WO2007062028A2

WO2007062028A2 - Treatment of qt interval prolongation and diseases associated therewith

Info

Publication number: WO2007062028A2
Application number: PCT/US2006/045042
Authority: WO
Inventors: Lin Zhao; Arthur Brown; Glenn Kirsch; Antonio Lacerda
Original assignee: Tap Pharmaceutical Products, Inc.
Priority date: 2005-11-21
Filing date: 2006-11-20
Publication date: 2007-05-31
Also published as: EP1957064A4; WO2007062028A8; JP2009516691A; EP1957064A2; CA2630639A1; WO2007062028A3

Abstract

The present invention relates methods for treating QT interval prolongation and diseases associated therewith, such as, but not limited to, congenital long QT syndrome, acquired long QT syndrome, myocardial ischemia, heart failure, diabetes or stroke.

Description

TREATMENT OF QT INTERVAL PROLONGATION AND DISEASES

ASSOCIATED THEREWITH

Field of the Invention

The present invention relates to cardiology and, in particular, methods for treating QT interval prolongation and diseases associated therewith, such as, but not limited to, congenital long QT syndrome, acquired long QT syndrome, myocardial ischemia, heart failure, diabetes or stroke.

Background of the Invention

Ion channels are macromolecular aqueous protein tunnels that span cell membranes. A vast number of ion channels are known to exist. These channels generate and orchestrate a variety of electrical signals that pass through the brain, heart and muscle each second of life. Ion channels are classified based on the type of ion that they allow to pass — e.g. sodium, potassium, calcium or chloride and their gating properties. Often times there are different channels for each type of ion. The direction of ionic movement in an ion channel is governed by electrical and chemical concentration gradients. In many channels the movement of ions is controlled by gating structures that form the basis for a broad classification of gated ion channels into mechano-, voltage- and ligand-gated subtypes. Thus, ion channels are not opened continuously. Instead, they have "gates" which open briefly and then close again. The synchronized activity of gated ion channels within individual cells produces the complex and vital voltage waveforms characteristic of excitable tissues.

During the resting stage, cells maintain an electrical potential difference (voltage) across their cell membranes depending on whether the ion channels on their cell membrane are open or closed. Typically, the interior of cells (cytoplasm) is electrically negative relative to the extracellular fluid, so the cells are polarized during the resting stage. This electrical potential difference across the cell membrane is called the resting membrane potential. For cardiac muscle cells, the resting membrane potential is about -90 mV. Electrically excitable cells become excited when they are exposed to different stimuli which can cause the ion channels to open or close. The main types of stimuli that are known to change (or gate) ion channel activity are a change in the voltage across the membrane (i.e., voltage-gated channels), a mechanical stress (i.e., mechanically gated channels) or the binding of a ligand (i.e., ligand-gated channels). When a cell is excited, it undergoes a cycle of transmembrane potential change which is referred to as the action potential.

In the heart, the action potential in a heart ventricular cell comprises five phases. Phase 0 is the rapid depolarization phase when the cell membrane rapidly transits from the negative resting potential to a positive potential due to an almost exclusive influx of positively charged sodium ions into the cell. This influx causes the membrane potential to become positive. Phase 0 is also referred to as the "upstroke" of the action potential because it lasts less than one millisecond and is the fastest phase. During depolarization, the potential difference is actually reversed, so that the potential of the cytoplasm exceeds that of the extracellular fluid by about 20 mV. The upstroke is immediately followed by a brief period of partial, or early repolarization (Phase 1) which is mediated mainly by the transient efflux of potassium ions, which is then followed by a plateau phase (Phase 2). During the plateau phase, there is an influx of positively charged calcium ions which is counterbalanced by the efflux of positive charged potassium ions. Following the plateau phase, the membrane repolarizes (Phase 3) back to the resting state of polarization (i.e, the change in membrane potential back to an negative value after depolarization). This final repolarization occurs when the efflux of potassium ions begins to exceed the influx of calcium ions. The Phase 3 repolarization develops more slowly than does the depolarization Phase 0. The potassium currents through the potassium channels play a major role in determining the duration of Phase 3 and thus, the duration of the action potential. The last phase of the action potential (Phase 4) is silent in terms of the membrane potential changes. Phase 4 is the phase during which the ionic concentrations are restored via the elimination of the sodium and calcium ions that entered into the cell in exchange with the potassium ions that exited the cell during the action potential. Each action potential of a cardiac muscle cell causes a contraction of that cell. The contraction of all the cardiac muscle cells in concert forms a coordinated heart contraction or a heart beat. At the same time, an integrated electrical signal (action potentials) from all the cardiac muscle cells is emitted to the surface of the body. This signal can be recorded on an electrocardiogram (such as an ECG or EKG) which produces a characteristic wave form. The different parts of the wave form are designated by the letters - P, Q, R, S and T, which represent sum of the action potentials from different regions of the heart. Certain intervals of time between the different parts of the wave provide valuable information about the condition of the heart. For example, the period of time from the beginning of the QRS complex of the wave to the end of the T wave (known as the "QT" interval) provides a measure of the duration of ventricular depolarization and repolarization. In other words, it is a measurement of the duration of cardiac ventricular action potential.

Unfortunately, in some individuals, the duration of the QT interval is prolonged. Prolongation of the QT interval, which is clinically referred to as "long QT syndrome" (hereinafter "LQTS"), has been associated with increasing the risk of certain medical conditions, such as ventricular tachyarrhythmia, particularly, torsade de pointes, which can lead to sudden cardiac death. QT interval prolongation or an increase in action potential duration can result from an increase of the inward (or influx) sodium or calcium currents, or inhibition of one or more of the outward (or efflux) potassium currents. Two of the potassium channels involved in the Phase 3 repolarization of the heart are referred to as the rapidly and slowly activating components of the delayed rectifier potassium channels, Iκ_r and Iκ_s- These channels have a significant role in determining the duration of the action potential and thus, the QT interval. ^' Any defect or blockage of either of these channels slows the repolarization Phase 3, thereby prolonging the duration of the action potential and the QT interval. The rapidly delayed rectifier potassium channel /_KΓ is encoded by the human ether-a-go-go-τύ&teά gene (hereinafter referred to as "hERG"). Thus, the channel is also known as a "hERG" channel. The prolongation of the QT interval is generally believed to result from one or more genetic defects (which are referred to as "congenital LQTS") in these ion channels, or through the action of one or more drugs (which is referred to herein as "acquired LQTS"). Nonetheless, despite this, prolonged QT interval has been found in a number of cardiovascular or other related diseases, including myocardial ischemia (Puddu, et al., Journal of Electrocardiology, 19(3):203-l 1 (1986)), heart failure (Brooksby et al., European Heart Journal, 20(18):1335-41 (1999)), diabetes (Veglio et al., Journal of Internal Medicine, 251(4):317-24 (2002)) and stroke (Wong et al., Heart (British Cardiac Society), 89(4):377-81 (2003)), all of which are associated with high rates of death.

The measurement and determination of baseline QT intervals has been shown, albeit not consistently, to be a prognostic indicator of the mortality in patients with myocardial ischemia, heart failure and stroke. This suggests that QT prolongation may be a factor for mortality associated with the above mentioned diseases. Indeed, electrophysiological studies using isolated myocytes from heart failure patients (Beuckelmann, et al., Circulation Research, 73(2):379-85 (1993)) and from animals with myocardial infarction (Kaprrielian et al., American Journal of Physiology, 283:H1157- Hl 168 (2002)) and experimentally induced heart failure (Despa et al., Circulation, 105:2543-2548 (2002); Rose et al., American Journal of Physiology, 288:H2077-H2087 (2005)) have shown that currents through the transient outward (I_to), the inward rectifier (I_K1) K⁺ channels, and delayed rectifier currents can be reduced as compared to healthy normal controls (Janse, Cardiovascular Research, 61: 208-217 (2004)).

Treatment options for congenital LQTS include reduction of the QT interval directly and indirectly through β-blocker therapy, cardiac pacing and implantable cardioverter defibrillators, (Ackerman, MJ., Mayo Clin. Proc, 73:250-269 (1998); Wehrens et al., Ann. Intern. Med., 137:981-992 (2002); Khan, Am. Heart J. 143:7-14 (2002)). Pharmacological modulation of ion channels has had some success. Sodium channel blockers can reduce the QT interval directly in patients with LQT3 since QT prolongation is due to a defect in sodium channel inactivation causing the mutated sodium channels to be overactive during the cardiac action potential plateau. This "gain of function" can be reversed pharmacologically with sodium channel blockers such as mexiletine and ilecainide (Schwartz et al., Circulation, 92:3381-3386 (1995); Wang et al., J. Clin. Invest. 99:1714-1720 (1997); Windle et al., ElectrocardioL, 6:153-158 (2001); Liu et al., J. Pharmacogenomics, 3:173-179 (2003)). This approach is mechanism based and effective but limited to the minority of congenital LQTS patients with LQT3. However, the potential for cardiac toxicity is well established for sodium channel blockers (which are referred to as Class I anti-arrhythmic drugs) due to their slowing of conduction in potential reentrant circuits and triggering arrhythmias (Nattel, Cardiovasc. Res., 37:567-577 (1998)). Methods based on increasing extracellular potassium concentration, such as intravenous (i.v.) infusion of potassium, shorten the QT interval by increasing the activity of repolarizing potassium channels. QT intervals have been significantly shortened in patients that received this treatment (Compton et al., Circulation, 94:1018-1022 (1996)). However, this therapy is not widely used because it requires incontinent i.v. infusion and achieving sufficiently high long-term potassium levels is difficult (Etheridge et al., J. Am. Coll. Cardiol, 42: 1777-1782 (2003)). The ATP-sensitive potassium (K_ATP) channel opener, nicorandil, has been shown to normalize the congenitally prolonged QT interval in patients (Shimizu et al., Curr. Pharm. Des. 11 : 1561-1572 (2005)). However, K_AT_P channel openers are all associated with unwanted vasodilating activities (e.g. hypotension) due to the presence of KA_TP channels not only in the heart, but also in vascular smooth muscle (Quast et al., Cardiovasc. Res., 28:805-810 (1994)). Therapies targeting ion channels directly can be successful, as is the case for LQT3, and are likely to be useful generally.

It is also known in the art that intracellular calcium overloads contribute to myocardial ischemia injury (Farber J.L., Laboratory Investigation, 47(2): 114-23 (1982)). Increased cytosolic calcium concentrations (calcium overload) result in irreversible increases in the resting tension of the cardiac muscle and thus prevent normal relaxation of the heart (Lowe et al., Journal of Molecular & Cellular Cardiology, 11(10): 1017-31 (1979)). The opening of potassium channels could provide cardioprotection via shortening the action potential plateau and speeding repolarization, thus leading to a reduction of the influx of calcium through L-type calcium channels. Efforts have been made to identify potassium channel openers that are safe and effective for the treatment of myocardial ischemia (Gomma et al., Drugs, 61(12):1705-10 (2001)). So far only KA_TP openers have been pursued. As discussed above, the prevalence of K_ATP channels in non- target tissues increases the likelihood of unwanted side effects with these agents. Moreover, KA_TP openers can significantly shorten QT intervals and be arrhythmogenic under certain circumstances.

Reversing QT prolongation or calcium overload by increasing the activity of Iκ> potassium channels may also be beneficial in treatment of the acquired and inherited LQTS, myocardial ischemia, heart failure, diabetes and stroke. Two recent reports (Kang et al., MoI Pharmacol, 67:827-836 (2005); Zhou et al., MoI. Pharmacol, 68:876-884 (2005)) describe drug molecules that act as hERG agonists and may permit development of a drug therapy for reversal of prolonged QT. However, micromolar concentrations of these compounds are required to increase hERG current (which may not be achievable in human after oral dosing) and they shorten normal action potential duration and QT intervals (which may translate into a proarrhythmic risk).

Thereupon, there is a need in the art for pharmaceutically acceptable potassium channel openers that can be used to safely shorten prolonged QT intervals and reduce the calcium overload. Such agents would be useful in treating a variety of cardiovascular or other related diseases such as LQTS, heart failure, diabetes, stroke, myocardial ischemia etc.

Summary of the Present Invention

In one embodiment, the present invention relates to methods for shortening a QT interval in a patient suffering from QT prolongation. The methods involve the following steps:

administering to a patient suffering from QT prolongation a therapeutically effective amount of at least one pharmaceutically acceptable human ether-a-go-go- related gene ("hERG") channel agonist wherein said at least one hERG channel agonist does not shorten the QT interval when administered to a patient that is not suffering from QT prolongation. The patient suffering from QT prolongation to be treated by the above method may be suffering from congenital long QT syndrome, acquired long QT syndrome, myocardial ischemia, heart failure, diabetes or stroke.

