12.5mM KCI; further. preferably from 45, to 55mM Tris.Cl and from 8.5.to 11.SmM KCI.
It is particularly preferred that the assay buffer comprise or consist of 50mM
Tris.Cl and 1 OmM KCI. , . . .
'If MgCl2 is included in the assay buffer, the concentration of MgCl2 is preferably from 0.6mM to 2.OmM MgCl2, more preferably from 0.6mM, to l.6mM MgCl2, ,yet more preferably.yfrom 0.8mM to .1.4mM , MgCl2, , further., preferably from 0.9mM
,to 1.3mM
MgCl2,_ yet further preferably from l,.OmM to 1.2mM MgCl2,. most preferably 1.OmM, or 1.2mM MgCl2,, in a preferred embodiment the assay buffer used comprises or consists of from 30 to 1 OOmM Tris.Cl, from 5, to ,20mM KCI, and from 0.6 to 2.OmM MgCl2;, preferably from 30 to 100mM Tris.Cl or from 30 to 70mM Tris.Cl, from 6 to lSmM KCI, and from 0.6 to l.6mM MgCl2; yet more preferably from 40 to 60mM Tris.Cl, from 7.5 to 12.5mM
KCI
and from 0.8 to 1.4mM MgCl2; further preferably from 45 to 55mM Tris.Cl, from.
8.5 to 11.SmM KCI and from 0.9 to 1.3mM MgCl2 or from 1.0 to 1.2mM MgCl2.
The assay buffer may comprise or consist of 50mM Tris.Cl, lOmM KCI and I.OmM
MgCl2; or 50mM Tris.Cl, lOmM KCI and 1.2mM MgCl2.
Tris.Cl, more preferably from 30mM to 70mM Tris.Cl, yet more preferably from 40mM to 60mM Tris.Cl, further preferably from 45mM to 55 mM Tris.Cl, most preferably 50mM
Tris.Cl.
The concentration of KCI in the assay buffer is preferably from 5 to 20mM KCI, more preferably from .6 to l5mM KCI, yet more preferably from 7.5 to 12.5mM KCI, further preferably from 8.5 to 11.SmM KCI, most preferably lOmM KCI.
In a particularly preferred embodiment, the assay buffer comprises or consists of from 30 to 1 OOmM Tris.Cl and from 5 to 20mM KCI, preferably from 30 to 70mM or from 30 to 100mM .Tris.Cl and from 6 to l5mM KCI; yet more preferably from 40 to 60mM
Tris.Cl and from 7.5 to 12.5mM KCI; further. preferably from 45, to 55mM Tris.Cl and from 8.5.to 11.SmM KCI.
It is particularly preferred that the assay buffer comprise or consist of 50mM
Tris.Cl and 1 OmM KCI. , . . .
'If MgCl2 is included in the assay buffer, the concentration of MgCl2 is preferably from 0.6mM to 2.OmM MgCl2, more preferably from 0.6mM, to l.6mM MgCl2, ,yet more preferably.yfrom 0.8mM to .1.4mM , MgCl2, , further., preferably from 0.9mM
,to 1.3mM
MgCl2,_ yet further preferably from l,.OmM to 1.2mM MgCl2,. most preferably 1.OmM, or 1.2mM MgCl2,, in a preferred embodiment the assay buffer used comprises or consists of from 30 to 1 OOmM Tris.Cl, from 5, to ,20mM KCI, and from 0.6 to 2.OmM MgCl2;, preferably from 30 to 100mM Tris.Cl or from 30 to 70mM Tris.Cl, from 6 to lSmM KCI, and from 0.6 to l.6mM MgCl2; yet more preferably from 40 to 60mM Tris.Cl, from 7.5 to 12.5mM
KCI
and from 0.8 to 1.4mM MgCl2; further preferably from 45 to 55mM Tris.Cl, from.
8.5 to 11.SmM KCI and from 0.9 to 1.3mM MgCl2 or from 1.0 to 1.2mM MgCl2.
The assay buffer may comprise or consist of 50mM Tris.Cl, lOmM KCI and I.OmM
MgCl2; or 50mM Tris.Cl, lOmM KCI and 1.2mM MgCl2.
It is preferred that the assay buffer be at a pH between 7.2 and 7.6 at room temperature; it is particularly preferred that the assay buffer be at pH 7.4 at room temperature.
The ERG gene (cDNA) can be from a vertebrate or invertebrate source; for vertebrates the ERG gene may be from a mammalian source (e.g. human, simian, bovine, porcine, canine, rabbit, guinea pig, rat, or mouse) or an invertebrate source such as an insect source (e.g. drosophila). A prokaryotic homologue of mammalian ERG may be used. It is preferred that the ERG gene be mammalian ERG, in particular human ERG
(hERG) or canine ERG (cERG).
The ERG , gene.. may be expressed in a mammalian cell line e.g. HEK-293 (Human embryonic kidney) cells, CHO (Chinese hamster ovary) cells; CHL (Chinese hamster lung) cells, COS (monkey) cells; or in an insect cell line e.g. SF9. A
baculovirus vector system, can be used for expression of ERG in a compatible insect cell. line.
Alternatively,, ERG may be expressed in yeast or bacterial cells. It is preferred that the ERG, gene is hERG or cERG and is expressed in either-HEK-293, CHO or CHL.
cells.
The,., assay may, be performed using whole cells expressing ERG or membrane preparations derived from cells expressing ERG, or membrane preparations derived from tissue expressing ERG.
Dofeti,lide.,is':an .IKR b.locker (selective inhibitor of the rapid component of the delayed rectifierpotassium, current), .which prolongs the action potential duration and the effective refractory period in a concentration-dependent manner. Clinical studies have demonstrated that dofetilide is effective in treating patients with atrial as well as ventricular arrhythmias. Dofetilide has formula I below.
Formula I
\ NHS O
// \
\ O~N / O
~S o ' /
/ ~N
O H
Dofetilide is claimed arid its preparation is described in European patent EP
0245997.
MK-499 (Merck) is methylsulphonamide antiarrhythmic drug that acts as an IKR
blocker. MK-499 has formula II shown below.
The ERG gene (cDNA) can be from a vertebrate or invertebrate source; for vertebrates the ERG gene may be from a mammalian source (e.g. human, simian, bovine, porcine, canine, rabbit, guinea pig, rat, or mouse) or an invertebrate source such as an insect source (e.g. drosophila). A prokaryotic homologue of mammalian ERG may be used. It is preferred that the ERG gene be mammalian ERG, in particular human ERG
(hERG) or canine ERG (cERG).
The ERG , gene.. may be expressed in a mammalian cell line e.g. HEK-293 (Human embryonic kidney) cells, CHO (Chinese hamster ovary) cells; CHL (Chinese hamster lung) cells, COS (monkey) cells; or in an insect cell line e.g. SF9. A
baculovirus vector system, can be used for expression of ERG in a compatible insect cell. line.
Alternatively,, ERG may be expressed in yeast or bacterial cells. It is preferred that the ERG, gene is hERG or cERG and is expressed in either-HEK-293, CHO or CHL.
cells.
The,., assay may, be performed using whole cells expressing ERG or membrane preparations derived from cells expressing ERG, or membrane preparations derived from tissue expressing ERG.
Dofeti,lide.,is':an .IKR b.locker (selective inhibitor of the rapid component of the delayed rectifierpotassium, current), .which prolongs the action potential duration and the effective refractory period in a concentration-dependent manner. Clinical studies have demonstrated that dofetilide is effective in treating patients with atrial as well as ventricular arrhythmias. Dofetilide has formula I below.
Formula I
\ NHS O
// \
\ O~N / O
~S o ' /
/ ~N
O H
Dofetilide is claimed arid its preparation is described in European patent EP
0245997.
MK-499 (Merck) is methylsulphonamide antiarrhythmic drug that acts as an IKR
blocker. MK-499 has formula II shown below.
Formula II
°\ , ~s~
The IKR blocker used in the assay is labelled with a detectable label, for example a ~radiolabel or fluorescent tag. In a preferred embodiment of the invention, the labelled IKR blocker used in the assay is labelled dofetilide, preferably radiolabelled dofetilide, most preferably tritiated dofetilide ([3H]-dofetilide). In another embodiment of the invention, the labelled IKR blocker used in the assay is labelled MK-499, preferably radiolabelled MK-499, most preferably tritiated MK-499 ([3H]-MK-499).
Preferred assay formats include the filter binding technique, whereby bound and unbound labelled IKR blocker e.g. labelled dofetilide or labelled MK-499;
preferably radiolabelled dofetilide or radiolabelled MK-499; most preferably [3H]-dofetilide or [3H]-MK-499, are separated by filtration. The assay can. be . performed utilising the sciritillation proximity assay (SPA) technique, using radiolabelled IKR
blocker e.g.
radiolabelled dofetilide or radiolabelled MK-499, .preferably [3H]-dofetilide or [3H]-MK-499.
In the filter binding, technique, cells expressing ERG or membranes derived from cells expressing ERG or membranes derived from tissue expressing ERG are incubated in assay buffer with labelled IKR blocker e.g. [3H]-dofetilide or [3H]-MK-499, in the presence (test) or absence (control) of the test compound or mixture , of test compounds. Incubations are preferably carried out at room temperature for from 60 to :120.minutes, preferably for 90 minutes. Non-specific binding is determined in the presence of unlabelled IKR blocker, e.g. lOp.M dofetilide or lOp.M MK-499.
Bound labelled IKR blocker is separated from unbound IKR blocker by filtration through filter mats, or onto multiwell filter plates. Filter mats or plates are washed to remove unbound labelled IKR blocker, bound labelled IKR blocker is quantified e.g.
for tritiated IKR blocker such as [3H]-dofetilide or [3H]-MK-499 by scintillation spectroscopy using an appropriate counter for radioactivity.
In the scintillation proximity assayT"' (SPA) system (Amersham Biosciences), beads are used to bind cells expressing ERG or membranes derived from cells expressing ERG or membranes derived from tissue expressing ERG. A variety of bead types are suitable for use. in a SPA. assay according to the invention, these .include PVT wheat germ agglutinin,. ;yttrium oxide polylysine beads, or yttrium silicate beads (YSi) ; (Amersham BiVosciences) such as YSi. polylysine or YSi wheat.. germ agglutinin. The optimum bead type for ,,use in, a SPA assay of ,the invention depends on the cells, or cell. membranes used; :bead .to. cell or :bead. .to. membrane binding, may be assessed to identify the optimum bead type for the,cell. or cell membrane used. Beads bound to ERG
material (whole cell, cell membrane preparation or tissue membrane preparation) are incubated in assay buffer .with labelled IKR blocker, e.g. [3H]-dofetilide or [3H]-MK-499 in the presence (test) or ,absence (control) of the test compound or mixture of test compounds. The ability of. the test compound or mixture of test compounds to displace bound' radiolabelled IKR, blocker is determined by detecting light emissions, for example using,standard,cou,nters that, can be used, with SPA technology. ' , The assay may.. also include one or more of the steps of: calculation of the concentration of the test compounds) that gives 20% inhibition of dofetilide binding (I~C2o),,calculation of the concentration of, the test compounds) that.gives 50% inhibition of.dofetilide binding (ICSO), calculation of the compound affinity as Ki or calculation of the compound affinity as pKi,.