The administration of the hERG channel agonist in the above-described method increases the currents of the hERG channel in said patient. Specifically, the increase in the current of the hERG channel was found to be voltage dependent. More specifically, it was found that the at least one hERG channel agonist increases the current of the hERG channel at a positive transmembrane potential. This positive transmembrane potential is between about +0.1 mV and about +50 mV, preferably between about +5 mV and about +30 mV and most preferably from about +10 mV and about +20 mV.

hERG channel agonists that can be used in the above-described method include those having the following formula:

wherein R₁ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR, where n is an integer from 0 to 5; or

NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group, wherein R₂ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR, where n is an integer from 0 to 5; or

NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group, wherein R₃ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR, where n is an integer from 0 to 5; or

NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group, wherein R₅ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR, where n is an integer from 0 to 5; or

NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 — membered cyclic amino group, wherein R₆ is

R₇ is hydrogen, a C₁-C₄ alkyl, carboxyl, COO-Glucoronide or COO-Sulfate, a C₁- C₅ alkoxycarbonyl, carbamoyl or C₁-C₄ alkyl aminocarbonyl group; and R₈ is hydrogen, a C₁-C₄ alkyl, carboxyl, COO-Glucoronide or COO-Sulfate, a C₁- C₅ alkoxycarbonyl, carbamoyl or C₁-C₄ alkyl aminocarbonyl group.

Examples of hERG channel agonists having the above formula are selected from the group consisting of: 2-[3-cyano-4-(2-methylpropoxy)phenyl]-4-methylthiazole-5- carboxylic acid, 2-[3-cyano-4-(3-hydroxy-2~methylpropoxy)phenyl]-4-methyl-5- thiazolecarboxylic acid, 2-[3-cyano-4-(2-hydroxy-2-methylpropoxy)phenyl]-4-methyl-5- thiazolecarboxylic acid, 2-(3-cyano-4-hydroxyphenyl)-4-methyl-5-thiazolecarboxylic acid, 2-[4-(2-carboxypropoxy)-3-cyanophenyl]-4-methyl-5-thiazolecarboxylic acid and a pharmaceutically acceptable salt thereof.

hi another embodiment, the present invention relates to a method of treating a patient suffering from myocardial ischemia, heart failure, diabetes or stroke. The method involves the steps of:

administering to a patient suffering from myocardial ischemia, heart failure, diabetes or stroke a therapeutically effective amount of at least one pharmaceutically acceptable human et/ze/'-α-go-go-related gene ("hERG") channel agonist wherein said at least one hERG channel agonist does not shorten the QT interval when administered to a patient that is not suffering from QT prolongation.

wherein R₁ is a hydrogen; a carhoxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR, where n is an integer from 0 to 5; or

NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group. wherein R₂ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted Ci-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR, where n is an integer from 0 to 5; or

NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-Ci₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl^'amino group, or a 5- to 7 - membered cyclic amino group, wherein R₃ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted Ci-Cio haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR, where n is an integer from 0 to 5; or

NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-Ci₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-Ci_O alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted Ci-Ci₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted Ci-Ci₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group, wherein R₅ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted Ci-Qo alkyl; an unsubstituted or substituted C₁-Ci₀ haloalkyl; > an unsubstituted or substituted C₁-Ci_O alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR, where n is an integer from 0 to 5; or

NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-Ci₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group, wherein R₆ is

R₇ is hydrogen, a C₁-C₄ alkyl, carboxyl, COO-Glucoronide or COO-Sulfate, a C₁- C₅ alkoxycarbonyl, carbamoyl or C₁-C₄ alkyl aminocarbonyl group; and

R₈ is hydrogen, a C₁-C₄ alkyl, carboxyl, COO-Glucoronide or COO-Sulfate, a C₁- C₅ alkoxycarbonyl, carbamoyl or C₁-C₄ alkyl aminocarbonyl group.

Examples of hERG channel agonists having the above formula are selected from the group consisting of: 2-[3-cyano-4-(2-methylpropoxy)phenyl]-4-methylthiazole-5- carboxylic acid, 2-[3-cyano-4-(3-hydroxy-2-methylpropoxy)phenyl]-4-methyl-5- thiazolecarboxylic acid, 2-[3-cyano-4-(2-hydroxy-2-methylpropoxy)phenyl]-4-methyl-5- thiazolecarboxylic acid, 2-(3-cyano-4-hydroxyphenyl)-4-methyl-5-thiazolecarboxylic acid, 2-[4-(2-carboxypropoxy)-3-cyanophenyl]-4-methyl-5-thiazolecarboxylic acid and a pharmaceutically acceptable salt thereof.

Brief Description of the Drawings

Figure 1 shows sample HEK/hERG current trace before and after 50 and 500 μM application of 2-[3-cyano-4-(2-methylpropoxy)phenyl]-4-methylthiazole-5-carboxylic acid (hereinafter referred to as "febuxostat"). HEK/hERG currents [Current (pA); Time (ms)] were obtained using the voltage procedure [Voltage (mV)] described for concentration-response (shown in the lower panel). Current records in the presence of febuxostat were obtained after three minutes of equilibration at the indicated concentration.

Figure 2 shows a sample HEK/hERG current trace before and after 0.1 and 1 μM febuxostat application. HEK/hERG currents [Current (pA); Time (ms)] were obtained using the voltage procedure [Voltage (mV)] described for concentration-response (shown in the lower panel). The current records in the presence of 0.1 and 1 μM febuxostat was obtained after at least three minutes of exposure to febuxostat.

Figure 3 shows the sample time course of HEKThERG current measured before, during and after 50 μM febuxostat application at +20 mV.

Figure 4 shows the use- or frequency-dependence of febuxostat effect on HEK/HERG peak tail current. Before and after 500 μM febuxostat equilibration, repetitive test pulses at frequencies 0.3 Hz (Top Panel) and 3.0 Hz (Bottom Panel) were applied. Current amplitudes were normalized to the first pulse and plotted against time. Data are the average of two cells.

Figure 5 shows sample CHO/hERG current trace before and during febuxostat application. CHO/hERG currents [Current (pA); Time (ms)] were obtained using the voltage procedure described for concentration-response and is shown in the lower panel. The steady state effect record was obtained 7 minutes after the start of febuxostat application.

Figure 6 shows a sample time course of CHO/hERG current measured before and after 1 μM febuxostat application at +20 mV. The cell was superfused with Tyrode's solution from an array of three capillary tubes placed adjacent to the cell. To control for solution flow artifacts, control solution was switched between two capillary tubes (Control 1 and Control 2) before switching to febuxostat containing solution. Figure 7 shows a sample time course of CHO/hERG current measured before and after 0.1 μM febuxostat application at +20 mV. The cell was superfused with Tyrode's solution from an array of three capillary tubes placed adjacent to the cell. To control for solution flow artifacts, control solution was switched between two capillary tubes (Control 1 and Control 2) before switching to febuxostat containing solution.

Figure 8 shows the concentration-response of the initial maximum effect of febuxostat (also known as "TMX-67") on hERG current at +20 mV. The mean fractional currents present after application of febuxostat (circles) ± S.E.M. were fit to a simple binding equation (Solid Line). The calculated EC_5O was 0.003 μM. Number of observations is shown in parentheses.

Figure 9 shows the concentration-response of the steady state effect of febuxostat on hERG current at +20 mV. The mean fractional currents present after application of febuxostat (circles) + S.E.M. were fit to a simple binding equation (Solid Line). The calculated ECs₀ was 0.070 μM. Number of observations is shown in parentheses.

Figure 10 shows the use-dependence of febuxostat agonist effect measured at +60 mV. Before and after 1 μM febuxostat equilibration repetitive test pulses at frequencies of 0.3 Hz (Top Panel) and 3.0 Hz (Bottom Panel) were applied. The train of pulses was generated by repetition of this step waveform: depolarization +60 mV for 250 ms; repolarization: -50 mV for 70 ms; followed by return to the holding potential of -80 mV. Peak current amplitudes were measured at the onset of the +60 mV step. Peak currents were normalized to the train second pulse amplitude in control and in febuxostat solution so that steady state drug effects before the start of the train did not overlap the frequency dependent effects. Normalized currents were plotted against time. Data are the average of three cells. Figure 11 shows the use-dependence of febuxostat agonist effect measured at -50 mV. Before and after 1 μM febuxostat equilibration repetitive test pulses at frequencies of 0.3 Hz (Top Panel) and 3.0 Hz (Bottom Panel) were applied. The train of pulses was generated by repetition of this step waveform: depolarization +60 mV for 250 ms; repolarization: -50 mV for 70 ms; followed by return to the holding potential of -80 mV. Peak tail current amplitudes were measured at -50 mV following channel activation and inactivation at +60 mV. Peak tail currents were normalized to the train first pulse amplitude in control and in febuxostat solution so that steady state drug effects before the start of the train did not overlap the frequency dependent effects. Normalized currents were plotted against time. Data are the average of three cells.

Figure 12 shows the effect of febuxostat on voltage-dependence of activity and steady state I- V relation. Current values (Mean + S.E.M.) measured in 3 cells at the end of the 4-second activating voltage step in control and after equilibration with 1 μM febuxostat are plotted for each voltage step. Data were normalized to the maximum value in control for each cell.

Figure 13 shows the effect of febuxostat on the steady state I-V current records. Each panel shows 16 superimposed current records from a CHO/hERG cell produced by the voltage protocol diagrammed below the currents in control (upper panel) and 1 μM febuxostat (lower panel).

Figure 14 shows the steady state G-V relation in control and febuxostat treated cells. Normalized conductance measured from peak tail current amplitude values (Mean ± S.E.M.) in 3 CHO/hERG cells during the -50 mV repolarizing voltage step of the steady-state I-V relation protocol. Measurements in control and after equilibration with 1 μM febuxostat are plotted for each voltage during the preceding variable voltage step. Data in control and in febuxostat were fit to a Boltzmann equation of the form

Normalized Current = l/(l+e-(^v"^vi/2)/Kv) where V is the voltage of the 4 second activating voltage step of the steady- state I- V relation protocol preceding the —50 mV repolarizing step, V_1Z2 is the potential at which half maximal conductance occurs, and K_v is the exponential slope factor setting the steepness of the curve. Values for Vy₂ in control and febuxostat were 0.9 and -2.1 mV, respectively. Values for K_v in control and febuxostat were 9.9 and 9.8 mV, respectively.

Figure 15 shows the fully activated I- V relation in control and febuxostat treated cells. Normalized peak current values (Mean + S.E.M.) measured in 3 cells in control and after equilibration with 1 μM febuxostat are plotted for each voltage step. Peak current measurements were made during the second, 5-second duration variable voltage step of the voltage protocol. Data were normalized to the maximum value in control for each cell.

Figure 16 shows the effect of febuxostat on the fully activated I-V current records. Each panel shows 15 superimposed current records from a CHO/hERG cell produced by the voltage protocol diagrammed below the currents in control (upper panel) and 1 μM febuxostat (lower panel).

Figure 17 shows the effect of febuxostat on voltage-dependence of inactivation. Normalized channel availability values (Mean ± S.E.M.) measured in 3 CHO/hERG cells in control and after equilibration with 1 μM febuxostat are plotted for each voltage step. Peak current measurements were made during the second, 1 second duration, variable voltage step of the voltage protocol. Data were normalized to the maximum value in control for each cell. Data were fit to an equation of the form:

Channel Availability = l/(l+e(^v-^vi/2)/K_v)) where V is the voltage of the variable voltage step in the protocol, Vy₂ is the voltage for half-maximal channel availability, K_v is the exponential slope factor setting the steepness of the curve. Values for Vy₂ in control and febuxostat were -68 and -67 mV, respectively. Values for K_v in control and febuxostat were 28 and 30 mV per e-fold change, respectively. The fraction of channels inactivated is "1 - channel availability".

Figure 18 shows the effect of febuxostat on the alternate I-V relation. Each panel shows 17 superimposed current records from a CHO/hERG cell produced by the voltage protocol diagrammed below the currents in control (upper panel) and 1 μM febuxostat (lower panel).

Figure 19 shows the effect of febuxostat on instantaneous I-V relation. Current values (Mean ± S.E.M.) measured in 3 cells at the beginning of the 1 second variable voltage step in the alternate I-V relationship voltage protocol, in control and after equilibration with 1 μM febuxostat, are plotted for each voltage step. Data were normalized to the 0 mV value in control for each cell.

Figure 20 shows the effect of febuxostat on action potential. Superimposed records before (control) and after equilibration with increasing concentrations of febuxostat (10, 100 and 1000 nM). Febuxostat did not cause significant changes in any of the action potential parameters. Temperature was maintained at 37 ± 1°C and the BCL was set to 2s.

Figure 21 shows the effect of 50 μM sotalol on action potential. Superimposed records before (control) and after equilibration of 50 μM sotalol at 23 min (1), 50 μM sotalol at 46 min (2) and 50 μM sotalol at 69 min (3). Temperature = 37 ± 1° C, BCL = 2s. Sotalol significantly prolonged APD.

Figure 22 shows the effect of 20 nM ATX II on action potential.. Superimposed records before (control) and after equilibration of 20 nM ATX II at 23 min (1), at 46 min (2) and at 69 min (3). Temperature = 37 + 1° C, BCL = 2s. ATX II significantly prolonged APD.

Figure 23 shows the effects of febuxostat (also known as TMX) and sotalol on action potential duration. Percent change in APD90 (BCL = 2s) relative to baseline was plotted versus exposure period. In the febuxostat group (open diamonds, n = 4) 10, 100 and 1000 nM febuxostat concentrations were applied cumulatively during exposure periods 1, 2 and 3, respectively. In the sotalol group (filled squares, n = 4), 50 μM sotalol was applied continuously throughout exposure periods 1, 2 and 3. In the combined sotalol and febuxostat group (open triangles, n = 4) 50 μM sotalol, 50 μM sotalol + 100 nM febuxostat, and 50 μM sotalol + 1000 nM febuxostat were applied cumulatively during exposure periods 1, 2 and 3, respectively. The sotalol data was overlaid on the sotalol + febuxostat data by normalization of the sotalol data by the sotalol + febuxostat exposure period 1 value. The normalized sotalol data (crosses) overlays the period 2 and 3 sotalol + febuxostat data, indicating that febuxostat at 100 and 1000 nM concentration had no effect on the time course of sotalol APD90 prolongation.

Figure 24 shows the effect of febuxostat on ATX Il-induced APD₉₀ prolongation. Measurements were made at BCLs of 2 s (A), 1 s (B) and 0.34 s (C). The percent change in APD90 (Mean ± SEM) at each BCL was plotted versus exposure period during sequential applications of 20 nM ATX 11 + 100 nM febuxostat (exposure periods 1 and 2), 20 nM ATX II + 1000 nM febuxostat (exposure period 3). Data graphed with diamond, triangle and square symbols were obtained at BCLs of 2, 1 and 0.34 s, respectively. Filled and open symbols represented data obtained in ATX II alone (n = 4 fibers) and ATX II + febuxostat (n= 7 fibers). * Statistically significant difference between ATX II and ATX II + febuxostat groups (PO.05, Student's t-test). Detailed Description of the Invention

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an active agent" includes a single active agent as well two or more different active agents in combination.

Definitions

In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

The term "acquired LQTS" refers to the prolongation of the QT interval in a patient that is believed to be the result of the action of one or more drugs.

The terms "administer", "administering", "administered" or "administration" refer to any manner of providing a drug to a subject or patient. Routes of administration can be accomplished through any means known by those skilled in the art. Such means include, but are not limited to, oral, buccal, intravenous, subcutaneous, intramuscular, by inhalation and the like.

The term "congenital LQTS" refers to the prolongation of the QT interval in a patient that is believed to be the result of one or more genetic defects.

The term "dosage form" refers to any solid object, semi-solid, or liquid pharmaceutical composition designed to contain a specific pre-determined amount (i.e. dose) of a certain active ingredient (i.e, at least one hERG channel agonist). Suitable dosage forms may be pharmaceutical drug delivery systems, including those for oral administration, buccal administration, rectal administration, topical or mucosal delivery or subcutaneous implants, or other implanted drug delivery systems and the like. Preferably, the dosage form of the pharmaceutical composition of the present invention is considered to be solid, however, they may containing liquid or semi-solid components. More preferably, the dosage form is an orally administered system for delivering an active ingredient to a patient.

By an "effective amount" or a "therapeutically effective amount" of an active ingredient (i.e., at least one hERG channel agonist) is meant a nontoxic but sufficient amount of the active ingredient to provide the desired effect. The amount of active ingredient that is "effective" will vary from subject to subject, depending on the age and general condition of the individual, the particular active ingredient or active ingredient, and the like. Thus, it is not always possible to specify an exact "effective amount." However, an appropriate "effective amount" in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

The term "human eύher-a-go-go-related gene ("hERG") channel agonist" refers to a compound, peptide, active ingredient or drug that potentiates or increases the current in a hERG channel in the heart of a patient that is suffering from QT prolongation thereby reversing or shortening the QT interval in said patient. Additionally, the hERG channel agonist used in the methods of the present invention does not shorten the QT interval when administered to a patient that is not suffering from QT prolongation.

Examples of hERG channel agonists that can be used in the present invention are those compounds having the below formula I:

Formula I wherein R₁ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy; OR; S(O)_nR₅ where n is an integer from 0 to 5; or NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C_1O alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group.

wherein R₂ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C_1O alkoxy; an unsubstituted or substituted hydroxyalkoxy; OR; S(O)_nR, where n is an integer from 0 to 5; or NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted Ci-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁Q alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group. wherein R₃ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy; OR; S(O)_nR, where n is an integer from 0 to 5; or NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group. wherein R₅ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy; OR; S(O)_nR, where n is an integer from 0 to 5; or NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group. wherein R₆ is

In formula I above, substituents which may have further substituent(s), namely, a pyridyl, thienyl, furyl or naphthyl group; Ci-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group; 5- to 7- membered heterocyclic ring; C₁-C₁₀ alkoxy, aryloxy or aralkyloxy group; a unsubstituted or substituted hydroxyalkoxy; and C₁-Ci₀ alkyl (mono- or di-substituted) amino, aryl (mono- or disubstituted) amino group, on chain or cyclic moiety thereof, can be substituted by one or more of: a C₁-C₄ halogenated alkyl, carboxyl, alkylcarbonyl, alkyloxy, alkylcarbonyloxy, hydroxyl, mono- or di-substituted alkylamino, amino, nitro, cyano or formyl group, or halogen atom, heterocyclic ring such as 5- to 7- membered cyclic-secondary amino group, etc. Preferred substituents are a halogen atom, methyl group, ethyl group, methoxy group and ethoxy group.

As used herein, the term "C₁-C₄ alkyl" refers to a methyl group, ethyl group, propyl (iso- or n-) group and butyl (iso-, n-, tert- or sec-) group.

As used herein, the term "C₁-C₄ alkyl aminocarbonyl" refers to a group comprising an alkyl group of one to four carbon atoms and an aminocarbonyl group. As used herein, the term " unsubstituted or substituted C₁-C_1O alkyl" group refers to a Ci-C₁₀ straight-chain or branched aliphatic hydrocarbon residue, cyclic aliphatic hydrocarbon residue or chain-cyclic aliphatic hydrocarbon residue which can be mono- or di-substituted. Examples include, but are not limited to, methyl, ethyl, n-propyl, iso- propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, neo-pentyl, n-hexyl, n-octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, cyclohexylmethyl, cyclohexylpropyl, methoxyethyl, ethoxyethyl, and the like.

As used herein, the term "unsubstituted or substituted C₁-C₁₀ alkoxy" refers to an alkyl group (which can be mono- or di-substituted) in which one hydrogen atom has been replaced by an oxy group. Examples include, but are not limited to, methoxy, ethoxy, propoxy (n- or iso-), butoxy (n-, iso-, sec- or tert-), 3-methylbutoxy, 2-ethylbutoxy, pentyloxy, hexyloxyl, 3-methyl-2-butenyloxy, geranyloxy, cyclopentyloxy, cyclohexyloxy, cyclohexyl- C₁-C₁₀ -alkyloxy (e.g., cyclohexylmethyloxy), and the like.

As used herein, the term "C₁-C₅ alkoxycarbonyl" refers to a group comprising an alkoxy group having one to five carbon atoms and a carbonyl group.

As used herein, the term "unsubstituted or substituted C₁-Ci₀ alkyl amino" refers to a group comprising an alkyl (which can be mono- or di-substituted) group and an amino group. Examples include, but are not limited to, methylamino, ethylamino, dimethylamino, diethylamino groups, and the like.

As used herein, the term "aminocarbonyl" refers to a group comprising an amino group and a carbonyl group.

As used herein, the term "aryl" group refers to aromatic hydrocarbon residues or aromatic heterocyclic ring groups comprising a 5- or 6-membered monocyclic or fused ring. Examples include, but are not limited to, phenyl, 1-naphthyl, 2-naphthyl, 2- pyrrolyl, 2-furyl, 2-thienyl, 2-pyridyl, and the like. If said aromatic hydrocarbon resisdue or aromatic heterocyclic ring or fused ring is mono- or di-substituted than said aryl group can be considered to be a "substituted" aryl group.

As used herein, the term "unsubstituted or substituted aryl amino" refers to a group that comprises an aryl (which can be mono- or di-substituted) group and an amino group. Examples include, but are not limited to, phenylamino, methylphenylamino, and the like.

As used herein, the term "aryloxy" group refers to an aryl group and an oxy group. Examples include, but are not limited to, phenoxy, 1-naphthoxy, and the like.

As used herein, the term "aralkyl" refers to an alkyl group (such as any of a C₁- C₁₀ alkyl group) that is substituted by an aryl group. Examples include, but are not limited to, benzyl, 1-phenylethyl, 1 -methyl- 1-phenylethyl, 2-phenylethyl, 3- phenylpropyl, cinnamyl, 2-pyrrolylmethyl, furfuryl, thenyl, and the like, and a benzyl group is preferred.

As used herein, the term "unsubstituted or substituted aralkyl amino" refers to a group that comprises an aralkyl (mono- or di-substituted) group and an amino group. Examples include, but are not limited to, benzylamino, methylbenzylamino, and the like.

As used herein, the term "alkylcarbonyl" refers to a group comprising an alkyl group and a carbonyl group. Examples include, but are not limited to, C₂-C₇ lower aliphatic acyl groups such as acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 2-methylbutanoyl, 3-methylbutanoyl, pivaloyl, hexanoyl, cyclopropylcarbonyl, and the like.

As used herein, the term "arylcarbonyl" refers to a group comprising an aryl group and a carbonyl group. Examples include, but are not limited to, benzoyl, toluoyl, 2-pyrrolcarbonyl, 2-fluoyl, 2-thiophenecarbonyl, and the like. As used herein, the term aralkylcarbonyl" refers to a group comprising an aralkyl group and a carbonyl group. Examples include, but are not limited to, C₅-C₁₀ aralkylcarbonyl groups such as phenylacetyl, 3-phenylpropanoyl, 4-phenylbutanoyl, cinnamoyl, 2-pyrrolylacetyl, 2-furylacetyl, 2-thienylacetyl, and the like.

As used herein, the term "aralkyloxy" refers to a group comprising an aralkyl group and an oxy group. Examples include, but are not limited to, benzyloxy, 1- phenylethoxy, 1 -methyl- 1-phenylethoxy, and the like.

As used herein, the term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine atoms. Chlorine and fluorine are particularly preferred.

As used herein, the term "haloalkyl" refers to a group comprising a halogen atom and an alkyl group.

As used herein, the term "unsubstituted or substituted C₁-C₁₀ haloalkyl" refers to a haloalkyl group comprising from one to ten carbon atoms, in which the alkyl group can be mono- or di-substituted.

As used herein, the term "hydroxyalkoxy" refers to alkoxy group in which one hydrogen atom has been replaced by a hydroxy group. Examples, include, but are not limited to, hydroxymethoxy and 2-hydroxyethoxy.

In formula I above, examples of OR include, but are not limited to, ethoxy, propoxy (n- or iso-), butoxy (n-, iso-, sec- or tert-), pentyloxy, n-hexyloxy, cyclopropylmethyloxy, cyclohexyloxy, phenyloxy, benzyloxy, phenetyloxy, methoxethyloxy, ethoxyethyloxy, acetoxy, propanoyloxy, butanoyloxy, benzoyloxy, and the like.

In formula I above, examples of S(O)_n R include, but are not limited to, ethylthio, isopropylthio, isopropylsulfmyl, isopropylsulfonyl, pentylsulfonyl, phenylthio, phenylsulfmyl, phenylsulfonyl, and the like.

In formula I above, examples of NRR' include, but are not limited to, dimethylamino, diethylamino, benzylamino, phenethylamino, and the like.

In formula I above, where R and R' taken together with each other nitrogen atom bonded thereof, represent atoms can form an unsubstituted or substituted 5- to 7- membered heterocyclic ring. Examples of a heterocyclic ring include, but are not limited to, morpholino, 1-pyrrolyl, 1-pyrrolidinyl, piperidino, piperazino, and the like.