The IC2o values generated from competitive displacement of IKR blocker binding, e.g.
[3H]-dofetilide binding, ,using the assay of the invention are comparable to th,e free drug concentration associated with QT prolongation in man. Thus the assay can be used to predict the concentration of a compound liable to cause undesirable cardiac side effects.
°\ , ~s~
The IKR blocker used in the assay is labelled with a detectable label, for example a ~radiolabel or fluorescent tag. In a preferred embodiment of the invention, the labelled IKR blocker used in the assay is labelled dofetilide, preferably radiolabelled dofetilide, most preferably tritiated dofetilide ([3H]-dofetilide). In another embodiment of the invention, the labelled IKR blocker used in the assay is labelled MK-499, preferably radiolabelled MK-499, most preferably tritiated MK-499 ([3H]-MK-499).
Preferred assay formats include the filter binding technique, whereby bound and unbound labelled IKR blocker e.g. labelled dofetilide or labelled MK-499;
preferably radiolabelled dofetilide or radiolabelled MK-499; most preferably [3H]-dofetilide or [3H]-MK-499, are separated by filtration. The assay can. be . performed utilising the sciritillation proximity assay (SPA) technique, using radiolabelled IKR
blocker e.g.
radiolabelled dofetilide or radiolabelled MK-499, .preferably [3H]-dofetilide or [3H]-MK-499.
In the filter binding, technique, cells expressing ERG or membranes derived from cells expressing ERG or membranes derived from tissue expressing ERG are incubated in assay buffer with labelled IKR blocker e.g. [3H]-dofetilide or [3H]-MK-499, in the presence (test) or absence (control) of the test compound or mixture , of test compounds. Incubations are preferably carried out at room temperature for from 60 to :120.minutes, preferably for 90 minutes. Non-specific binding is determined in the presence of unlabelled IKR blocker, e.g. lOp.M dofetilide or lOp.M MK-499.
Bound labelled IKR blocker is separated from unbound IKR blocker by filtration through filter mats, or onto multiwell filter plates. Filter mats or plates are washed to remove unbound labelled IKR blocker, bound labelled IKR blocker is quantified e.g.
for tritiated IKR blocker such as [3H]-dofetilide or [3H]-MK-499 by scintillation spectroscopy using an appropriate counter for radioactivity.
In the scintillation proximity assayT"' (SPA) system (Amersham Biosciences), beads are used to bind cells expressing ERG or membranes derived from cells expressing ERG or membranes derived from tissue expressing ERG. A variety of bead types are suitable for use. in a SPA. assay according to the invention, these .include PVT wheat germ agglutinin,. ;yttrium oxide polylysine beads, or yttrium silicate beads (YSi) ; (Amersham BiVosciences) such as YSi. polylysine or YSi wheat.. germ agglutinin. The optimum bead type for ,,use in, a SPA assay of ,the invention depends on the cells, or cell. membranes used; :bead .to. cell or :bead. .to. membrane binding, may be assessed to identify the optimum bead type for the,cell. or cell membrane used. Beads bound to ERG
material (whole cell, cell membrane preparation or tissue membrane preparation) are incubated in assay buffer .with labelled IKR blocker, e.g. [3H]-dofetilide or [3H]-MK-499 in the presence (test) or ,absence (control) of the test compound or mixture of test compounds. The ability of. the test compound or mixture of test compounds to displace bound' radiolabelled IKR, blocker is determined by detecting light emissions, for example using,standard,cou,nters that, can be used, with SPA technology. ' , The assay may.. also include one or more of the steps of: calculation of the concentration of the test compounds) that gives 20% inhibition of dofetilide binding (I~C2o),,calculation of the concentration of, the test compounds) that.gives 50% inhibition of.dofetilide binding (ICSO), calculation of the compound affinity as Ki or calculation of the compound affinity as pKi,.
The IC2o values generated from competitive displacement of IKR blocker binding, e.g.
[3H]-dofetilide binding, ,using the assay of the invention are comparable to th,e free drug concentration associated with QT prolongation in man. Thus the assay can be used to predict the concentration of a compound liable to cause undesirable cardiac side effects.
To assess whether a compound, or mixture of compounds, is likely to prolong the QT
interval in the electrocardiogram in man, the following steps are carried out:
a) An assay is carried out according to the invention.
b) An IC2o value is obtained; this indicates the real or predicted free drug concentration at which QT prolongation will occur in man;
c) The IC2o value is compared with the free drug concentration required for the desired therapeutic effect of the compound or mixture of compounds in vivo.
If the free drug concentration required for the desired therapeutic effect of the compound or mixture of compounds is within 10 to 30 fold of the IC2o of the compound or mixture of compounds in the assay, the compound or mixture of compounds is likely to show QTinterval prolongation in man.
The assay of. the invention is a better predictor of in vivo QT prolongation effect of drug molecules than existing assays such as .the HERG patch clamp assay.
List of.Figures Figure 1: Representative saturation curve data for [3H]-dofetilide binding to HERG in (a) filter 'binding, (b) SPA 96 well format and (c) SPA 384 well format:
Figure ~2: Correlation plots comparing pK values obtained from filter binding' and SPA
binding assays: (a) correlation between 96 well hERG [3H] dofetilide SPA assay and radioligand binding assay, (b) correlation between 96 well and 384 well hERG
[3H]
dofetilide SPA assay:
Figure 3: Comparison of inhibition of [3H]-dofetilide binding to hERG, hERG
patch clamp, and free drug concentration known to induce QT interval prolongation in man, for (a) E-4031, (b) dofetilide, (c) terfenadine and (d) cisapride. , ., Figure 4: Comparison of the dofetilide ICSO in 'the dofetilide binding assay carried out in cell membranes from HEK-293 cells.transfected with human ERG (hERG (.1,)) or.with canine. ERG.(cERG (/)) "~~.~Na~.son of terfenadine ICSO in the dofetilide binding assay in cERG or hERG transfected HEK-293 cell membranes Figure 6: Comparison of E4031 ICSO in the dofetilide binding assay in cERG or hERG
transfected cell HEK-293 membranes Figure 7: , Mean (n = 2) concentration effect curves for (a) dofetilide and,(b) terodili.ne in tritiated dofetilide SPA assays using assay buffer 50mM Tris CI, lOmM KCI, at ~pH7.4.
Examples Example 1: Preparation of membranes from HEK-293 cells expressing human or canine ERG
An adherent HEK-293 cell line expressing human ERG (Zhou, Z et al (1998) Biophys.
J. 74, 230-241) was provided by Dr. Craig January, University of Wisconsin, USA; this cell line was designated the "January" cell line. An alternative adherent HEK-293 cell line, designated cell line 15 (293S-HERG clone 15) was produced by the method described in Zhou, Z et al (1998). Full length cDNA for human ERG was inserted downstream of the CMV promoter in pcDNA3.1 (Invitrogen), the vector also has a promoter that drives expression of a neomycin resistance gene. The construct was transfected into human embryonic kidney 293S (HEK-293) cells. Stable transformants were selected using 6418 (Gibco). Although cell line 15 has slightly lower expression of hERG than the January cell line, it has improved growth characteristics.
Cell line 15 (293S-HERG (Clone 15)) was deposited on 26 June 2002 with the ECACC
(CAMR Salisbury, Wiltshire, SP4 OJG, UK) in accordance with the terms of the Budapest Treaty 1977 under deposit accession number 02062678.
Adherent HEK-293 cells expressing human ERG, were grown in MEM Earles medium (Life Technologies) supplemented with 10% foetal calf serum (PAA
Laboratories), 2 mM L-glutamine (Sigma), 1 mM sodium pyruvate (Sigma), 0.4 mg/ml 6418 (Life Technologies) and an addition of 1x non-essential amino acids (Life Technologies). The cells were grown at 37°C in a humidified atmosphere with 5% C02 in T225 cm3 flasks.
interval in the electrocardiogram in man, the following steps are carried out:
a) An assay is carried out according to the invention.
b) An IC2o value is obtained; this indicates the real or predicted free drug concentration at which QT prolongation will occur in man;
c) The IC2o value is compared with the free drug concentration required for the desired therapeutic effect of the compound or mixture of compounds in vivo.
If the free drug concentration required for the desired therapeutic effect of the compound or mixture of compounds is within 10 to 30 fold of the IC2o of the compound or mixture of compounds in the assay, the compound or mixture of compounds is likely to show QTinterval prolongation in man.
The assay of. the invention is a better predictor of in vivo QT prolongation effect of drug molecules than existing assays such as .the HERG patch clamp assay.
List of.Figures Figure 1: Representative saturation curve data for [3H]-dofetilide binding to HERG in (a) filter 'binding, (b) SPA 96 well format and (c) SPA 384 well format:
Figure ~2: Correlation plots comparing pK values obtained from filter binding' and SPA
binding assays: (a) correlation between 96 well hERG [3H] dofetilide SPA assay and radioligand binding assay, (b) correlation between 96 well and 384 well hERG
[3H]
dofetilide SPA assay:
Figure 3: Comparison of inhibition of [3H]-dofetilide binding to hERG, hERG
patch clamp, and free drug concentration known to induce QT interval prolongation in man, for (a) E-4031, (b) dofetilide, (c) terfenadine and (d) cisapride. , ., Figure 4: Comparison of the dofetilide ICSO in 'the dofetilide binding assay carried out in cell membranes from HEK-293 cells.transfected with human ERG (hERG (.1,)) or.with canine. ERG.(cERG (/)) "~~.~Na~.son of terfenadine ICSO in the dofetilide binding assay in cERG or hERG transfected HEK-293 cell membranes Figure 6: Comparison of E4031 ICSO in the dofetilide binding assay in cERG or hERG
transfected cell HEK-293 membranes Figure 7: , Mean (n = 2) concentration effect curves for (a) dofetilide and,(b) terodili.ne in tritiated dofetilide SPA assays using assay buffer 50mM Tris CI, lOmM KCI, at ~pH7.4.
Examples Example 1: Preparation of membranes from HEK-293 cells expressing human or canine ERG
An adherent HEK-293 cell line expressing human ERG (Zhou, Z et al (1998) Biophys.
J. 74, 230-241) was provided by Dr. Craig January, University of Wisconsin, USA; this cell line was designated the "January" cell line. An alternative adherent HEK-293 cell line, designated cell line 15 (293S-HERG clone 15) was produced by the method described in Zhou, Z et al (1998). Full length cDNA for human ERG was inserted downstream of the CMV promoter in pcDNA3.1 (Invitrogen), the vector also has a promoter that drives expression of a neomycin resistance gene. The construct was transfected into human embryonic kidney 293S (HEK-293) cells. Stable transformants were selected using 6418 (Gibco). Although cell line 15 has slightly lower expression of hERG than the January cell line, it has improved growth characteristics.
Cell line 15 (293S-HERG (Clone 15)) was deposited on 26 June 2002 with the ECACC
(CAMR Salisbury, Wiltshire, SP4 OJG, UK) in accordance with the terms of the Budapest Treaty 1977 under deposit accession number 02062678.