In formula I above, examples of the 5- to 7- membered cyclic-secondary amino group include, but are not limited to, morphorino, 1-pyrrolyl, 1-pyrrolidino, piperidino, and the like.

Examples of hERG channel agonists having the above-described formula I include, but are not limited to, 2-[3-cyano-4-(2-methylpropoxy)phenyl]-4-methylthiazole- 5-carboxylic acid (which shall also be referred to herein as "febuxostat"), 2-[3-cyano-4- (3 -hydroxy-2-methylpropoxy)phenyl] -4-methyl-5 -thiazolecarboxylic acid, 2- [3 -cyano-4- (2-hydroxy-2-methylpropoxy)phenyl] -4-methyl-5 -thiazolecarboxylic acid, 2-(3 -cyano-4- hydroxyphenyl)-4-methyl-5 -thiazolecarboxylic acid, 2-[4-(2-carboxypropoxy)-3- cyanophenyl]-4-methyl-5-thiazolecarboxylic acid or pharmaceutically acceptable salts thereof. Methods for making these compounds are described in U.S. Patent No. 5,614,520, which is herein incorporated by reference. Additionally, it is known in the art that febuxostat does not prolong the QT interval in healthy subjects (See, Yu, P., et al.₅ J Clin. Pharmacol, 44(10): 1195 (2004)).

The term "long QT syndrome" or "LQTS" refers to prolongation of the QT interval in a patient. The term "patient" refers to an animal, preferably a mammal, including a human or non-human. The terms patient and subject may be used interchangeably herein.

By "pharmaceutically acceptable," such as in the recitation of a "pharmaceutically acceptable excipient," or a "pharmaceutically acceptable additive," is meant a material that is not biologically or otherwise undesirable, i.e., the material maybe incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects.

The terms "treating" and "treatment" refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage. Thus, for example, "treating" a patient involves prevention of a particular disorder or adverse physiological event in a susceptible individual as well as treatment of a clinically symptomatic individual by inhibiting or causing regression of a disorder or disease.

The Invention

As mentioned briefly above, the present invention relates to methods for reversing or shortening the QT interval of a patient suffering from QT prolongation. Specifically, the methods of the present invention can be used to treat patients suffering from congenital or acquired LQTS, myocardial ischemia, heart failure, diabetes or stroke (methods for determining whether a patient is suffering from any of the aforementioned are well known to those skilled in the art). Additionally, the methods of the present invention can also be used to reduce intracellular calcium overload in patients suffering diseases with intracellular calcium overload such as myocardial ischemia and in need of a therapy.

The methods of the present invention will generally comprise administering to a patient in need of such therapy a therapeutically effective amount of at least one pharmaceutically acceptable hERG channel agonist. As will be discussed in more detail herein, the administration of at least one hERG channel agonist potentiates or increases the currents of the hERG channel in a heart of a patient suffering from QT prolongation (such as in a patient suffering from congenital or acquired LQTS, myocardial ischemia, heart failure, diabetes or stroke), thereby shortening the QT interval of said patient. However, the hERG channel agonists of the present invention are different from other hERG channel agonists known in the art in that they do not shorten the QT interval when administered to a patient that is not suffering from QT prolongation.

As mentioned briefly above, using the methods of the present invention, QT prolongation can be reversed (i.e., shortened) in patients suffering from QT prolongation (congenital or acquired LQTS, myocardial ischemia, heart failure, diabetes or stroke) by increasing the activity of repolarizing potassium channels in particular, the hERG channel or Iκ_rVia the administration to said patients of a therapeutically effective amount at least one pharmaceutically acceptable hERG channel agonist. Specifically, administration of a therapeutically effective amount of at least one pharmaceutically acceptable hERG channel agonist to a patient suffering from QT prolongation potentiates or increases the outward potassium currents, particularly the currents in the hERG channel (i.e., the Iκ_r), thus reversing or shortening the QT interval in said patient. However, not only do the hERG channel agonists described for use in the methods herein increase the current in the hERG channel in patients suffering from QT prolongation, but, most importantly, the hERG channel agonists of the present invention do not shorten the QT interval in normal patients that do not suffer from a prolonged QT interval (such as, for example, a normal, healthy patient).

Additionally, the potentiation or increase in the current in the hERG channel after administration of at least one hERG channel agonist to a patient suffering from QT prolongation was found to be voltage dependent. More specifically, the increase in the current of a hERG channel was found to occur at positive transmembrane potentials, specifically, from about +0.1 mV to about +50 mv, more preferably at from about +5 mV to about +30 mV, and even more preferably, at about +10 mV to about +20 mV. The hERG channel agonists of the present invention potentiate or increase the currents in the hERG channel during the action potential plateau (in patients suffering from QT prolongation). Normally, hERG channels are mostly inactivated at plateau potentials, whereas the hERG channel agonists of the present invention potentiate or increase the currents during this period.

Methods for identifying hERG channel agonists that can be used in the methods of the present invention can be readily achieved using routine techniques known to those skilled in the art. For example, as described in the examples herein, whole cell patch clamp measurements can be performed on cell lines (such as HEK293 and CHO cells) that have been transfected with hERG cDNA to screen for hERG channel agonists that increase the currents in the hERG channel as described herein. Once such hERG channel agonists have been identified, they can be further screened to determine whether or not these compounds reduce or reverse QT prolongation in patients that suffer from QT prolongation. This can be achieved by administering such hERG channel agonists to a patient that suffer from QT prolongation and then taking an ECG/EKG of said patient during the time when the at least one hERG channel agonist is circulating in the blood. One skilled in the art could easily determine, by reading the ECG/EKG, whether the administration of the hERG channel agonist to said patient has shortened the patient's prolonged QT interval.

For patients suffering from myocardial ischemia, heart failure, diabetes or stroke, one skilled in the art could easily monitor, using routine techniques, the reduction of mortality (death) or frequency of a disease event (i.e., meaning how often a patient may experience a stroke and/or heart attack) associated with any of the above diseases, and/or an improvement in the symptoms, biochemical markers (i.e., for a patient suffering from myocardial ischemia a reduction creatine phosphate kinase (CPK), a reduction in C reactive protein (CRP) in patient in suffering from myocardial ischemia or stroke, etc.) and/or ECG/EKG abnormality associated with these diseases, after administration of the hERG channel agonist to said patient. In addition to the methods described above, some pharmaceutical compounds that have shown utility in preventing a broad variety of disease states never benefit the public because these compounds are prone to prolonging the QT interval thus causing acquired LQTS. With the discovery of the methods described herein, these drugs may now be available to benefit the public. In particular, a hERG channel agonist that is capable of selectively shortening the QT interval (the hERG channel agonists described herein are selective in that these compounds shorten or reverse the QT interval only in patients suffering from QT prolongation and not in normal patients that do not suffer from QT prolongation) can be co-administered with compounds that would otherwise benefit the public but for the fact that these compounds prolong the QT interval. By coadministration of a hERG channel agonist, the detrimental effects of these compounds can be assuaged to make them useful for their intended purpose. Such drugs may come from a wide variety of compound classes and include but are not limited to antihistamines, antidepressants, neuroleptics, antimalaria drugs, macrolide antibiotics, serotonin antagonists and calcium antagonists.

Compositions containing at least one hERG channel agonist in combination with another pharmaceutical compound are therefore part of the present invention. Using the excipients and dosage forms described below, formulations containing such combinations are a matter of choice for those skilled in the art. Further, those skilled in the art will recognize that various coatings or other separation techniques may be used in cases where the combination of compounds are incompatible.

The hERG channel agonists used in accordance with the methods of the present invention can be provided in the form of pharmaceutically acceptable salts derived from inorganic or organic acids. Pharmaceutically acceptable salts are well-known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1 et seq. (1977). The salts can be prepared in situ during the final isolation and purification of the compounds or separately by reacting a free base function with a suitable organic acid. Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphor sulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isothionate), lactate, maleate, methane sulfonate, nicotinate, 2- naphthalene sulfonate, oxalate, palmitoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Also, basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained. Examples of acids which can be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid.

Basic addition salts can be prepared in situ during the final isolation and purification of compounds by reacting a carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium and aluminum salts and- the like and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium, triethylammonium, diethylammonium, and ethylammonium among others. Other representative organic amines useful for the formation of base addition salts include ethyl enediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like.

The at least one hERG channel agonist may be formulated in a variety of ways that is largely a matter of choice depending upon the delivery route desired. For example, solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the hERG channel agonist can be mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders, such as, but not limited to, starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders, such as, but not limited to, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants, such as, but not limited to glycerol; d) disintegrating agents, such as, but not limited to, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents, such as, but not limited to, paraffin; f) absorption accelerators, such as, but not limited to, quaternary ammonium compounds; g) wetting agents, such as, but not limited to, cetyl alcohol and glycerol monostearate; h) absorbents, such as, but not limited to, kaolin and bentonite clay; and i) lubricants, such as, but not limited to, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

Solid dosage forms comprising tablets, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings well-known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the hERG channel agonist, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as, but not limited to, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof.

The compositions can also be delivered through a catheter for local delivery at a target site, via an intracoronary stent (a tubular device composed of a fine wire mesh), or via a biodegradable polymer.

Compositions suitable for parenteral injection may comprise physiologically acceptable, sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include, but are not limited to, water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, and suitable mixtures thereof.

These compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Suspensions, in addition to the active agent (i.e., hERG channel agonist), may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

In some cases, in order to prolong the effect of the drug (i.e. hERG channel agonist), it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

Dosage forms for topical administration of the compounds of this present invention include powders, sprays, ointments and inhalants. The active compound(s) is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers or propellants that can be required. Opthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.

It will be understood that formulations to be used in the methods of the present invention generally will comprise a therapeutically effective amount of one or more hERG channel agonists. The phrase "therapeutically effective amount" as used herein means a sufficient amount of, for example, the composition, hERG channel agonist, or formulation necessary to treat the desired disorder (i.e., the prolonged QT interval), at a reasonable benefit/risk ratio applicable to any medical treatment. As with other pharmaceuticals, it will be understood that the total daily usage of a pharmaceutical composition of the invention will be decided by a patient's attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and other factors known to those of ordinary skill in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

Formulations of the present invention are administered and dosed in accordance with sound medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, and other factors known to medical practitioners.

Therapeutically effective amounts for purposes herein thus can readily be determined by such considerations as are known in the art. The daily pharmaceutically effective amount of the hERG channel agonist administered to a patient in single or divided doses range from about 0.01 to about 750 milligram per kilogram of body weight per day (mg/kg/day). More specifically, a patient may be administered from about 5.0 mg to about 1000 mg daily, preferably from about 20 mg to about 500 mg daily and most preferably from about 40 mg to about 300 mg daily of a KERG channel agonist.

As mentioned previously, the present invention relates to methods for reversing or shortening a QT interval in a patient suffering from QT prolongation. The methods of the present invention involve increasing the currents of the hERG channel in a heart of a patient suffering from QT prolongation by administering to a patient a therapeutically effective amount of at least one pharmaceutically acceptable hERG channel agonist. Once a patient has been administered at least one hERG channel agonist as described herein, the effectiveness and progress of the treatment in reversing or shortening the QT interval can be monitored by performing an ECG/EKG on said patient and determining the QT interval of said patient using routine techniques known to those skilled in the art. An ECG/EKG can be repeated as many times as necessary until the QT interval has been reversed or shortened to the satisfaction of the treating physician.

For patients suffering from myocardial ischemia, heart failure, diabetes or stroke, one skilled in the art could easily monitor, using routine techniques, the reduction of mortality (death) or frequency of a disease event (i.e., meaning how often a patient may experience a stroke and/or heart attack) associated with any of the above diseases, and/or an improvement in the symptoms, biochemical markers (i.e., for a patient suffering from myocardial ischemia a reduction creatine phosphate kinase (CPK), a reduction in C reactive protein (CRP) in patient in suffering, from myocardial ischemia or stroke, etc.) and/or ECG/EKG abnormality associated with these diseases, after administration of the hERG channel agonist to said patient.

By way of example and not of limitation, examples of the present invention shall now be given.

EXAMPLE 1: Effect of febuxostat on cloned hERG channels expressed in Human Embryo Kidney (HEK)293 cells: whole cell patch clamp measurements focusing on peak tail current Materials and Methods

Solutions and Chemicals

All chemicals used in preparation of bath and electrode solutions were purchased from Sigma (St. Louis, MO) unless otherwise noted and were of ACS reagent grade purity or higher. Febuxostat was obtained from Teijin Limited (Yamaguchi, Japan). All solutions containing febuxostat were prepared in glass containers whenever possible. Test solutions of febuxostat and terfenadine (positive control) were prepared daily using a modified HEPES-buffered Tyrode's (HBT) solution (composition in mM): NaCl, 137; KCl, 5.4; CaCl₂, 1.8; MgCl₂, 1; HEPES, 10; Glucose, 10; pH adjusted to 7.4 with NaOH. The HBT solution was freshly prepared weekly. Terfenadine solution was prepared in HBT at a concentration of 60 nM. The HBT solution was warmed to room temperature before preparing febuxostat or terfenadine solutions. Fresh test and control solutions were prepared on each experimental day. Pipette solution for whole cell recordings was (composition in mM): K-aspartate, 130; MgCl₂, 5; EGTA, 5; ATP, 4; HEPES, 10; pH adjusted to 7.2 with KOH. The pipette solution was prepared in batches, stored at -20⁰C, and was freshly thawed each day of use.

Cell Culture

HEK293 cells were stably transfected with hERG cDNA. Stable transfectants were selected by coexpression of the hERG cDNA and G418 gene incorporated into the expression plasmid. Selection pressure was maintained by including G418 in the culture medium. Cells were cultured in Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (D-MEM/F-12) supplemented with 10% fetal bovine serum, 100 U/mL penicillin G sodium, 100 μg/mL streptomycin sulfate and 500 μg/mL G418. Cells were maintained in tissue culture incubators at 37°C in a humidified 5% CO2 atmosphere, with stocks maintained in cryogenic storage. Cells used for electrophysiology were plated on 35 mm tissue culture dishes or glass coverslips. AU experiments were performed at room temperature (22 C - 25 ⁰C) unless otherwise noted. Each cell acted as its own control. Electrophysiology

Warner PC501A and Axon Instruments Axopatch 200B patch clamp amplifiers were used for whole cell patch clamp recordings. Current records were analog filtered at 0.2 of the sampling frequency for digital conversion by Axon Instruments Digidata 1320A AD/DA converters attached to PC-compatible desktop computers. Axon Instruments Clampex 8.2 software was used to acquire data and generate stimulus voltage waveforms. The suite of Axon Instruments pCLAMP8.2 applications (Molecular Devices Corp., Sunnyvale, CA) and Microsoft Excel 2000 spreadsheet software were used to analyze the data.

Cells attached to glass coverslips or plastic 35mm Petri dishes were transferred to the recording chamber and superfused with HBT solution. Patch pipettes were fabricated from TW150-F glass capillaries on a P 97 horizontal puller (Sutter Instrument Co., Novato, CA) to generate pipettes with 1 - 5 MΩ resistances after fire polishing. The concentration-response relationship for febuxostat modulation of hERG channel function was evaluated at concentrations of febuxostat ranging from 0.1 to 500 μM. These concentrations were applied cumulatively to cells expressing hERG channels. Each concentration had an n > 3 where n = number of measurements. Only one or two concentrations of febuxostat were applied to each cell. Terfenadine (60 nM) was applied to two cells as a positive control.

Patch Clamp Voltage Protocols Concentration-Response

Cells stably expressing hERG were held at -80 mV. Onset and steady state modulation of hERG current due to febuxostat or terfenadine was measured using a pulse pattern with fixed amplitudes (depolarization: +20 mV for 2 s; repolarization: -50 mV for 2 s) repeated at 10 s intervals. Peak tail current was measured during the 2 s step to -50 mV. A steady state was maintained for at least 30 s before applying febuxostat or terfenadine. Peak tail currents after application of febuxostat or terfenadine were measured until a new steady state was achieved. Frequency-Dependence

Cells were held at -80 mV for at least 1 minute. A train of pulses (depolarization: +60 mV for 250 ms; repolarization: -50 mV for 70 ms) sufficient to reach a steady state value (typically in the range of 20 to 30 pulses) was then applied with pulses in the train repeated at frequencies of 0.3 Hz and 3 Hz. Frequency-dependence of febuxostat's effect on peak tail currents was measured before and after equilibration with 500 μM febuxostat. Peak tail current was measured during the step to -50 mV in each pulse of the train.

Data Analysis

Data acquisition and analyses were performed using the suite of pCLAMP8.2 applications (Molecular Devices Corp., Sunnyvale, CA). Steady state was defined as a limiting constant rate of change with time (linear time dependence). The steady states before and after test article application were used to measure drug effects.

Analysis of frequency-dependence of febuxostat hERG modulation

Data for analysis of frequency-dependence (use-dependence) were normalized to the peak current of the first pulse in each train of pulses and normalized data from trains at each frequency were pooled to construct average time courses.

Analysis of temperature-dependence

Data for analysis of temperature-dependence compared the currents in the presence of 500 μM febuxostat at room temperature (22°C - 25°C) and at near physiological temperature (35 ± 2⁰C) in at least two cells at each temperature

Results

Febuxostat modulation of HEK/hERG peak tail currents.

These initial patch-clamp measurements were to evaluate potential hERG blocking effect of febuxostat. In contrast, the steady state effect of febuxostat on the time course of normalized peak tail currents over the concentration range tested (0.1 to 500 μM) revealed a slight increase from 1.01 to 1.09 in mean current and no change in tail current kinetics (Table 1). Moreover, febuxostat produced a voltage-dependent increase in the hERG currents that was prominent during the +20 mV step. Individual HEK/hERG voltage-clamp current-time (I-T) records acquired before and following equilibration with 500, 50, 1 and 0.1 μM febuxostat are shown superimposed in Figures 1 and 2. The magnitude of the effect at +20 mV was variable, but consistently present. The time course of the normalized peak current response to febuxostat application at +20 mV, measured in consecutive records repeated at 10 second intervals, consisted of an initial rapid increase that declined to a smaller steady state effect (Figure 3). These results indicate that febuxostat is not a hERG blocker and will not cause QT prolongation, instead, it is a novel hERG agonist.

Evaluation of the use- or frequency-dependence of 500 μM febuxostat modulation of HEKJhEKG peak tail current magnitude (measured at -50 mV) showed no difference in the time course of normalized peak tail current amplitudes in control and after equilibration with febuxostat when activating pulses were repeated at a frequency of 0.3 Hz. At the higher activation frequency of 3 Hz, febuxostat produced a small, insignificant reduction in the time course of normalized peak tail current magnitude relative to control (Figure 4). This suggests that difference in heart rate will not significantly affect the lack of blocking activity of febuxostat on hERG channel.

Raising the temperature of the bath to 35 ± 2⁰C did not significantly change the effect on peak tail current magnitude caused by application of 500 μM febuxostat from that obtained at room temperature (22°C -25°C) (Table 2). The agonist effect on the hERG current at +20 mV was still present at 35 ± 2°C and was qualitatively similar. These results demonstrate that the effects of febuxostat observed at room temperature mentioned above should also occur in the body at body temperature.

Terfenadine is an established and potent hERG blocker and was used as a positive control. The effect of application of 60 nM terfenadine on peak tail current was measured. As expected, 60 nM terfenadine blocked 77 ± 3 % (n=2) of HEK/hERG peak tail current (Table 3).

In summary, these results indicate that febuxostat at concentrations up to 500 μM has no undesirable blocking effect on the HEK/hERG peak tail current. And the lack of a blocking effect of 500 μM febuxostat on peak tail current was not use- or temperature- dependent. Instead, a voltage dependent increase in bERG currents during +20 mV was observed, suggesting that febuxostat is a novel hERG agonist.

Table 1: Effect of febuxostat on HEK/HERG peak tail current

^a Mean fraction of current (Iτe_st/Icontr_oi) at each febuxostat concentration, standard error of the mean (SEM), and ^c number of observations (n) for each febuxostat concentration.

Table 2: Comparison of fraction of HEK/hERG peak tail current after application of febuxostat at room tem erature and 35⁰C

^a Mean fraction of current (Iτ_est/Icontroi) with febuxostat at 500 μM, standard error of the mean (SEM).

Table 3: Fraction of HEK/hERG current after a lication of terfenadine

^a Mean fraction of current (Iτ_est/Ic_ontroi) with terfenadine at 60 nM, standard error of the mean (SEM).

EXAMPLE 2: Effect of febuxostat on cloned hERG channels expressed in Chinese Hamster Ovary (CHO) cells: whole cell patch clamp measurements focusing on agonist effects at positive potential.

Material and Methods Solutions and Chemicals

All chemicals used in preparation of bath and electrode solutions were purchased from Sigma (St. Louis, MO) unless otherwise noted and were of ACS reagent grade purity or higher. Febuxostat was obtained from Teijin Limited (Yamaguchi, Japan). All solutions containing febuxostat were prepared in glass containers whenever possible. Test solutions of febuxostat and terfenadine (positive control) were prepared daily using a modified HEPES-buffered Tyrode's (HBT) solution (composition in mM): NaCl₃ 137; KCl, 5.4; CaCl₂, 1.8; MgCl₂, 1; HEPES, 10; Glucose, 10; pH adjusted to 7.4 with NaOH. The HBT solution was freshly prepared weekly. Terfenadine solution was prepared in HBT at a concentration of 60 nM. The HBT solution was warmed to room temperature before preparing febuxostat or terfenadine solutions. Fresh test and control solutions were prepared on each experimental day. Pipette solution for whole cell recordings was (composition in mM): K-aspartate, 130; MgCl₂, 5; EGTA, 5; ATP, 4; HEPES₅ 10; pH adjusted to 7.2 with KOH. The pipette solution was prepared in batches, stored at -20⁰C, and was freshly thawed each day of use.

Cell Culture

CHO cells were stably transfected with hERG cDNA. Stable transfectants were selected by coexpression of the hERG cDNA and G418 gene incorporated into the expression plasmid. Selection pressure was maintained by including G418 in the culture medium. Cells were cultured in Dulbecco's Modified Eagle Medium / Nutrient Mixture F- 12 (D-MEM/F-12) supplemented with 10% fetal bovine serum, 100 LVmL penicillin G sodium, 100 μg/mL streptomycin sulfate and 500 μg/mL G418. Cells were maintained in tissue culture incubators at 37°C in a humidified 5% CO2 atmosphere, with stocks maintained in cryogenic storage. Cells used for electrophysiology were plated on 35 mm tissue culture dishes or glass coverslips. AU experiments were performed at room temperature (22 C — 25 C) unless otherwise noted. Each cell acted as its own control.

Electrophysiology

Cells attached to glass coverslips or plastic 35mm Petri dishes were transferred to the recording chamber and superfused with HBT solution. Patch pipettes were fabricated from TW150-F glass capillaries on a P 97 horizontal puller (Sutter Instrument Co., Novato, CA) to generate pipettes with 1 - 5 MΩ resistances after fire polishing. The concentration-response relationship for febuxostatmodulation of hERG channel function was evaluated at concentrations of febuxostat ranging from 0.0001 to 10 μM. These concentrations were applied cumulatively to cells expressing-JhERG channels. Each concentration had an n > 3 where n = number of measurements. Only one or two concentrations of febuxostat were applied to each cell. Terfenadine (60 nM) was applied to two cells as a positive control.

Patch Clamp Voltage Protocols Concentration-Response

Cells stably expressing hERG were held at -80 mV. Onset and steady state modulation of hERG current due to febuxostat (0.0001 to 10 μM) or terfenadine (60 nM) was measured using a pulse pattern with fixed amplitudes (depolarization: +20 mV for 2 s; repolarization: -50 mV for 2 s) repeated at 10 s intervals. Peak tail current was measured during the 2 s step to -50 mV. For modulation of hERG current at positive potentials, peak current during the step to +20 mV was measured. A steady state was maintained for at least 30 s before applying febuxostat or terfenadine. Peak currents after application of febuxostat or terfenadine were measured until a new steady state was achieved.

Frequency-Dependence

Cells were held at -80 mV for at least 1 minute. A train of pulses (depolarization: +60 mV for 250 ms; repolarization: -50 mV for 70 ms) sufficient to reach a steady state value (typically in the range of 20 to 30 pulses) was then applied with pulses in the train repeated at frequencies of 0.3 Hz and 3 Hz. Frequency-dependence of febuxostat modulation of hERG channel function was measured before and after equilibration with 1 μM febuxostat. Febuxostat frequency-dependent modulation of hERG channels was . measured as the time course of peak current magnitude measured during the steps to +60 mV and -50 mV in each pulse of the train.

Steady State I-V Relation

From the holding potential of -80 mV, 4 s depolarizing voltage steps to voltages from -70 to +80 mV in 10 mV increments, followed by repolarization to -50 mV for 5 s, were used to measure the steady-state I-V relation in control and in the presence of 1 μM febuxostat. The voltage protocol was repeated at 15 s intervals. A normalized steady-state I-V relation was generated using the current amplitude at the end of the depolarizing pulse for normalization.