Adherent HEK-293 cells expressing human ERG, were grown in MEM Earles medium (Life Technologies) supplemented with 10% foetal calf serum (PAA
Laboratories), 2 mM L-glutamine (Sigma), 1 mM sodium pyruvate (Sigma), 0.4 mg/ml 6418 (Life Technologies) and an addition of 1x non-essential amino acids (Life Technologies). The cells were grown at 37°C in a humidified atmosphere with 5% C02 in T225 cm3 flasks.
The cells were split 1:3 to 1:5 after reaching 80% confluence using cell dissociation solution (Sigma, cat no: C5914 in 2001 ) and later seeded into 850 cm2 C02 gassed roller bottles (Corning, cat no: 430849 in 2001 ) in the absence of 6418.
For the preparation of membranes, cells were harvested from the roller bottles by scraping and resuspended in PBS (Life Technologies, cat no: 14190-094 in 2001 ). All cells were pelleted, washed twice with PBS and snap-frozen on dry ice prior to storage at -80°C until required.
A NEK-293 cell line expressing canine ERG was produced by transient transfection of HEK-..293 cells. ; The complete coding sequence of cERG cDNA (Zehelein et al ..(2001).
,. . . . . : ~ . .
Pflugers Archiv. European Journal of. Physiology. 442(2): 188 - 191 ) was provided .in the . pBluescript~ vector (Stratagene) by Professor Zehelein University of Heidelberg, Germany. In the pBluescript construct, the cERG cDNA was flanked by BamHl Sites.
Initial. experiments. indicated poor insertion efficiency; for direct insertion of cERG.BamHl fragment into the desired vector, pcDNA3.1. To overcome this, .an indirect cloning method was . devised using the cloning vector ,pSl'73 (Promega). The cERG/pBluescript ;.co;nstruct and pSP73 vector were subjected to BamHl digestion, to reduce interference by the presence of pBluescript BamHl fragments in the ligation reaction;. -the, cERG/pBluescript BamHl digested material .was. also ,subjected to. Scal digestion to cleave, pBluescript and ensure more effective separation of the cERG
BamNl fragment .on agarose gel. The restriction mixtures were subjected.to agarose gel. electrophoresis, bands ,containing the cERG and pSP73 BamHl, fragments .were visualized following staining with ethidium bromide and UV illumination. The .cERG and pSP73, bands were excised and. eluted from the gel using a QIAgen MinELute Gel extraction kit according to the manufacturers instructions. To prevent religation of. the - BamHl ends of the pSP73 DNA during the ligation reaction, the plasmid DNA
fragments were .subjected to CIP treatment using a standard protocol. The cERG BamHl fragments were ligated into the pSP73 BamHl fragments using a standard ligation protocol. After the reaction, the, ligation mixture , was transformed, into, cJM109 competent E, coli cells using a standard transformation protocol. _ Transformants. were selected by plating on LB agar (Millers) containing ampicillin ~.(50Ng/ml) and incubated overnight at 37°C. Overnight cultures of the transformed .cells-were used to produce mini preparations of cERG/pSP73 DNA using a QIAgen Miniprep kit according to the manufacturer's instructions. The resulting DNA was subjected to restriction digestion and agarose gel electrophoresis to ideritify positive clones.
The cERG cDNA was excised from cERG/pSP73 as an Xhol (5') EcoRl (3') fragment, 5 this fragment was ligated into an XhollEcoRl fragment of the reverse poly linker form of pcDNA3.1, pcDNA3.1(-) XhollEcoRl. In this instance the reverse polylinker form was used because the cERG/pSP73 clone selected contained the reverse orientation of cERG. After ligation into pcDNA3.1 (-), the 5' end of cERG was located adjacent to the enhancer-promoter sequence from human cytomegalovirus (CMV). The ligation 10 mixture was transformed into cJM109 competent E. coli cells using a standard transformation protocol, transformants were selected via plating onto. LB agar (Millers) containing: ampicillin .(50~g/ml) and incubating overnight at 37°C
along with required controls. Colonies picked at random from the cERG/pcDNA3.1 (-) plates were inoculated into :5m1 of LB. media containing ampicillin (50~g/ml) and incubated at, 37°C, 200rpm overnight. These, overnight cultures were subsequently used to produce mini-preps of DNA using, a QIAgen Minip,rep Kit. The resulting DNA was subjected to a Xhol and: EcoRl .double . digestion and analysis on 1% agarose gel. No positive cERG/pcDNA3.1 (-) clones, were identified because of low insertion efficiency in the ligation reaction coupled with the fact that DNA from only a small number of clones was analysed; using ;the mini prep..method: A colony PCR method was thus used to screen a.:larger, number of colonies for,positive clones. , . , , .
..
The colony .PCR protocol permitted rapid detection of .cERG/pcDNA3.1(-) clones.
Three primers were designed and made for use. in the PCR protocol: , -..:.. :. .. _. ; .. , 2b ; , . . . .
Primer 1: 'C.ERG01' .(SEQ ID NO: 1 ) which hybridises to cERG at nucleotide .positions 601-620 of.the coding sequence:
:. ... . . . .. -- . . . .
5'-ACCACATCCACCAGGCACAG-3' Primer 2: 'NHE PCDNA3' , (SEQ ID NO: 2), which hybridises to pcDNA3.1 (-) at nucleotide positions 886-910 (within the multicloning site flanking the Nhe1 cloning site):
5'-CCCAAGCTGGCTAGCGTTTAAACGG-3' Primer 3: 'T7 SP73' (SEQ ID N0: 3) which was used as a control and used against a colony known to produce cERG/pSP73. This hybridised to pSP73 at nucleotide positions 98-121, within the T7 polymerase promoter sequence:
5'-TAATACGACTCACTATAGGGAGA-3' Ninety-five cJM109 colonies were picked from the LB agar transformation plates and transferred to a sterile deep well 96-well plate containing 1 ml/well LB broth supplemented with ampicillin (50pg/ml). As a control, a colony known to contain the cE.RG/pSP73 plasmid was transferred to the final 96t" well containing LB-amp broth.
The; plate was .covered and incubated. at 37°C :overnight at 200rpm. An aliquot of 7,0,1 of, each .mini-culture .was transferred ~to a 96-well. PCR.plate. (0.5m1/well)-;and placed- in a Beckman.,~Allegra .,6R. centrifuge for 2800rpm, room temperature for 10 minutes. The supernatant was discarded and the plate drained for 3 minutes. The PCR
reaction mixes. were vset pup and -added ~to the PCR plate containing the bacterial pellets as follows:
. . Test wells ,Control well cERG/pSP73, .
Taqrnan Gold buffer (X1,0) .. 1 O.Op.I. -10.x,1 r .
dNTPs-.(X10, 2mM/dNTP) , .-. 2.0p,1 2~1 - . - , Taqman, Gold Polymerase. 0.5p1; 0.5w1 : -(5u/p.l) .
C.ERG01,.; (primer. ~l~=. ; : 2~.1. . 2p:1; ,, . . . . .-. w 25~,M) . .
NHE.:PCDNA3 (primer 2- 25~M) ~..2~1. . . , . . . . :~ .
. . . .
T7.:SP73 (primer3-~=25p,M) ~ - :.' .,~. . :. 2w1 : - . , Nuclease free water . . 83.5p;1w ~ :83.5.1 w v 'r...: : y: :;::.. a : - :.
,-,:
The bacterial pellet was resuspended in the PCR reaction mixture. The PCR
reaction was performed as specified by the manufacturers protocol for the Taqman GoId..PCR kit (Applied Biosystems, 1999 edition) thus:
. , " ., Temperature Time Step 1. - hot start . : .95.°C . . 6 minutes Step,2 - denaturation. 95°.C 1 minute Step, 3,- annealing 60°C _ , 1 minute Step.4.- .extension 72°C 1 minute To step 2 for 35 cycles, then step 5.
For the preparation of membranes, cells were harvested from the roller bottles by scraping and resuspended in PBS (Life Technologies, cat no: 14190-094 in 2001 ). All cells were pelleted, washed twice with PBS and snap-frozen on dry ice prior to storage at -80°C until required.
A NEK-293 cell line expressing canine ERG was produced by transient transfection of HEK-..293 cells. ; The complete coding sequence of cERG cDNA (Zehelein et al ..(2001).
,. . . . . : ~ . .
Pflugers Archiv. European Journal of. Physiology. 442(2): 188 - 191 ) was provided .in the . pBluescript~ vector (Stratagene) by Professor Zehelein University of Heidelberg, Germany. In the pBluescript construct, the cERG cDNA was flanked by BamHl Sites.
Initial. experiments. indicated poor insertion efficiency; for direct insertion of cERG.BamHl fragment into the desired vector, pcDNA3.1. To overcome this, .an indirect cloning method was . devised using the cloning vector ,pSl'73 (Promega). The cERG/pBluescript ;.co;nstruct and pSP73 vector were subjected to BamHl digestion, to reduce interference by the presence of pBluescript BamHl fragments in the ligation reaction;. -the, cERG/pBluescript BamHl digested material .was. also ,subjected to. Scal digestion to cleave, pBluescript and ensure more effective separation of the cERG
BamNl fragment .on agarose gel. The restriction mixtures were subjected.to agarose gel. electrophoresis, bands ,containing the cERG and pSP73 BamHl, fragments .were visualized following staining with ethidium bromide and UV illumination. The .cERG and pSP73, bands were excised and. eluted from the gel using a QIAgen MinELute Gel extraction kit according to the manufacturers instructions. To prevent religation of. the - BamHl ends of the pSP73 DNA during the ligation reaction, the plasmid DNA
fragments were .subjected to CIP treatment using a standard protocol. The cERG BamHl fragments were ligated into the pSP73 BamHl fragments using a standard ligation protocol. After the reaction, the, ligation mixture , was transformed, into, cJM109 competent E, coli cells using a standard transformation protocol. _ Transformants. were selected by plating on LB agar (Millers) containing ampicillin ~.(50Ng/ml) and incubated overnight at 37°C. Overnight cultures of the transformed .cells-were used to produce mini preparations of cERG/pSP73 DNA using a QIAgen Miniprep kit according to the manufacturer's instructions. The resulting DNA was subjected to restriction digestion and agarose gel electrophoresis to ideritify positive clones.
The cERG cDNA was excised from cERG/pSP73 as an Xhol (5') EcoRl (3') fragment, 5 this fragment was ligated into an XhollEcoRl fragment of the reverse poly linker form of pcDNA3.1, pcDNA3.1(-) XhollEcoRl. In this instance the reverse polylinker form was used because the cERG/pSP73 clone selected contained the reverse orientation of cERG. After ligation into pcDNA3.1 (-), the 5' end of cERG was located adjacent to the enhancer-promoter sequence from human cytomegalovirus (CMV). The ligation 10 mixture was transformed into cJM109 competent E. coli cells using a standard transformation protocol, transformants were selected via plating onto. LB agar (Millers) containing: ampicillin .(50~g/ml) and incubating overnight at 37°C
along with required controls. Colonies picked at random from the cERG/pcDNA3.1 (-) plates were inoculated into :5m1 of LB. media containing ampicillin (50~g/ml) and incubated at, 37°C, 200rpm overnight. These, overnight cultures were subsequently used to produce mini-preps of DNA using, a QIAgen Minip,rep Kit. The resulting DNA was subjected to a Xhol and: EcoRl .double . digestion and analysis on 1% agarose gel. No positive cERG/pcDNA3.1 (-) clones, were identified because of low insertion efficiency in the ligation reaction coupled with the fact that DNA from only a small number of clones was analysed; using ;the mini prep..method: A colony PCR method was thus used to screen a.:larger, number of colonies for,positive clones. , . , , .