Voltage-Dependence of Activation (G-V relationship)

Peak tail currents were measured during the repolarization step (-50 mV) in the steady-state I-V protocol (above) in control and in the presence of 1 μM febuxostat. Fully activated JtERG I-V Relationship

From the holding potential of -80 mV, cells were depolarized to +60 mV for 1 sec to fully activate and partially inactivate hERG currents, and then repolarized for 5 sec to voltages ranging from -100 to +40 mV in 10 mV increments. The interval between voltage protocol repetitions was 15 s. Peak currents were measured during the repolarizing step and plotted as a function of voltage. The fully activated hERG I-V relation was measured in control and in the presence of 1 μM febuxostat.

Alternate I-V Relationship (Conservative Protocol)

From a holding potential of 0 mV, a 25 ms hyperpolarizing pulse to -80 mV was followed by a 1 s depolarizing step to potentials from -120 mV to +40 mV in 10 mV increments. The voltage protocol was repeated at 10 s intervals. A normalized peak I-V relation was generated using the peak current amplitude during the variable voltage step plotted as a function of voltage. The alternate hERG I-V relation was measured in control and in the presence of 1 μM febuxostat.

Voltage-Dependence of Inactivation i

The steady state inactivation- voltage relation was measured by calculating the ratio of the initial current to the steady state current in the 1 s variable voltage step at each step voltage from the Alternate I-V Relationship protocol.

Data Analysis

Data acquisition and analyses were performed using the suite of pCLAMP8.2 applications (Molecular Devices Corp., Sunnyvale, CA). Steady state was defined as a limiting constant rate of change with time (linear time dependence). The steady states before and after test article application were used to measure drug effects. Analysis ofECso data

To quantify febuxostat's agonist effects, a Hill equation of the following form was used:

WIcontroi = W{l+( EC₅₀/[Test])^N} + I₀, (1) where EC₅₀ is the concentration of febuxostat that produces half-maximal stimulation, I_max is the maximum stimulation value, I₀ is the initial, control current, [Test] is the concentration of febuxostat, Iτest/Icontroi is the ratio of steady state channel current amplitudes in test and control solutions and N, the Hill coefficient, is a measure of cooperativity. IfN is fixed at 1, equation (1) becomes a simple one-to-one binding model for current stimulation.

Analysis of frequency-dependence of febuxostat ItERG modulation

Data for analysis of frequency-dependence (use-dependence) were normalized to the peak current at -50 mV of the first pulse and to the peak current at +60 mV of the second pulse in each train of pulses and normalized data from trains at each frequency were pooled to construct average time courses.

Analysis of voltage dependence ofhERG activation

Voltage dependence of activation was fit with a single Boltzmann distribution of the form:

Iτ_ai_l(V)/Iτ_ai_{l M}ax = l/{l+e-(^V"Vl/2)^/Kv} (2)

Where Iτ_aii_(V) is the peak tail current elicited by the variable voltage V activating step in the steady state I-V relation protocol. Iτ_aii_Max was calculated as the average of the peak values for currents during voltage steps to 60, 70 and 80 mV. Vy₂ and K_v are the midpoint potential and the exponential slope factor for this Boltzmann distribution. Analysis of voltage dependence ofhERG inactivation

An equation similar to equation (2) was used to fit the voltage dependence of inactivation with a single Boltzmann distribution of the form:

(Ipeak(V)-Is_teady(V))/IPeak₍V) = l/{ l+e(V-V_1/2)/K_v}₅ (₃) where Is_teady(v₎ is the current at the end of the 1 s variable voltage step when steady state inactivation is attained for potentials greater than -80 mV in the alternate I-V relationship and Ip_eak(V) is the current at the beginning of the 1 s step at each voltage V. For potentials equal to or less than -80 mV, Isteady(v) was the extrapolated value of the current at the beginning of the 1 s variable voltage step. The extrapolated value was obtained by fitting a single exponential function to the decaying phase of the current transient. All initial measures for inactivation were relative to the inactivation present at -80 mV (value of

This relative measure was renormalized so that the asymptotic value for channel availability at negative potentials was 1. The best fit value OfVy₂ and K_v was determined by nonlinear least squares fitting. Vy₂ and K_v are the midpoint potential and the exponential slope factor for this Boltzmann distribution. The term on the left of the equal sign is the channel availability and channel inactivation is defined as "1 - channel availability".

Results

HEK293 cells have an endogenous delayed rectifier current that overlaps the heterologously expressed hERG currents at positive potentials. In order to characterize the agonist effects of febuxostat at positive potentials, CHO cells were used to heterologously express hERG channels (CHO/hERG), since untransfected CHO cells have only small time-independent background currents over the range of potentials at which febuxostat modulated hERG channel activity as observed in the above HEK/hERG example. Agonist effect of febuxostat on CHO/hERG current at +20 mV

When KERG was expressed in CHO cells, febuxostat again produced a voltage- dependent increase in KERG currents that was prominent during the +20 mV step but much more reduced during the -50 mV step. Sample CHO/hERG voltage-clamp I-T records acquired during control and after application of 1 μM febuxostat are shown in Figure 5. The time course of the agonist action of febuxostat application, measured as the maximum current at +20 mV in consecutive records acquired at 0.1 Hz, was comprised of an initial rapid increase in current that rose to a maximum during the first 1- 2 minutes followed by a slow decline to a smaller steady state (maintained) current that was established after at least 3 minutes of febuxostat application. The time course in two cells of the peak current evoked by the +20 mV voltage step before (Control 1 and Control 2) and following application of 1 μM (Figure 6) and 0.1 μM (Figure 7) febuxostat both show the initial and steady state agonist responses and washout of the effect at 0.1 μM. Summary statistics for the initial current increase (Table 4) and for the steady- state component (Table 5) measured from the time course of normalized peak currents at +20 mV showed the agonist effect febuxostat was concentration dependent. The concentration-response relations for the initial and steady state response components measured from the time course of peak currents at +20 mV gave EC₅₀ values of 0.003 for the initial component (Figure 8) and 0.070 μM for the steady-state component (Figure 9).

Table 4: Initial current increase at +20 mV followin febuxostat a lication

^a Mean fraction of current (Iτ_est/Ic_ontroi) at each febuxostat concentration, standard error of the mean (SEM), and number of observations (n) for each febuxostat concentration. Table 5: Steady-state current increase at +20 mV followin febuxostat a lication

^a Mean fraction of current (Iτ_est/Icontroi) at each febuxostat concentration, standard error of the mean (SEM)₅ and number of observations (n) for each febuxostat concentration.

Effect of febuxostat on CHO/hERG current at -50 mV (tail current)

Summary statistics for the peak tail current (Table 6) and tail current at the end of the two-second step to -50 mV (Table 7) confirmed that the agonist effect of febuxostat retained the voltage sensitivity identified in HEK/hERG cells. The measurements presented in Tables 4 - 7 were obtained from the same set of CHO/hERG cells. The effects of febuxostat on hERG peak tail currents during the -50 mV voltage step were too small to fit EC₅₀ values. Application of the positive control (60 nM terfenadine) blocked CHO/hERG peak tail currents by 76 ± 5% (Table 8), as expected and similar to block of HEK/hERG by terfenadine (see above in Example 1).

Table 6: Peak tail current increase at -50 mV followin febuxostat a lication

^a Mean fraction of current (Iτest/Ico_ntroi) at each febuxostat concentration, standard error of the mean (SEM), and number of observations (n) for each febuxostat concentration. Table7: End tail current increase at —50 mV following febuxostat ap lication.

^a Mean fraction of current (Iτ_est/Icontroi) at each febuxostat concentration, standard error of the mean (SEM), and number of observations (n) for each febuxostat concentration. End tail currents were measured at the end of a two second voltage step to -50 mV.

Table 8: Fraction of CHO/hERG current after a lication of terfenadine

Frequency- or use-dependence of the effect with febuxostat on CHO/HERG current hi experiments to measure frequency-dependence of the agonist effect with 1 μM febuxostat, enhancement of CHO/hERG peak current measured at +60 mV and -50 mV was not observed at 0.3 Hz stimulus repetition frequency but was pronounced at 3 Hz frequency (Figures 10 and 11). These results indicate that agonist effect of febuxostat was frequency or use-dependent. The effect may be more pronounced at higher frequencies.

Febuxostat modulation of CHO/hERG voltage gating parameters

The families of current traces from one cell, as an example, analyzed to produce the steady state I-V relation in Figure 12 are shown superimposed in Figure 13. The steady-state current- voltage (I-V) relation was measured in three cells before and after exposure to febuxostat (Figure 12). The maximum increase of CHO/hERG current by 1 μM febuxostat occurs at +10 and +20 mV. This result is consistent with the pronounced effect on current at +20 mV and the relative lack of effect on current at -50 mV seen in Figures I₅ 2 and 5. The steady state conductance- voltage (G-V) relation (Figure 14) was constructed from measurements of the peak tail current at voltages less than +60 mV normalized by the average of peak tail current measured at +60, +70 and +80 mV. The values for the midpoint potential (Vm) were 0.9 and -2.1 mV in the absence and presence of febuxostat, respectively. The slope factors (K_v) were 9.9 and 9.8 mV per e-fold change during equilibration with control and 1 μM febuxostat, respectively. The differences in the absence and presence of febuxostat for the V^₂ and K_v values were insignificant and small, indicating that the agonist effect of febuxostat is not the result of a simple shift in the voltage dependence of hERG activation to more negative potentials.

The families of current traces from a CHO/hERG cell, as an example, analyzed to produce Figure 15 are shown superimposed in Figure 16. The fully-activated I- V relation (Figure 15) shows the agonist effect of febuxostat developed at +60 mV was abolished by repolarizing to potentials of -60 mV or less. The stimulation persists through the potential range where inactivation gating causes rectification of the fully activated I-V relation (positive to -50 mV). This is consistent with the reduced agonist effect seen in peak tail current measurement during negative potentials.

The families of current traces from a CHO/hERG cell, as an example, analyzed to produce Figure 17 are shown superimposed in Figure 18. The channel availability- voltage relationship (Figure 17), like the normalized G-V relationship, changes little in response to application of febuxostat. Channel inactivation is equal to "1 - channel availability". The gating parameter values for the midpoint potential Vy₂ in the absence and presence of 1 μM febuxostat were -67.6 and -67.3 mV, respectively. The values for K_v were 27.9 and 29.6 mV per e-fold change for control and 1 μM febuxostat, respectively. These results suggest that a simple voltage shift in channel availability to more positive potentials does not explain the agonist effect of febuxostat. The instantaneous 1- V relation (Figure 19) measures the conductance properties of open hERG channels. This measurement is based on the ability of the voltage clamp to change membrane potential much faster than channels gate, so that the number of channels open at -80 mV remains unchanged for a short time after the change in membrane potential specified in the voltage protocol. The current measured immediately after the change in voltage from -80 mV is free of channel gating and reflects only the conductance properties of the open hERG channel. Like many potassium channels, the I- V relation measured for the open channel is linear over the voltage range measured and all the rectification associated with the hERG channel is derived from voltage-dependent gating. There is a small increase in the slope of the instantaneous I- V for hERG channels indicating a small increase in the number of open channels in the presence of febuxostat, but the linearity of the I-V relation is unaffected, demonstrating that febuxostat does not alter the conductance properties of open hERG channels.

In summary, febuxostat had an agonist effect on hERG currents measured with the whole cell patch clamp method in CHO cells stably expressing cloned hERG channels. The agonist effect was voltage dependent and more pronounced at positive potentials with maximal effect occurring at +10 and +20 mV. The agonist response was biphasic with an initial maximum and a smaller steady state effect during maintained application of febuxostat to the cells. The concentration dependence of the initial maximum and steady state effects yielded EC₅₀ values of 0.003 μM and 0.070 μM, respectively.

The agonist effect of febuxostat is voltage dependent and occurs rapidly. Closed channels are much less stimulated by febuxostat. Open channels and depolarized potentials are required for stimulation and the stimulatory effect equilibrates with open channels rapidly. The agonist effect of febuxostat is not the result of a simple shift in the voltage dependence of hERG activation to more negative potentials, nor is a simple voltage shift in channel inactivation to more positive potentials. Febuxostat does not alter the conductance properties of open hERG channels. While not wishing to be bound any theory, the inventors believe that one possible mechanism for the agonist effect consistent with these observations could be to increase burst duration of hERG channel openings.