..
The colony .PCR protocol permitted rapid detection of .cERG/pcDNA3.1(-) clones.
Three primers were designed and made for use. in the PCR protocol: , -..:.. :. .. _. ; .. , 2b ; , . . . .
Primer 1: 'C.ERG01' .(SEQ ID NO: 1 ) which hybridises to cERG at nucleotide .positions 601-620 of.the coding sequence:
:. ... . . . .. -- . . . .
5'-ACCACATCCACCAGGCACAG-3' Primer 2: 'NHE PCDNA3' , (SEQ ID NO: 2), which hybridises to pcDNA3.1 (-) at nucleotide positions 886-910 (within the multicloning site flanking the Nhe1 cloning site):
5'-CCCAAGCTGGCTAGCGTTTAAACGG-3' Primer 3: 'T7 SP73' (SEQ ID N0: 3) which was used as a control and used against a colony known to produce cERG/pSP73. This hybridised to pSP73 at nucleotide positions 98-121, within the T7 polymerase promoter sequence:
5'-TAATACGACTCACTATAGGGAGA-3' Ninety-five cJM109 colonies were picked from the LB agar transformation plates and transferred to a sterile deep well 96-well plate containing 1 ml/well LB broth supplemented with ampicillin (50pg/ml). As a control, a colony known to contain the cE.RG/pSP73 plasmid was transferred to the final 96t" well containing LB-amp broth.
The; plate was .covered and incubated. at 37°C :overnight at 200rpm. An aliquot of 7,0,1 of, each .mini-culture .was transferred ~to a 96-well. PCR.plate. (0.5m1/well)-;and placed- in a Beckman.,~Allegra .,6R. centrifuge for 2800rpm, room temperature for 10 minutes. The supernatant was discarded and the plate drained for 3 minutes. The PCR
reaction mixes. were vset pup and -added ~to the PCR plate containing the bacterial pellets as follows:
. . Test wells ,Control well cERG/pSP73, .
Taqrnan Gold buffer (X1,0) .. 1 O.Op.I. -10.x,1 r .
dNTPs-.(X10, 2mM/dNTP) , .-. 2.0p,1 2~1 - . - , Taqman, Gold Polymerase. 0.5p1; 0.5w1 : -(5u/p.l) .
C.ERG01,.; (primer. ~l~=. ; : 2~.1. . 2p:1; ,, . . . . .-. w 25~,M) . .
NHE.:PCDNA3 (primer 2- 25~M) ~..2~1. . . , . . . . :~ .
. . . .
T7.:SP73 (primer3-~=25p,M) ~ - :.' .,~. . :. 2w1 : - . , Nuclease free water . . 83.5p;1w ~ :83.5.1 w v 'r...: : y: :;::.. a : - :.
,-,:
The bacterial pellet was resuspended in the PCR reaction mixture. The PCR
reaction was performed as specified by the manufacturers protocol for the Taqman GoId..PCR kit (Applied Biosystems, 1999 edition) thus:
. , " ., Temperature Time Step 1. - hot start . : .95.°C . . 6 minutes Step,2 - denaturation. 95°.C 1 minute Step, 3,- annealing 60°C _ , 1 minute Step.4.- .extension 72°C 1 minute To step 2 for 35 cycles, then step 5.
Step 5 - aenaiuration 95°C 45 secs Step 6 - annealing 60°C 45 secs Step 7 - extension 72°C 5 minutes The PCR products for each well were then separated by electrophoresis on a 1.5%
agarose gel using a 1 OObp DNA ladder marker at 1 OOV for 25 minutes in 1 X
TAE buffer and visualised using UV light. Putative positive clones were identified and samples from these PCR .reaction. mixtures were. run on a second separate 1.5% agarose gel at 100V for one hour to examine the sizes of the PCR products.
, The mini-cultures which. gave an amplified a PCR product were each seeded from the original deep-well 96-well plate into sterile tubes with 5m1 LB broth containing 50~g/ml ampicillin, and incubated. at 37°C overnight at 200rpm. The overnight cultures were then used to produce mini-preps of DNA using a QIAgen Miniprep Kit. The resulting DNA
was subjected .to an Xhol and .EcoRl. double digestion to check.,for. the .presence. of cERG/pcDNA3...1 (-).. The restriction digest was analysed via a 1 % agarose gel run. for 1 hour at 1:00V with .1 kb DNA ,ladder markers (201 sample loading, with 2~.1 gel, loading solution):, ,Further ..restriction :digestion analysis was performed to confirm that ,the purified plasmids from, the transformants were indeed cERG/pcDNA3.1 (-).
Untransfected HEK-293 cells were routinely maintained in 50m1 Minimum Essential Medium (MEM)supplemented with 1~0% (v/v) foetal calf serum (FCS), 2mM ~L-glutamine, 1 m~M sodium pyruvate and 1 mM non-essential amino acids. Cells were seeded irito 225cm2 ventilated cap flasks - and were maintained in a humidified atmosphere containing 5% C02. The HEK-293 cells used in this study were between passage' numbers 39-48. Cells were passaged typically every Three days iri a ratio of 1:3 froim a flask of 80-90% confluency; fresh medium was added~after washing twice with l Orril PBS and dissociating from the flask using cell 'dissociation fluid. ~ ~ ' Tfie cERGIpcDNA3.1 (-) construct was transfected into HEK-293 cells grown to 80-95%
confluency , in 225cm2 ventilated flasks using the following method. Endotoxin free cERG/pcDNA3.1(-). DNA (94~g) and Lipofectamine2000 (Gibco BRL) (94~.g), were added to 2.25m1 of OPTIMEM-I media (Gibco BRL) in sterile 10m1 centrifuge tubes;
mixing was carried out after incubation at room temperature for five minutes.
The uporecramme~uuulDNA/OPTIMEM-I mix was then incubated at room temperature for twenty minutes before the addition of a further 10.5m1 OPTIMEM-I. HEK-293 cells were washed with 10m1 PBS and the Lipofectamine2000/DNA/OPTIMEM-I mixture added and incubated for 3.5 hours at 37°C in a humidified atmosphere containing 5% C02.
After incubation, 50m1 of MEM (supplemented with 10% (v/v) FCS, 2mM L-glutamine, 1 mM sodium pyruvate and 1 mM non-essential amino acids) was added. The. HEK-cells were incubated for 24 hours at 37.°C. . Transfected.cells were.harvested after..24 hours by washing with PBS, scraping the cells into lOml PBS and centrifuging at.
1 OOOrpm for 5 minutes at room temperature. The resulting cERG/pcDNA3.1 (-) transfected HEK-293 cell pellet was stored at -80°C until required.
,. . ,: . ,. . , , .._ Preparation:of .membranes from HEK-293 cells express,in.g ,human or canine ERG.
Cell membrane .fractions were prepared.from frozen aliquots of cells., All procedures were carried out at 4°C ,unless otherwise stated. .Frozen aliquots of cells were,.thawed at room temperature.. and resuspe.nded in assay buffer (e..g. 50mM Tris.Cl, IOmM. KCI,, 1, to.."1.2mM MgCl2, pH7.4, or, 50mM Tris.Cl, lOmM KCI,, pH7.4). . The cells, were then disrupted by homogenisation in an .Omni LabTek homogenises at .20,000 rpm for seconds. The: homogenate was. centrifuged for 20 minutes at 48,OOOxg (4°C,, Sorvall RCSB centrifuge) . and the supernatant removed. The resulting pellets were resuspended in assay buffer and homogenised as above for 10 seconds. The pellets were".collected by, centrifugation and .the final pellet resuspended in assay buffer.
Protein content was determined using a Coomassie Blue based protein assay kit.
Aliquots were stored. at.,-80°C until needed; when. stored. in these conditions, the binding ability of the, cell membrane fractions proved to, be stable for at least 4 .months. _ , .
Example.2: Filter binding,assaywith [3H]-dofetillide ,, ,. ., [3HJ-dofetilide (80-83 Ci/mmol) was synthesized by catalytic tritiation (a custom service provided, for example, by. Amersham Life Science). However, other detectable labels known to the skilled person can be used instead of 3H, e.g. fluorescent tags, other radiolabels, antibodies etc. .
On the day.of the assay, test compounds were dissolved at 1 mM in 50% DMSO or 100% DMSO, and then diluted to the desired concentrations (e.g. up to 100~.M, or up to the boundaries of solubility for the compound) in assay buffer. The final DMSO
concentration in assay incubations is preferably 1.0 to 1.5% or less for optimal assay conditions.
Incubations included membrane homogenate at 50Ng/ml in assay buffer (50 mM
Tris.Cl, lOmM KCI, I.OmM to l.2mM MgCl2, pH7.4) unless otherwise indicated, [3H]-dofetilide (4 to 7nM) and test compound or mixture of test compounds or control vehicle. Filtration assays were incubated at room temperature for 90 minutes.
Non-specific binding was determined in the presence of 10 ~.M dofetilide and was usually les.s_,than. l5 % of total binding. Bound ligand was separated from free ligand by rapid filtration ,.through GF/B ,glass , fibre filter ,mats using, for example,; , a Brande.l. .cell harvester, ,o,r onto GF/B .Unifilter 96-.well ,filter. plates _(Packard).
using a, Packard Filtermate 96 harvester. Filter mats and plates were pre-soaked in 5% PEI
(w/v) for 60 minutes and washed after harvesting with 3 x 1 ml washes of ice-cold assay buffer.
U,nifilter plates,were,air dried for a minimum of 1.5 hours at 37.°C, prior to the addition of Microscint-0... (Packard). Bound [3H]-dofetilide was determined. by liquid scintillation spectroscopy.. .using , an appropriate, counter, for example in a Packard TopCount Scintillation;Cou,nter:(NXT,Counte.r),or.Wallac Counter. (Trilux) for Unifilter plates and in a Wa,Ilac,.B.ig Spot Counter,when filter mats were used. , ._, ,".,,., : : ; : ; : '::: v. . - .. ~ . , _ ., , .
In. each experiment, .triplicate assays were routinely, performed and the.
data were ;:,..:r.:. : ...... ; ::.... ~ .. , .. :.. .. .:.:,: ....:
averaged. Specific binding was analysed by nonlinear regression fit using ,GraphPad Prism software (GraphPad, San Diego). ICSO values were derived from a~ 4 parameter . . ~ ... . , ;
logistic fit, using PRISM and converted, to Ki values by use of the Cheng &
Prusoff equation; .IC2o values were extrapolated from the graph.
. . ... ~ .:
Example 3: Scintillation proximity assay The scintillation proximity assay (SPA) was carried out in assay buffer consisting of 50mM Tris.Cl, lOmM KCI, I.OmM to l.2mM MgCl2, pH7.4, or using assay buffer consisting, of 50mM Tris base, lOmM KCI, pH7.4. Bead to membrane binding was assessed to determine the optimum bead type for the cell line used. YSi wheatgerm agglutinin beads were used with cell membranes derived from the January HEK-hERG expressing cell line; YSi polylysine beads were used in studies using membranes derived from CeII Line 15 (HEK-293 hERG expressing cell line). ~ Conditions were optimised with respect to bead and cell membrane homogenate concentration, prior to characterising ERG pharmacology. The incubations (200 NI total per well for 96 well plates and 60 p1 total per well for 384 well plates) included 25 pg of cell membrane 5 homogenate per mg of bead. The membrane homogenate was precoupled with the YSi Wheatgerm Agglutinin or YSi polylysine bead suspension at 4°C on a roller shaker for approximately 2 hours. For competition binding assays, membrane homogenate bead suspension was incubated in white clear bottom 96 or 384 well plates with 5nM
[3H]-dofetilide in the absence and presence of competitor i.e. the test compound or 10 mixture of test compounds. The plates were incubated at room temperature and shaken for approximately,1 ,hour. : Beads were allowed .to settle for a minimum of, 30 .,. . .. .~ : , . .. . ...
minutes.,.before, plates ,were counted .for retained radioactivity on a TopCount ., NXT
scintillation. counter. Nonspecific binding i.e. background.count, was determined by the addition of lOwM dofetilide.. Background counts were usually. less than 15% of the total 15 binding. For saturation: studies, specific binding of .[3.H]-dofetilide was determined over.a range , of . concentrations (5, to .500nM) in the. absence or presence. of cold ..(i.e.
unlabelled) 1ONM dof_etilide. , . , , Example.4: Assay. optimisation a) Effect of Hepes- and Tris-based buffers on dofetil.ide binding : , To optimise , the specific binding of dofetilide , to homogenates of cell membranes containing ERG, the interaction of [3H]-dofetilide with the cell membrane preparation was .examined in the presence of Hepes-based buffer (25mM Hepes, 135mM . NaCI, 5mM KCI, 1 mM MgS04, 50mM CaCl2, pH7.4) and Tris-based buffer (50mM Tris.Cl, 1 OmM KCI, 1 mM or 1.2mM MgCl2). Comparison of the specific binding in these buffers revealed that percentage specific binding was similar in both Tris-based and Hepes-based buffers. However, as shown in Table 1, specific counts were twice as high in the presence of Tris-based buffer compared to those detected in Hepes-based buffer.
agarose gel using a 1 OObp DNA ladder marker at 1 OOV for 25 minutes in 1 X
TAE buffer and visualised using UV light. Putative positive clones were identified and samples from these PCR .reaction. mixtures were. run on a second separate 1.5% agarose gel at 100V for one hour to examine the sizes of the PCR products.
, The mini-cultures which. gave an amplified a PCR product were each seeded from the original deep-well 96-well plate into sterile tubes with 5m1 LB broth containing 50~g/ml ampicillin, and incubated. at 37°C overnight at 200rpm. The overnight cultures were then used to produce mini-preps of DNA using a QIAgen Miniprep Kit. The resulting DNA
was subjected .to an Xhol and .EcoRl. double digestion to check.,for. the .presence. of cERG/pcDNA3...1 (-).. The restriction digest was analysed via a 1 % agarose gel run. for 1 hour at 1:00V with .1 kb DNA ,ladder markers (201 sample loading, with 2~.1 gel, loading solution):, ,Further ..restriction :digestion analysis was performed to confirm that ,the purified plasmids from, the transformants were indeed cERG/pcDNA3.1 (-).
Untransfected HEK-293 cells were routinely maintained in 50m1 Minimum Essential Medium (MEM)supplemented with 1~0% (v/v) foetal calf serum (FCS), 2mM ~L-glutamine, 1 m~M sodium pyruvate and 1 mM non-essential amino acids. Cells were seeded irito 225cm2 ventilated cap flasks - and were maintained in a humidified atmosphere containing 5% C02. The HEK-293 cells used in this study were between passage' numbers 39-48. Cells were passaged typically every Three days iri a ratio of 1:3 froim a flask of 80-90% confluency; fresh medium was added~after washing twice with l Orril PBS and dissociating from the flask using cell 'dissociation fluid. ~ ~ ' Tfie cERGIpcDNA3.1 (-) construct was transfected into HEK-293 cells grown to 80-95%
confluency , in 225cm2 ventilated flasks using the following method. Endotoxin free cERG/pcDNA3.1(-). DNA (94~g) and Lipofectamine2000 (Gibco BRL) (94~.g), were added to 2.25m1 of OPTIMEM-I media (Gibco BRL) in sterile 10m1 centrifuge tubes;
mixing was carried out after incubation at room temperature for five minutes.
The uporecramme~uuulDNA/OPTIMEM-I mix was then incubated at room temperature for twenty minutes before the addition of a further 10.5m1 OPTIMEM-I. HEK-293 cells were washed with 10m1 PBS and the Lipofectamine2000/DNA/OPTIMEM-I mixture added and incubated for 3.5 hours at 37°C in a humidified atmosphere containing 5% C02.
After incubation, 50m1 of MEM (supplemented with 10% (v/v) FCS, 2mM L-glutamine, 1 mM sodium pyruvate and 1 mM non-essential amino acids) was added. The. HEK-cells were incubated for 24 hours at 37.°C. . Transfected.cells were.harvested after..24 hours by washing with PBS, scraping the cells into lOml PBS and centrifuging at.
1 OOOrpm for 5 minutes at room temperature. The resulting cERG/pcDNA3.1 (-) transfected HEK-293 cell pellet was stored at -80°C until required.
,. . ,: . ,. . , , .._ Preparation:of .membranes from HEK-293 cells express,in.g ,human or canine ERG.
Cell membrane .fractions were prepared.from frozen aliquots of cells., All procedures were carried out at 4°C ,unless otherwise stated. .Frozen aliquots of cells were,.thawed at room temperature.. and resuspe.nded in assay buffer (e..g. 50mM Tris.Cl, IOmM. KCI,, 1, to.."1.2mM MgCl2, pH7.4, or, 50mM Tris.Cl, lOmM KCI,, pH7.4). . The cells, were then disrupted by homogenisation in an .Omni LabTek homogenises at .20,000 rpm for seconds. The: homogenate was. centrifuged for 20 minutes at 48,OOOxg (4°C,, Sorvall RCSB centrifuge) . and the supernatant removed. The resulting pellets were resuspended in assay buffer and homogenised as above for 10 seconds. The pellets were".collected by, centrifugation and .the final pellet resuspended in assay buffer.
Protein content was determined using a Coomassie Blue based protein assay kit.
Aliquots were stored. at.,-80°C until needed; when. stored. in these conditions, the binding ability of the, cell membrane fractions proved to, be stable for at least 4 .months. _ , .
Example.2: Filter binding,assaywith [3H]-dofetillide ,, ,. ., [3HJ-dofetilide (80-83 Ci/mmol) was synthesized by catalytic tritiation (a custom service provided, for example, by. Amersham Life Science). However, other detectable labels known to the skilled person can be used instead of 3H, e.g. fluorescent tags, other radiolabels, antibodies etc. .
On the day.of the assay, test compounds were dissolved at 1 mM in 50% DMSO or 100% DMSO, and then diluted to the desired concentrations (e.g. up to 100~.M, or up to the boundaries of solubility for the compound) in assay buffer. The final DMSO
concentration in assay incubations is preferably 1.0 to 1.5% or less for optimal assay conditions.
Incubations included membrane homogenate at 50Ng/ml in assay buffer (50 mM
Tris.Cl, lOmM KCI, I.OmM to l.2mM MgCl2, pH7.4) unless otherwise indicated, [3H]-dofetilide (4 to 7nM) and test compound or mixture of test compounds or control vehicle. Filtration assays were incubated at room temperature for 90 minutes.
Non-specific binding was determined in the presence of 10 ~.M dofetilide and was usually les.s_,than. l5 % of total binding. Bound ligand was separated from free ligand by rapid filtration ,.through GF/B ,glass , fibre filter ,mats using, for example,; , a Brande.l. .cell harvester, ,o,r onto GF/B .Unifilter 96-.well ,filter. plates _(Packard).
using a, Packard Filtermate 96 harvester. Filter mats and plates were pre-soaked in 5% PEI
(w/v) for 60 minutes and washed after harvesting with 3 x 1 ml washes of ice-cold assay buffer.
U,nifilter plates,were,air dried for a minimum of 1.5 hours at 37.°C, prior to the addition of Microscint-0... (Packard). Bound [3H]-dofetilide was determined. by liquid scintillation spectroscopy.. .using , an appropriate, counter, for example in a Packard TopCount Scintillation;Cou,nter:(NXT,Counte.r),or.Wallac Counter. (Trilux) for Unifilter plates and in a Wa,Ilac,.B.ig Spot Counter,when filter mats were used. , ._, ,".,,., : : ; : ; : '::: v. . - .. ~ . , _ ., , .
In. each experiment, .triplicate assays were routinely, performed and the.
data were ;:,..:r.:. : ...... ; ::.... ~ .. , .. :.. .. .:.:,: ....:
averaged. Specific binding was analysed by nonlinear regression fit using ,GraphPad Prism software (GraphPad, San Diego). ICSO values were derived from a~ 4 parameter . . ~ ... . , ;
logistic fit, using PRISM and converted, to Ki values by use of the Cheng &
Prusoff equation; .IC2o values were extrapolated from the graph.
. . ... ~ .:
Example 3: Scintillation proximity assay The scintillation proximity assay (SPA) was carried out in assay buffer consisting of 50mM Tris.Cl, lOmM KCI, I.OmM to l.2mM MgCl2, pH7.4, or using assay buffer consisting, of 50mM Tris base, lOmM KCI, pH7.4. Bead to membrane binding was assessed to determine the optimum bead type for the cell line used. YSi wheatgerm agglutinin beads were used with cell membranes derived from the January HEK-hERG expressing cell line; YSi polylysine beads were used in studies using membranes derived from CeII Line 15 (HEK-293 hERG expressing cell line). ~ Conditions were optimised with respect to bead and cell membrane homogenate concentration, prior to characterising ERG pharmacology. The incubations (200 NI total per well for 96 well plates and 60 p1 total per well for 384 well plates) included 25 pg of cell membrane 5 homogenate per mg of bead. The membrane homogenate was precoupled with the YSi Wheatgerm Agglutinin or YSi polylysine bead suspension at 4°C on a roller shaker for approximately 2 hours. For competition binding assays, membrane homogenate bead suspension was incubated in white clear bottom 96 or 384 well plates with 5nM
[3H]-dofetilide in the absence and presence of competitor i.e. the test compound or 10 mixture of test compounds. The plates were incubated at room temperature and shaken for approximately,1 ,hour. : Beads were allowed .to settle for a minimum of, 30 .,. . .. .~ : , . .. . ...
minutes.,.before, plates ,were counted .for retained radioactivity on a TopCount ., NXT
scintillation. counter. Nonspecific binding i.e. background.count, was determined by the addition of lOwM dofetilide.. Background counts were usually. less than 15% of the total 15 binding. For saturation: studies, specific binding of .[3.H]-dofetilide was determined over.a range , of . concentrations (5, to .500nM) in the. absence or presence. of cold ..(i.e.
unlabelled) 1ONM dof_etilide. , . , , Example.4: Assay. optimisation a) Effect of Hepes- and Tris-based buffers on dofetil.ide binding : , To optimise , the specific binding of dofetilide , to homogenates of cell membranes containing ERG, the interaction of [3H]-dofetilide with the cell membrane preparation was .examined in the presence of Hepes-based buffer (25mM Hepes, 135mM . NaCI, 5mM KCI, 1 mM MgS04, 50mM CaCl2, pH7.4) and Tris-based buffer (50mM Tris.Cl, 1 OmM KCI, 1 mM or 1.2mM MgCl2). Comparison of the specific binding in these buffers revealed that percentage specific binding was similar in both Tris-based and Hepes-based buffers. However, as shown in Table 1, specific counts were twice as high in the presence of Tris-based buffer compared to those detected in Hepes-based buffer.