EXAMPLE 3: Effect of febuxostat on action potentials and prolongation of action potential duration induced by rf/-sotalol and ATX II in isolated cardiac Purkinje fibers

Material and Methods

Solutions and Chemicals

Chemicals used in preparation of experimental solutions were obtained from Sigma- Aldrich (St. Louis, MO) or Calbiochem (San Diego, CA) and were of ACS reagent grade purity. All solutions containing febuxostat were prepared in glass containers whenever possible. Test solutions of febuxostat were prepared daily by diluting stock solutions into a modified Tyrode's solution prepared fresh weekly and refrigerated, (composition in mM): NaCl, 131; KCl, 4.0; CaCl₂, 2.0; MgCl₂, 0.5; NaHCO₃, 18.0; NaH₂PO₄, 1.8; Glucose, 5.5. Before use, the Tyrode's solution was aerated with a mixture of 95% O₂ and 5% CO₂ (pH 7.2 at room temperature). Febuxostat concentrations were prepared by serially diluting a 1000 μM stock solution in Tyrode's solution. The Tyrode's solution was warmed to room temperature before preparing febuxostat or positive control solutions. Febuxostat solutions were prepared freshly no more than six hours before use and protected from light. Febuxostat was tested at concentrations of 10, 100, and 1000 nM in the Purkinje fiber assay.

J/-Sotalol (Sigma- Aldrich) is a potent β-adrenergic receptor antagonist with class III antiarrhythmic properties. The drug prolongs the cardiac action potential duration (APD) by selectively blocking the rapid delayed rectifier potassium current, IK_J. dl- Sotalol solutions were prepared fresh daily by directly dissolving the chemical into Tyrode's solution. ATX II (toxin II, Anemonia sulcata) was obtained from Calbiochem and is a toxic polypeptide component of sea anemone venom. ATX II acts specifically on voltage- gated Na⁺ channels of excitable membranes to induce persistent non-inactivating Na⁺ currents. These persistent Na⁺ currents cause APD prolongation. Test solutions were prepared by dilution with Tyrode's solution of a 1000-fold concentrated stock prepared in distilled water.

Purkinje Fiber Electrophysiology Fiber Preparation

Purkinje fibers were excised from canine ventricles by standard methods (Gintant et al., 2001). Briefly, 5-7 purpose-bred Beagle dogs (young adult female, Marshall Farms USA Inc., NY) were housed in AAALAC accredited facilities. On each test day a dog was anesthetized with sodium pentobarbital (30 mg/kg i.v.). The heart was rapidly removed through a left lateral thoracotomy, placed in a container with chilled, oxygenated, storage Tyrode's solution (8 mM KCl), and transported to ChanTest on wet ice. All usable free-running Purkinje fibers from both ventricles were removed along with their muscle attachments. The fibers were stored at room temperature in oxygenated standard Tyrode's solution (4 mM KCl) until use.

Electrophysiological Recording

Purkinje fibers were mounted in a glass-bottomed Plexiglas chamber (approximate volume, 1 ml) affixed to a heated platform, and superfused at approximately 4 ml/min with standard Tyrode's solution. The bath temperature was maintained at 37 ± 1° C using a combination of SH-27B in-line solution pre-heater, Series 20 chamber platform heater, and TC-344B dual channel feedback temperature controller (Warner Instruments, Inc., Hamden, CT). Bath temperature was recorded using a thermistor probe. Intracellular membrane potentials were recorded using conventional intracellular microelectrodes pulled from borosilicate glass capillary tubing on a Sutter Instruments P-97 horizontal puller (Sutter Instrument Co., Novato, CA), filled with 3 M KCl solution and connected via Ag-AgCl wire to a Warner Instruments IE 210 intracellular electrometer amplifier (Warner Instruments, Inc., Hamden, CT). Membrane potential was referenced to a Ag-AgCl wire electrode in contact with the Tyrode's solution via a 3 M KCl-agar bridge.

Action potentials were evoked by repetitive electrical stimuli (0.1-3 ms duration, approximately 1.5 times threshold amplitude). A bipolar, insulated (except at the tip) platinum wire electrode was used to deliver pulses generated by a Dagan Corp. S-900 photo-isolated, electronic stimulator (Dagan Corp., Minneapolis, MN). Analog signals were low-pass filtered at 20 kHz before digitization at 50 kHz with a DT3010 AD/DA board (Data Translation, Inc., Marlboro, MA), and stored on hard disk using a PC- compatible computer controlled by NOTOCORD-HEM 3.5 software (Notocord Systems SA, Croissy sur Seine, France).

Concentration-response and rate-dependence were determined by the following test procedure. Purkiηje fibers were paced continuously at a BCL of 2 s (equivalent to stimulation frequency of 0.5 Hz) during a stabilization period of at least 25 minutes before obtaining control AP responses. Only fibers with resting potentials more negative than -80 mV and normal AP morphology (APD₉₀ = 250-450 ms) were used. Acceptable fibers were stimulated continuously at BCL of 2 s for 20 minutes. At the end of this period, baseline APD rate- or frequency- dependence under control conditions was measured using stimulus pulse trains consisting of approximately 50 pulses at BCL of 2, 1 and 0.34 s (equivalent to stimulation frequency of 1 and 3Hz, respectively). After returning to BCL of 2 s, test solution at the lowest concentration was applied for 20 minutes to allow equilibration, and the stimulus trains repeated. The entire sequence (20 minutes of equilibration followed by three cycles of stimulus trains at decreasing BCL, a total of 23-minuts per cycle) was repeated at increased drug concentration cumulatively. The average responses from the last five recorded action potentials from each stimulus train were analyzed for each test condition. Purkinje Fiber Electrophysiological Response to Febuxostat

Three concentrations of febuxostat (10, 100 and 1000 nM) were applied cumulatively (e.g. three 23-minute exposure periods) to a group of four Purkinje fibers as outlined above to examine the effect of febuxostat on action potential parameters and rate-dependence of these effects.

Modulation of Purkinje Fiber Electrophysiological Response to Sotalol or ATX π by Febuxostat

Modulation of Purkinje fiber response to sotalol by febuxostat was assayed by measuring responses in fibers exposed to both compounds and comparing to the response to sotalol alone. In the sotalol alone group, sotalol at 50 μM was applied to four Purkinje fibers with exposure times in each fiber approximately the same as in the febuxostat test group (three 23-minute exposure periods). In the sotalol plus febuxostat group, sotalol at 50 μM was applied throughout the measurement periods and febuxostat at 100 and 1000 nM was applied during the second and third, respectively, 23-minute exposure periods.

In a similar series of experiments with ATX II, the responses of four Purkinje fibers to application of 20 nM ATX II alone for all three 23-minute exposure periods were measured. The experiment was repeated in seven fibers but with addition of 100 (exposure periods 1 and 2) and 1000 nM (exposure period 3) febuxostat to ATX II.

Data Analysis

Action Potential Analysis

Data were analyzed with the AP analysis module of Notocord-Hem version 3.5 and Microsoft Excel 2000. The following parameters were determined: RMP (resting membrane potential, mV), APA (action potential amplitude, mV), Vmax (maximum rate of rise V/s), APD₆₀ and APD90 (action potential duration at 60 and 90% repolarization, respectively, ms). Concentration-response data are presented relative to baseline before test article application. APD₆₀, APD₉0 and Vmax at each stimulus frequency are presented as percent change (Δ%) from baseline at each concentration. RMP and APA data are presented as absolute change in membrane potential (ΔmV).

Statistical Analysis

Data were reported as mean ± SEM. Pooled data were tabulated for each condition: control baseline, drug concentration and stimulus frequency. Changes in action potential parameters induced by febuxostat, 50 μM sotalol or 20 nM ATX II were evaluated using a two-tailed Student's t-test for paired samples to determine whether the means obtained during the drug-free control period are significantly different (PO.05) from those obtained after equilibration in each drug concentration. The effects of febuxostat on sotalol- or ATX II-induced changes in action potential parameters were evaluated by a Student's t-test comparing the data obtained in the presence of 20 nM ATX-II or 50 μM sotalol alone, and the data from time-matched experiments performed in the presence of one of these agents together with febuxostat. Statistical analyses were performed in Microsoft Excel 2000.

Results

Effect of febuxostat on action potential parameters

At BCL that simulates bradycardia (BCL=2s), the average change in APD₉₀ was -2.4 + 0.8%, -1.9 ± 0.7% and -6.8 + 3.7%, respectively, at febuxostat concentrations 10, 100 and 100O nM. (Table 9, Figure 20). At shorter cycle lengths of Is and 0.34s (simulating normocardia and tachycardia, respectively) the average change in APD₉₀ was -2.4 ± 1.0% and -2.0±1.0%, respectively, at 1000 nM febuxostat (Table 10 and 11). None of these small effects were statistically significant (PO.05), and also they are not considered to be biologically significant. As shown in Tables 12-14, febuxostat did not significantly change the maximum rate of rise (Vmax), action potential amplitude or resting potential amplitude at any concentrations or BCL regimens.

In summary, despite the fact that febuxostat is a hERG agonist, it had no effect by itself on action potential parameters. Table 9: Effect of febuxostat on action potential duration at 2 second BCL

319.7

15.6

BCL, basic cycle length. Δ%, Percent change from control values. NA, not applicable. Table 10: Effect of febuxostat on action potential duration at 1 second BCL

287.6

13.8

BCL, basic cycle length. Δ%, Percent change from control values. NA, not applicable. Table 11: Effect of febuxostat on action potential duration at 0.34 second BCL

208.2

5.3

Δ%, Percent change from control values. NA, not applicable. Table 12: Effect of febuxostat on resting and action potential amplitudes and maximum action potential rate of rise at 2 second basic cycle length

Febuxostat RMP APA Vmax

(nM) (mV) (ΔmV) (mV) (ΔmV) (V/s) (Δ%)

0 -89.8 ± 0.2 NA 109.8± 2.7 NA 349. l± 75.5 NA

10 -90.6 ± 0.6 -0 8 ± 0.6 109.2± 3.1 -0.7 ± 1.3 336.9± 79.1 -4.5 ± 5.7

100 109.8± 2.7 -0 7 ± 0.4 108.4± 3.4 1.5 ± 0.8 311. l± 76.4 -12.3 ± 4.0

1000 109.8± 2.7 0. 5 ± 1.5 101.3± 4.4 -8.5 ± 4.4 255.1± 44.1 -20.5 ± 5.8

Data are expressed as mean ± SEM from n= 4 fibers. Δ%, Percent change from baseline values; ΔmV, absolute change from baseline in millivolts; NA, not applicable; RMP, resting membrane potential; APA, action potential amplitude; Vmax, maximum action potential rate of rise.

Table 13: Effect of febuxostat on resting and action potential amplitudes and maximum action potential rate of rise at 1 second basic cycle length

Febuxostat RMP APA Vmax

(nM) (mV) (ΔmV) (mV) (ΔmV) (V/s) (Δ%)

0 -91.6 ± 0.8 NA 112.7± 2.4 NA 363.8 ± 90.6 NA

10 -92.3 ± 0.6 -0 .7 ± 1.3 112.0± 2.7 -0 7 ± 1. 2 357.7± 96.7 -3.0 ± 5. 3

100 -92.3 ± 0.5 -0 .7 ± 0.9 111.7± 3.1 -1 O ± O. 8 340.6± 101.5 -9.2 ± 5. 5

1000 -91.3 ± 1.2 0 3 ± 1.8 106.6± 2.6 -6 1 ± 2. 7 268.6± 57.3 -24.8 ± 4 .8

Table 14: Effect of febuxostat on resting and action potential amplitudes and maximum action potential rate of rise at 0.34 second basic cycle length

Febuxostat RMP APA Vmax

(nM) (mV) (ΔmV) (mV) (ΔmV) (V/s) ( Δ%)

0 -90.6 ± 0.8 NA 113.7± 1.7 NA 351.6± 89.5 NA

10 -92.3 ± 0.6 -1 7 ± 0. 6 114.2± 2.1 0.4 ± 0.8 360.1± 110.4 -1. 2 ± 5. 9

100 -92.0 ± 0.9 -1 .4 ± 1. 1 113. l± 2.5 -0.6 ± 1.5 347.9± 113.3 -5. 5 ± 5. 9

1000 -91.2 ± 1.0 -0 .6 ± 1. 3 110.3± 1.9 -3.4 ± 2.0 272.2± 64.2 -22 .0 ± 3 .2

Data are expressed as mean ± SEM from n= 4 fibers. Δ%, Percent change from baseline values; ΔmV, absolute change from baseline in millivolts; NA, not applicable; RMP, resting membrane potential; APA, action potential amplitude; Vmax, maximum action potential rate of rise. Febuxostat modulation of prolonged action potential duration induced by dl-sotalol and ATX π

In contract to febuxostat, under identical recording conditions, the positive control dl-sotdlol at 50 μM produced significant APD prolongation (Figure 21). The effect of the same concentration of d7-sotalol increased overtime during each 23-minute exposure period, reflecting a slow component of sotalol equilibration with Purkinje fiber tissue. At the end of the third 23-minute exposure period, there were APD₉₀ prolongation of 40.9 ± 8.8%, 34.8 ± 7.5% and 14.6 ± 7.2% at BCL of 2s (Figure 21, Table 15), Is (Table 16), and 0.34s (Table 17), respectively. Sotalol at 50 μM did not significantly change the maximum rate of rise (Vmax), action potential amplitude or resting potential amplitude at BCL 2s, Is and 0.34s. Figure 23 shows addition of 100 and 1000 nM febuxostat together with 50 μM ύ?/-sotalol did not change the prolongation of action potential duration at 2s, (Table 18, and Figure 23) or at Is and 0.34s BCL (Tables 19 and 20). Febuxostat together with sotalol did not change the maximum rate of rise (Vmax), action potential amplitude or resting potential amplitude.