Table 1. Comparative effects of Tris-based and Hepes-based buffers on [3H]-dofetilide binding to cell membrane homogenate expressing hERG.
Buffer 25mM HEPES free acid 50mM Tris 135mM NaCI, 5mM KCI lOmM KCI and 1 mM MgS04, 50 pM CaCl2 1.0 or 1.2mM MgCl2 pH 7.4 at room temp pH 7.4 at room temp Total Binding (ccpm) 8510 ~ 669 19627 ~ 1189 Non-specific Binding (ccpm) 321 ~ 27 315 ~ 23 Specific Binding (ccpm) 8189 19312 Specific Binding 96 98 Total: and non-specific biriding data represent arithmetic mean' ~ standard error rriean of '14 ~individual:wells.per buffer split-over two assays, performed at a protein concentration of 75Ng/ml and a mean [3H]-dofetilide concentration of 6.7nM. Incubation was carried out~for 60 minutes at room temperature. ccpm=corrected counts,per ri~inute., , So that the maximum specific binding.window could be achieved; the assay buffer used in 'Examples: 1 ..to 8 was the Tcis-based incubation buffer (50mM Tris:Cl,' lOmM KCI, 1 mM ~MgCl2).~ r Additionally, experiments were performed to optimise the cell membrane protein,concentration a:nd bead concentration for filter and SPA binding assays.
b) Saturation binding .:..-: ~ ., ::. ~:; . .~~.~ ._. ;; ,-, . :. :. ,_ .... .:., ~-..:: : .... :
.,:.. :...
'fim~e~'cou~rse's ' viie~re ~ perfornied ~to deterriiiiie .~ ~optiinal incubation time for binding activities. ~ Incubation times' were similar for both filter binding and SPA
assays. The filter binding assay reached equilibrium in ~90 minutes, SPA required 60 minutes. [3H]-dofetilide binding. to ERG in.. both filter .binding: ~and scintillation proximity assays ,was saturable. with a Kp of 5.08 ~ 1.OnM for filter binding.and Ko values of 8.9 +0.6nM and 9..1,,~1~.8nM for .96 .and 384 format scintillation proximity, assays, respectively (Figure 1 a-c, with. Fig,:.1 a..showing the results of the filter binding assay, Fig. .1 b the results of the SPA in 96-well format, and Fig. 1 c showing the results of the SPA in 384 well format).
Non-linear curve fitting of this data indicated that binding was to a single site. A BmaX of 7.4 ~ 0.7pmol/mg protein for [3H]-dofetilide was obtained -from filter binding (Figure 1 ).
As scintillation proximity assays do. not give an accurate determination of dpm (disintegrations.per minute) values, a BmaX is not quoted for.SPA.
Buffer 25mM HEPES free acid 50mM Tris 135mM NaCI, 5mM KCI lOmM KCI and 1 mM MgS04, 50 pM CaCl2 1.0 or 1.2mM MgCl2 pH 7.4 at room temp pH 7.4 at room temp Total Binding (ccpm) 8510 ~ 669 19627 ~ 1189 Non-specific Binding (ccpm) 321 ~ 27 315 ~ 23 Specific Binding (ccpm) 8189 19312 Specific Binding 96 98 Total: and non-specific biriding data represent arithmetic mean' ~ standard error rriean of '14 ~individual:wells.per buffer split-over two assays, performed at a protein concentration of 75Ng/ml and a mean [3H]-dofetilide concentration of 6.7nM. Incubation was carried out~for 60 minutes at room temperature. ccpm=corrected counts,per ri~inute., , So that the maximum specific binding.window could be achieved; the assay buffer used in 'Examples: 1 ..to 8 was the Tcis-based incubation buffer (50mM Tris:Cl,' lOmM KCI, 1 mM ~MgCl2).~ r Additionally, experiments were performed to optimise the cell membrane protein,concentration a:nd bead concentration for filter and SPA binding assays.
b) Saturation binding .:..-: ~ ., ::. ~:; . .~~.~ ._. ;; ,-, . :. :. ,_ .... .:., ~-..:: : .... :
.,:.. :...
'fim~e~'cou~rse's ' viie~re ~ perfornied ~to deterriiiiie .~ ~optiinal incubation time for binding activities. ~ Incubation times' were similar for both filter binding and SPA
assays. The filter binding assay reached equilibrium in ~90 minutes, SPA required 60 minutes. [3H]-dofetilide binding. to ERG in.. both filter .binding: ~and scintillation proximity assays ,was saturable. with a Kp of 5.08 ~ 1.OnM for filter binding.and Ko values of 8.9 +0.6nM and 9..1,,~1~.8nM for .96 .and 384 format scintillation proximity, assays, respectively (Figure 1 a-c, with. Fig,:.1 a..showing the results of the filter binding assay, Fig. .1 b the results of the SPA in 96-well format, and Fig. 1 c showing the results of the SPA in 384 well format).
Non-linear curve fitting of this data indicated that binding was to a single site. A BmaX of 7.4 ~ 0.7pmol/mg protein for [3H]-dofetilide was obtained -from filter binding (Figure 1 ).
As scintillation proximity assays do. not give an accurate determination of dpm (disintegrations.per minute) values, a BmaX is not quoted for.SPA.
c) Comparison of SPA and filter binding techniques A comparison of SPA and filter binding techniques revealed excellent concordance of results. Affinity values displayed excellent correlation between the two assay types and the rank order of compound affinity is identical, as is shown in Figure 2 (correlation plots comparing pK values obtained from filter binding and SPA binding assays).
d) Competitive binding studies A. range- of compounds, including hERG blockers known to prolong the QT
interval in man, was examined for competitive .displacement., of. [3H]-dofetilide. E4031, dofetilide, terfenadine, and cisapride produced complete inhibition of specific binding with a range of calculated affinity values that are summarised in Table 2. -Table- 2.. ,Affinity; values for compounds tested- against [3H]-dofetilide filter , and. S,PA
binding. assaysao HERE. , . . . .
Corripound filter SPA 96 SPA 384 binding pK pK pK
~ " Dofefilide 8.22 0.04 8.05 0.54 8.26 0.12 E4031 ' , . . .. . 7.82 0.03 - 7.81 ~ ' 0.057.89 0.11 . -Terfenadine 7.53 0.09 7.75 0.07 7.72 t 0.41 Cisapride 7.34 0.05 7.15 0.04 7.55 t 0.22 - -_ ,.;Glibenclamide,< 5 - - , < 5 . , < 5 -, .
.
. .: D-Sotalo,l < 5 < 5 < 5 . -. . . ~ .
Data.-expressed as pK values (the negative logarithm of molar concentration of competing ligand to displace 50% of 5nM [3H]-dofetilide binding). Data are the mean of at least n = 3 experiments.
d) Competitive binding studies A. range- of compounds, including hERG blockers known to prolong the QT
interval in man, was examined for competitive .displacement., of. [3H]-dofetilide. E4031, dofetilide, terfenadine, and cisapride produced complete inhibition of specific binding with a range of calculated affinity values that are summarised in Table 2. -Table- 2.. ,Affinity; values for compounds tested- against [3H]-dofetilide filter , and. S,PA
binding. assaysao HERE. , . . . .
Corripound filter SPA 96 SPA 384 binding pK pK pK
~ " Dofefilide 8.22 0.04 8.05 0.54 8.26 0.12 E4031 ' , . . .. . 7.82 0.03 - 7.81 ~ ' 0.057.89 0.11 . -Terfenadine 7.53 0.09 7.75 0.07 7.72 t 0.41 Cisapride 7.34 0.05 7.15 0.04 7.55 t 0.22 - -_ ,.;Glibenclamide,< 5 - - , < 5 . , < 5 -, .
.
. .: D-Sotalo,l < 5 < 5 < 5 . -. . . ~ .
Data.-expressed as pK values (the negative logarithm of molar concentration of competing ligand to displace 50% of 5nM [3H]-dofetilide binding). Data are the mean of at least n = 3 experiments.
example ~: rrea~ction of QT interval prolongation effect of compounds in man The IC2o values generated from competitive displacement of [3H]-dofetilide binding using the assay of the invention are comparable to the free drug concentration associated with QT prolongation in man as is shown in Figure 3 for a range of compounds, including E-4031 (Figure 3a), dofetilide (Figure 3b), terfenadine (Figure 3c) and cisapride (Figure 3d). For each compound, the inhibition of dofetilide binding in the binding assay (filter binding technique), and in a hERG patch clamp assay is compared with the concentration of free drug associated with QT interval prolongation in man ,(Fu.liki,A,:,et al. (1994) Cardiovascular Pharmacol, 23: 374-378; Van Haarst AD et al.
(1998) :Clin Pharmacol. The.r. 64: 542-546; Honig..PK, et. al. (1993) J.A.M.A.
269: .1513-1518).
The . ERG patch clamp assay ,provides a measure of, the current through the.
ERG
channel and indicates.the. number of ion channels present in a cell. However, due to the"phenomena of. state dependent block observed in, patch clamp studies (Walker, B.D.~ et al (1,999,) British. J.. Pharmacol 128, 444-450) exhibited by a number of known hERG blockers with.the propensity to prolong the QT interval ,in vivo,.the ligand binding assay provides..a .better ,predictor of in vivo QT prolongation effect of a drug than. the hER,G patch,clamp technique..,(Figure 3d). , , ' .. f. ; ~ ~~'.,,._ ~ ....., . .:.' ;
To.assess whether a compound or mixture of compounds is likely to prolong the QT
interval in the electrocardiogram in man, the following steps are carried out:
a) . A binding assay is carried out according to the invention, for, example as, as described .in Example 2 or Example 3, to test the affinity of the compound or .. ....~ . ; . ~ . .
mixture of compounds for ERG, preferably hERG or cERG;
_. ;: ~ : : :. . . . a b). The IC2o is obtained, e.g. as described at the end of Example 2; the IC2o being . the real or predicted free drug, concentration at which QT prolongation occurs in man;
c) ' The IC2o value is compared with the free drug concentration required for the desired therapeutic~effect of the compound in vivo.
(1998) :Clin Pharmacol. The.r. 64: 542-546; Honig..PK, et. al. (1993) J.A.M.A.
269: .1513-1518).
The . ERG patch clamp assay ,provides a measure of, the current through the.
ERG
channel and indicates.the. number of ion channels present in a cell. However, due to the"phenomena of. state dependent block observed in, patch clamp studies (Walker, B.D.~ et al (1,999,) British. J.. Pharmacol 128, 444-450) exhibited by a number of known hERG blockers with.the propensity to prolong the QT interval ,in vivo,.the ligand binding assay provides..a .better ,predictor of in vivo QT prolongation effect of a drug than. the hER,G patch,clamp technique..,(Figure 3d). , , ' .. f. ; ~ ~~'.,,._ ~ ....., . .:.' ;
To.assess whether a compound or mixture of compounds is likely to prolong the QT
interval in the electrocardiogram in man, the following steps are carried out:
a) . A binding assay is carried out according to the invention, for, example as, as described .in Example 2 or Example 3, to test the affinity of the compound or .. ....~ . ; . ~ . .
mixture of compounds for ERG, preferably hERG or cERG;
_. ;: ~ : : :. . . . a b). The IC2o is obtained, e.g. as described at the end of Example 2; the IC2o being . the real or predicted free drug, concentration at which QT prolongation occurs in man;
c) ' The IC2o value is compared with the free drug concentration required for the desired therapeutic~effect of the compound in vivo.
If the free drug concentration required for the desired therapeutic effect of the compound is within 10 to 30 fold of the IC2o of the compound in the assay of the invention, the compound is highly likely to cause QT interval prolongation in man.
Example 6: Comparison of dofetilide binding assay carried out HEK-293 cells transfected with cERG or hERG.
The dofetilide binding assay was carried out as described Example 2 using HEK
cells transfected with either human ERG or canine ERG. The results are shown in figure,4, from which it can be seen that the ICSO for dofetilide is similar for canine and human ERG,,being.13.9nM.and 15.6nM respectively. IC2o values for dofetilide were 1.92nM and 2.15nM fo,r canine and human ERG, respectively. . , , Example 7: Comparison of terfenadine competition assay using HEK-293 cells w transfected with~cERG or hERG .
The dofetilide binding assay was carried out using terfenadine as the test compound.
Transiently trarisfected cERG HEK=293 cell membranes (200~,g/well), or stable hERG
HEK-293' ' cell r meimbrahes (1 OO~.g/well) were 'iricubated with twelve different cortcen~trations~ ~~of terfenadine ' and 5nM [3H]-dofetilide for ~90 minutes at room ferripe~ature.' Total and"non-specific binding were i-neasured by incubating with '10%
DIVISO ~. arid ~' 1 OfiNt unlabelled dofetilide- to a total assay volume of 200,1. The membranes were harvested by filtration with a Packard Unifilter cell harvester and radioactivity (cprn)ywas measured.. Two saturation experiments were carried,out each fo,r;BERG.~and;:hERG;y.expressing',cell membrane samples. Each experiment was carried out in triplicate. Figure 5 shows 'the mean values of the experiments for each cell type (cERG or hERG 'transfected) and indicates that the. ICSO for terfenadine is similar ,for cERG:and; hERG,, being .77.2,nM:.:and 88.;9nM respectively.. IC2o,~yalues for;terfenadine were 10.7nM_ and 12.3nM .for canine and human ERG, respectively.
' ' ~ ' . .. _ . ..
txampie ~: c;omparison of E4031 competition assay in HEK-293 cells transfected with cERG or hERG
The dofetilide binding assay was carried out using E4031 as the test compound.
5 Transiently transfected cERG HEK-293 cell membranes (200~.g/well) or stable hERG
HEK-293 cell membranes (100~,g/well) were incubated with twelve different concentrations of E4031 and 5nM [3H]-dofetilide for 90 minutes at room temperature.
Total and non-specific binding values were measured by incubation with 10%
DMSO
and 10~.M unlabelled dofetilide in a total assay volume of 200.1. The membranes were 10 harvested-by filtration with a Packard Unifilter cell harvester and radioactivity (cpm) was measur..ed:~" Two saturation experiments were carried :out each for cERG and hERG
expressing cell :membrane samples. Each experiment was carried out in triplicate.
Figure 6 shows the mean values of the experiments for each cell membrane type (cERG or :hERG transfected) and. indicates that the IC5o for E4031 is similar for-.cERG
15 and hERG, 'be,ing 27.3 nM and 35.4 nM respectively.. IC2o values for E4031.
were 3.8 nM and.4.9 nM for canine and human ,ERG, respectively.
When_ICSO, (or LC2o) values are compared for the compounds.tested, they were found to be,.very .similar. for cERG and hERG. This indicates that either hERG, or cERG
can be 20 ,used in the, assay of the invention to predict the onset of QT
prolongation in man.
'. ':w'~r~'. ":''. ~:~..~ :.. ~ .~; . ~ ~ . ' , ..:' , . . : ~ I .: '~ ~. .. .
. ,_ _ ,,, . .. ~i.. : ; .,' ~ ~ ..'. ... '. w : . '. ~ : ..: ~ , <; r , . ~. ~ . .
~~ : : : ~, .. ., ~: : ~, ~ . : . ~ . . ; . . : .. - , y . . y, ' _ :, , . '..
' -' : , ' .. , Examp.le,9: Further, assay optimisation studies . '~;:: ~.-. _ ..: ~ - : :~ ..~ : . : ..:~
T.o_further o,ptimise_the assay for.specific,binding of dofetilide to homogenates of cell. ,.' membrane containing hERG, the interaction of [3H]-dofetilide with cell membrane , preparations was examined in the SPA assay format using a Tris based buffer containing either KCI or MgCl2. SPA assays were performed according to example 3 in 50mM Tris.C.l, 1 OmM KCI at pH7.4 or in 50mMTris.Cl, 1 mM MgCl2 at p.H 7.4 as the assay buffer. , Assays were performed using dofetilide or terodiline as the test , compound. Comparison of specific binding detected in these buffer conditions revealed that specific binding was not observed when the assay buffer used was 50mMTris.Cl, 1 mM MgCl2 at pH 7.4; specific binding was observed in assay buffer consisting of 50mM Tris.Cl, lOmM KCI at pH7.4. For the assays carried out in 50mM Tris.Cl, 1 OmM
KCI at pH7.4 as the assay buffer the ICSO and IC2o values were generated for each test compound. 1 ne mean ICSO value for dofetilide was 8.6910.45nM, the mean ICSO
value for terodiline was 1.87 t 0.00 ~M. The mean IC2o value for dofetilide was 1.2nM, the mean IC2o value for terodiline was 0.248,uM.
Sequence Listing Information <110> Pfizer Inc (CA, EP except EP(GB), JP, US. Pfizer Ltd (EP(GB)) <120> Assay <130> PCS 22042 <150> GB 0121440.2 ~< 15.1 > :2001-09-04 .. 4 . .. ~ : , . . . . - . . . . .
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<150>~ US (07323973 . . . ~ , <~:1~:5:1~>.200.1..=09-20 .;.:: - .
<160> 3 w::~.:~-.:_:__s='~..i-=s=--:=~ :w;_~.~ ~;<~::~:
<170> FastSEQ for Windows Version 4.0 <210> '1 2y ~' ' °~ '' "' . ' ' ; : : . - . .
<211 > 20 <212> DNA' <213> :Canine <400> 1 .
accacatcca ccaggcacag 20 . V -'. , '.. . ~~,, ~ y ;.'.. ....
. .. ~. ..
<2.10> 2;.; , . , <211 > 25 '. . .~ .
r, 1Y
<2'12> ~DNA~ ~ ' <213> pcDNA3.1 (-) vector ' -<400> 2 . , cccaagctgg~ctagcgttta aacgg~ ~ ' 25 <210> 3.
<211 > 23 .
<212> DNA
<213> pSP73 vector <400> 3 taatacgact cactataggg aga 23 <110> Pfizer Inc (CA, EP except EP(GB), JP, US.) Pfizer Ltd (EP(GB)).
<120> Assay <130> PCS 22042 <150> GB 0121440.2 < 151 > 2001-09-04 <150> US 60/323973 <151> 2001-09-20 <160> 3 <170> FastSEQ for Windows Version 4.0 <210> 1 <211 > 20 <212> DNA
<213> Canine <400> 1 accacatcca ccaggcacag 20 <210> 2 <211 > 25 <212> DNA
<213> pcDNA3.1 (-) vector <400> 2 cccaagctgg ctagcgttta aacgg 25 <210> 3 <211 > 23 <212> DNA
<213> pSP73 vector <400> 3 taatacgact cactataggg aga 23
Example 6: Comparison of dofetilide binding assay carried out HEK-293 cells transfected with cERG or hERG.
The dofetilide binding assay was carried out as described Example 2 using HEK
cells transfected with either human ERG or canine ERG. The results are shown in figure,4, from which it can be seen that the ICSO for dofetilide is similar for canine and human ERG,,being.13.9nM.and 15.6nM respectively. IC2o values for dofetilide were 1.92nM and 2.15nM fo,r canine and human ERG, respectively. . , , Example 7: Comparison of terfenadine competition assay using HEK-293 cells w transfected with~cERG or hERG .
The dofetilide binding assay was carried out using terfenadine as the test compound.
Transiently trarisfected cERG HEK=293 cell membranes (200~,g/well), or stable hERG
HEK-293' ' cell r meimbrahes (1 OO~.g/well) were 'iricubated with twelve different cortcen~trations~ ~~of terfenadine ' and 5nM [3H]-dofetilide for ~90 minutes at room ferripe~ature.' Total and"non-specific binding were i-neasured by incubating with '10%
DIVISO ~. arid ~' 1 OfiNt unlabelled dofetilide- to a total assay volume of 200,1. The membranes were harvested by filtration with a Packard Unifilter cell harvester and radioactivity (cprn)ywas measured.. Two saturation experiments were carried,out each fo,r;BERG.~and;:hERG;y.expressing',cell membrane samples. Each experiment was carried out in triplicate. Figure 5 shows 'the mean values of the experiments for each cell type (cERG or hERG 'transfected) and indicates that the. ICSO for terfenadine is similar ,for cERG:and; hERG,, being .77.2,nM:.:and 88.;9nM respectively.. IC2o,~yalues for;terfenadine were 10.7nM_ and 12.3nM .for canine and human ERG, respectively.
' ' ~ ' . .. _ . ..
txampie ~: c;omparison of E4031 competition assay in HEK-293 cells transfected with cERG or hERG
The dofetilide binding assay was carried out using E4031 as the test compound.
5 Transiently transfected cERG HEK-293 cell membranes (200~.g/well) or stable hERG
HEK-293 cell membranes (100~,g/well) were incubated with twelve different concentrations of E4031 and 5nM [3H]-dofetilide for 90 minutes at room temperature.
Total and non-specific binding values were measured by incubation with 10%
DMSO
and 10~.M unlabelled dofetilide in a total assay volume of 200.1. The membranes were 10 harvested-by filtration with a Packard Unifilter cell harvester and radioactivity (cpm) was measur..ed:~" Two saturation experiments were carried :out each for cERG and hERG
expressing cell :membrane samples. Each experiment was carried out in triplicate.
Figure 6 shows the mean values of the experiments for each cell membrane type (cERG or :hERG transfected) and. indicates that the IC5o for E4031 is similar for-.cERG
15 and hERG, 'be,ing 27.3 nM and 35.4 nM respectively.. IC2o values for E4031.
were 3.8 nM and.4.9 nM for canine and human ,ERG, respectively.