Table 15: Effect of sotalol on action potential duration at 2 s basic c cle len th

Data are expressed as mean ± SEM from n= 4 fibers.

Δ%, Percent change from baseline control values. NA, not applicable. *Statistically significant difference between baseline control and sotalol mean values (P<0.05, t-test paired samples).

Table 16: Effect of sotalol on action otential duration at 1 s basic cycle length

Data are expressed as mean ± SEM from n= 4 fibers.

Δ%, Percent change from baseline control values. NA, not applicable. * Statistically significant difference between control and sotalol mean values (P<0.05, t-test paired samples).

Table 17: Effect of sotalol on action potential duration at 0.34 s basic cycle length

Data are expressed as mean ± SEM from n= 4 fibers.

Δ%, Percent change from baseline control values. NA, not applicable.

Table 18: Effect of febuxostat on sotalol-induced action potential duration prolongation at 2 s basic cycle length

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Data are expressed as mean ± SEM from n= 4 fibers.

Δ%, Percent change from baseline control values. NA, not applicable. *Statistically significant difference between control and sotalol mean values (P<0.05, t-test paired samples).

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Data are expressed as mean ± SEM from n= 4 fibers.

Δ%, Percent change from baseline control values. NA₅ not applicable. * Statistically significant difference between control and sotalol mean values (P<0.05, t-test paired samples). Table 20: Effect of febuxostat on sotaloϊ-induced action potential duration rolon ation at 0.34 s basic c cle len th

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Δ%, Percent change from control values. NA, not applicable.

Like sotalol, 20 nM ATX II induced significant APD prolongation, which was increased during each 23 -minute exposure period, reflecting a slow component of ATX II equilibration with Purkinje fiber tissue, hi addition, it elevated the plateau potential as well (Figure 22). At the end of the third 23-minute exposure period, ATX II at 20 μM induced a maximum prolongation of APDg₀ to 75.1 ± 8.1% at 2s BCL (Table 21, Figure 22), 46.4 + 6.2% at Is BCL (Table 22), and 13.6 ± 2.8% at 034s BCL (Table 23). Febuxostat at 1000 nM reduced the maximum ATX Il-induced prolongation OfAPDp₀ to 37.2 ± 3.6% (Δ% = -50% vs ATX II alone) at 2s BCL (Figure 24, Table 24), 27.7 ± 2.5% (Δ% = -40% vs ATX II alone) at Is BCL (Table 25), and 9.0 ± 1.2% (Δ% = -34% vs ATX II alone) at 0.34s BCL (Table 26). The blunting effect of 1000 nM febuxostat on ATX Il-induced prolongation was statistically significant at BCL 2s and Is (Figure 24). Febuxostat at 100 nM moderately shortened APD prolongation induced by ATX-II at all stimulus intervals tested. However, the effect was only statistically significant at BCL 0.34s (Figure 24). ATX II by itself or in association with febuxostat did not alter the maximum rate of rise (Vmax), action potential amplitude or resting potential amplitude.

Table 21: Effect of ATX II on action potential duration at 2 s basic cycle length

Data are expressed as mean =fc SEM from n= 4 fibers.

Δ%, Percent change from baseline control values. NA, not applicable. * Statistically significant difference between control and ATX II mean values (PO.05, t-test paired samples).

Table 22: Effect of ATX II on action potential duration at 1 s basic cycle length

Data are expressed as mean ± SEM from n= 4 fibers.

Δ%, Percent change from baseline control values. NA, not applicable. * Statistically significant difference between control and ATX II mean values (P<0.05, t-test paired samples). Table 23: Effect of ATX II on action potential duration at 0.34 s basic cycle length

Data are expressed as mean ± SEM from n= 4 fibers.

Δ%, Percent change from baseline control values. NA, not applicable. * Statistically significant difference between control and ATX II mean values (P<0.05, t-test paired samples).

Table 24: Effect of febuxostat on ATX Il-induced action potential duration rolon ation at 2 basic c cle len th

Mean 261.5 322.8

SEM 16.7 15.2

Mean

SEM

Mean

SEM

Δ%, Percent change from control values. NA, not applicable.

Table 25: Effect of febuxostat on ATX II-induced action potential duration prolongation at 1 basic c cle len th

Mean

SEM

SEM 16.4 5.9 18.4 3.8

Δ%, Percent change from control values. NA, not applicable.

Table 26: Effect of febuxostat on ATX It-induced action potential duration prolongation at 0.34 basic cycle length

Δ%, Percent change from control values. NA, not applicable.

In conclusion, febuxostat at 10, 100 and 1000 nM did not have any effects on action potential parameters by itself. Febuxostat had no effect on sotalol-induced action potential prolongation. However, febuxostat at 100 and 1000 nM dose-dependently shortened the ATX II-induced action potential prolongation.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The molecular complexes and the methods, procedures, treatments, molecules, specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. AU patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of and "consisting of maybe replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for shortening a QT interval in a patient suffering from QT prolongation, the method comprising the steps of: administering to a patient suffering from QT prolongation a therapeutically effective amount of at least one pharmaceutically acceptable human ether-a-go-go- related gene ("hERG") channel agonist wherein said at least one hERG channel agonist does not shorten the QT interval when administered to a patient that is not suffering from QT prolongation.

2. The method of claim 1, wherein the administration of the hERG channel agonist to the patient increases the currents of the hERG channel of said patient.

3. The method of claim 2, wherein the increase in the currents in the hERG channel caused by the at least one hERG channel agonist are voltage dependent.

4. The method of claim 3, wherein at least one hERG channel agonist increases the current of a hERG channel at a positive transmembrane potential.

5. The method of claim 4, wherein the positive transmembrane potential is between about +0.1 mV and about +50 mV.

6. The method of claim 5, wherein the positive transmembrane potential is between about +5 mV and about +30 mV.

7. The method of claim 6, wherein the positive transmembrane potential is between about +10 mV and about +20 mV.

8. The method of claim 1, wherein said patient is suffering from congenital prolonged QT syndrome.

9. The method of claim 1, wherein said patient is suffering from acquired prolonged QT syndrome.

10. The method of claim 1, wherein said patient is suffering myocardial ischemia, heart failure, diabetes or stroke.

11. The method of claim 1 , wherein the hERG channel agonist is at least one compound having the following formula:

wherein R₁ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁O haloalkyl; an unsubstituted or substituted Ci-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR, where n is an integer from 0 to 5; or NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group, wherein R₂ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR, where n is an integer from 0 to 5; or

NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group, wherein R₃ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-Ci₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR, where n is an integer from 0 to 5; or

NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C_1O alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 — membered cyclic amino group, wherein R₅ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR, where n is an integer from 0 to 5; or

NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group, wherein R₆ is

12. The method of claim 11, wherein the hERG channel agonist is selected from the group consisting of: 2-[3-cyano-4-(2-methylpropoxy)phenyl]-4-methylthiazole- 5-carboxylic acid, 2-{3-cyano-4-(3-hydroxy-2-meraylpropoxy)phenyl]-4-methyl-5~ thiazolecarboxylic acid, 2~[3 -cyano-4-(2-hydroxy-2-methylpropoxy)phenyl]-4-methyl-5- thiazolecarboxylic acid, 2-(3-cyano-4-hydroxyphenyl)-4-methyl-5-thiazolecarboxylic acid, 2-[4-(2-carboxypropoxy)-3-cyanophenyl]-4-methyl-5-thiazolecarboxylic acid and a pharmaceutically acceptable salt thereof.

13. A method of treating a patient suffering from myocardial ischemia, heart failure, diabetes or stroke, the method comprising the steps of: administering to a patent suffering from myocardial ischemia, heart failure, diabetes or stroke a therapeutically effective amount of at least one pharmaceutically acceptable human ether-a-go-go-related gene ("hERG") channel agonist wherein said at least one hERG channel agonist does not shorten QT interval when administered to a patient that is not suffering from QT prolongation.

14. The method of claim 13, wherein the administration of the hERG channel agonist to the patient increases the currents of the hERG channel of said patient.

15. The method of claim 14, wherein the increase in the currents in the hERG channel caused by the at least one hERG channel agonist are voltage dependent.

16. The method of claim 15, wherein at least one hERG channel agonist increases the current of a HERG channel at a positive transmembrane potential.

17. The method of claim 16, wherein the positive transmembrane potential is between about +0.1 mV and about +50 mV.

18. The method of claim 17, wherein the positive transmembrane potential is between about +5 mV and about +30 mV.

19. The method of claim 18, wherein the positive transmembrane potential is between about +10 mV and about +20 mV.

20. The method of claim 19, wherein the hERG channel agonist is at least one compound having the following formula:

wherein R₁ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-Cj₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR₅ where n is an integer from 0 to 5; or

NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 — membered cyclic amino group, wherein R₂ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR, where n is an integer from 0 to 5; or

NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C₁₀ alkyl, aryl or aralkyl group, a hydroxy! group; a unsubstituted or substituted C₁-C₁₀ alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C₁₀ alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group, wherein R₃ is a hydrogen; a carboxyl; a halogen atom; a nitro group; a cyano group; a formyl group; an unsubstituted or substituted C₁-C₁₀ alkyl; an unsubstituted or substituted C₁-C₁₀ haloalkyl; an unsubstituted or substituted C₁-C₁₀ alkoxy; an unsubstituted or substituted hydroxyalkoxy;

OR;

S(O)_nR, where n is an integer from 0 to 5; or

OR; S(O)_nR, where n is an integer from 0 to 5; or

NRR'; where R or R' is each independently a hydrogen, a unsubstituted or substituted C₁-C₁₀ alkyl, aryl, aralkyl, alkylcarbonyl, arylcarbonyl or aralkylcarbonyl group or where R and R' taken together with a nitrogen atom bonded thereto form an unsubstituted or substituted 5- to 7- membered heterocyclic ring; or COR" where R" is a unsubstituted or substituted C₁-C_1O alkyl, aryl or aralkyl group, a hydroxyl group; a unsubstituted or substituted C₁-C_1O alkoxy, aryloxy, aralkyloxy, an amino group, an unsubstituted or substituted C₁-C_1O alkyl amino, an unsubstituted or substituted aryl amino group, a substituted or unsubstituted aralkyl amino group, or a 5- to 7 - membered cyclic amino group, wherein R₆ is

R₇ is hydrogen, a C₁-C₄ alkyl, carboxyl, COO-Glucoronide or COO-Sulfate, a C₁- C₅ alkoxycarbonyl, carbamoyl or C₁-C₄ alkyl aminocarbonyl group; and R₈ is hydrogen, a C₁-C₄ alkyl, carboxyl, COO-Glucoronide or COO-Sulfate, a C₁-C₅ alkoxycarbonyl, carbamoyl or C₁-C₄ alkyl aminocarbonyl group.

21. The method of claim 20, wherein the hERG channel agonist is selected from the group consisting of: 2-[3-cyano-4-(2-methylpropoxy)phenyl]-4-methylthiazole- 5-carboxylic acid, 2-[3-cyano-4-(3-hydroxy-2-methylpropoxy)phenyl]-4-methyl-5- thiazolecarboxylic acid, 2-[3-cyano-4-(2-hydroxy-2-methylpropoxy)phenyl]-4-methyl-5- thiazolecarboxylic acid, 2-(3-cyano-4-hydroxyphenyl)-4-methyl-5-thiazolecarboxylic acid, 2-[4-(2-carboxypropoxy)-3-cyanophenyl]-4-methyl-5-thiazolecarboxylic acid and a pharmaceutically acceptable salt thereof.