When_ICSO, (or LC2o) values are compared for the compounds.tested, they were found to be,.very .similar. for cERG and hERG. This indicates that either hERG, or cERG
can be 20 ,used in the, assay of the invention to predict the onset of QT
prolongation in man.
'. ':w'~r~'. ":''. ~:~..~ :.. ~ .~; . ~ ~ . ' , ..:' , . . : ~ I .: '~ ~. .. .
. ,_ _ ,,, . .. ~i.. : ; .,' ~ ~ ..'. ... '. w : . '. ~ : ..: ~ , <; r , . ~. ~ . .
~~ : : : ~, .. ., ~: : ~, ~ . : . ~ . . ; . . : .. - , y . . y, ' _ :, , . '..
' -' : , ' .. , Examp.le,9: Further, assay optimisation studies . '~;:: ~.-. _ ..: ~ - : :~ ..~ : . : ..:~
T.o_further o,ptimise_the assay for.specific,binding of dofetilide to homogenates of cell. ,.' membrane containing hERG, the interaction of [3H]-dofetilide with cell membrane , preparations was examined in the SPA assay format using a Tris based buffer containing either KCI or MgCl2. SPA assays were performed according to example 3 in 50mM Tris.C.l, 1 OmM KCI at pH7.4 or in 50mMTris.Cl, 1 mM MgCl2 at p.H 7.4 as the assay buffer. , Assays were performed using dofetilide or terodiline as the test , compound. Comparison of specific binding detected in these buffer conditions revealed that specific binding was not observed when the assay buffer used was 50mMTris.Cl, 1 mM MgCl2 at pH 7.4; specific binding was observed in assay buffer consisting of 50mM Tris.Cl, lOmM KCI at pH7.4. For the assays carried out in 50mM Tris.Cl, 1 OmM
KCI at pH7.4 as the assay buffer the ICSO and IC2o values were generated for each test compound. 1 ne mean ICSO value for dofetilide was 8.6910.45nM, the mean ICSO
value for terodiline was 1.87 t 0.00 ~M. The mean IC2o value for dofetilide was 1.2nM, the mean IC2o value for terodiline was 0.248,uM.
Sequence Listing Information <110> Pfizer Inc (CA, EP except EP(GB), JP, US. Pfizer Ltd (EP(GB)) <120> Assay <130> PCS 22042 <150> GB 0121440.2 ~< 15.1 > :2001-09-04 .. 4 . .. ~ : , . . . . - . . . . .
' ~ .
<150>~ US (07323973 . . . ~ , <~:1~:5:1~>.200.1..=09-20 .;.:: - .
<160> 3 w::~.:~-.:_:__s='~..i-=s=--:=~ :w;_~.~ ~;<~::~:
<170> FastSEQ for Windows Version 4.0 <210> '1 2y ~' ' °~ '' "' . ' ' ; : : . - . .
<211 > 20 <212> DNA' <213> :Canine <400> 1 .
accacatcca ccaggcacag 20 . V -'. , '.. . ~~,, ~ y ;.'.. ....
. .. ~. ..
<2.10> 2;.; , . , <211 > 25 '. . .~ .
r, 1Y
<2'12> ~DNA~ ~ ' <213> pcDNA3.1 (-) vector ' -<400> 2 . , cccaagctgg~ctagcgttta aacgg~ ~ ' 25 <210> 3.
<211 > 23 .
<212> DNA
<213> pSP73 vector <400> 3 taatacgact cactataggg aga 23 <110> Pfizer Inc (CA, EP except EP(GB), JP, US.) Pfizer Ltd (EP(GB)).
<120> Assay <130> PCS 22042 <150> GB 0121440.2 < 151 > 2001-09-04 <150> US 60/323973 <151> 2001-09-20 <160> 3 <170> FastSEQ for Windows Version 4.0 <210> 1 <211 > 20 <212> DNA
<213> Canine <400> 1 accacatcca ccaggcacag 20 <210> 2 <211 > 25 <212> DNA
<213> pcDNA3.1 (-) vector <400> 2 cccaagctgg ctagcgttta aacgg 25 <210> 3 <211 > 23 <212> DNA
<213> pSP73 vector <400> 3 taatacgact cactataggg aga 23
Claims (17)
1. An assay comprising or consisting of the following steps:
(a) incubation of cells expressing ERG, or membranes derived from cells expressing ERG, or membranes derived from tissue expressing ERG, with labelled IKR blocker in assay buffer in the presence or absence of a test compound or a mixture of test compounds;
(b) determination of specifically bound labelled IKR blocker;
(c) calculation of the inhibition of labelled IKR blocker binding by the test compound or mixture of test compounds.
(a) incubation of cells expressing ERG, or membranes derived from cells expressing ERG, or membranes derived from tissue expressing ERG, with labelled IKR blocker in assay buffer in the presence or absence of a test compound or a mixture of test compounds;
(b) determination of specifically bound labelled IKR blocker;
(c) calculation of the inhibition of labelled IKR blocker binding by the test compound or mixture of test compounds.
2. An assay according to claim 1, wherein the assay buffer is a Tris based buffer containing KCI.
3. An assay according to claim. 2, wherein the assay buffer comprises or consists of from 30 to 100mM Tris Cl, from 5 to 20mM KCI, and optionally from 0.6 to 2.0mM
MgCI2.
MgCI2.
4. An assay according to claim 2, wherein the assay buffer comprises or consists of from 30 to 70mM Tris Cl, from 6 to 15mM KCI, and optionally from 0.6 to 1.6mM
MgCl2.
MgCl2.
5. An assay according to claim 2, wherein the assay buffer comprises or consists of from 40 to 60mM Tris Cl from 7.5 to 12.5mM KCI and optionally from 0.8 to 1.4mM MgCl2.
6. An assay according to claim 2, wherein the assay buffer comprises or consists of from 45 to 55mM Tris Cl, from 8.5 to 11.5mM. KCI and optionally from 0.9 to 1.3 mM MgCl2 or from 1.0 to 1.2mM MgCl2.
7. An assay according to claim 2, wherein the assay buffer comprises or consists of 50mM Tris and 10mM KCI.
8. An assay according to claim 2, wherein the assay buffer comprises or consists of 50mM Tris, 10mM KCI and 1.0mM MgCl2; or 50mM Tris, 10mM KCI and 1.2 mM
MgCl2.
MgCl2.
9. An assay according to any one of the preceding claims wherein the assay buffer is at a pH between pH7.2 and pH7.6 at room temperature.
10. An assay according to claim 9, wherein the assay buffer is at pH7.4.
11. An assay according to any one of the preceding claims wherein the ERG is human ERG.
12. An assay according to any one of the preceding claims, wherein the labelled IKR
blocker is labelled dofetilide or labelled MK-499.
blocker is labelled dofetilide or labelled MK-499.
13. An assay according to claim 12, wherein the labelled dofetilide or labelled MK-499 is radiolabelled.
14. An assay according to claim 13, wherein the radiolabel is tritium (3H).
15. An assay according to any one of the preceding claims having the following additional step(s):
(d) calculation of the IC20 for the test compound or mixture of test compounds, and optionally, (e) comparison of the IC20 value of the test compound or mixture of test compounds with the concentration required for the desired therapeutic effect of the. compound in vivo.
(d) calculation of the IC20 for the test compound or mixture of test compounds, and optionally, (e) comparison of the IC20 value of the test compound or mixture of test compounds with the concentration required for the desired therapeutic effect of the. compound in vivo.
16. An assay according to any one of the preceding claims wherein the assay is performed as a filter binding assay.
17. An assay according to anyone of claims 1 to 15 wherein, the assay is performed as a scintillation proximity assay.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0121440.2 | 2001-09-04 | ||
| GBGB0121440.2A GB0121440D0 (en) | 2001-09-04 | 2001-09-04 | Assay |
| US32397301P | 2001-09-20 | 2001-09-20 | |
| US60/323,973 | 2001-09-20 | ||
| PCT/IB2002/003618 WO2003021271A2 (en) | 2001-09-04 | 2002-09-04 | Affinity-assay for the human erg potassium channel |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2459265A1 true CA2459265A1 (en) | 2003-03-13 |
Family
ID=26246508
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002459265A Abandoned CA2459265A1 (en) | 2001-09-04 | 2002-09-04 | Affinity-assay for the human erg potassium channel |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP1423700A2 (en) |
| JP (1) | JP2005510691A (en) |
| BR (1) | BR0212295A (en) |
| CA (1) | CA2459265A1 (en) |
| IL (1) | IL160674A0 (en) |
| MX (1) | MXPA04002058A (en) |
| PL (1) | PL370539A1 (en) |
| WO (1) | WO2003021271A2 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE9701144D0 (en) † | 1997-03-27 | 1997-03-27 | Pharmacia & Upjohn Ab | Novel compounds, their use and preparation |
| EP0957073A1 (en) | 1998-05-12 | 1999-11-17 | Schwarz Pharma Ag | Novel derivatives of 3,3-diphenylpropylamines |
| DE10315917A1 (en) * | 2003-04-08 | 2004-11-18 | Schwarz Pharma Ag | Highly pure bases of 3,3-diphenylpropylamine monoesters |
| SE0302775D0 (en) * | 2003-10-20 | 2003-10-20 | Astrazeneca Ab | Chemical compound and assay |
| US7927820B2 (en) | 2004-07-20 | 2011-04-19 | Janssen Pharmaceutica Nv | Assay systems and methods for detecting molecules that interact with membrane channels |
| US7326772B2 (en) * | 2005-05-12 | 2008-02-05 | Penta Biotech, Inc. | Peptide for assaying hERG channel binding |
| EP1801200B1 (en) | 2005-12-21 | 2010-03-31 | F. Hoffmann-La Roche Ag | Stable cell lines expressing hERG |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2415295C (en) * | 2000-07-14 | 2010-02-16 | Merck & Co., Inc. | Radioligand and binding assay |
-
2002
- 2002-09-04 IL IL16067402A patent/IL160674A0/en unknown
- 2002-09-04 JP JP2003525303A patent/JP2005510691A/en not_active Withdrawn
- 2002-09-04 CA CA002459265A patent/CA2459265A1/en not_active Abandoned
- 2002-09-04 MX MXPA04002058A patent/MXPA04002058A/en not_active Application Discontinuation
- 2002-09-04 BR BR0212295-2A patent/BR0212295A/en not_active IP Right Cessation
- 2002-09-04 PL PL02370539A patent/PL370539A1/en not_active Application Discontinuation
- 2002-09-04 EP EP02762684A patent/EP1423700A2/en not_active Withdrawn
- 2002-09-04 WO PCT/IB2002/003618 patent/WO2003021271A2/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| BR0212295A (en) | 2004-10-13 |
| MXPA04002058A (en) | 2004-06-07 |
| WO2003021271A3 (en) | 2003-12-18 |
| JP2005510691A (en) | 2005-04-21 |
| WO2003021271A2 (en) | 2003-03-13 |
| PL370539A1 (en) | 2005-05-30 |
| IL160674A0 (en) | 2004-08-31 |
| EP1423700A2 (en) | 2004-06-02 |
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| EEER | Examination request | ||
| FZDE | Discontinued |